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Herbal medicines are extensively utilized across the world. In Ethiopia, people use Ajuga remota Benth to treat high blood pressure, hyperlipidaemia, diabetes, malaria, fever, ascariasis, stomach pain, diarrhoea, evil eye, and retained placenta. However, work has not been done on standardizing the herb. Thus, the aim of this study was to standardize the leaves of A. remota. Fresh leaves, powdered leaves, 80% methanol, chloroform, and methanol extracts of leaves of A. remota were evaluated using macroscopic and microscopic characterization, fluorescence analysis, physicochemical characterization, phytochemical analysis and residual analysis. Macroscopically, fresh leaves of A. remota were 17 - 62 mm (length) × 7.5 - 23 mm (width); 1 - 2 mm thick and dark sea green coloured, soft, fibrous, had a very bitter taste and a fruity odour. Microscopic characterization showed turgid guard cells, anisocytic stomata, epidermal cells (stratum spinosum), parenchymatous (aerenchyma) cells, pointed trichomes, spiral tracheids, and xylem fibre in powdered leaves of A. remota. The powder of the plant gave characteristic colours in fluorescence analysis under visible and 366 nm UV light. The powder was also studied for swelling index (2.392), foaming index (100), bitterness value (8400), extractive value (aqueous: 34.33% and methanol: 21.16%), moisture content (6.43%), total ash value (27.10%), water-soluble ash (11.43%), acid-insoluble ash (7.38%), tannin content (0.016%), and powder fineness (moderately fine). TLC, LC-MS, GC-MS, and FT-IR fingerprinting were produced. Ajugarin I, dodecyl acrylate, and hexacosane were identified as analytical marker compounds. In the residual analysis of the leaves of A. remota, soil took the highest percent (0.83%) among foreign matters. Heavy metals (Cd, Co, Pb, Cr, Ni, Cu, Zn, Fe, and Mn) and aerobic bacterial count, mould, yeast, coliform, Escherichia coli, Salmonella and Shigella species were quantified. 228Ac (7 ± 1.8 Bq kg-1) and 40K (530 ± 34 Bq kg-1) radionuclides with a low dose were detected. The findings provide the standards for identification and authentication of the plant.

In spite of an increase in the health service coverage of the country, studies reported that significant proportion of the Ethiopian population still relies on traditional medicine, particularly on the herbal aspect, for their healthcare. The aim of the present study was to determine the extent of the concomitant use of herbal medicines with modern antiretroviral medicines and identify the commonly used herbs among patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in Ethiopia. A cross-sectional study was conducted from May to June, 2017 among HIV/AIDS patients in five hospitals selected from four regions and one city Administration (Addis Ababa). A total of 768 HIV/AIDS patients were successfully interviewed and included in the analysis. Most 480 (62.5%) of them were on tenofovir-disoproxil fumarate (TDF) plus lamivudine (3TC)and efavirenz (EFV) regimen. Two hundred nineteen (28.5%) patients reported that they had used herbal medicines to treat their illness; of these 145 (66.2%) used herbal products along with modern medicine. Of those who reported to have used herbal medicine with modern medicine, 53 (40.8%) claimed that they took the herbal medicines for the treatment of HIV/AIDS, while 51(39.2%) used for treating co-morbid diseases. Only 33 (15.0%) of those who used herbal medicines along with modern drugs discussed the issue either with their physicians or pharmacists. A total of 31 plant species were reported to be used for the treatment as well as alleviation of symptoms associated with the disease. The most commonly cited herbal medicines that have been used by HIV/AIDS patients to treat their comorbid diseases were Moringa stenopetala (Baker f.) Cufod. (Moringaceae) (35.6%) followed by Ocimum lamiifolium Hochst. (Lamiaceae) (18.0%), Zheneria scabra Sond. (Cucurbitaceae) (7.2%), Allium sativum L. (Alliaceae) (5.7%), and Ruta chalapensis L. (Rutaceae) (5.7%). The study revealed that close to one-third of HIV/AIDS patients interviewed reported use of herbal medicine and almost two-thirds of them did so concomitantly with modern medicine. Only small percentage of the patients who used herbal medicines along with modern drugs disclose the use either to their treating physicians or to counselling pharmacists. Practitioners involved in antiretroviral therapy (ART) clinics, therefore, need to inquire information related to the use of herbal medicines and accordingly advise the patients. Keywords: HIV/AIDS, medicinal plant, cross-sectional study, concomitant use, Ethiopia

