Five different methods were carried out for the preparation of the antineoplastic alkaloids from Catharanthus roseus powdered plant. Method IV depends on partition between solvents and the fifth one depends on adsorption chromatography. The highest percentage of total alkaloids have been obtained by method no. III, but the purest mixture was obtained from method no V, also the two target alkaloids (viz. vinblastine and vincristine) were found together in fraction (I). showed that, the maximum quantitative yield of vinblastine rich fraction was obtained from method III. Vincristine was obtained in a maximum quantitative yield from method I. Moreover, a qualitative and quantitative HPLC method was adopted for the determination of vinblastine and vincristine. The different methods applied for the preparation of the alkaloids were examined by HPLC and HPTLC techniques to evaluate the most reliable one.
Quantitative determination of alkaloidal fractions of C. roseus (VB & VC rich fractions)
High performance liquid chromatography HPLC and high performance thin layer chromatography HPTLC studies of different fractions; obtained from the five methods used in the preparation of total alkaloids; revealed the presence of vinblastine (Rf 0.54) in fractions B, D, F and fraction H while vincristine (Rf 0.21) was found in fractions C and E. whereas fraction I was found to contain both of vinblastine and vincristine as main constituents ().
All the fractions obtained from the five methods for preparing total alkaloids were subjected to HPLC analyses using isocratic elution with phosphate buffer solution of pH 6.5, acetonitril (55 : 45) as mobile phase, according to the conditions mentioned before and the results were shown in , these conditions showed difference in retention times of the target authentic alkaloids viz. vinblastine and vincristine of 3.771 mins.
From HPLC and HPTLC analyses of the total alkaloids prepared by the five methods, it was illustrated that fraction eluted from charcoal column by 70% methanol (K) gave mixture relatively pure fraction containing the target alkaloids vinblastine (Vb) and vincristine (Vc) with only six other alkaloids, these fraction was subjected to VLC (5g on mixed bed column of equi portions of silica gel g for TLC and neutral Alumina (250 g packed in glass column 120 × 5 cm, i.d.) eluted with chloroform containing increasing proportions of methanol with an increament of 2.5 %, resulted in ten fractions. Fraction eluted by chloroform /methanol 85 : 15 (v/v) was found to contain the target alkaloids with only two more contaminants. The collected fractions containing the target alkaloids (750 mg) were subjected to further purification using the centrifugally accelerated radial chromatography (chromatotrone) using silica gel 2mm thickness (type 7749, Merck, Darmstadt, Germany) eluted by chloroform : methanol 95 : 5 (v/v) with flow rate of 6ml/min. to give pure Vb and Vc. Their identity and purity were established by co-chromatography by TLC, HPTLC and HPLC with authentic samples.
In conclusion: charcoal column is very simple, easy, cheap, perfect and reliable for isolation of highly purified form of the total alkaloids especially Vb and Vc and proved to be very reproducible particularly for the production scale. Also, the results obtained from HPTLC analysis were promising and in the range of those obtained from HPLC.