Development of a Reverse-Phase Hplc Method for the Quantification of Cryptolepine in the Dry Roots of Cryptolepis Sanguinolenta
Cryptolepis sanguinolenta is a slender, thin-stemmed climbing shrub with orange-coloured juice in the cut stem which is distributed throughout the west coast of Africa. The aqueous root extract is a popular antimalarial in West African ethnomedicine. It is commonly known in Ghana as Ghana quinine or “Nibima” (Twi language). The dry roots of Cryptolepis sanguinolenta were obtained from Center for Scientific Research into Plant Medicine (CSRPM), Akropong-Mampong. Roots were authenticated by comparing with herbarium sample at the Department of Pharmacognosy, KNUST. The major alkaloid in the roots of Cryptolepis sanguinolenta, cryptolepine, was isolated by means of gravity column chromatography of the total alkaloidal extract followed by preparative TLC (Mobile phase: ethylacetate:methanol:ammonia (35%) 80:15:5) using a reference cryptolepine(CLP-R) sample. The authenticity of the isolated cryptolepine was confirmed by determining the UV, Infrared, 13C and 1H Nuclear Magnetic Resonance (NMR) and mass spectrometric data and the assignments were in agreement with those reported by by Grellier et al., (1996) and Dwuma-Badu, et al., (1978). As a step towards efforts to standardize and to ensure quality in herbal preparations containing extracts of the roots of Cryptolepis sanguinolenta, a simple reverse-phase HPLC method with UV detection was developed and validated to quantify the levels of cryptolepine in the roots of the Cryptolepis plant. An isocratic elution of extractable cryptolepine in methanolic extract of dry roots of Cryptolepis was performed using methanol: water (90:9) modified with trifluoroacetic acid(TFA,98%) to a pH of 2.4 as mobile phase set at flow rate of 1ml/min on a Hichrom Zorbax C8 Column (5micron 15cm×4.6mm id) and cryptolepine monitored at 366nm. The average retention time of cryptolepine was found to be 2.723±0.069 min(n=8). The method developed was found to be precise since the intra-day and inter-day precision were <2% RSD in the concentration range of 1.02μg/ml-10.2μg/ml. Since the coefficient of correlation(r2) of 0.9976 for the regression line of peak area(y) against % v concentration of cryptolepine(x) was greater than 0.995, the method was said to be linear in the concentration range. The method was also found to be robust since deliberate alteration of pH, flow rate and wavelength of detection in the range of 2.4±0.2units , 1±0.3ml/min and 366±2nm respectively did not affect the precision of the method. Hence, it could also be inferred that the method was accurate. The Limit of detection(LOD) and Limit of Quantitation(LOQ) of the method were found to be 0.574μg/ml and 1.740μg/ml respectively. The developed method was used to determine the percentage content of extractable cryptolepine in the roots of the Cryptolepis plant and it was found to be 2.763±0.185% w/w (n=9). This quantity of cryptolepine is the highest reported so far in the roots of the Cryptolepis plant.
A Thesis Submitted In Partial Fulfilment Of The Requirements for the Degree Of Master of Science In Pharmaceutical Analysis and Quality Control.