Satureja punctata Benth. Briq. (Lamiaceae) is a small shrub known by its vernacular name “Lomishet”. It is used in Ethiopian traditional medicine for the treatment of hypertension, diabetes, liver disease, headache and febrile illness. Mean Arterial Blood Pressure (MABP) lowering effect and vasorelaxant activity of the aqueous extract of aerial parts of the plant were studied. Intravenous administration of the aqueous extract at different doses brought a fall in MABP in normotensive guinea pigs and also in renovascular hypertensive guinea pigs. The extract also caused a dose-dependent relaxation of precontracted aorta; with a maximum relaxation of 98.19% achieved at 40 mg/ml. Preparative reversed-phase HPLC analyses of the extract resulted in the isolation of two phenolic compounds, namely, rosmarinic acid and linarin. The structures of these compounds were elucidated using UV, IR, MS, and NMR (1D and 2D) techniques. Furthermore, rosmarinic acid showed significant (p < 0.01) reduction of MABP in normotensive guinea pigs at doses of 0.75, 1.50 and 3.00 mg/kg. The findings of this study suggest that the aerial parts of S. punctata have genuine antihypertensive activity.

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.

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, Ethiopia

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.

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of ...

Cyclotides are a family of ultra-stable, head-to-tail cyclic mini-proteins from plants, with each member comprising about 30 amino acid residues. Their stability derives from the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring, which is knotted by a third disulfide bond. The cyclotides find potential applications in the pharmaceutical industry as stable peptide-based scaffolds for unstable drugs, and also as medicinal agents, due to the wide range of their inherent pharmacological activities. However, there is a lack of fundamental toxicological studies on this type of compound. The current study determined the possible DNA-damaging effects of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1, in human lymphoma cells by use of the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sub-lethal concentration, cycloviolacin O2 and vaby D gave a bell-shaped dose–response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub-cytotoxic concentrations. Single-cell micro-autoradiography was carried out on tritium-labeled cycloviolacin O2 in order to understand the mechanism behind the dose–response curve. The results revealed that the peptide is taken up into the cell, both at cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms and targets still remain unknown, the current study has unequivocally demonstrated that cyclotides also must have other dose-dependent modes of action.

During the last decade there has been increasing interest in small circular proteins found in plants of the violet family (Violaceae). These so-called cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines forming three disulfide bonds, arranged in a cyclic cystine knot (CCK) motif. In this study we map the occurrence and distribution of cyclotides throughout the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years of age. Even the oldest specimens contained cyclotides in the preserved leaves, with no degradation products observable, confirming their place as one of the most stable proteins in nature. Over 200 samples covering 17 of the 23-31 genera in Violaceae were analyzed, and cyclotides were positively identified in 150 species. Each species contained a unique set of between one and 25 cyclotides, with many exclusive to individual plant species. We estimate the number of different cyclotides in the Violaceae to be 5000-25,000, and propose that cyclotides are ubiquitous among all Violaceae species. Twelve new cyclotides from six phylogenetically dispersed genera were sequenced. Furthermore, the first glycosylated derivatives of cyclotides were identified and characterized, further increasing the diversity and complexity of this unique protein family.

Bioassay-guided fractionation using the human colorectal adenocarcinoma (HT-29) cell line of the methanol extract of dried roots of Podocarpus falcatus led to the isolation of two new type C nagilactones, 16-hydroxynagilactone F (1) and 2β,16-dihydroxynagilactone F (2), and the new totarane-type bisditerpenoid 7β-hydroxymacrophyllic acid (4), along with the seven known compounds 2β-hydroxynagilactone F (3), macrophyllic acid (5), nagilactone D (6), 15-hydroxynagilactone D (7), nagilactone I (8), inumakiol D (9), and ponasterone A (10). 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 3 and 8 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 (1) and 2β,16-dihydroxynagilactone F (2), were the most active (IC50 0.3-5.1 μM range) against the HT-29 cell line, whereas the bisditerpenoids (4 and 5) and the other known compounds 9 and 10 were inactive. The presence of the bioactive nagilactones in P. falcatus supports its traditional use.

Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), is a global pandemic causing millions of deaths annually. Highly active antiretroviral therapy (HAART) greatly enhances lifespan but eventually causes debilitating side effects, in part, due to their chronic administration required to suppress HIV-1 replication. If treatment is discontinued, viral suppression is lost and dormant replication-competent monocytic cell reservoirs become reactivated, leading to viral recrudescence and progression to AIDS. Therefore, novel strategies to circumvent obstacles to HIV-1 therapy are critically needed. We evaluated the potentially therapeutic effects of cycloviolacin O2 (CyO2) on cell viability (MTT assay), membrane disruption (SYTOX Green uptake), p24 production [enzyme-linked immunosorbent assays (ELISA)], and proviral integration (PCR amplification) in U1 cells; a monocytic cell model of HIV-1 latency and reactivation. We demonstrate, for the first time, that CyO2 (0.5-5.0 μM) kills productively infected cells. Sub-toxic concentrations (

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.

