CATHARANTHUS ROSEUS MICROPROPAGATION & PRODUCTION OF ANTI-CANCER DRUGS

SUBMITTED BYP.HANUMANTHA RAO,MSc GENETICS II YR,ROLL NO:1007-14-517-008,OSMANIA UNIVERSITY.

INTRODUCTION;

Catharanthus is a perennial tropical medicinal plant belonging to the Family Apocynaceae which comprises eight species, seven endemic to Madagascar (C. coriaceus, C. lanceus, C. longifolius, C. ovalis, C. roseus, C. scitulus, C. trichophyllus), and one, C. pusillus, from India commonly known as the Madagascar periwinkle or rosy periwinkle. The species has long been cultivated for herbal medicine and as an 

ornamental plant. In Ayurveda (Indian traditional medicine) the extracts of its roots and shoots, though poisonous, is used against several diseases.( diabetes, malaria, and Hodgkin's lymphoma.)

vinca alkaloids were first isolated from Catharanthus roseus. The substances vinblastine and vincristine(it shows a strong antimicrobial activity )extracted from the plant are used in the treatment of leukemia and Hodgkin's lymphoma.

Specifically, C. roseus is a decorative and curing plant ,it producing more than 130 different terpenoid indole alkaloids (TIAs)

vinblastine and vincristine are commercial TIAs used in anticancer chemotherapy, and together with a number of related semi-synthetic compounds, are collectively named the Vinca alkaloids.

In addition, C. roseus also produces ajmalicine and serpentine, which are monoterpenic indole alkaloids used as anti-hypertensive and anti-neuro-inflammatory agents, yohimbine which is mainly used in treatments for erectile dysfunction, and vindolicine used for the development of antidiabetic therapeutics.

This plant is being exploited on a large scale on commercial basis for its medicinal property.

Due to low productivity and high production costs of these alkaloids by cultures of Catharanthus roseus, non-conventional methods have to be employed for better production.

Catharanthus roseus: micropropagation

Axillary node or shoot tip ↓

Rince with Teepol solN(2 Min),70%Ethanol(v/v 1 min)&0.1% HgCl2 (5min) ↓

Wash with water(2-4 times) ↓

Axillary bud were cultured on sterile M.S medium ↓ Shoot multiplication on MS medium supplemented with 1.0 mg/l BAP(A) 1.0 mg/l Kn(B) 2.0 mg/l NAA(C) 0.5 mg/l BAP+1.0 mg/l NAA(D) ↓ In vitro root induction on ¼ of MS medium supplemented with 5.0 mg/l IBA(E) ↓

4 weeks old rooted plant for hardening(F) ↓ well growing plant in green house(G)

Wash with tap water 5 min

Stage-

0

Stage-1

Stage-2

Stage-3

Stage-4S-0 Selection of mother plant and its

maintenance

S-1 Initiation & establishment of culture

S-2 Shoot multiplication (2.0 mg/l NAA)

S-3 root induction (¼ of MS with 5.0 mg/l IBA )

S-4 (F) rooted plant for hardening (G)well growing plant in green house.

Major stages involved in

micropropagation

High in vitro production of anti-canceric indole alkaloids from periwinkle (Catharanthus roseus) tissue culture

The periwinkle, Catharanthus roseus L. (G.) Don, produces several commercially valuable secondary metabolites including the anticancer agent, vinblastine, vincristine and the hypertension drugs, ajmalicine and serpentine.

These are dimeric indole alkaloids that are formed in vivo by condensation of vindoline and catharanthine.

Tabersonine, a key precursor of vindoline, has been reported occasionally in tissue cultures and only recently measured extensively in C. roseus hairy root cultures.

the ability of certain cell lines to accumulate high levels of catharanthine and serpentine indicates that they have a high potential for the production of some monoterpenoid indole alkaloids

vinblastine and vincristine can increase in new roots from tissue culture of C. roseus petioles of intact plants.

MATERIALS AND METHODSPlant material and induction of callus and roots cultures

Leaves of six week old seedlings, germinated in green house were used, and petiole segments (ca.2 cm) were cut out and used for initiation of tissue cultures.

