Chitosan Elicitation for Enhancing of Vincristine and Vinblastine Accumulation in Cell Culture of Catharanthus roseus ( L . ) G . Don

Catharanthus roseus (L.) G. Don is an important herbal plant. There are two important alkaloids, vinblastine and vincristine, use in anti-cancer drugs. In this study production of the two alkaloids was enhanced in C. roseus cell cultures, in a Murashige and Skoog (MS) liquid medium supplemented with 1.5 mg/L 2,4-D, 0.5 mg/L kinetin and 30 g/L sucrose, by adding 0, 50, 100, 250 or 500 mg/L medium molecular weight chitosan or chitosan derived from shrimp shell. After 14 days of culture, the cell suspension at stationary phase in the 100 mg/L medium molecular weight chitosan could produce the highest amounts of vinblastine and vincristine at 4.15 and 5.48 μg/mg cell dry weight, respectively. At the same time, the controls (0 mg/L chitosan) produced the two alkaloids at only 2.43 and 2.49 μg/mg cell dry weight, respectively. For chitosan from shrimp shell, it was found that 100 mg/L chitosan could lead to the highest quantity of 4.09 μg vinblastine/mg cell dry weight. The highest amount of 5.47 μg vincristine/mg cell dry weight was obtained when 250 mg/L chitosan was added.


Introduction
Catharanthus roseus (L.) G. Don is commonly known as the Madagascar periwinkle (Gajalakshmi et al., 2013).It synthesized terpenoid indole alkaloids were used in medicine such as vinblastine and vincristine (Mujib et al., 2014).These alkaloids have commercially important chemotherapy drugs (Zhou et al., 2010).Normally, vinblastine and vincristine could be produced very low approximately 0.0003% of 2.56% total alkaloid content (Shukla et al., 2006), Although, C. roseus has turn into one of the best studied herbal plants, in the use of cell suspension cultures for the production of valuable secondary metabolites.
Plant cell suspension culture technologies can be established for production of secondary metabolites.In vitro production of secondary metabolites in plant cell suspensions cultures has been reported from various medicinal plants (Zhao et al., 2005).Several strategies, such as manipulating the nutrient, optimizing the culture conditions, feeding of precursor, environmental stress and elicitation, can be applied in order to substantially increase the yields of secondary metabolites in plant cell cultures (Bourgaud et al., 2001;Zhang et al., 2004).Enhancement of secondary metabolites by elicitation is one of the few strategies recently finding commercial application (Namdeo, 2007).
Elicitors are molecules that stimulate defense or stress induced responses in plants.On the basis of their nature, elicitors can be divided into 2 types such as biotic and abiotic (Sreedhar et al., 2009).Both biotic and abiotic elicitors are known to stimulate synthesis of secondary metabolite in plant cells as a result of their defensive reaction against pathogen attack (García-Brugger et al., 2006).The use of both elicitors to activate product formation has become an important develop strategy and has been very useful in reducing the process time required to achieve high product concentrations and increasing product quantity (Cai et al., 2011).Natural elicitors include polysaccharides from insect and fungal pathogen such as chitosan (Brasili et al., 2014) are frequently used in a many plant cell suspension cultures for efficient induction of pharmaceutical secondary metabolites.
Chitosan is the major component of exoskeletons of insects, crustacean and fungal cell wall (Yin et al., 2010).As a natural substance, chitosan mimics the effects of some pathogen to stimulate plant secondary metabolites production Chitosan h in cell cult elicitation the feasibl chitosan el

Plant M
A cultivar length, we for 15 min

Callus
The shoot They were

Elicito
In the pres were disso the solutio

Elicita
The cell su supplemen medium m Each expe 25±2 °C u
Individual  on of e cell 7 day ht.In ned at nt, in after ences

