Effects of Cor and US on Taxol Production of Taxus x Media

Effects of Cor and US on Taxol Production of genus Taxus x MediaSUMMARY OF PROPOSED investigate INCLUDING KEY GOALSFor scientifically qualified assessorsTaxol is a inhering diterpenoid alkaloid first isolated from the inner talk of pacific yew, Taxus brevifolia (Wani et al., 1971) and widely affair in the pharmaceutical persistence as the antitumor agents. However, number one taxol content, high cost of rootage and the end of yew trees spark a major environmental controversy and cause a problem to fulfil its growing demand. The application of set out life tissue burnish technique has appeared as an eco-friendly method and feasible burn down for commercial supply, nevertheless the production rate of this petty(a) metabolite are still very low. notwithstanding 10% of taxol secreted in the extra stallphoneular middling of T x media jailbreak closes (Wickremesinhe and Arteca, 1993). These problems prompted a great effort to discover and identify the alternative way of life to upraise the lot production of taxol in make cell coatings. In this study, elicitation utilise coronatine (Cor) and ultrasound (US) ordain be used for discount and transport of taxol. Cor is a toxin cookd by Pseudomonas syringae and sight act as a deeds harvest-feast regulator and consumes the defense lawyers mechanism in bes. Meanwhile, Low-intensity US is a physical or mechanical excitant that green goddess enhance the cellular transporter natural process and trigger the pathway of defence-related subaltern metabolites. It is hypothesized that the taxol ingathering in Taxus x media conclusion is high than potency (unelicited) and single discourse after tempered with Cor and low- energy US. The experiment pass on use two-stage shift culture and longitudinally young stalking as the explant. The effect of these two elicitors on biomass production and taxol production leave be analysed. At the end of this study, it is expected that the taxol produc tion and cell growth pull up stakes show positive result after elicited with Cor and low-energy US. This study testament be a probative endeavour in enhance the oust of taxol by dint of elicitation approach.For lay readersSecondary metabolites are chemics produced by plants and do not have any physiological functions, but use as flavour, food colours, fragrances and pharmaceutical study. Taxus species are one of the medicinal plants that whoremaster produce natural anti-cancer agents known as taxol. However, over exploitation of natural witnesss of taxol from bark, curtain call, roots and needles put yew trees as an endangered species. Besides, the low give up and fall growth of yew trees limit its potential as a viable groundwork of taxol. Thus, it is around(prenominal)-valuable to check alternative method to attach the taxol production in Taxus sp. The production of taxol from Taxus x media can be greatly enhanced through elicitation. Elicitation is a method to s timulate the gene expression of enzymes responsible for the synthesis of junior-grade coil metabolites. The substance used in stimulating the production of exuded compound is cal lead elicitor. Elicitor is formed inside or outback(a) of plant cells and imitate as a stress to activate the plant defence systems and cause qualitative and quantitative changes in the secondary winding metabolite content. two elicitors that result be used in this study are Coronatine (Cor) and low-energy ultrasound (US). Plants use Cor to build defensive compounds and as growth regulators. US, on the opposite hand can stimulated the cell membrane permeabilization and encourage the red ink of taxol to culture modal(a). Plant cell cultures lead be used to mobilise the secondary metabolite on demand by maintain the growth of plant cells in aseptic condition with appropriate supply of nutrients. It is expected that the accumulation of taxol treated with elicitors is higher than the untreated cultur e. This study will provide a good grounds about the effect of elicitors in activate the chemic defence system in plants and designately stimulate the production of taxol. look for Outcomes / ProductsWhat is the proposed outcome of your projectIt is expected that all treatments will give significant adjoin of taxol accumulation when compared with control. Combination between Cor and US film will improve the extracellular and cell-associated taxol production. The expected taxol production from the treatment of both Cor and US is 20 to 50% higher than control. Elicitation of Taxus x media using Cor and US also will enhance the cell growth. The use of fence in hanging culture will be one of the reasons that improve the accumulation of taxol and can be a good strategy for the continous production of taxol.Why is it important?Taxol is well known anti-cancer drug derived from natural source of Taxus species. However, direct extraction from yew trees has subjected them as an endange red species. On the other hand, the process of extraction and purification of taxol is also time consuming and not economically feasible. Thus, plant tissue culture is a promising approach to obtain this secondary metabolite. However, one of the main problems in cast up the taxol production is instability in the synthesis of secondary metabolite. In order to maximize the yield of taxol, the gang of biotic and abiotic elicitor can be a good strategy. Besides, the elicitation approach is also much economical proficient compare with other methods such(prenominal) as precursor feeding, phytohormone regulation and biotransformation. US is a honest but potent elicitor while Cor is expected has the similar