Background and objectives: Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as hypertension, diabetes, dyslipidemia, obesity, and hyperuricemia. We aim to investigate the relationship between uric acid and NAFLD in a non-obese and young population. Materials and Methods: This study was performed in January 2010–2019 with a group of 367 (225 patients in the NAFLD group and 142 in the control group) patients with liver biopsy-proven NAFLD or no NAFLD. Patients with NAFLD were classified according to the percentage of steatosis as follows, group I had 1–20% and group II >20%. Demographic, clinical, and laboratory  features were collected retrospectively. Results: The mean body mass index (BMI) and age of the patients were 26.41 ± 3.42 and 32.27 ± 8.85, respectively. The BMI, homeostatic model of assessment (HOMA-IR), and uric acid (UA) values of the NAFLD group were found to be significantly higher than those of the controls. A positive correlation was found between the NAFLD stage and UA. The following factors were independently associated with NAFLD: BMI, HOMA-IR, and UA. In addition, the cut-off value of UA was 4.75 mg/dl with a sensitivity of 45.8% and a specificity of 80.3%. Conclusions: UA is a simple, non-invasive, cheap, and useful marker that may be used to predict steatosis in patients with NAFLD. Nonalcoholic fatty liver disease (NAFLD) is defined as a condition where more than 5% of the body’s hepatic cells contain fatty deposits without a history of alcohol consumption. This deposition of fat can cause a variety of diseases ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, liver failure, and hepatocellular carcinoma . NAFLD iUric acid (UA) is a natural end product of purine metabolism. While it is naturally found in blood in certain amounts, its deviation can indicate a metabolic disorder. Lately, links have been suggested between the UA level and diabetes mellitus, obesity, cardiovascular disease, and renal disease ,12,13,14In this study, the association between serum UA and the severity of liver damage was evaluated in a large cohort of biopsy-proven, non-obese, and young Turkish individuals with NAFLD.This study was performed in a group of 367 patients with liver-biopsy-proven NAFLD or no NAFLD between January 2010 and January 2019 at the Istanbul Demiroglu Bilim University Gastroenterology Department. The study population included 225 patients with biopsy-proven NAFLD and 142 control subjects without NAFLD. NAFLD was diagnosed by liver biopsy. Liver histology was evaluated by an experienced liver pathologist, and specimens were classified according to the NASH Clinical Research Network guidelines . PatientThe inclusion criteria were for male or female individuals aged between 18 and 65 years with no history of excessive alcohol drinking , negative tests for the presence of the hepatitis B surface antigen and the antibody to hepatitis C virus, absence of a history of cirrhosis and other causes of liver disease , and no treatment with drugs that are known to cause liver steatosis . The exclusion criteria were the presence of any other chronic liver disease, HIV infection, diabetes mellitus, heart failure, valvular disease, asthma, chronic obstructive pulmonary disease, peripheral and cerebral vascular disease, hematological disorders, acute or chronic infections, cancer history involving liver transplantation, previous exposure to drugs associated with fatty liver, or refusal to participate in the study. Demographic and anthropometric data were obtained ) and serum biochemistry was assessed including total cholesterol, triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), total bilirubin, albumin, blood urea nitrogen (BUN), creatinine, and UA concentrations. The evaluation of insulin resistance relied on the homeostatic model of assessment (HOMA-IR) calculation (Fasting insulin (mU/mL) × glucose (mmol/L) / 22.5). A score of HOMA-IR ≥2.5 was used to define insulin resistance [2 test for categorical parameters; comparisons between more than two subgroups were performed by ANOVA and Kruskal–Wallis h tests. Bivariate correlations were explored by Pearson’s (continuous variables) or Spearman’s  coefficients. Logistic regression analysis was performed as multivariate analysis on parameters with significant differences observed in the univariate analysis. The ability of UA to predict NAFLD was evaluated using the receiver operating characteristic (ROC) curve analysis. The recommended cut-off value of UA for the optimum sensitivity and specificity ratio of the diagnostic test was determined. Differences were considered statistically significant if the two-tailed 𝑃 value was less than 0.05.Data are expressed as the mean ± standard deviation. A statistical analysis was performed using SPSS 21.0 . Basic descriptive statistics were measured including the means, standard deviations, ranges, and percentages. The normality of the distribution was examined by the Kolmogorov–Smirnov test. Mean values between two independent groups were compared by the Mann–Whitney U test for continuous variables and by the χThere were 367 patients in total (225 patients in NAFLD group and 142 in control group). The mean BMI of the patients was 27.25 ± 4.02 in the NAFLD group and 24.71 ± 3.34 in the control group. The mean age of the patients was 34.08 ± 9.08 years in the NAFLD group and 34.24 ± 8.72 years in the control group. There were 187 males (61.4%) in the NAFLD group and 81 males (57.1%) in the control group. To evaluate the independence of associations between NAFLD and anthropometric and biochemical parameters, we undertook a regression analysis with NAFLD/non-NAFLD as the binary outcome . This anIn addition, a comparison of the mentioned variables between three groups  is presented in With this study, we found that in non-obese and young patients with NAFLD, UA levels were significantly higher than those of individuals in a healthy control group. The UA level also showed a positive correlation with the degree of hepatic steatosis and NAFLD. Furthermore, UA was found to be an independent risk factor for NAFLD. In addition, creatinine levels were significantly higher than those of individuals in the healthy control group, but the difference in the BUN level between groups was insignificant.Nutritional, metabolic, and genetic factors contribute to the development of NAFLD. NAFLD is among the most common causes of chronic liver disease worldwide. It was shown to be the most important cause of chronic liver disease and cirrhosis in a recent large study . Not onlUA is an end product of purine metabolism. Its level in blood can increase with insulin resistance, atherosclerosis, hypertension, and obesity. The factors resulting in this relationship are hypothesized to be inflammation and oxidative stress ,26. MoreIn our study, we found that UA levels were higher in NAFLD patients than in the control group, and we also found a positive correlation between the NAFLD stage and UA. Similar findings were reported in some studies ,24. HoweMany studies showed that the elevation of UA levels is an independent predictor of an increased risk for NAFLD ,25,33. SBUN and creatinine are parameters that are used to predict kidney function in daily practice . SimilarTo date, the association between UA and NAFLD has been demonstrated in many clinical studies. However, in these studies, UA levels were over the normal range ,24. DiffA possible limitation of our study may be a reporting or recall bias caused by patient histories, most of which reported a relatively healthy diet. The retrospective aspect of the study and the retrieval of data only from records may be limitations of the study. Finally, in our study, the BMI values of patients with NAFLD were significantly higher than those in the control group. However, differing from published studies, we excluded obese patients with BMI values of 30 and over. Therefore, the BMI difference between the control and NAFLD groups might not have affected our results.In summary, the UA level was found to be significantly and independently associated with hepatocellular steatosis and the NAFLD stage in biopsy-proven NAFLD patients. UA is a simple, non-invasive, cheap, and useful marker that could be used to predict steatosis in patients with NAFLD. In addition, we suggest that caution should be exercised in terms of NAFLD progression when the UA level is greater than 4.75 mg/dl. Further investigations in larger prospective studies are needed to validate our study results.


Sun-dried beef is a frequently consumed and valued product in Brazil, however, there have been no scientific studies on its texture. To assess the tenderness of sun-dried beef, an instrumental analysis , a sensory analysis  and the sarcomere length (SL) were used as indicators. Significant differences were observed among the sun-dried beef samples. Sample 3 (composed of sun-dried meat purchased at three fairs from Region 3 in the city of João Pessoa-PB) was considered the most tender by the assessors, with a score of 6.7, and its WBSF analysis revealed a maximum value of 2.70 kgf. Additionally, this sample exhibited the highest SL value (1.89 µm). Samples 1 and 2  exhibited very similar tenderness values (WBSF and QDA) but differed in their SL values, which suggested that sample 2 was the least tender. In conclusion, these results demonstrate that the studied parameters are complementary