A network pharmacology-based approach to explore the therapeutic potential of Sceletium tortuosum in the treatment of neurodegenerative disorders.

Sceletium tortuosum (SCT) has been utilized medicinally by indigenous Koi-San people purportedly for mood elevation. SCT extracts are reported to be neuroprotective and have efficacy in improving cognition. However, it is still unclear which of the pharmacological mechanisms of SCT contribute to the therapeutic potential for neurodegenerative disorders. Hence, this study investigated two aspects-firstly, the abilities of neuroprotective sub-fractions from SCT on scavenging radicals, inhibiting some usual targets relevant to Alzheimer's disease (AD) or Parkinson's disease (PD), and secondly utilizing the network pharmacology related methods to search probable mechanisms using Surflex-Dock program to show the key targets and corresponding SCT constituents. The results indicated sub-fractions from SCT could scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, inhibit acetylcholinesterase (AChE), monoamine oxidase type B (MAO-B) and N-methyl-D-aspartic acid receptor (NMDAR). Furthermore, the results of gene ontology and docking analyses indicated the key targets involved in the probable treatment of AD or PD might be AChE, MAO-B, NMDAR subunit2B (GluN2B-NMDAR), adenosine A2A receptor and cannabinoid receptor 2, and the corresponding constituents in Sceletium tortuosum might be N-trans-feruloyl-3-methyldopamine, dihydrojoubertiamine and other mesembrine type alkaloids. In summary, this study has provided new evidence for the therapeutic potential of SCT in the treatment of AD or PD, as well as the key targets and notable constituents in SCT. Therefore, we propose SCT could be a natural chemical resource for lead compounds in the treatment of neurodegenerative 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.

Investigation of Biowaivers for Immediate Release Formulations Containing BCS III Drugs, Acyclovir, Atenolol, and Ciprofloxacin Hydrochloride, Using Dissolution Testing.

The dissolution of several products containing Biopharmaceutical Classification System (BCS) class III drugs, acyclovir, atenolol, and ciprofloxacin hydrochloride, listed in the WHO essential drug list (EDL), was tested and compared with their respective comparator pharmaceutical products (CPPs) marketed in South Africa and India. US Pharmacopeia (USP) buffers of pH 1.2, 4.5, and 6.8 were used as dissolution media and tested using USP apparatus 2 at 75 rpm and 900 ml. Nine acyclovir products were tested, and only three dissolved very rapidly in all media; i.e., they showed a release of >85% in 15 min. Eight atenolol products tested were all very rapidly dissolving in all three pH media. Ten ciprofloxacin hydrochloride products were tested, and the results showed that only five products met the WHO biowaiver criteria. This study indicates that not all marketed products containing the same BCS III active pharmaceutical ingredient (API) in similar strength and dosage form are necessarily in vitro equivalent as per the WHO biowaiver criteria. Furthermore, selection and availability of an innovator product as CPP are important considerations that can affect the outcomes of such studies.

Medicinal use of Sceletium: Characterization of Phytochemical Components of Sceletium Plant Species using HPLC with UV and Electrospray Ionization--Tandem Mass Spectroscopy.

PURPOSE: Sceletium plants have been used for its medicinal properties for centuries. However, there is a wide range of Sceletium plant species in which various alkaloidal components such as ∆7mesembrenone, mesembrenol, mesembranol, mesembrenone, mesembrine hydrochloride, epimesembranol and, sceletium A4 differ between species. Hence, to ensure the quality of Sceletium products used as a medicine, it is imperative to identify the appropriate species using both botanical and chemical methods. The chemical approach to identify and characterize the phytochemical composition of a particular species facilitates the choice of species that will provide the purported therapeutic outcome. Hence, specific analytical methods to identify relevant constituents from complex matrices are necessary. Although HPLC-UV detection is commonly used to identify and estimate phytochemical content of medicinal plants, use of mass spectroscopy (MS) and tandem mass spectroscopy (MS/MS) can unequivocally confirm their presence/absence based on characteristic ions and fragmentation patterns. METHODS: The various alkaloidal components were characterized by electrospray ionization (ESI) MS and MS/MS using an ionizing medium of 0.1% ammonium hydroxide in water mixed with acetonitrile. Compounds were purified and characterized for use as reference standards to identify the relevant alkaloidal constituents of several Sceletium plant species using HPLC with on-line UV-MS detection. RESULTS: ESI-MS provided the [M+H](+) ions with respective m/z values that related to the respective molecular weights 287, 289, 291, 287, 289, 324 and 291 for the above mentioned alkaloids, whereas, ESI MS/MS provided the characteristic fragment ions to confirm the structural identity of the individual alkaloids and subsequently used to confirm the presence and/or absence of specific alkaloids in various Sceletium plant samples. CONCLUSIONS: Whilst HPLC-UV detection has been a widely-used conventional analytical technique for both qualitative and quantitative analyses, the results highlight the necessity of ESI-MS detection to avoid erroneous identification of phytochemical components, particularly with mesembrine-type compounds which have closely related chemical structures. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

In vitro dissolution of generic immediate-release solid oral dosage forms containing BCS class I drugs: comparative assessment of metronidazole, zidovudine, and amoxicillin versus relevant comparator pharmaceutical products in South Africa and India.