↓ Petiole segments

↓ cultured on MS agar medium

↓ placed in a growth chamber

↓ sub cultured to MS medium

↓ The samples were used for alkaloid assay after 6 weeks.

sterilized by hypochlorite 5% for 45 min and were washed by sterile water

supplemented with naphthalene acetic acid (NAA 0.1mg/l) and kinetin (Kin 0.1mg/l) treatments

at dark period for first of two weeks and 24 h light period for second of 4 weeks at 35ºC.

0.1 mg/l of NAA supplemented with 0.1 mg/l Kin, 24 h light period&hormone free

Alkaloids extraction :- New callus, root-callus and roots from 3-5 g fresh weight were

homogenized (1 min in 60-100 ml ethanol) and were set in 60ºC for 10 min.

Following filtrated, alkaloids were extracted & purified . For purification, ethanolic extracts were dried by vacuum evaporator

at 60ºC and used. The resulting alkaloids extracts were dissolved in 1 ml ethanol used

for alkaloids assay. Alkaloids assay vindoline (vin), catharanthine (cat), vinblastine (vib), vincristine (vcr), ajmalicine (ajc) and serpentine (ser) of callus and roots analyzed by TLC and HPLC at 6 weeks after the initiation of each culture.TLC: 50µl alkaloids were applied onto thin layer chromatography (TLC) plates.HPLC In HPLC analysis, alkaloids were detected by retention time at 254 nm.

Table1 . Removing factor (RF) and standards alkaloids color reaction by TLC analysis. Color of alkaloids with UV (λ =366, λ =254) and ceric ammonium sulfate (CAS) reagent.

Table . Retention time and concentration for some standards alkaloids by HPLC analyze& the relative amount of indole alkaloids in the new organs were formed from petiole

Alkaloid Rf λ =366 λ =254 CASSerpentine 0.1 Blue Blue NonVincristine 0.25 Non Dark LavanderVinblastine 0.35 Non Dark LavanderCataranthine 0.75 Non Dark blueVindoline 0.85 Non Dark Dark-

LavanderAjmalicine 0.95 Apple-green Dark Apple-green

Alkaloid Retention time (min)

Concentration (mg)

petiole

Serpentine 20.525 2.5×10-³ 0.08Vincristine 24.2 5 2.5×10-³ 0.54Vinblastine 28.290 2.5×10-³ 0.085 Cataranthine 28.290 2.5×10-³ 0.07Vindoline 35.36 2.5×10-³ 1.81 Ajmalicine 41.42 2.5×10-³ 7.32

SMT’S PRODUCTION USING BIOREACTORS: A number of indole alkaloids are produced by in vitro cultures of

Catharanthus roseus Since C. roseus cells enlarge as they age, they may become more shear

sensitive, and this increased shear sensitivity may explains low production of secondary metabolites.

Due to this shear sensitivity of cells, several workers have adopted pneumatically agitated type bioreactors for culturing C. roseus cells

For the production of Vinblastine & Vincristine a two-stage culture system was used similar to that for the production of shikonin.

Since the growth of cells was low in the medium (designated as M-9) which supports high levels of indole alkaloids production

vinblastin vinblasti

n

cells are first grown in a growth promoting medium ↓ filtered and pumped the M-9 production medium is added

↓ filtration

↓ Indole alkaloids were extracted from plant cells

(designated as MG-5) for 9 days

cells are cultured for 14 additional days

VINBLASTINE & VINCRISTINE:- VINBLASTINE VINCRISTINE♣used to treat a number of types of cancer - Hodgkin's lymphoma, non-small cell lung cancer,bladder cancer, brain cancer, and testicular cancer 

 ♣also known as leurocristine♣used to treat a number of types of cancer-This includes acute lymphocytic leukemia, acute myeloid leukemia,Hodgkin's disease, neuroblastoma, and small cell lung cancer 

♣Vinblastine was first isolated in 1958 ♣Vincristine was first isolated in 1961 MEDICAL USES:♣Vinblastine is a component of a number of chemotherapyregimens, including  ABVD forHogkin lymphoma.♣It is also used to treat histiocytosis according to the established protocols of the Histiocytosis Association