and can be used as tenderness indicators for sun-dried beef. However, although the difference was beyond the detection limit of the assessors and the texturometer, the SL analysis appears to have been the most effective. Sun-dried beef is a characteristic product of Brazil, where refrigerated meat, typically from cattle, is lightly salted and marketed. Although it is salted, sun-dried beef has characteristics more similar to those of fresh, refrigerated beef than to those of salted products, such as charque (CH) and jerked beef (JB). Because CH and JB are considered foods with intermediate moisture content, they have water activity (Aw) values between 0.60 and 0.90 ,2 and unLike CH and JB, sun-dried beef undergoes salting and drying steps; however, the latter only reduces surface humidity because it is performed in the shade and for a short period of time. Thus, the shelf life of sun-dried beef under ambient conditions ranges from 2 days to 4 days ,4, whichThe composition of charque and jerked beef is 15% to 20% salt and approximately 45% to 50% moisture. In contrast, sun-dried beef is 5% to 6% salt and 64% to 70% moisture . The proStudies on beef tenderness are common because tenderness is a determining factor in the acceptability of the product by consumers. Indicators, such as sensory evaluation and instrumental analysis by shear force, have been reported in several studies ,9,10,11.However, no research has been undertaken to specifically assess the tenderness of sun-dried beef. Thus, this study aims to fill this information gap by using instrumental, sensory and sarcomere length evaluations as indicators of the tenderness of this meat product.Differences were observed in the compositions of the studied sun-dried beef samples . VariatiThe results displayed in et al. [The results from the WBSF analysis of the sun-dried beef samples, described in et al. , beef caWhile developing a product similar to sun-dried beef, with the addition of 4% sodium chloride, Carvalho Jr.  obtainedWhile investigating the effect of maturation in the processing of sun-dried beef, intermediate values, compared with the present study (ranging from 3.08 kgf to 3.51 kgf), were obtained .The sensory panel identified differences among the sun-dried beef samples, as shown in p < 0.01) was observed between the WBSF and the QDA. A strong correlation  between the SF and the tenderness values, which were assigned by a trained panel, was observed for the samples that were removed in a direction parallel to the longissimus muscle fiber axis [p < 0.01) between the shear force and the consumer response was observed. Other studies have also linked the sensory attributes reported by a trained panel of assessors with the Warner-Bratzler shear force [A significant correlation  [The most desirable sarcomere shortening is a percentage decrease on the order of 0% to 40% relative to the length under resting conditions . Based ofemoris) , values semimembranosus post-rigor, in which an average value of 1.82 µm was observed for 18 sarcomeres [longissimus muscle, in which an average value of 1.79 µm was observed for 15 analyzed sarcomeres [longissimus and semimembranosus muscles, and 1.90 µm was observed for the adductor muscle [These results are consistent with the results for the sarcomere length of the rcomeres . These rrcomeres . In anotr muscle .In the micrographs shown in Tenderness in meat products is the result of multiple factors, including muscle contraction, protease activities and both fat and moisture content ; howeverA quantitative survey was conducted on the marketing of sun-dried beef in all fairs in the city of João Pessoa, which are traditional places that sell this product. The city was divided into three regions  in which three fairs with the highest sale volumes of sun-dried beef were surveyed. Samples 1, 2 and 3 were from sun-dried beef purchased at three fairs from Regions 1, 2 and 3, respectively.semimembranosus and adductor femoris, which, in Brazil, are popularly known as coxão mole, from zebu breeds (4 to 5 years old) and weighed approximately 6.0 kg each. The meat was sliced (approximately 5 cm thick), salted , washed under running water, shade dried for 30 min and cooled. This sun-dried meat process was reported by traders that, in addition to marketing, also produced sun-dried beef.All sun-dried beef samples were processed from et al. [The moisture, protein, ash and sodium chloride contents of the beef were determined according to the Association of Official Analytical Chemists (AOAC) methods . Total let al. . Water aThe instrumental texture of the sun-dried beef was evaluated by the shear force, which was measured using a TAXT-2i texturometer coupled