Srinivas Patnala

Interactions between Phytochemical Components of Sutherlandia Frutescens and the Antiretroviral, Atazanavir In Vitro: Implications for Absorption and Metabolism

PURPOSE African traditional medicinal plants, such as Sutherlandia frutescens have the potential to interact pharmacokinetically with the protease inhibitor class of antiretrovirals, thereby impacting on their safety and efficacy. The effects of extracts and phytochemical components of Sutherlandia frutescens, on the in vitro absorption and metabolism of the protease inhibitor, atazanavir were thus investigated. METHODS Aqueous and methanolic extracts of Sutherlandia frutescens were prepared by freeze-drying of hot water and methanol decoctions of Sutherlandia frutescens plant material respectively, whilst crude triterpenoid glycoside and flavonol glycoside fractions were isolated by solvent extraction and subsequent column chromatography. Atazanavir was quantitated in the absence or presence of these compounds as well as commercially available purported constituents of Sutherlandia frutescens, namely, L-canavanine, L-GABA and D-pinitol, after a one hour co-incubation in Caco-2 cell monolayers and human liver microsomes. RESULTS The triterpenoid and flavonol glycoside fractions were found to be present in the aqueous and methanolic extracts of Sutherlandia frutescens and were shown to contain the sutherlandiosides and sutherlandins known to be present in Sutherlandia frutescens. The aqueous extract and D-pinitol significantly reduced atazanavir accumulation by Caco-2 cells, implying a decrease in atazanavir absorption, whilst the opposite was true for the triterpenoid glycoside fraction. Both the aqueous and methanolic extracts inhibited atazanavir metabolism in human liver microsomes, whilst enhanced atazanavir metabolism was exhibited by the triterpenoid glycoside fraction. CONCLUSIONS The extracts and phytochemical components of Sutherlandia frutescens influenced the accumulation of atazanavir by Caco-2 cells and also affected ATV metabolism in human liver microsomes. These interactions may have important implications on the absorption and metabolism and thus the overall oral bioavailability of atazanavir.

A capillary zone electrophoresis method for the assay and quality control of mesembrine in Sceletium tablets

The Sceletium plant has been reported to contain psychoactive alkaloids, specifically mesembrine, mesembrenone, mesembrenol and other related alkaloids. Sceletium is marketed through health shops and on the internet as dried plant powder and as pharmaceutical dosage forms. The objectives of this research was to develop and validate a capillary zone electrophoresis (CZE) method to identify five alkaloids and quantitatively determine the content of the important alkaloid, mesembrine in Sceletium tablets. Since reference standards of the relevant alkaloids are not commercially available for use in quality control of Sceletium products, it was necessary to isolate and characterize an appropriate analytical marker for use in the assay and additional markers for fingerprinting by CZE. The separation of the relevant alkaloids was carried out by CZE on a 50cm effective length, fused silica capillary tubing (50microm i.d.x360microm o.d.) using 50mM of sodium dihydrogen orthophosphate dihydrate at pH 1.5 as the background electrolyte and monitored at a UV wavelength of 228nm. All the marker alkaloids were found to be well resolved and were identified in the plant material and in commercially available Sceletium tablets based on the relative migration times (MTs) with respect to quinine hydrochloride that was used as an internal standard. The method was validated and used to assay the mesembrine content in Sceletium tablets. Calibration curves were found to be linear over the entire concentration range of 2.5-80microg/ml with correlation coefficients >0.995. The accuracy was found to be 92.5 and 104.5% (R.S.D.<3.5%) and the R.S.D.s of the inter-day precision at low, medium and high tablet masses were better than 0.9, 2.2 and 2.7%, respectively. The recoveries were all within the range of 91.8 and 105.8% (R.S.D.<8.5%) and the limit of quantitation (LOQ) and limit of detection (LOD) values were found to be 2.5 and 1.5microg/ml, respectively.

