Industrial production, estimation of VINCRISTINE AND VINBLASTINE.pptx

Industrial production, estimation

1. INDUSTRIAL PRODUCTION , ESTIMATION, AND UTILIZATION VINCRISTINE AND VINBLASTINE

2. INTRODUCTION Plant materials have been used in the treatment of malignant diseases for centuries Vincristine and Vinblastine are two anticancer drugs obtained from vinca. Very successful higher plant materials used in cancer chemotherapy are the alkaloids of Catharanthus roseus. Research on this plant, the Madagascan periwinkle, was stimulated by its mention in folklore. The search for naturally derived anticancer agents not as a cure for cancer, but in the treatment of diabetes. No hypoglycaemic activity was detected, but treated test animals became suscepctible to bacterial infection, and this led the researchers to undertake extensive examination for possible immunosuppressive principles causing these effects

3. Vinca rosea,Catharanthus , Madagascar periwinkle,Barmasi,Sadabahar ,Vinca etc SYNONYMS It is obtained from the whole plant of Catharanthus roseus belonging to family Apocyanaceae . BIOLOGICAL SOURCE ALKALOID ( Bis- indole) CLASS OF COMPOUND It is indigenous to Madagascar. GEOGRAPHICAL SOURCE Widely distributed throughout warm regions . Commercial supplies of the drug are obtained from both wild and cultivated plants. Produced in various locations, including Africa, India, Thailand, Taiwan, eastern Europe, Spain, USA and Australia.

4. Sources of Vincristine and Vinblastine Dark green coloured glossy leaves A number of other Catharanthus species have been investigated and found to contain vindoline-type alkaloids; some (e.g. C. longifolius , C. trichophyllus and C. lanceus ) contain dimeric alkaloids similar to vincristine and vinblastine

5. CONSTITUENTS OF Catharanthus roseus About 150 alkaloids have now been isolated from C. roseus; some, for example, ajmalicine, lochnerine , serpentine and tetrahydroalstonine . Particular interest is a group of about 20 bisindole alkaloids which contains those having antineoplastic activity(neutropenic, depressing immune system ): Leurocristine (vincristine ) and Vincaleukoblastine (vinblastine) Vinblastine is produced by coupling of the indole alkaloids catharanthine and vindoline, both of which occur free in the plant. Vincristine is structurally similar to vinblastine, but has a formyl (CHO) group rather than a methyl( CH 3 ) on the indole nitrogen in the vindoline-derived portion. Because these alkaloids are only minor constituents of the plant (vincristine is obtained in about 0.0002 % yield from the crude drug ), large quantities of raw material are required and chromatographic fractionations are extensively employed in the isolation procedures. In addition, there is a growing demand for vincristine rather than vinblastine, but the plant produces a much higher proportion of vinblastine. Fortunately, it is now possible to convert vinblastine into vincristine either chemically, or via a microbiological N-demethylation using Streptomyces albogriseolus .

6. INDUSTRIAL PRODUCTION

7. Purification

8. Cell lines to enhance yield In efforts to improve the production of alkaloids, cell cultures of C. roseus have received considerable attention .Success has been achieved in obtaining total alkaloid yields corresponding to 0.1–1.5% dry weight cultured cells, but cultures produced catharanthine and tabersonine , and not vindoline, so lacked one of the essential precursors for formation of the bisindole alkaloids. A similar problem arises with transformed root cultures although the feeding of loganin alone at the early stationary phase has been shown to increase the ajmalicine production 2.3-fold and the serpentine 1.8 fold when compared with control cultures; catharanthine levels are unaffected by a single feed of the precursor. Research is still necessary to find means of inducing the production of the useful alkaloids Ellicitors used are : Acetylsalicylic acid =Increased production of tumour cell suspensions (505%), total phenolics (1587%), furanocoumarins (612%), anthocyanins (1476%)

9. IDENTIFICATION TESTS Sr no. Reagent used Chemical composition Observation 1 Mayer’s reagent Potassium Mercuric Iodide Solution Cream coloured ppt 2 Dragendroff’s reagent Potassium Bismuth Iodide Solutiom Reddish Brown ppt 3 Wagner’s reagent Iodine Potassium Iodide Solution Reddish Brown ppt 4 Hager’s reagent Picric Acid Yellow coloured ppt 1. Qualitative Chemical tests of alkaloids

10. 2. TLC Method Stationary Phase Precoated Silica gel GF 254 Mobile Phase Toulene : Ethyl Acetate : Benzene (6:3:1) Visualization Iodine chamber / UV at 366 nm   R f value 0.36 vincristine 0.48 vinblastine SAMPLE PREPARATION : 1mg extract + 10 mL ethanol STANDARD PREPARATION : 1mg of standard vincristine & vinblastine + 5 mL ethanol

11. ESTIMATION 1. HPTLC METHOD STATIONARY PHASE Precoated silica gel aluminium plates 60 F254 MOBILE PHASE Toulene : methanol : diethylamine (8.75: 0.75:0.5)(by vol.) DETETCTION UV at 307nm(vincristine) UV at 225nm(vinblastine) SCANNING SPEED 20mm/s R f value 0.39(vincristine) 0.49(vinblastine) Sample Preparation: 50 g Vinca leaves boiled for 2 hours → mixed with alcoholic KOH → dried at 100 °C. 2 g leaves sonicated with 4 mL methanol for 20 min → extracted with 150 mL methanol using Soxhlet for 6 hours. Methanol extract shaken with dilute H₂SO₄ (3 portions × 5 mL). Acid extracts combined and filtered. Alkaloids precipitated by adding ammonia → filtered and dried. Precipitate dissolved in methanol (200 mg/mL). Standard Preparation: 10 mg vincristine + 10 mg vinblastine in 10 mL methanol (1000 µg/mL).

12. 2. HPLC METHOD COLUMN Chromolith performance RP-18e MOBILE PHASE Acetonitrile : (0.1M) Phosphate buffer (glacial acetic acid;3.5pH) 21:79 DETETCTION UV 254 nm FLOW RATE 1.2mL/min Injection volume 20 20 µL R t value (Retention time) 16.75min(vincristine) 26.93min(vinblastine) Sample Preparation: Powdered leaves extracted thrice with 90% ethanol. Extract concentrated, diluted with water, acidified with 3% HCl, washed with hexane. Aqueous layer basified (pH 8.5) with ammonia → extracted with chloroform. Chloroform extract washed, dried, concentrated, redissolved in methanol. Standard Preparation: 0.25 mg/mL each of vincristine and vinblastine in methanol .

13. UTILIZATION DOSE: Vincristine sulphate : 10-30g/kg of body weight Vinblastine:100 g/kg of body weight

14. MARKETED PREPARATION s

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