ABSTRACT
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 possesses several promising pharmacological activities, such as hepatoprotective effects, and antiinflammatory, antioxidant, or anticancer activities. OBJECTIVE: The objective of the present work is to establish cell suspension culture of P. fruticosa, investigate the influence of several factors such as plant growth regulators and carbon source on cell growth, and determine their oleanolic acid content. METHODS: Cell culture was established 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 220 rpm. The culture was then incubated at 25±2ºC with a shaking speed of 120 rpm in the period of 12 h daylight at a light intensity of about 6.75 µmol/m2/s. Cell growth was measured by fresh and dry biomass at 16 h 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-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.
Subject(s)
Araliaceae , Oleanolic Acid , Saponins , Triterpenes , Cell Culture Techniques , Oleanolic Acid/pharmacologyABSTRACT
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, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. 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 the transgenic centella plant. The 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 transformed into centella plants by the 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.
Subject(s)
Bacterial Toxins/genetics , Biolistics/methods , Centella/genetics , Enterotoxins/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Plants, Genetically Modified/genetics , Animals , Bacterial Toxins/immunology , Bacterial Toxins/metabolism , Centella/metabolism , Enterotoxins/chemistry , Enterotoxins/immunology , Enterotoxins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/immunology , Escherichia coli Proteins/metabolism , Gene Expression , Hot Temperature , Plant Extracts , Plants, Genetically Modified/metabolism , TriterpenesABSTRACT
BACKGROUND: Fibrinolytic enzymes, such as Nattokinases from Bacillus species are known to degrade the fibrin blood clots. They belong to serine protease group having commercial applications, such as therapeutic agents and functional food formulation. OBJECTIVE: The present study reports some characteristics and fibrinolytic activity of serine protease from B. subtilis C10 strain that was isolated from shrimp shell. METHODS: Extracellular enzyme from B. subtilis C10 culture was harvested and partially purified by ammonium sulphate precipitation. Fibrinolytic activity of the enzyme was determined by zymography and measured by spectrophotometry with fibrinogen and thrombin used as substrates. The optimal temperature and pH for fibrinolytic activity were studied in the range of 31-43ºC and 5-10, respectively. The thermal and pH stability of enzyme was studied by incubating enzyme for 30 min in the same range of temperature and pH as above. The effect of some metal ions and reagents on fibrinolytic activity of enzyme was evaluated by concentrations of 5 mM and 5%, respectively. RESULTS: Zymogram analysis indicated the presence of four fibrinolytic enzymes with molecular weights of approximately 69, 67, 39 and 36 kDa. The optimal temperature and pH for enzyme activity were 37°C and 9, respectively. The thermal and pH stability ranged from 35-39°C and 8-10, respectively. Fibrinolytic activity reached a maximum value of about 400 U/mg protein after 16 h of C10 strain culture. Enzyme has been drastically inhibited by PMSF and SDS, and partially inhibited by EDTA, while Triton X-100 has significantly increased enzyme activity. Effects of ions such as Mg2+, Ca2+ and Mn2+ on enzyme were negligible, except Cu2+ and Zn2+ have strongly decreased its activity. CONCLUSION: Results from the present study suggested that enzyme obtained from B. subtilis C10 could be serine protease that has a high fibrinolytic activity up to about 400 U/mg protein at the most appropriate temperature and pH of 37ºC and 9. This activity can be improved up to 142% by incubating enzyme with 5% Triton X-100 for 30 min.
