Cyclotides : Tuning Parameters Toward Their Use in Drug Design

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned. An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method. Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m. One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile. Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action.

Cyclotides : Tuning Parameters Toward Their Use in Drug Design

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned. An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method. Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m. One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile. Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action.

Cyclotides : Tuning Parameters Toward Their Use in Drug Design

Cyclotides are plant proteins with a unique topology, defined as the cyclic cystine knot motif. The motif endows cyclotides with exceptional chemical and biological stability. They also exhibit a wide range of biological activities including insecticidal, cytotoxic, anti-HIV and antimicrobial effects. Hence, cyclotides have become potential candidates in the development of peptide-based drugs; either as scaffolds to stabilize susceptible peptide sequences or as drugs by their own right. In this thesis, important parameters that could be inputs toward this development have been tuned. An extraction protocol that can be extended to industrial scale production of the cyclotides from natural sources was developed; accordingly, a single maceration with hydroalcoholic solutions of medium polarity represented an optimum extraction method. Moreover, it was shown that investigating the cyclotide content of cyclotide-bearing plants from diverse environments is a promising approach for extending the knowledge of both structural and biological diversity of these proteins. Five novel cyclotides with new sequence diversity were isolated and characterized from a violet that grows on Ethiopian highlands at an altitude of 3400 m. One of the areas where the cyclotide framework has attracted interest is the development of stable antimicrobial peptides. A stability study was carried out to determine the stability of the cyclotide framework in a cocktail of bacterial proteases and serum where the native forms of tested cyclotides exhibited high stability profile. Understanding the modes of cyclotide-cell interaction is certainly an important factor for the potential development of cyclotide-based drugs. Cellular studies were carried out using the comet assay and microautoradiography. A bell-shaped dose response curve was obtained for the DNA damaging effect of the cyclotides in the comet assay, which was the first toxicological assay of its kind on this class of proteins. The microautoradiography study revealed that the cyclotides penetrate into the cells even at cytotoxic concentrations. From previous reports, it was known that the cyclotides interact with membranes; the cellular studies in this thesis added to this knowledge by clearly demonstrating that these proteins have multiple modes of action.

Antimalarial Activity of the Chemical Constituents of the Leaf Latex of Aloe pulcherrima Gilbert and Sebsebe

Malaria is one of the three major global public health threats due to a wide spread resistance of the parasites to the standard antimalarial drugs. Considering this growing problem, the ethnomedicinal approach in the search for new antimalarial drugs from plant sources has proven to be more effective and inexpensive. The leaves of Aloe pulcherrima Gilbert and Sebsebe, an endemic Ethiopian plant, are locally used for the treatment of malaria and other infectious diseases. Application of the leaf latex of A. pulcherrima on preparative silica gel TLC led to the isolation of two C-glycosylated anthrones, identified as nataloin (1) and 7-hydroxyaloin (2) by spectroscopic techniques (UV, IR, 1H- and 13C-NMR, HR-ESIMS). Both the latex and isolated compounds displayed antimalarial activity in a dose-independent manner using a four-day suppressive test, with the highest percent suppression of 56.2% achieved at 200 mg/kg/day for 2. The results indicate that both the leaf latex of A. pulcherrima and its two major constituents are endowed with antiplasmodial activities, which support the traditional use of the leaves of the plant for the treatment of malaria

Antimalarial Activity of Acetylenic Thiophenes from Echinops hoehnelii Schweinf

Malaria is one of the world’s most severe endemic diseases and due to the emergence of resistance to the currently available medicines, the need for new targets and relevant antimalarial drugs remains acute. The crude extract, four solvent fractions and two isolated compounds from the roots of Echinops hoehnelii were tested for their antimalarial activity using the standard four-day suppressive method in Plasmodium berghei-infected mice. The 80% methanol extract exhibited suppression of 4.6%, 27.8%, 68.5% and 78.7% at dose of 50, 100, 200 and 400 mg/kg respectively. The dichloromethane fraction displayed chemosuppression of 24.9, 33.5 and 43.0% dose of 100, 200 and 400 mg/kg of body weight. Five acetylenicthiophenes were isolated from the dichloromethane fraction of which 5-(penta-1,3-diynyl)-2-(3,4-dihydroxybut-1-ynyl)-thiophene decreased the level of parasitaemia by 43.2% and 50.2% while 5-(penta-1,3-diynyl)-2-(3-chloro-4-acetoxy-but-1-yn)-thiophene suppressed by 18.8% and 32.7% at 50 and 100 mg/kg, respectively. The study confirmed the traditional claim of the plant to treat malaria and could be used as a new lead for the development of antimalarial drugs

Concomitant Use of Herbal and Conventional Medicines among Patients with Diabetes Mellitus in Public Hospitals of Addis Ababa, Ethiopia: A Cross-Sectional Study