(A)driamycin (B)leomycin(V)inblastine(D)acarbazine 

MEDICAL USES;♣Vincristine is delivered via intravenous infusion for use in various types of chemotherapy regimens.♣Its main uses are in non-Hodgkin's lymphoma as part of the chemotherapy regimen CHOP, Hodgkin's lymphoma  or the less popular Stanford V  chemotherapy regimen in acute lymphoblastic leukemia (ALL), and in treatment for nephroblastoma

1kg vinblastine costs around $US 20 million 1kg vincristine costs around US$ 94.07 million

MECHANISM OF ACTION VINBLASTINE&VINCRICRISTINE ↓ BINDS TO BEATA-TUBULIN(drug-tubulin complex) ↓ DISRUPTION OF MITOTIC SPINDLE ↓ CHROMOSOMES FAIL TO MOVE APART DURING MITOSIS ↓ METAPAHASE ARREST ↓ CELL DIVISION IS INHIBITED

Catharanthus roseus alkaloids: application of biotechnology for improving yield:

♣Vincristine and vinblastine - are two very powerful anticancerous compounds of terpenoidindole alkaloids Among other components, (Table 1) ajmalicine and serpentine are used in relieving hypertension and related cardiac disorders. The yield of these two compounds (vincristine and vinblastine) is very low, therefore is expensive.♣ This poses a serious challenge to all including biotechnologists as to improve the yield and to reduce the unit cost.♣ In biotechnology based alkaloid enrichment program, the following two approaches have currently been adopted: - (1) use of superior cell lines from various sources and optimization of cultural conditions/factors that control synthesis - (2) metabolic engineering and/or over expression of key enzymes

(1)Raw materials for improving yield of alkaloids(superior cell lines)♣Explant and other primary cultures ♣Shoot and root culture♣Culture of callus ♣Hairy root culture♣Cell suspension ♣In vitro embryogenesis and alkaloid synthesis

Table 1 Some important anticancerous alkaloids/derivatives 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, shoot Catharanthine Suspension, shoot, root Vinblastine Callus, crown gall, shoot, somatic embryo Vincristine Shoot, somatic embryo

(2) metabolic engineering and/or over expression of key enzymes to produce high yield alkaloids

16 Hydroxytabersonine

Deacetylvindoline

Vindoline

T16H

DAT

D4HLochnericine

Horhammericine

Serpentine

Vincristine,vinblastine

Fig;Schematic steps of indole alkaloid synthesis pathway

Therapeutic Properties/ Uses:of C.roseus:-1. Anti cancer property- anticancer alkaloids Vinblastine and Vincristine are

derived from stem and leaf2. Anti diabetic property- ethanolic extracts of the leaves and flower 3. Anti bacterial property- Crude extracts from different parts of the plant 4. Anti oxidant property- ethanolic extract of the roots5. Anti helminthic property- ethanolic extract of the concentration of 250 mg/ml6. Anti ulcer property- Vincamine and Vindoline alkaloids of the plant showed anti ulcer property. 7. Hyptensive property- Extract of leaves of th plant 8. Anti diarrheal property- ethanolic leaf extracts 9. Phyto remediation- Exposed C. roseus bioaccumulates heavy metal like cd etc., so it is used in phyto remediation. 10.Folkloric uses- juice of leaves is used as application to bee sting/ wasp sting(IND)

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

important medicinal plant with immense anticancerous potentiality.

Various in vitro techniques as micropropagation from existing and adventitious meristems or organ, tissue and cell cultures provide a bigger amount of Catharanthus roseus plant material for the isolation of mono- and dimeric indole alkaloids having therapeutic properties

The plant also possess various property such as anti cancerous, anti diabetic, anti helmintic, anti diarrheal, anti microbial etc. Hence most work could be done on the above plant to reveal the unknown mysteries which would help the need of the present pharmaceutical world.

C. roseus is an amazing chemical factory, producing more than 130 TIAs, some of which exhibit strong pharmacological activities