biological activity as methyl jasmonate (MeJA), the most common elicitor used in stimulate secondary metabolites. The results from this study can provide a fundamental understanding of the effects of US in cabal with Cor on the production of secondary metabolite and plant cell cul ture. Understanding how Taxus x media cell culture react with these two elicitors can provide a a beneficial in link the growth rate with the production of taxol.DETAILS OF RESEARCH PROJECTIntroduction and BackgroundTaxus species are medicinal plants and members of the yew family family that can produce taxol, a chemotheraphy drug used widely in cancers treatment. FDA approval of taxol in 1992 and a series of success of taxol in clinical test for various cancers led to massive demand for this supple substance. The increment of demand was expected since taxol also useful to combact non-canceorus disorders, such as polycystic kidney malady (Woo et al., 1994). However, it is difficult to perform large-scale culture because of the difficulty to gain this active compound from natural source. Three mature, century-old yew trees would produce only 1 g of taxol. It is estimated that about 10 tons of bark or felling of 3000 trees was needed to yield 1 kg of purified taxol to treat 500 can cer patients. Besides, the natural source of taxol from yew trees is only about 0.01% to 0.06% (Sung et al., 2005). All these c oncerns have prompted extensive efforts in find the method that can subjoin the mass production taxol. Taxus x media is choose for this study since it is good starting materials for the taxol production via biotechnological technique. Besides, the amount of taxol produce by this cultivar is the highest among other Taxus species (Table 1).The elicitation approach has been one of the most promising strategies in improve the mass production of taxol and other secondary metabolites in cell cultures (Roberts ND Shuler, 1997). The idea of using elicitor arise from the defence pathway utilize by plant in deal with environmental stress, pathogenic microorganisms, insects and higher herbivorous predators by stimulate the production of stinking chemics like secondary metabolites. One of the most commonly studied and effective biotic elicitor is methyl jasmonate (M eJA). However, the report that Cor resemble the biological activity of the jasmonic demigod gives an idea that the mode of action between these two compounds could be similar. The chemical structure of Cor is more stable than MeJA because of the rigid cis-orientation in its bi-cyclic skeleton. This lead to the higher levels of induction in the production of secondary metabolites by plant cells treated with Cor when compare with those treated with natural JA. Although Cor stimulated paclitaxel production with less talent than MeJA, it resulted in higher paclitaxel excretion to the liquid phases of culture middling. Besides that, at that place are not many reports about the influence of Cor on the production of secondary metabolite in plant cell shift culture.Table 1 Content (wt %) of selected taxanes in different species of Taxus plants (Lin Lidong, 2003)The combination of biotic and abiotic elicitor had interactional effects to taxol production in many reports. Thus, it is exp ected that the combine use of Cor with a physical stimulus like US might append the yield of taxol in the Taxus culture. Generally, the abiotic elicitors use in experimental studies is chemical agents, heavy metals and biochemical agents like polysaccharides and organic acid. The use of US to ergonomics is relatively new and not many reports can be set in motion on the effects of US in increase the useful secondary metabolites. This simple and effective elicitor can activate cells or enzymes and induced plant defence metabolism by ultrasonic waves. High intensity US waves can bring detrimental effect to cells, enzymes and other biological materials. Meanwhile, low intensity US waves can change the cellular metabolism, improve the mass transfer of reagents and enhance the release of intracellular products from cultured cells by increase the membrane permeability. The use of US will not disrupt the culture process and allow continuous use of cells biosynthetic capacity (Kilby and Hu nter, 1990). US deserve an extensive research regarding its effects in plant secondary metabolite due to its wide application in biological field. The combination of Cor and US has great prospect and economic benefits in enhance the taxol production at low cost. Until now, there is no study or information about the effect of Cor and US and their combination on the Taxus species.Work which has led up to the projectThe production of taxol using cell cultures were first reported by Christen et al., in 1989. Cell open frame cultures can emit secondary metabolites into growth medium. For instance, the production of resveratrol in the medium was higher than in the cell (Zamboni et al., 2009). Work by Kajani et al., in 2010 on Taxus baccata L cell culture showed that 74.9 % of total taxanes was exuded to growth medium (5. 