with a Warner-Bratzler blade . The sun-dried beef samples were cooked according to the recommendations of the American Beef Science Association (AMSA) . AccordiQuantitative descriptive analysis (QDA) was used for sensory evaluation of the sun-dried beef samples. The samples were prepared according to the methodology used in the instrumental determination of the texture. The sun-dried beef samples were served to a sensory panel composed of six judges who had been previously selected and trained in three sessions.The sensory panel initially consisted of 17 volunteers . The pre-selection of the candidates was performed based on their interest, availability, discriminative ability related to beef, familiarity with the product and ability to express proportionality using scales. The candidates then underwent basic taste and odor recognition tests as well as triangular tests to evaluate their discriminative ability .The selection of assessors was based on their discriminative ability  and consensus with the panel in more than 80% of the descriptors, in accordance with the methodology proposed in . Thus, aThe candidates signed a consent form, as required by Resolution No. 196 of 10/10/1996, from the National Health Council . This st3 cubes were removed and immersed in Karnovsky’s fixative solution, which consisted of 2.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M sodium cacodylate. Each cube was cut to approximately 2 × 2 × 2 mm3, washed successively with cacodylate buffer to remove the fixative and post-fixed with 1% osmium tetroxide, 10 mM calcium chloride, 1.6% potassium ferrocyanide and 0.2 M sodium cacodylate buffer. The samples were then washed with 5% uranyl acetate, dehydrated with increasing concentrations of acetone and embedded in resin. Semi-thin cuts were made with an ultramicrotome (LEICA EM UC6 MZ6) to determine the direction of the muscle fibers. The blocks from each sample were then cut into ultrathin sections. The sections were treated with 5% uranyl acetate and lead citrate. For sarcomere identification, myofibril images were viewed with an FEI Morgagni 286D microscope operating at 80 kV. To determine the sarcomere length, the ITEM software installed in the FEI Morgagni 286D microscope was used. For each sample, 25 randomly chosen myofibril fragments were evaluated, and the lengths of 6 sarcomere units of each fragment were measured [To visualize the sarcomere length, 0.5 cmmeasured ,34.Statistical analyses were performed using an analysis of variance (ANOVA), and a comparison of means was performed using Tukey’s test at 5% significance. The statistical analyses were performed with the SAS software . A correBased on our results, it can be concluded that there were differences in the processing of the sun-dried meat, which is evidenced by variations in the composition of the data, indicating the need for specific legislation to standardize the processing of this product and parameters to identity the product and its quality. Differences in the tenderness of the samples were also observed. Although fat influences meat tenderness, tenderness did not vary with fat content in this study, likely due to the low fat content of the samples. The shear force showed variations from hard (sample 2) to very soft (sample 3). All softness indicators demonstrated that sample 3 was the softest. Samples 1 and 2 differed only in their sarcomere lengths, which indicated that sample 2 was the softest, suggesting an improved accuracy with this analysis because the length displayed a higher sensitivity than the sensory analysis and shear force. In this context, it can be inferred that the softness indicators used in this study were satisfactory and complementary when applied to sun-dried meat.


Strychnos pseudoquina was the species with the highest amount of use indications. The diversity index was 4.5 nats/ind-1. The body system with the most citations was the code XVIII of ICD 10, corresponding to the species: alfavaca, mentraste, terramicina, angelim, fedegoso. Medicinal species with AMU values higher than 25% were: Strychnos pseudoquina, Plectranthus barbatus, Citrus sinensis cv. pera, Cymbopogon citratus. There was a relationship between the number of useful plants and the residence time of the participants. The Bananal community revealed high species richness and the relationship of knowledge showed that the older the residents and the longer their residence time in the community, the more knowledge they acquired.Studies in rural communities are important to maintain popular knowledge between generations, as well as to identify new species for pharmaceutical production. Thus, the objectives of this study were to determine which plant