Biowaivers are recommended for immediate-release solid oral dosage forms using dissolution testing as a surrogate for in vivo bioequivalence studies. Several guidance are currently available (the World Health Organization (WHO), the US FDA, and the EMEA) where the conditions are described. In this study, definitions, criteria, and methodologies according to the WHO have been applied. The dissolution performances of immediate-release metronidazole, zidovudine, and amoxicillin products purchased in South African and Indian markets were compared to the relevant comparator pharmaceutical product (CPP)/reference product. The dissolution performances were studied using US Pharmacopeia (USP) apparatus 2 (paddle) set at 75 rpm in each of three dissolution media (pH1.2, 4.5, and 6.8). Concentrations of metronidazole, zidovudine, and amoxicillin in each dissolution media were determined by HPLC. Of the 11 metronidazole products tested, only 8 could be considered as very rapidly dissolving products as defined by the WHO, whereas 2 of those products could be considered as rapidly dissolving products but did not comply with the f 2 acceptance criteria in pH 6.8. All 11 zidovudine products were very rapidly dissolving, whereas in the case of the 14 amoxicillin products tested, none of those products met any of the WHO criteria. This study indicates that not all generic products containing the same biopharmaceutics classification system (BCS) I drug and in similar strength and dosage form are necessarily in vitro equivalent. Hence, there is a need for ongoing market surveillance to determine whether marketed generic products containing BCS I drugs meet the release requirements to confirm their in vitro bioequivalence to the respective reference product.

Comparison of in vitro release rates of acyclovir from cream formulations using vertical diffusion cells.

Acyclovir, indicated in the treatment of herpes labialis ("cold sores"), is formulated as semisolid topical dosage forms and marketed in numerous countries. Since the formulations of the various acyclovir products may differ from country to country, this study was undertaken to compare the in vitro release of acyclovir from various generic cream products available on the South African and Indian markets using the respective brand/innovator product as the reference product. The in vitro studies were carried out using vertical diffusion cells with a diffusional surface area of 1.767 cm(2) and various commercially available membranes. Normal saline was used as receptor fluid and the temperature maintained at 32 ± 0.5°C. The in vitro release comparisons were based on the recommendations described in the US Food and Drug Administration Draft Guidance for acyclovir ointment and the SUPAC-SS Guidance for non-sterile semisolid dosage forms. The release rates (slope) of the test (T) and the relevant reference product (R) were monitored and compared. The comparative release of acyclovir from the various generic formulations compared with the reference product was found to be within the limits of 75-133.33% with a 90% confidence interval. These experiments indicate that the generic acyclovir cream formulations exhibited release rates that were comparable to the innovator product and could be considered to be bioequivalent.

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.

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 RSD's 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.

Investigations of the phytochemical content of Sceletium tortuosum following the preparation of "Kougoed" by fermentation of plant material.

AIM OF THE STUDY: Sceletium plant species that contain alkaloids are claimed to have mood elevation and anti-anxiety properties, especially after the plant material has been fermented. The fermented preparation is locally known as "kougoed" or "channa" and has been emphasized and advertised for its increased potency when incorporated in commercial products. The aim of the study was to investigate quantitative and qualitative changes in alkaloidal content following fermentation of plant samples carried out under controlled conditions and also on pure mesembrine hydrochloride (MHCl). MATERIALS AND METHODS: Samples were prepared from the aerial parts of Sceletium tortuosum. Studies were also conducted on mesembrine hydrochloride (MHCl) in aqueous and methanolic solutions under similar conditions of exposure to sunlight as well as under ambient and elevated temperature (40+/-2 degrees C). Quantitative and qualitative changes in alkaloidal content were monitored by HPLC and LC-MS, respectively. RESULTS AND CONCLUSIONS: The initial fermentation study showed transformation of mesembrine to Delta(7)mesembrenone, where the content of the former decreased from a concentration of 1.33% to 0.05% whilst the latter increased from below its limit of quantitation (LoQ) to 0.11% on the 10th day. The experiments on pure MHCl revealed similar transformations in aqueous solutions whereas no change was seen in methanolic solutions. Sunlight and aqueous conditions appear necessary to facilitate the transformation, which was confirmed by the absence of such a transformation when solutions of MHCl were kept in the dark.

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.