Introduction. The majority of the population in developing countries including Ethiopia still relies on traditional medicines (TMs). Patients with chronic illness like diabetes mellitus (DM) are dissatisfied with conventional medicines and thus are more likely to simultaneously use herbal medicines (HMs). However, such practice could result in potential herb-drug interaction. This study aimed to identify the commonly used HMs among patients with DM and determine the magnitude of concomitant use of herbal and conventional antidiabetic medicines. Method. A health facility-based cross-sectional study design was employed using both quantitative and qualitative data collection methods to determine the magnitude of concomitant use. Patients with DM and prescribers from four public hospitals were the study population for the quantitative and qualitative study, respectively. Simple descriptive statistics were used to describe variables for the quantitative data, and content analysis had been conducted manually for qualitative data. Result. Out of 791 respondents, 409 (51.7%) used traditional medicine at least once in their life time, and 357 (45.1%) used traditional medicine in the last six months prior to data collection. A majority (288 (80.7%)) of the respondents used HMs after starting the conventional antidiabetic medicines within the last six months. Moringa stenopetala, Thymus vulgaris, Trigonella foenum-graecum, Nigella sativa, and Allium sativum were among the frequently mentioned HMs. Prescribers were requesting patients’ HM use when they saw sign of liver toxicity and skin disease, and they were not documenting their history in the patient’s chart. Conclusion. Concomitant use of herbal and conventional antidiabetic medicines was a common practice. Cognizant of its potentially serious herb-drug interactions, efforts should be made to improve awareness and knowledge of healthcare providers about HM potential effects. Further studies on dose, frequency, duration, and modes of interaction are recommended

Concomitant use of medicinal plants and conventional medicines among adult patients with diabetes in Ethiopia: a cross-sectional study

Majority of the Ethiopian population relies on medicinal plants for managing their health conditions. However, there are few studies that assessed the use of these medicinal plants especially for diabetes mellitus where the concomitant use of these remedies with modern medicines may lead to negative health consequences. The aim of this study was therefore to assess the prevalence of concomitant use of medicinal plants with conventional medicines among patients with diabetes attending treatment in five referral hospitals across five regions of the country and to identify the commonly used medicinal plants for diabetes. The study used a cross-sectional design where structured interview questionnaires and data abstraction forms were used to collect the data which was then analyzed using descriptive statistics. The findings revealed that 47.8% of the study participants had ‘ever used medicinal plants’ to treat their diabetes; of whom 74.7% reported to have co-used them with modern medicines. Of the participants who reported concomitant use, only 24.0% knew of the possible safety concern of medicinal plants or their possible interactions with modern medicines. Furthermore, only 15.7% of these participants had ever discussed their use of medicinal plants with their physicians or pharmacists. Twenty-nine plant species were listed by the study participants that were reportedly used for diabetes and related conditions. The most frequently mentioned plants were Shiferaw (Moringa stenopetala), Damakase (Ocimum lamiifolium), Nech-Shinkurt (Allium sativum), Tenadam (Ruta chalapensis), Abish (Trigonella foenum-graecum), Tosign (Thymus schimperi), Tikur-Azimud (Nigella sativa), and Haregresa (Zheneria scabra). The overall findings are indicative of the need to educate patients and providers about the potential implications of the concomitant use of medicinal plants with modern medicines, and the need for further research to establish the potential interactions of some of these medicinal plants with limited or no reported data.Keywords: medicinal plants, modern medicine, diabetes mellitus, cross-sectional study, Ethiopi

Antiproliferative Constituents of the Roots of Ethiopian <i>Podocarpus falcatus</i> and Structure Revision of 2α-Hydroxynagilactone F and Nagilactone I

Bioassay-guided fractionation using the human colorectal adenocarcinoma (HT-29) cell line of the methanol extract of dried roots of <i>Podocarpus falcatus</i> led to the isolation of two new type C nagilactones, 16-hydroxynagilactone F (<b>1</b>) and 2β,16-dihydroxynagilactone F (<b>2</b>), and the new totarane-type bisditerpenoid 7β-hydroxymacrophyllic acid (<b>4</b>), along with the seven known compounds 2β-hydroxynagilactone F (<b>3</b>), macrophyllic acid (<b>5</b>), nagilactone D (<b>6</b>), 15-hydroxynagilactone D (<b>7</b>), nagilactone I (<b>8</b>), inumakiol D (<b>9</b>), and ponasterone A (<b>10</b>). The structures of the new compounds were determined by 1D and 2D NMR, HRESIMS, UV, and IR and by comparison with the reported spectroscopic data of their congeners. The orientation of the C-2 hydroxy group of <b>3</b> and <b>8</b> was revised to be β based on evidence from detailed analysis of 1D and 2D NMR data and single-crystal X-ray diffraction studies. Among the isolated compounds, the nagilactones, including the new dilactones 16-hydroxynagilactone F (<b>1</b>) and 2β,16-dihydroxynagilactone F (<b>2</b>), were the most active (IC₅₀ 0.3–5.1 μM range) against the HT-29 cell line, whereas the bisditerpenoids (<b>4</b> and <b>5</b>) and the other known compounds <b>9</b> and <b>10</b> were inactive. The presence of the bioactive nagilactones in <i>P. falcatus</i> supports its traditional use