species the rural community of Bananal, Mato Grosso, Brazil, uses by calculating the levels of fidelity and concordance regarding species uses among residents and to determine if there is a relationship between the number of known useful plants and levels of education, age, and residence time. Ethnobotanical data was collected from residents of the community through semi-structured interviews in January/December/2016. Species diversity was calculated using Shannon-Wiener, Level of Fidelity (LF), Correction Factor, and the Percentage of Agreement regarding the Main Uses (AMU). Statistical tests were performed using generalized linear models (GLM) in the R environment. The plant use indications were grouped according to the International Classification of Diseases and Related Health Problems (ICD 10). We found 152 species belonging to 130 genera and 67 families. The most frequently used plant parts were leaves, and decoction was the most frequent preparation mode.  Ethnobotanical studies have been carried out throughout human history. Such studies started off as qualitative descriptions, but later on focused on quantitative analyses that evaluated levels of fidelity and agreement of use of the species, and now used as statistics with inferencial analyses. Quantitatively, it is possible to demonstrate the importance of different plant characteristics by investigating the knowledge, uses of such plants within a society , and praIn developed countries, there are less ethnobotanical studies in comparison to developing countries. Some recent works include studies from Italy ,4, from In light of these surveys, ethnobotanical studies are important throughout the world because of communities that use plant resources for subsistencc. Dissemination of such data favors the exchange of knowledge between communities from different places in the world, forming a knowledge network. Currently in Brazil, most of the population still uses medicinal plants to alleviate or even cure certain diseases due to their low cost and/or effective results –18. The Such knowledge about useful plants is only orally passed down from one generation to the next , 20, 21 This study aimed to: (i) determine the plant species used by the rural community of Bananal, Mato Grosso, Brazil, (ii) verify the levels of fidelity and agreement of uses among the local inhabitants, (iii) determine the relationship between the number of known medicinal plants and the levels of education, age, and residence time.The study was conducted in a rural area in the Bananal Community, located in the northern region of the city of Rondonópolis, Mato Grosso, Brazil, at the coordinates 16°8’58.68”S and 54°35’10.63”W . The comAccording to Köppen, the local climate is Tropical Aw. The average annual temperature is 27°C, with a maximum of 40°C in September and a minimum of 9°C in July. The highest average rainfall occurs in January, with 320mm, and the lowest in June .The vegetation is divided into regions of gallery forests, savannas, and grasslands, and presents a transition between savanna woodland (Cerradão) and riparian forest. The range of riparian forest vegetation runs parallel to the watercourse and represents the largest part of the remnant native vegetation. The areas of Cerrado present woody vegetation with trees, shrubs, and native herbaceous plants.Historically, the colonization of the Bananal region began around 1920, with the migration and occupation of landless workers in the Rondonópolis territory . In the The community is assisted by programs that provide courses, lectures, and workshops that invest in quality of life and the implementation of family agriculture, such as ponds for fish farming, spring recovery, guidance for field activities, hydroponics implementation, as well as vegetable seeds for families interested in growing vegetable crops .After the determination of the study site, contact was made with the nurse and Community Health Agent (CHA), who are members of the FHS care team. The research was explained and invited to CHA to accompany the researcher in recognizing the area and visiting the homes of people in the rural community, since the residences are far from each other. Follow-up by a local representative helped establish a good relationship between researcher and community residents. They were informed and invited to participate in the survey. Data collection was between January and December 2016, which consisted of semi-structured interviews in the participants' homes.The following requirements were established for all research interviews: prior consent, availability of participant, all participants were 18 years old or older, participants were residents and registered