Isolierung, Strukturaufklärung, Quantifizierung und Formulierung der Saponine und Flavonoide der Samen von Glinus Lotoides

Suitable extraction methods for the seeds of Glinus lotoides have been developed and the major saponins and flavonoids have been isolated. In this, four new hopane-type saponins, glinusides F, G, H and K, and the known succulentoside B as well as the two known flavones, 5,7,4'f-trihydroxyflavone-6,8-di-C-glucoside (vicenin-2) and 5,7,4'f-trihydroxy-flavone-8-C-sophoroside (vitexin-2''-O-glucoside), have been isolated from the seeds of the plant. Based on the spectral analyses including 2D NMR and HRESI mass spectroscopy, the new structures have been characterized as 3alpha-O-alpha-Dxylopyranosyl-6beta-O-alpha-D-xylopyranosyl-16alpha-O-alpha-D-xylopyranosyl-22-hydroxy-hopane (glinuside F), 3alpha-O-beta-L-rhamnopyranosyl-(1->2)-alpha-D-xylopyranosyl-6beta,16alpha-dihydroxy-22-O-beta-L-rhamnopyranosyl-hopane (glinuside G), 3alpha-O-beta-l(TM)hamnopyranosyl-( 1->2)-alpha-D-xylopyranosyl-6beta-O-alpha-D-xylopyranosyl-16alpha-hydroxy-22-O-beta-L-rhamnopyranosyl-hopane (glinuside H) and 3alpha-O-beta-L-rhamnopyranosyl-(1->2)-alpha-D-xylopyranosyl-6beta-O-alpha-D-xylopyranosyl-16alpha-O-alpha-D-xylopyranosyl-22-hopane (glinuside I). The total flavonoids and saponins of the seeds of G. lotoides in the crude extracts and tablet formulations thereof have been quantified by reversed-phase high performance liquid chromatographic (RP-HPLC) methods with UV detection. The saponins were analyzed after acid hydrolysis in 3M HCl at 100 °C for 1 hour. Vicenin-2 and mollugogenol B have been isolated and used as reference substances for the quantification of total flavonoids and saponins, respectively. The identities of vicenin-2 and mollugogenol B have been confirmed using UV, MS and NMR spectral analyses and comparison with respective published data. The purity of the isolated mollugogenol B has been determined by TLC, HPLC and UV spectrophotometry as 98%, calculated from HPLC peak area. The molar absorptivity of the methanol solution of mollugogenol B at a concentration of 0.948 mg/100ml was found to be 3.98, 4.16 and 4.12 at ƒÉmax of 260sh, 251 and 243 nm, respectively, which is in good agreement with literature values, indicating the purity of mollugogenol B. Similarly, the purity of vicenin-2 has been determined as 97% using HPLC, which is confirmed by TLC and UV methods. Satisfactory separation of the components of the flavonoids and saponins compounds has been obtained in less than 30 and 25 min, for the flavonoids and saponins, respectively. The HPLC methods were validated for linearity, repeatability, limits of detection (LOD) and quantification (LOQ). Repeatability (inter- and intra-day, n = 6 and 9, respectively) showed less than 2% relative standard deviation (RSD). The LOD and LOQ were found to be 0.075 and 0.225 mg/mL, respectively, for vicenin-2 and 0.027 and 0.082 mg/100mL, respectively, for mollugogenol B. The total amounts of saponins of the crude extract and tablet formulation have been determined as glinuside G equivalent (mol. Wt. 900) which approximates the average molecular weight of the saponins of G. lotoides. The content of flavonoids and saponins of six single tablets has been found to be between 95 and 103% for flavonoids and 94 to 98% for saponins. The validated HPLC methods could be employed to standardize a laboratory produced purified extract, which could be used as a secondary standard for the routine quality control. Accordingly, the secondary standard (extract F) has been found to contain 10% vicenin-2 and 21.3% total flavonoids calculated as vicenin-2 equivalent. The amount of saponins in the secondary standard was 6.3% mollugogenol B and 12.4% total aglycons calculated as mollugogenol B, 14.2% glinuside G and 25.4% total saponins calculated as glinuside G equivalent. Admixing of the crude liquid extract of the seeds of G. lotoides with Aeroperl(TM) 300 Pharma, as an inert carrier material, and subsequent drying the mixture has provided a non-adherent and free-flowing powder. The required amount of Aeroperl(TM) 300 Pharma has been optimized as 30%, based on the physicochemical properties such as particle size distribution, surface morphology, moisture content and sorption kinetics of the dry extract preparations containing 10, 20, 30, 40, 50 and 60% w/w Aeroperl(TM) 300 Pharma. A tablet formulation containing granules of the dry extract preparation (947 mg), Avicel(TM) PH101 (363 mg) and Ac-Di-sol (90 mg) provided the best tablet properties such as disintegration time of 2.4 min at a tablet hardness of 73 N. Granulation by roller compaction of the dry extract preparation has improved the disintegration time of the tablets. Oblong tablets which could administer the traditional dose as a single tablet have been developed and enteric coated. Enteric coated tablets of G. lotoides