CATHARANTHUS ROSEUSBIOTECHNOLOGY

FOR IMPROVING

ALKALOIDS YIEID

SEMINAR PRESENTED BY P.HANUMANTHA RAO

H.T NO:1007-14-517-008 DATE:-30/12/2015

Introduction:-• Family- Apocynaceae

• Dicotyledonous medicinal plant

• Distribution -Africa, America, Asia, Australia, Southern Europe

• Propagated by seeds or by stem

cutttings

MEDICINE SOURCES:-• C. roseus- studied extensively for its remarkable anticancer

constituents i.e; Vincristine & vinblastine

• Other compounds- ajmalicine and serpentine used for hypertension and related cardiac disorders

• LOW yield(V.cristine, V.blastine)

• These compounds is expensive

CHEMICAL STRUCTURES OF VINBLASTINE&VINCRISTINE

Some important anticancerous alkaloids isolated from cell cultures of C. roseus

Alkaloid Sources of isolationAjmalicine Callus, suspension, shoot, hairy rootSerpentine Callus, suspension, shoot, hairy root Tabersonine Callus, suspension Vindoline Suspension, shoot, crown gall Vindolinine Suspension, shoot3,4 anhydro vinblastine Shoot Leurosine Shoot Catharanthine Suspension, shoot, root Vinblastine Callus, crown gall, shoot, somatic embryo Vincristine Shoot, somatic embryo

OBJECTIVE:-In this seminar, the current development of secondary metabolite synthesis in C. roseus involving biotechnology has been discussed the various basic factors that influence yield.

OUTLINE :-approaches aiming at higher production of alkaloids

• Explant and other primary cultures• Culture of callus• Cell suspension• Shoot and root culture• Hairy root culture• In vitro embryogenesis and alkaloid synthesis• Bioreactors application• Alkaloids biosynthesis-metabolic engineering• Precursor feeding• Cryopreservation of cell lines• Synthetic&semi-synthetic alkaloid preparation

Explant and other primary cultures• Undifferentiated and differentiated tissues callus, suspension, shoot,

hairy root has improve the yield

• where several plant parts such as stem-node, root, leaf, flower, anther and seed(cultured as 1° explants sources)

Culture of callus

• Is an undifferentiated tissue

• Callus is a good source of 2° Metabolites & natural products

Cell suspension

• The cell suspension has often been used for alkaloid synthesis studies in C. roseus as the extraction of alkaloids is far more simple, it also uptake precursors and other nutrients more rapidly that favours enhanced accumulation of alkaloids.

Shoot and root culture

• A high accumulation of ajmalicine in multiple shoot cultures of C. roseus in MS medium high concentration of IAA (11.42 µM) and a low (2.22 µM) concentration of BA.• Vindoline synthesis in multiple shoot culture were detected. • Leaves and flowers of two different C. roseus (pink flower and

white flower) were variable amounts of vincristine and vinblastine were detected.• Yield variation is also observed in callus

Hairy root culture

• Higher concentration of ajmalicine and catharanthine was found in genetically transformed hairy root cultures.

• vindoline and catharanthine producing hairy roots were thin and slow in growth.

In vitro embryogenesis and alkaloid synthesis

• somaclones to be used as raw material for 2° metabolite production

• in vitro embryogenesis was earlier categorized into three distinct stages: (1) initiation and proliferation of SEs

(2) maturation (3) germination or plantlet conversion

Culture conditions:-• Medium composition

• Effect of PGRs

• Temperature

• pH

• Influence of light

• Oxygenation

Bioreactor application:-• Alkaloids production has conducted on CELL SUSPENSION CULTURE

using a ROTARY SHAKER• For mass scale –bioreactors, fermentars

vinblastinvinblastin

Alkaloids biosynthesis-metabolic engineering:-

Fig;Schematic steps

of indole alkaloid synthesis pathway

Precursor feeding:-• precursors enhanced secondary metabolites synthesis

• These compounds used as precursors L-tryptophan, loganin, loganic acid, secologanin

• The amino acids added to culture media for enhanced production of

indole template based alkaloids

Biotransformation:-• Catharanthus, biotransformation used for the synthesis of Vinblastine

• Use two precursor compounds Catharathine and Vindoline • The AVLB(3’4’-anhydrovinblastine) biotransformed to produce

vincristine- antitumour activity

Elicitor’s role on alkaloid accumulation

Fig. Model for the elicitor mediated signal transduction leading to activation of genes in

indole alkaloid synthesis pathways

Cryopreservation of cell lines:-• Superior cell-lines capable of high production of secondary

metabolites require screening and preserving

• To maintain secondary metabolite synthesis for long term using different pre-treatments, cryo-protectants, cooling and thawing processes.

• C. roseus LD50/ h13 cell lines were preserved for over 6 months in the presence of sorbitol and 5 % DMSO

Synthetic- and semi synthetic alkaloid preparation:-

• Vinblastine was produced semi synthetically by incubating the two monomers catharanthine & vindoline

CONCLUSION:-• Catharanthus roseus has been investigated clinically as it is an

important medicinal plant with immense anticancerous potentiality• A treasure of information about the biosynthetic pathways, the

genes, the enzymes and other factors’ role has now been known.• Studies clearly indicate that the synthetic pathway is very long and

branched.• The presence of important key steps are therefore many that may

complex the entire process of metabolic engineering, targeting to synthesize enhanced level of yield.

ACKNOWLDGEMENTS:-• I would like to thanks sincerely to HOD Dept of Genetics

• I would like to thanks sincerely to BOS Dept of Genetics

• I would like to thanks sincerely to Guide Dept of Genetics

• And I would like to thanks our faculty

BIBILOGRAPHY:-• References :-Abdul Mujib et al.,(2012) Catharanthus roseus alkaloids: application of biotechnology for improving yield. Review paper

Azra Ataei-Azim et al.,(2008) High in vitro production of ant-canceric indole alkaloids from periwinkle (Catharanthus roseus) tissue culture. 1Biological Department, Islamic Azad University of Saveh Branch, Iran♦WEBSITES:-Wikipedia.com

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