Background: Oleanolic acid is an oleanane triterpene found in many plant species all over the world. This compound is also a major saponin in leaves of Polyscias fruticosa and possess several promising pharmacological activities, such as hepatoprotective effects, and anti-inflammatory, antioxidant, or anticancer activities. Objective: The objective of the present work is to establish cell suspension culture of P. fruticosa, investigate influence of several factors such as plant growth regulators and carbon source on cell growth, and determine their oleanolic acid content. Method: Cell culture was carried out by using 2 g fresh weight of 30 day old friable callus derived from in vitro stem segment in 50 mL of liquid medium with a shaking speed of 120 rpm. Culture was then incubated at 25±2ºC with a shaking speed of 120 rpm in the period of 12 h daylight at light intensity of about 6.75 µmol/m2 /s. Cell growth was measured by fresh and dry biomass at 16h day. Oleanolic acid content was determined using HPLC analysis. Results & Discussion: The study results showed that MS medium containing 2% sucrose as a carbon source, supplemented with 1 mg/L 6-benzylaminopurine and 0.5 mg/L 2,4-D dichlorophenoxyacetic acid was the most appropriate growth medium. Cell biomass and oleanolic acid content reached the highest values of 0.43 g dry weight/flask and 25.4 mg/g dry weight, respectively. Conclusion: These results indicated the potential production of oleanolic acid, a compound with high pharmacological value, from P. fruticosa cell culture.
Background: Heat-labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. Objective: In the present study, we want to produce LTB protein which encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as a antigen in next studies. Methods: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in transgenic centella plant. Expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. Results: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. Conclusion: The s-ltb gene that was successfully transformated into centella plants by biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.
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