with pharmacopoeial requirements could be prepared using methacrylic acid/ ethylacrylate co-polymer (Eudragit(TM) L 100/55 or Kollicoat(TM) MAE 100P). Propylene glycol and acetyl tributyl citrate could be used as plasticizers at concentrations of about 11% in the dry film.Glinus lotoides wird in Athiopien traditionell als Anthelminthikum eingesetzt. Das Ziel der vorliegenden Arbeit ist es, Auszuge von G. lotoides herzustellen, diese zu charakterisieren und zu standardisieren. In einem weiteren Abschnitt sollen aus den Auszügen Arzneiformulierungen hergestellt werden. Um die Ziele der Arbeit zu erreichen, muss ein standardisiertes Extraktionsverfahren entwickelt werden. Des Weiteren ist ein umfangreiches Screening der Inhaltsstoffe durchzuführen, was die Isolierung und Identifizierung der pharmakologisch aktiven Substanzen einschliesst. Die Isolierung und die Strukturaufklarung der Saponine und der Flavonoide als wirksame Inhaltsstoffgruppen von G. lotoides liefern vier neue Hopan-Saponine, die Glinuside F, G, H und K, das bekannte Succulentosid B sowie zwei bekannte Flavonoidglykoside; 5,7,4'-trihydroxyflavon-6,8-di-C-glucosid (Vicenin-2) und 5,7,4'-trihydroxy-flavon-8-C-sophorosid (Vitexin-2"-O-glucosid). Die Strukturen der Saponine werden mittels NMR, HRESI Massenspektroskopie und Zucker-Analytik als 3beta-O-beta-D-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-O-beta-D-xylopyranosyl-22-hydroxy-hopan (Glinusid F), 3beta-O-alpha-L-rhamnopyranosyl-(1->2)-beta-D-xylopyranosyl-6alpha,16beta-dihydroxy-22-O-alpha-L-rhamnopyranosyl-hopan (Glinusid G), 3beta-O-alpha-l(TM)hamnopyranosyl-( 1->2)-beta-D-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-hydroxy-22-O-alpha-L-rhamnopyranosyl-hopan (Glinusid H) and 3beta-O-alpha-L-rhamnopyranosyl-(1->2)-beta-D-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-O-beta-D-xylopyranosyl-22-hopan (Glinusid I) aufgeklart. Die Gesamtsaponin- und Favonidgehalte der Samen von G. lotoides werden mittels RPHPLC in den Extrakten und in den Tabletten bestimmt. Die quantitative Bestimmung der Saponine wird nach saurer Hydrolyse (3M HCl, 100 °C fur 1 Stunde) durchgeführt, wobei Mollugogenol B entstehen. Für die quantitative Bestimmung der Flavonoide wird Vicenin-2 als externer Standard eingesetzt. Mittels DC und HPLC unter verschiedenen Bedingungen wird eine mindestens 97 %ige Reinheit des isolierten Vicenin-2 und Mollugogenol B gefunden. Die Berechnung der Konzentrationen erfolgt über die Peakflachen von Vicenin-2 und von Mollugogenol B nach der Methode des externen Standards. Der Gesamtflavonoidgehalt und der Gesamtsaponingehalt von G. lotoides werden über die Gesamtpeakfläche des jeweiligen Chromatogramms ermittelt. Im Rahmen der Validierung der Gehaltsbestimmungsmethode erfolgt die Bestimmung von Selektivität, Wiederfindung, System- und Methodepräzision, Bestimmungsgrenze und Nachweisgrenze. Als Sekundärstandard wird der aufgereinigte Extrakt F charakterisiert. Der Flavonoid- und Saponingehalt der Tabletten beträgt zwischen 95 und 103 % für die Flavonoide und zwischen 94 und 98 % für die Saponine. Eine zur Formulierung von Tabletten geeignete Extraktzubereitung wird durch Mischung des Extraktes mit Aeroperl(TM) 300 Pharma als Träger-Material hergestellt. Die benötigte Menge von Aeroperl(TM) 300 Pharma wird durch Untersuchungen zur Wasseraufnahme und REM-Untersuchungen optimiert. Hierbei ergibt eine Extraktzubereitung mit 30% (m/m) Aeroperl(TM) 300 Pharma die besten Eigenschaften im Hinblick auf die weitere Verarbeitung. Zusätzlich, durch Walzen-Kompaktierung der Extrakt-Aeroperl-Mischung wird die Fliessfähigkeit der Tablettiermischung und der Zerfall der Tabletten verbessert. Eine geeignete Tablettenformulierung, die 947 mg Extraktzubereitung, 363 mg Avicel(TM) PH101, und 90 mg Ac-Di-sol enthält, wird durch mehrere Optimierungsschritte entwickelt. Diese Tabletten zeigen einen Zerfall der Tabletten innerhalb von 2,4 Minuten bei einer Harte von 73 N. Zusätzlich erhalten die Oblong-Tabletten einen Magensaftresistenten Überzug, der den Arzneibuchanforderungen entspricht. Dieser Überzug wird mittels der hier für geeigneten Filmbildner Eudragit(TM) L 100/55 oder Kollicoat(TM) MAE 100P hergestellt. Der Filmüberzug enthält Propylenglykol und Acetyltributylcitrat als Weichmacher. Durch die Prüfung der Zerfallzeit wird sichergestellt, dass die überzogenen Tabletten in künstlichen Magensaft Ph. Eur. (0,1 M Salzsäure, pH 1,0) innerhalb 2 Stunden nicht zufallen jedoch anschliessend in künstlichen Darmsaft (Phosphat-Pufferlosung, pH 6,8) innerhalb von 15 Minuten zufallen. Zusammenfassend kann festgestellt werden, dass es gelungen ist, einen Auszug von G. lotoides herzustellen, dieser zu einer zur Formulierung von Tabletten geeigneten Extraktzubereitung zu verarbeiten, eine Tablettenformulierung zu entwickeln und die Tabletten mit einem magensaftresistenten Überzug zu versehen