HPLC Analysis of Mesembrine-Type Alkaloids in Sceletium Plant Material Used as An African Traditional Medicine

PURPOSE Sceletium plant species have been reported to contain psychoactive alkaloids, specifically belonging to mesembrine-type alkaloids. Sceletium is presently marketed through health shops and on the internet as dried plant powder and as pharmaceutical dosage forms and purported to be useful in the treatment of psychological disorders. However, there are no validated analytical methods and reference standards of the relevant alkaloids are not commercially available for use in the analysis and quality control of Sceletium products and dosage forms. Hence, the objective of this research was to isolate and characterize appropriate analytical markers for use in the assay and as well as markers for fingerprinting by high performance liquid chromatography (HPLC). METHODS The separation of the relevant alkaloids was carried out on a C18 column and detected at a UV wavelength of 228 nm. The method was validated and used to assay the mesembrine-type alkaloids namely Δ(7)mesembrenone, mesembranol, mesembrenone, mesembrine and epimesembranol. RESULTS The calibration curves were found to be linear over the entire concentration range of 400-60,000 ng/ml with correlation coefficients >0.99. The accuracies of the relevant alkaloids were found to be between 94.8 and 103.6% with an inter-day relative standard deviation (RSD) of less than 2.8%. The precision studies showed inter-day RSDs of less than 3%. The recoveries were all within the range of 95 and 105% (RSD <4.5%) and the limits of quantitation (LOQ) and detection (LOD) were found to be 100 and 200 ng/ml respectively using the respective S/N ratios of 3 and 10. Conclusions. An HPLC method for the quantitative analysis of Δ(7)mesembrenone, mesembranol, mesembrenone, mesembrine and epimesembranol in Sceletium plant material has been developed and validated. This assay method can be applied for the quality control of Sceletium plant material which is used as an African Traditional Medicine for the treatment of psychological disorders.

Application of an Optimized Tape Stripping Method for the Bioequivalence Assessment of Topical Acyclovir Creams

This study indicates the application of tape stripping (TS) for bioequivalence (BE) assessment of a topical cream product containing 5% acyclovir. A TS method, previously used successfully to assess BE of topical clobetasol propionate and clotrimazole formulations, was used to assess BE of an acyclovir cream (5%) formulation as well as a diluted acyclovir formulation (1.5%) applied to the skin of healthy humans. An appropriate application time was established by conducting a dose duration study using the innovator product, Zovirax® cream. Transepidermal water loss was measured and used to normalize thicknesses between subjects. The area under the curve (AUC) from a plot of amount of acyclovir/strip vs cumulative fraction of stratum corneum (SC) removed was calculated for each application site. BE was assessed using Fieller’s theorem in accordance with FDA’s guidance for assessment of BE of topical corticosteroids. Adco-acyclovir cream (5%) was found to be BE to Zovirax® cream, where the mean test/reference (T/R) ratio of the AUC’s was 0.96 and the bioequivalence interval using a 90% confidence interval was 0.91–1.01 with a statistical power > 95%, whereas the diluted test product fell outside the BE acceptance criteria with T/R ratio of AUC of 0.23 and a 90% CI of 0.20–0.26. This study indicates that the data resulting from the application of this TS procedure has reinforced the potential for its use to assess BE of topical drug products intended for local action, thereby obviating the necessity to undertake clinical trials in patients.

Sceletium Plant Species: Alkaloidal Components, Chemistry and Ethnopharmacology

The genus Sceletium, classified under the Aizoaceae family, is indigenous to the Western, Eastern and Northern Cape province of South Africa. There are currently eight reported species divided into two main “types” with five species in the tortuosum and three in the emarcidum type. It has been observed that, in general, mesembrine‐type alkaloids such as mesembrenol, Δ7mesembrenone, mesembranol, mesembrenone, mesembrine and epime‐ sembranol as well as some non‐mesembrine type such as Sceletium A4, tortuosamine and joubertiamine occur in the tortuosum type; the emarcidum type is devoid of alkaloids. Morphological identification of species type presents a formidable challenge, where subtle differences are found in the secondary veins that branch off from the middle vein toward the leaf margin. In view of the fact that the plant contains a complex mixture of closely related compounds, in particular alkaloidal components, separation techniques and their application to evaluate specific chemical components are an important aspect which permits accurate characterization and quantification. In addition, the develop‐ ment of appropriate analytical methods for chemotaxonomic studies has provided valu‐ able information to confirm specific plant identity. Importantly, these methods are also required for the quality control of plant material used to manufacture complementary and traditional medicines containing Sceletium.