584 mg/ L). Roberts et al., in 2003 reported that suspension culture of Taxus canadensis secreted more than 90% of the total taxol to the medium after treated with cell wall enzymes. Cell culture of Taxus chinensis emitted 90% of the total paclitaxel in medium with chitosan (Zhang et al., 2007). Komaraiah et al., in 2003 reported that up to 70% of the plumbagin in Plumbago rosea cell culture were released into the medium. Taxol accumulate at high level (1.5 mg/L) in the second phase of growthPrevious research by J. Wu and L. Lin in 2003 on the application of US as a physical stimulus and MeJA in increase the mass production of taxol in Taxus chinensis becomes a fundamental reference for this study. Their study showed that the taxol yield was increased about 1.5- to 1.8-fold with 2 minutes US exposure once or twice during a 4 week culture period. Meanwhile, the total taxol after treated with MeJA increase about 5-fold. The percentage of extracellular taxol and cell-associated taxol also showed significant increase. The taxol production from combination between US and MeJA treatment was 20 to 50% higher than each(prenominal) of the treatment used al one. Another research by Wu and Lin in 2002 institute that low energy US can play a voice as abiotic elicitor to trigger the plant defence responses and induce the production of plant metabolite in cell cultures. Apart from that, US also can increase the cell membrane permeabilization to increase the efficiency of intracellular product release. In their previous study, they found that the combination of US stimulation and in situ resolving extraction in a Lithospermum erythrorhizan cell culture led to 2 to 3-fold increases in the yield of shikonin.Tamogami and Kodama reported in 2000 that Accumulation of flavonoid phytoalexins in rice occurred when treated with Cor. This shown that the effect of Cor on flavonoid production was greater than that of JA or its precursor, 12-oxo-phytodienoic acid (OPDA). Cor also gave positive result in the alkaloid production in Eschscholzia californica cell cultures (Haider et al., 2000). The previous work by also showed the greater increase in the accumulation of glyceollins, the phytoalexins of soybean (Glycine max. L.), in soybean cell suspension cultures after treated with Cor. Besides, the effect of Cor in induced the phytoalexin productin was greatly break in when compared with JA and MeJA. (Fliegmann et al., 2003 Lauchli et al, 2002).Aims of the projectTo study the biological and synergistic effects of Cor and US on the taxol production of Taxus x media cell culture.(d) Experimental design and methods to be used in investigating this problemCell culture shapingApproximately 1.0 cm long section of longitudinal young stem of Taxus x media will be wash with distilled water and knock off in 70% (v/v) ethanol for 30 to 60 seconds. later on that, the stem will be surface sterilized by immersion in 1.5% sodium hypochlorite with a few drops of Tween 20 for 20 to 30 minutes. After sterilization, the explants will be rinsed 3 times with sterile distilled water. Next, the longitudinally halved stem sections will be placed wi th the inner cut surface in contact with the solid Gamborgs B5 media to induce calli. The media supplemented/ fortified with 3.0% sucrose (w/v), 0.8% agar-agar (w/v), 1 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L 6-Benzyladenine (BA), at pH 5.5. All of the cultures will be placed in nighttime at 25oC for 25 days. To obtain a homogenous callosity, several subcultures of calli will be done every 3 weeks. The snow, friable and massive callus will be used as the inoculums for cell suspension culture.Cor treatmentThe same media without agar will be used for cell suspension culture. About 10 g of fresh weight of callus will be used as the inoculums and inoculated into 300 mL conical flasks containing carbon mL liquid growth B5 medium and placed on orbital shakers (120 rpm) at 25oC in darkness. 1 uM Cor will be added to the shake-flask culture on day 7 post inoculation since the Taxus cell culture is expected to be in the middle of a rapid growth phase, which is usually a favourable time for elicitor treatment in most plant cell cultures.3) US treatmentAn ultrasonic cleaning vat will be used to sonicate the cells in Erlenmeyer flasks from the shake-flask culture (Lin et al., 2001). The bath will had a fixed frequency of 38.5 KHz and variable power levels. The bath temperature wwill be maintained at 250.5oC. The culture will be sonicated at level 2 US power (corresponding to 13.7 mW/ cm3 in the culture) once (on day 5, 9, or 16) or twice (day 5 and day 9) for 2 min each time (total US energy of each exposure = power x period = 1.64 J/cm3). The US power and exposure period will be selected based on the study by Lin et al., (2001) and Lin and Wu in 2002.Determination of cell densityThe cells in the shake-flasks will be separated from the medium by filtration through a Whatman filter cover under vacuum. The wet cell mass will be rinsed soundly with deionized water and then dried at 50oC in an oven to constant weight.Taxol extraction and determination The taxol content in powdered dried cell and medium in the culture flasks will be extracted by methods previously set forth by Wu and Lin (2003). The dried cells will pulverized and suspended in 10 mL methanol, filtered and air-dried. After that, the cells will be re-dissolved in dichloromethane water (11, v/v) followed by centrifugation at 5000 rpm. Dichloromethane phase will be collected, air-dried and re-dissolved in 100 L methanol (HPLC grade) and filtered passing through a 0.45 m syringe filter, before being injected to HPLC. The taxol content in the extract solution will be quantified by HPLC system, equipped with a C-18 column of 25 x 4.6 mm dimension. Taxol will be eluted with a linear gradient of acetonitrile and water (45 55, v/v)/ at a feed rate of 1 mL/min with UV detection at 227 nm. Identification of taxol will be accomplished by comparison of storage times with authentic regulation.Statistical analysisThe results will be examine statistically by one-way ANOVA ana lysis and Duncan multiple align tests. The data will be presented as the mean value standard deviation (SD) of three independent experiments.