Food insecurity in Farta District, Northwest Ethiopia: A community based cross–sectional study

Background Access to sufficient food is essential for household welfare as well as for accomplishing other development activities. Households with insufficient access to food often face other challenges related to food insecurity including poor health and a decline in productivity. These challenges can often create a vicious circle whereby households are unable to produce enough food even during a good crop season. Thus, this study aimed to determine the magnitude of food insecurity and its determinants in rural households of Farta District, Northwest Ethiopia. Methods A community based cross-sectional study was conducted from September to October 2012. Household heads were recruited using a multistage random sampling technique. Data were collected by face-to-face interviews using the Household Food Insecurity Access Scale (HFIAS) tool after verbal informed consent. Data were entered to Epi info 2002 and exported to SPSS version 16 for analysis. Multiple logistic regressions were fitted and odds ratios with 95% confidence intervals were calculated to identify associated factors and control confounding effect. Results A total of 836 households were included in this study. Nearly three quarters of the households (70.7%) had food insecurity. Households headed by females (AOR = 3.18, 95% CI:1.08, 15.21), lack of education (AOR = 2.59, 95% CI: 1.46, 4.60), family size of 4-7 (AOR = 2.39, 95% CI: 1.21,4.70), family size of >7 (AOR = 13.23,95% CI:6.18, 28.32), few or absence of livestock (AOR = 5.60, 95% CI:1.28, 24.43), absence of income from off-farm activities (AOR = 3.12, 95% CI:1.53, 6.36), lack of irrigation (AOR = 3.54, 95% CI:2.14, 5.18) and lack of perennial income (AOR = 3.15, 95% CI:1.88, 5.27) were factors associated with food insecurity. Conclusions This study revealed that most households of the district were food insecure. Hence, the promotion of contraceptive use, off-farm employment activities and the development of small scale irrigation are important recommendations to reduce food insecurity