by the FHS, and family member(s) who had knowledge of useful plants for any purpose. The study excluded residents who were not found after three attempts during the data collection period. The study was carried out with 50 residents from the rural community, the interviews were previously authorized by all the respondents, who signed a Term of Free and Informed Consent, and the CHA as the local representative of Health in the Community, signed an authorization for research on behalf of the entire community.The species of plants mentioned were defined as sampling units. The species were identified in the field and some collected and photographed for records and subsequent identification without the need of specific organs permissions, because they were on private land and have been authorized by the representative, not involving endangered or protected species. They were collected at several places in the following coordinates: place of collection 1: 16° 8'50.35"S and 54°34'58.64"W; place 2: 16° 8'57.66"S and 54°34'54.21"W; place 3: 16° 8'39.16"S and 54°34'59.71"W; place 4: 16° 7'24.03"S and 54°34'38.04"W; place 5: 16° 7'26.04"S and 54°34'49.02"W; place 6: 16° 7'16.10"S and 54°34'9.38"W; place 7: 16° 7'25.24"S and 54°34'2.24"W; place 8: 16° 7'49.98"S and 54°33'57.06"W.The collected material was herbarized and identified by consulting specialized literature and botanical experts, as well as through comparison with exsiccates. Of these, the reproductive stages were deposited into the UFMT Herbarium at the Universidade Federal de Mato Grosso (UFMT), Cuiabá, MT.The scientific names of the species were conferred using online databases of the Missouri Botanical Garden/Tropics  and the The empirical classification was used to describe the use category of the cited species. The cited species were grouped into seven categories: medicinal, medicinal and food, food, medicinal and other , ornamental and mystical, medicinal, food and lumber, and medicinal, ornamental, and mystical.Plant preparations were classified as follows: decoction (plant part was cooked) as tea, molasses, syrup, and drink with toasted plant, infusion (plant part was put in hot water), ingestion , bottled medicine, and others.Each plant use indicated by the participants was grouped according to the International Classification of Diseases and Related Health Problems (ICD 10), published by the World Health Organization .e To verify species diversity, we used the Shannon-Wiener index (H’) applied to ethnobotany as described by , which iS = Number of speciesI in relation to the total number of species cited.pi = Relative proportion of abundance of species Later, the Pielou evenness or uniformity measure was used to measure the distribution pattern in the community . This caH’ = Shannon-Wiener diversity indexMax = Maximum value of H’H’ One technique we used to analyze the relative importance of a plant in the community was the consensus among participants. The consensus analysis was based on the concordance between participant responses.For each plant, we also calculated an index called Level of Fidelity (LF) ,32, exprIp = number of informers that mentioned the main use of a particular species.Iu = number of participants that mentioned the species for any purpose.The Correction Factor (CF) was estimated to obtain consensus among the informants for each plant. Its value is derived from the number of citations of a certain species, i.e., the number of participants that mentioned each species divided by the number of citations for the most commonly mentioned species, i.e., the number of participants that mentioned the most commonly mentioned species.Calculating the CF:Then, the percentage of Agreement regarding the Main Uses (AMU) was calculated to neutralize the higher or lower popularity of the species. This calculation is based on the value found in the LF multiplied by the correction factor, expressed in the following formula:This analysis can be adapted to any use category, but was initially used in studies on medicinal plants . In thisStatistical models were used to explore how social and cultural variables interact with each other and with the knowledge of plant collection, use and management intensity . We use The research project was sent to the Committee of Ethics in Research with Human Beings of the University Hospital Julio Muller (CER / HUJM), Cuiabá / Mato Grosso, in compliance with Resolution 466, of December 12, 2012, of the National Health Council. The CER / HUJM approved all aspects of the research and issued a favorable opinion for the development under the number of CAAE: 48675315.0.0000.5541.The research in rural community was provided by the Municipal Health