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Theses and dissertations (Applied Sciences)

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Subject: search.filters.subject.Hairy root cultures

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    Genetic transformation of Datura stramonium for the production of tropane alkaloids in hairy root cultures
    (2021) Ally, Farnaaz; Odhav, Bharti; Mohanlall, Viresh
    Datura stramonium is well known for its tropane alkaloid content, specifically atropine and scopolamine. With the demand of tropane alkaloids in the pharmaceutical sector, as anti cholinergic agents, anesthetic or its use in transdermal patches, an alternate method to obtain higher yields is required. In addition, much interest has been developed around the plant for its potential biological profile due to its use in traditional medicine. This study investigated the potential production of tropane alkaloids in plant tissue cultures and the biological activity in crude extracts of D. stramonium. Elicited hairy root cultures had displayed the highest atropine and scopolamine content as compared to in vitro leaf and callus culture extracts. The concentrations of atropine and scopolamine was 5.2 µg.mL-1 and 5.01 µg.mL-1 respectively. This was an increase of 1.57 and 1.21 fold respectively as compared to wild plant extracts. The biological profile of D. stramonium was investigated by determining its antimicrobial activity and anti-oxidant activity. The anti bacterial activity wsas investigated by the Kirby-Bauer disc diffusion assay. Root extracts displayed a broader spectrum of bactericidal activity (7 mm – 33 mm) as compared to leaf extracts. No fungicidal activity was displayed by both extracts. The anti oxidant activity was investigated using 2-diphenyl-1-picrylhydrazyl (DPPH) photometric assay. The activity displayed was dose dependant, i.e.; as the extract concentration increased, so did the anti oxidant activity. At a concentration of 1 µg.mL-1 , the radical scavenging capacity of root and leaf extracts was 64.4% and 31.7% respectively whereas, at the highest concentration of 1000 µg.mL-1 , the radical scavenging capacity of root extracts and leaf extracts was 98.4% and 45.8% respectively. The biosafety evaluation of leaf and root extract demonstrated a linear relationship between the concentration of extracts and percentage toxicity. As the concentration and exposure period of the extracts increased, the toxicity towards A. salina had also increased. These results indicated the potential of plant, cell and tissue culture systems of D. stramonium to produce higher yields of atropine and scopolamine through optimization, and the potential of anti oxidant and anti bacterial components present in its leaf and root.
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    Genetic transformation of Ceratotheca triloba for the production of anthraquinones from hairy root cultures
    (2012) Naicker, Leeann; Odhav, Bharti; Mohanlall, Viresh
    Many secondary metabolites that have been extracted from medicinal plants have been used as source of clinical drugs. However, the concentration of the active metabolites in plants is generally low. An attractive alternative for producing these important secondary metabolites is via plant tissue culture technology. More particularly, the genetic transformation of a plant tissue by Agrobaterium rhizogenes has been employed for producing high yields of secondary metabolites. In a previous study, three structurally similar anthraquinones: 9,10-Anthracenedione, 1-Hydroxy-4-methylanthraquinone and 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, and one steroid; Androst-5-ene-3, 17, 19-triol were isolated from the root extracts of C. triloba. The anthraquinones have shown to exhibit the anticancer mechanism which involves the inhibition of the activity of the human topoisomerase II enzyme that transforms supercoiled DNA to linear DNA. However, these anthraquinones were found in very low concentrations. Therefore, in this study we used plant cell and tissue culture systems (cell suspension, shoot and hairy root cultures) of C. triloba to increase the production of anthraquinones. Since the establishment of C. triloba in vitro plant systems required a source sterile