Low level of knowledge about neonatal danger signs and its associated factors among postnatal mothers attending at Woldia general hospital, Ethiopia

Abstract Background Neonatal mortality has persisted high in Ethiopia in spite of many efforts being applied to decrease this adverse trend. Early detection of neonatal illness is an important step towards improving newborn survival. Toward this end, there is a need for the mothers to be able to identify signs in neonates that signify severe illnesses. The aim of this study was to assess knowledge about neonatal danger signs and its associated factors among postnatal mothers attending at Woldia general hospital, Ethiopian. Methods Institutional based cross-sectional study design was conducted from January–May, 2017. The hospital that provides antenatal care (ANC), delivery, and postnatal services was purposively sampled. Structured interviewer managed questionnaire was administered to postnatal mothers attending Woldia general hospital. Frequencies, bivariate and multivariate logistic regression were determined using the SPSS software (Version 20). Results During the study period 197 mothers attending postnatal care (PNC) service at Woldia general hospital were interviewed. Information on different neonatal danger signs was not provided to 92(46.7%) postnatal mothers during their antenatal clinic attendance by the healthcare providers. The majority of mothers, 174(88.3%) identified less than six neonatal danger signs. The hotness of the body of neonates was the commonly recognized danger sign by 106(53.8%) postnatal mothers. Of the total mothers, 67(34%), 60(30.5%), 56(28.4%), 44(22.3%) recognized unable to breastfeeding, convulsion, lethargy, difficulty in breathing as newly born danger signs, respectively. Out of 197 mothers, 32(16.2%) were giving birth at home. Mother’s age(AOR = 1.33, 95% CI: 1.99–3.08), marital status(AOR = 2.50, 95% CI: 0.29–4.31), mother’s education status(AOR = 3.48, 95% CI:1.57–8.72), husband’s education(AOR = 4.92, 95% CI: 1.29–12.81), attending ANC (AOR = 2.88, 95% CI: 1.15, 4.85), mother’s residence(AOR = 0.78, 95% CI: 0.47–1.65), information about neonatal danger signs(AOR = 3.48, 95% CI 1.40–9.49) had positive association with maternal level of knowledge to identify different neonatal danger signs. Conclusion Maternal knowledge level about neonatal danger signs was very low. Therefore, intervention modalities that focus on increasing level of parental education, access to ANC and PNC service are needed

Optimization of the Simple One-Step Stool Processing Method to Diagnose Tuberculosis: Evaluation of Robustness and Stool Transport Conditions for Global Implementation

ABSTRACT Stool is recommended as an alternative specimen for the diagnosis of tuberculosis (TB) in young children, as they cannot easily produce sputum. The Simple One-Step (SOS) stool processing method is a new and simple stool processing method for the detection of Mycobacterium tuberculosis (MTB) using Xpert MTB/RIF Ultra (Xpert-Ultra). We determined the robustness of the SOS stool processing method and stool specimen transport conditions in participants with confirmed TB. We processed stool using the standard protocol after simulated “transport,” varying time, and temperature, and experimented with slightly modified processing steps. We included 2,963 Xpert-Ultra test results from 132 stool specimens of 47 TB participants, including 11 children aged 0.8 g of stool. We found that almost all steps in the current SOS stool processing method provide optimal Xpert-Ultra results but recommend an adjustment to use a wider range of stool amounts (0.3 to 0.8 g) than advised previously (0.8 g). With this adaptation, stool-based diagnosis of TB using the SOS stool processing method can be scaled-up. IMPORTANCE The manuscript will support the global implementation and scale-up of the SOS stool method in routine settings. It also provides important insights on the optimal stool transport conditions and robustness of the SOS method, which can be used for bacteriological diagnosis of TB in children at the lowest levels of the healthcare system, avoiding lengthy healthcare-seeking pathways and additional costs