Effect of Chitosan Elicitors on Vinblastine and Vincristine Accumulation
The accumulation of vinblastine and vincristine from suspension cells were found to be stimulated by using two types of chitosan as elicitor stimulants.For medium molecular weight chitosan, it was found that the concentration of 100 mg/L was the most effective concentration, enhancing vinblastine and vincristine accumulation at 4.15 and 5.48 µg/mg cell dry weight respectively.,The molecular weights of 250, 500 and 50 mg/L were found to produce vinblastine at 3.8,5 2.96 and 2.68 µg/mg cell dry weight, respectively, and produce vincristine at 5.20, 3.91 and 3.66 respectively.,The controls produced vinblastine and vincristine only 2.43 and 2.49 µg/mg cell dry weight, respectively (Table 2).
For chitosan from shrimp shell, the most effective concentration for vinblastine induction was 100 mg/L.It led to the yield of vinblastine at 4.09 µg/mg cell dry weight.For vincristine induction, the most effective concentration was 250 mg/L.It could produce the highest amount of 5.47 µg vincristine /mg cell dry weight (Table 3).When comparing of both chitosan efficiencies on enhancing vinblastine and vincristine accumulation in cell suspension, it was found that there was no difference in the accumulation of vinblastine and vincristine.

Discussion
The present study was carried out to investigate the effect of chitosan on vincristine and vinblastine in cell suspension in C. roseus.We could enhance products of vincristine and vinblastine in response to chitosan elicitation.In this study, we found the distinction in the accummulation of vinblastine and vincristine during different development phases.It was found that both alkaloid were highly accumulated in the stationary phase because the substances were accumulated in vacuole and stationary phase had a large of vacuole than the other phase (Roytrakul & Verpoorte, 2007).
For the chitosan as a stimulant, chitosan is known to elicit activities leading to a variety of defensive responses in host plants to microbial infections, including the accumulation of phytoalexins, pathogen related proteins, callus formation and accumulation of secondary metabolite (Yin et al., 2012).This study found that the most effectivechitosan concentrations for inducing vinblastine and vincristine in C. roseus cell culture were 100 to 250 mg/L because itinduced programmed cell death and hypersensitive-associated responses in plants cell when concentration increased (Vasil'ev et al., 2009) or chitosan toxicity to the living cells or might be due to the phytotoxic action of vinblastine and vincristine on the cells (Amborabé et al., 2008).These results also agreed with the studies in Plumbago indica root cultures of Jaisi and Panichayupakaranant (2016).They found that treatment with chitosan at concentrations of 150 mg/L to 14 days old culture increased the production of plumbagin (13.08 mg/g dry weight) by up to 6.6-fold compared to the level of an untreated root culture (1.97 mg/g dry weight).The cell culture of Withania somnifera of Sivanandhan et al. (2012) reported that chitosan at 100 mg/L stimulated higher production of all withanolides about 323.85 mg/g dry weight when compared to control (19.05 mg/g dry weight).Ahmed and Baig (2014) showed that chitosan elicitors at 125 mg/L lead to 8-fold (7,982 µg/g dry weight) higher psoralen accumulation in Psoralea corylifolia L. cell culture over control cells (945 µg/g dry weight).

Conclusion
This is the first report of the effects of chitosan on vincristine and vinblastine yields in cell suspension cultures of C. roseus.The observation suggests that chitosan efficiently enhances vincristine and vinblastine yields especially when stimulated with 100 mg/L chitosan medium molecular weight.The identification of sign of chitosan in biosynthesis could be a very effective reach for large-scale development of both alkaloid yields for pharmaceutical industry.
represented mean±S.D.The different letters within the same column showed significant differences at P  0.01 analyzed by LSD.** = significant difference at P  0.01.Table 3. Vinblastine and vincristine accumulation in C. roseus cell suspension culture with chitosan from shrimp shell (12 days after culture) Chitosan (mg/L) Vinblastine 1 (µg/mg cell dry weight) Vincristine 1 (µg/mg cell dry weight) represented mean±S.D.The different letters within the same column showed significant differences at P  0.01 analyzed by LSD.** = significant difference at P  0.01

Table 2 .
Vinblastine and vincristine accumulation in C. roseus cell suspension culture with medium molecular weight chitosan (12 days after culture)