explants, a protocol that involved the use of NaCIO was optimized for the sterilization and subsequent germination of C. triloba seeds which were micro-propagated into shoot cultures. These cultures provided a source explants for the induction of callus and hairy root cultures. The biomass of these plant cell and tissue cultures were subsequently bulked up for the extraction for anthraquinones and the yields were compared followed by fractionation and identification of the major compounds. The bioactivity of the fractions was evaluated by testing their cytotoxicity on cancer cells and anti-topoisomerase activity. The sterilization protocol that provided sterile seeds was found to be a solution of 30% NaCIO at an exposure time of 10 minutes. From the sterilized seeds shoot cultures were established on MS medium. The leaf explants of the shoot cultures were then used to induce callus cultures which subsequently were transferred to liquid medium whereby the total biomass of suspension cultures increased from 4 g to 134.18 g (wet weight). Also hairy roots cultures were established from stem explants with a low cell density inoculum of A. rhizogenes at a transformation efficiency of 73%. The growth of these hairy roots was slow in hormone free medium. This was overcomed with the use NAA and IAA which increased the xvii biomass from 1.03 g in the control culture (without hormone) to 23.91 g and 46.13 g respectively. An evaluation of the anthraquinones in the field root and hairy root, cell suspension and shoot culture extracts was carried out by using their Thin Layer Chromatography profiles and the High Performance Liquid Chromatography profiles as well as the standards, 9,10-Anthracenedione and 1-Hydroxy-4-methylanthaquinone. TLC analysis showed that the RF values of the fractions CT01 and CT02 matched the RF values of anthraquinones standards while HPLC analysis revealed that hairy root cultures supplemented with IAA (125.03 μg.mg-1) or NAA (98.25 μg. mg-1) produced a higher concentration of anthraquinones than the control culture (without hormone) (13.33 μg.mg-1), the field roots (33.51 μg. mg-1) and the shoot (3.23 μg.mg-1) and cell suspension cultures (13.17 μg.mg-1). Due to co-elution of the compounds in HPLC analysis, six fractions were isolated by Preparative Thin Layer Chromatography from the hairy root extract (obtained from the culture supplemented with NAA) and were coded as CT01, CT02, CT03, CT04, CT05 and CT06. The compounds in these fractions were identified by Electron Ionization-Liquid chromatography-Mass Spectroscopy and it was found that the hairy roots produced one acridone derivative; 5-Methoxy-2-nitro-10H-acridin-9-one, one naphthoquinone derivative; 2H-Naphto[2,3-b]pyran-5,10-dione,3,4-dihydro-2,2-dimethyl- and seven anthracenedione derivatives. These were: i) 5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, ii) 9,10-Anthracenedione, 2-methyl-, iii) 1-Hydroxy-4-methylanthraquinone, iv) 9,10-Anthracenedione, 2-ethyl-, v) 1,5-Diaminoanthraquinone, vi) Phenanthrene, 3,6-dimethoxy-9-methyl-, vii) 9,10-Anthracenedione, 1,4-dimethyl-. Fractions CT01 (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 9,10-Anthracenedione, 2-methyl- and 1-Hydroxy-4-methylanthraquinone) and CT02 (9,10- Anthracenedione, 2-ethyl-) were cytotoxic to the DU-145 cancer cell line at concentrations of 125 μg.mg-1 to 1000 μg.mg-1. These fractions also showed anti-topoisomerase activity as they inhibited the conversion of supercoiled DNA into linear DNA. In conclusion this is the first study that describes the transformation of C. triloba by A. rhizogenes mediated transformation and compares the production of anthraquinones in C. triloba hairy roots to the field roots, shoot and cell suspension cultures. This study has xviii indicated that hairy root cultures is a high-yielding production system for anthraquinones (5,8-Dimethoxy-2,3,10,10a-tetrahydro-1H,4aH-phenanthrene-4,9-dione, 1-Hydroxy-4-methylanthraquinone, 9,10-Anthracenedione, 2-methyl- and 9,10- Anthracenedione, 2-ethyl-) which could have the potential to be used in cancer therapy. In addition the discovery of C. triloba hairy roots having the biosynthetic capacity to synthesize five valuable anthraquinone derivatives that are not found the field roots has also been revealed.