The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care

International audienceYoung children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care

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Risk perceptions and attitudinal responses to COVID-19 pandemic : an online survey in Ethiopia

Background Effective risk communication is one of the critical strategies in the response to COVID-19. This study examined risk perceptions and attitudinal responses to COVID-19 among the educated section of the society in Ethiopia. Methods An internet-based survey was conducted from April 22 to May 04, 2020, in Ethiopia. A questionnaire addressing the perception of health threat-combination of perceived vulnerability (PV) and perceived seriousness (PS), and perceived efficacy-combinations of perceived response efficacy (PRE), perceived self-efficacy (PSE), and perceived collective efficacy (PCE). The data were analyzed using SPSS 21.0. Descriptive statistics were computed after the standardization of the scores. The scores for overall efficacy and threat were split by median value and response classifications were generated through threat by efficacy interactions. For statistical significance, 95% CI and p-value < 0.05 were used. Results A total of 929 respondents submitted their responses. Eight hundred and twenty-eight (89.1%) of the respondents were male and 753 (81.1%) were Christian. The perceived threat to COVID-19 was generally low (median = 58.3). The median score for overall efficacy, PRE, and PSE were 79.8, 87.5, and 80.0, respectively. However, the median value (66.7) for PCE was relatively low. Perceived threat significantly varied by age, education, occupation, and place of residence (p < 0.05). Perceived efficacy significantly differed by gender, residence, and use of some sources of information (p < 0.05). In terms of response to COVID-19, 290 (31.2%), 239 (25.7%), 175 (18.8%) and 225 (24.2%) of the respondents were in the responsive, pro-active, avoidant, and indifferent attitudinal categories, respectively. The avoidant and indifferent groups constituted a fear control response (mal-adaptive motivation towards COVID-19 protective behavior) whereas responsive and pro-active categories formed a danger control response (self-protective motivation). These responses varied significantly by residence, region, religion, and sources of information (p < 0.05). Conclusions Understanding people's perceived health threat and efficacy is a critical step toward creating risk communication campaigns. Hence, this study provided an insight that has the potential to inform the COVID-19 risk communication campaigns targeting the educated section of the society, by ensuring a balanced combination of threat appeals and efficacy messages for improved self-protective responses

Simple Chromatographic Method for Simultaneous Analyses of Phosphatidylcholine, Lysophosphatidylcholine, and Free Fatty Acids

This study describes a simple chromatographic method for the simultaneous analyses of phosphatidylcholine (PC) and its hydrolytic degradation products: lysophosphatidylcholine (LPC) and free fatty acids (FFA). Quantitative determination of PC, LPC, and FFA is essential in order to assure safety and to accurately assess the shelf life of phospholipid-containing products. A single-run normal-phase high-performance liquid chromatography (HPLC) with evaporative light scattering detector has been developed. The method utilizes an Allsphere silica analytical column and a gradient elution with mobile phases consisting of chloroform: chloroform–methanol (70:30%, v/v) and chloroform–methanol–water–ammonia (45:45:9.5:0.5%, v/v/v/v). The method adequately resolves PC, LPC, and FFA within a run time of 25 min. The quantitative analysis of PC and LPC has been achieved with external standard method. The free fatty acids were analyzed as a group using linoleic acid as representative standard. Linear calibration curves were obtained for PC (1.64–16.3 μg, r2 = 0.9991) and LPC (0.6–5.0 μg, r2 = 0.9966), while a logarithmic calibration curve was obtained for linoleic acid (1.1–5.8 μg, r2 = 0.9967). The detection and quantification limits of LPC and FFA were 0.04 and 0.1 μg, respectively. As a means of validating the applicability of the assay to pharmaceutical products, PC liposome was subjected to alkaline hydrolytic degradation. Quantitative HPLC analysis showed that 97% of the total mass balance for PC could be accounted for in liposome formulation. The overall results show that the HPLC method could be a useful tool for chromatographic analysis, stability studies, and formulation characterization of phospholipid-based pharmaceuticals