Department of Rondonópolis, in Mato Grosso. In the interview, participants were informed about the research and their participation on a voluntary basis, they signed the Informed Consent Form authorizing the participation. In addition, a local representative, resident in the Community, also signed an authorization to conduct the survey on behalf of all, including the collection of some species for botanical identification on their properties, without the need for permissions of specific organs.Of the 50 interviewees, 28 were males and 22 were females; age ranged from 34 to 81, with a predominance of 50–59 year-olds. The most frequent level of education (34%) was incomplete elementary school, however, the educacional level of the interviewees varies from literate to higher with postgraduate studies. Family farming and retirement sustain the community. More than half of the interviewees (54%) were from the state of Mato Grosso.In relation to plants, the participants mentioned 152 species of useful plants, distributed in 130 genera and 67 families . The numThe botanical families with the highest number of useful species were Asteraceae (12), Fabaceae (11), Rutaceae (9), which represented 21% of all species recorded . Such reThis work evidences the ethnobotanical importance of the native Cerrado plant species and their relationship with the local inhabitants. Empirical knowledge of these species is high considering the number of species, and is capable of generating economic, social, and ecological values for these species. Such species are of fundamental importance for local development as a means of subsistence and immediate relief of health problems in the region, which clearly demonstrates the interaction between community and the environment.Through the interviews, we obtained 1,070 indications of use for the species reported. These values showed high ethnobotanical knowledge of plants, especially for medicinal plants, in this region. Several studies have been developed aiming to collect information regarding the knowledge of native plants use by different ethnic groups, such as ,44,48, iStrychnos pseudoquina A. St.–Hil (Loganiaceae) with 50 citations, followed by Hymenaea stigonocarpa Mart. Ex (Fabaceae) (47 cit.), Citrus sinensis Osbeck cv. Pera (Rutaceae) and Plectranthus amboinicus (Lour.) Spreng. (Lamiaceae) (29 cit.), and Senna occidentalis (Fabaceae) (L.) Link. (23 cit.)  .Strychnos pseudoquina (quina) was indicated for treatment of bodily problems, with several purposes including digestive, vermifuge, depurative, appetite stimulant, anemia, diabetes, cough, headache, however the most frequent citation was for problems with the digestive system  is distributed in South America in countries such as Brazil, Bolivia, Paraguay and Venezuela [Hymenaea spp. tested commercial sap samples and detected adulteration and microbiological contamination, which could be a risk to human health [enezuela  and is aenezuela . There aenezuela –64. Howen health . Thus, wCurcuma longa L. (acafrão) with European origin, Aloe vera L. Burm.f. (babosa) from Africa, Cymbopogon citratus (DC.) Stapf (capim-cidreira) from Asia, Punica granatum L. (romã) from the Mediterranean, and Ocimum basilicum L.  from India .Regarding the origins of the 152 identified useful species, 73% were native species and 27% were naturalized, cultivated or exotic. However, in relation to use indication, cultivated species stood out because of their availability and ease of collection, as they were often present in backyards or in vessels near homes. Among the cultivated and exotic species found in the community, we cite om India .-1 and species evenness was J’ = 0.9. Such high diversity values were also found in other studies [The species diversity was H’ = 4.5 nats individual studies –68. Dive studies  increaseStrychnos pseudoquina (50%), Plectranthus barbatus Andr. (40%), both mainly used for digestive problems, Citrus sinensis cv. pera (28%), primarily used for the flu, and Cymbopogon citratus (DC.) Stapf (26%), primarily used as a calmative  revealed four species with values higher than 25%, representing only 2.9% of the useful species. These species were almative .Strychnos pseudoquina, as all participants mentioned that it had at least one use and half of the participants indicated it for the same purpose. This reflects the species’ popularity and the knowledge the community has in relation to its therapeutic actions, with several studies [S. pseudoquina.This study highlightsthe species  studies –58 demonPlectranthus barbatus was only indicated for digestive problems by participants, a review of the species [Additionally, although  species  mentions species  identifi species  