As part of ongoing explorations of the structural diversity of cyclotides, the cyclotide content of a native violet of the East African highlands, Viola abyssinica (which grows at altitudes up to 3400 m), was studied. Six new cyclotides, vaby A-E (1-5) and varv E (6), were isolated and characterized by employing HPLC and MS techniques and quantitative amino acid analysis. Cyclotides 1-5 were found to have new sequences, and 1-3 have a further novel feature in their sequences, an alanine moiety in loop 2. Two of the cyclotides (1 and 4) also exhibited cytotoxic properties in a flourometric microculture cytotoxicity assay. The findings corroborate the hypothesis that investigating the cyclotide contents of violets growing in diverse environments is a promising approach for extending our knowledge of both the structural and biological diversity of cyclotides.

Since time immemorial food preservation has been a common practice. Most of these preservatives come form natural sources such as herbs and spices. Currently, synthetic antioxidants are extensively incorporated in foods and cosmetics for the prevention of lipid peroxidation. Some of these synthetic antioxidants, however, have been shown to be carcinogenic and since quite recently, efforts are underway to develop effective and safer natural antioxidants targeting mainly herbs and spices. In Ethiopia, the aerial parts of Lippia adoensis var. koseret and Ocimum basilicum var. purpurascens, rhizomes of Zingiber officinalis , and seeds of Aframomum corrorima are used to spice cooking butter and also to preserve its freshness for a long time. The volatile oils of these plants, obtained by hydrodistillation, and the methanolic extract of spiced butter were tested for their radical scavenging activity using TLC and 2,2- diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant principles of the examined volatile oils were detected in the methanolic extract of the cooking butter spiced with the four plants. Quantitative studies showed that the volatile oils were able to reduce the stable radical, DPPH to the yellow coloured 2,2-diphenylpicrayl-1-hydrazine in a dosedependent manner. The strongest effect was exhibited by the essential oil of the aerial parts of O. basilicum var. purpurascens (IC 50 = 0.04 µl/ml) whereas the weakest effect was shown by that of the seeds A. corrorima (IC 50 = 34.9 µl/ml). The antioxidant potentials of the oil obtained from the aerial parts of L. adoensis var. koseret and the rhizomes of Z. officinalis were somewhat intermediate (IC 50 = 6.13 and 9.66 µl/ml, respectively). The results of this study may provide a rational explanation for the traditional use of these plants to prevent rancidity and preserve the freshness of cooking butter, which could be attributed to their radical scavenging activity. Keywords : spiced butter, volatile oils, TLC analysis, antioxidant activity, DPPH assay Ethiopian Pharmaceutical Journal Vol. 23 2005: 7-14

In Ethiopia inflammatory skin diseases are among the most common health problems treated with traditional remedies which mainly comprise medicinal plants. In the present work, the anti-inflammatory and anti-nociceptive activities of Cheilanthes farinosa (Forsk.) Kaulf (Adianthaceae), a fern used in many parts of Ethiopia to treat inflammatory skin disorders, were studied using in vivo models of inflammation and pain. The results of the study showed that the fronds Cheilanthes farinosa possess strong anti-inflammatory and anti-nociceptive properties. It was further demonstrated that the active ingredients of the fern reside mainly in the methanol fraction from which three compounds viz. the flavonol glycoside rutin, and the natural cinnamic acids, caffeic acid and its quinic acid derivative chlorogenic acid have been isolated. The methanol extract was also shown to potentiate the anti-inflammatory activity of acetyl salicylic acid. At the tested concentrations, the methanol extract displayed a better anti-nociceptive activity than that of ASA in both the early and late phases of formalin induced nociception in mice. However, the activity of the extract was more pronounced in the late phase, which is commonly associated with inflammatory pain. Evaluation of the pharmacological properties of the compounds isolated from the active fractions pointed out that chlorogenic acid possesses strong anti-inflammatory and anti-nociceptive activities while caffeic acid and rutin were inactive. Moreover, on molar basis chlorogenic acid was proved to be superior in its anti-inflammatory action to acetyl salicylic acid. It was therefore concluded that chlorogenic acid contributes, in full or in part, to the anti-inflammatory and anti-nociceptive activities of Cheilanthes farinosa. Both the methanolic extract and pure chlorogenic acid failed to display anti-nociceptive activity when tested by the tail-flick test indicating that the plant is not a centrally acting analgesic but instead exerts its analgesic activity by way of its antinflammtory action.