referredA large proportion of species (97.5%) presented low AMU% (below 25%). However, this does not mean these species are irrelevant to the community, which is possibly related to the wide range of indication of species reported by informants, but few useful purposes. Even though the Bananal community expressesed species diversity, their use citations were restricted.According to participant indications, use categories were: medicinal, medicinal and food, food, medicinal and other , ornamental and mystical, medicinal, food and lumber, and medicinal, ornamental and mystical . Among tHymenaea stigonocarpa, which was mentioned as a wood source, food item, and for its medicinal purposes. Thus, 18.4% of the mentioned species had medicinal and food purposes and 9.2% were exclusively used for food industries  plant species as dustries .Dieffenbachia seguine (Jacq.) Schot (comigo-ninguém-pode) and Ruta graveolens L. (arruda) were claimed to protect the home and ward off evil, demonstrating the symbolic-mystical-religious of the local people [In the present study, food plants use was low. According to , food pul people .Regarding plant parts used to prepare medications, the most prominent parts were leaves 37.1%), bark (14.3%) and roots (13.1%) . Such da.1%, barkThe leaves were the most frequently mentioned parts in other studies with 72.5%, 68.2%, 61%, 40%, respectively ,67,83,84The research participants reported several preparation modes for the species with medicinal purposes. Decoction was the most common preparation mode (36.7%), followed by ingestion (22.4%), infusion (10.7%), and bottling (7.4%) .Decoction was also the most frequently used method by people in other communities ,44,42,86Digitaria insularis (L.) Fedde (capim-amargoso) added to Momordica charantia L. (melão-de-são-caetano) to treat renal infections. The practice of combining one or more plant species was expressed in other studies [The popular knowledge in the Bananal community is vast, as the participants reported that harvesting, as well as preparation and measuring, of plant material should be done carefully. They also mentioned combining some herbs to obtain more effective results, as  studies ,89 and p studies .Popular recipes depend heavily on the availability of local plants . KnowledGrouping by ICD-10 serves to inform the classification code of diseases and a wide variety of signs, symptoms, abnormal aspects, complaints, social circumstances, and external causes for injury or illnesses. Each state of health receives a bodily category or system, which corresponds to a code represented by roman numerals from I to XXII. This code is often used for classifying diseases. In this study, the classification was performed with the objective of evaluating the most affected diseases and body systems in the community.Ocimum basilicum L. , Angeratum conyzoides L. (mentraste), Alternanthera brasiliana (L.) Kuntze (terramicina), Vatairea macrocarpa (Benth.) Ducke (angelim), Senna occidentalis (L.) Link. (fedegoso). Following, code X represented 14.4% of citations and referred to diseases of the respiratory system, with the species: Anadenanthera peregrina (L.) Speg. (angico), Attalea phalerata Mart. ex. Spreng. (bacuri), Senna occidentalis (fedegoso) and Hymenaea stigonocarpa (jatobá). Code I (11.1% of citations) refers to infectious and parasitic diseases, represented by species: Dysphania ambrosioides (L.) Mosyakin & Clemants (erva-de-santa-maria), Momordica charantia L. (melão-de-são-caetano), Terminalia fagifolia Mart. (mussambé), and Leptolobium dasycarpum Vogel (unha-danta). Code XI (10.7% of citation) is related to diseases of the digestive system (Vatairea macrocarpa (angelim), Gymnanthemum amygdalinum (Delile) Sch. Bip. ex Walp. (caferana) and Leptolobium dasycarpum Vogel (unha-danta).The therapeutic indications of the136 medicinal species mentioned by the Bananal community were categorized according to the ICD-10. After organizing this information, we found that plants were indicated for 17 of the 22 bodily systems of ICD-10. The most frequent citation was code XVIII, with 16.8% , referrie system , and theThe high citation value for code XVIII may be caused by commonly occurring diseases and diseases with unknown causes . In placWhen verifying relationships of community knowledge about useful plants, inferential statistical tests showed that 73.1% of the use indications corresponded to 20 participants and approximately 26.9% of use indications to 30 participants. We infer that the community knowledge about useful plants was concentrated in a few residents of the community.P value = p ≤ 0.001  reinforced the interaction between residence time and age. Another study found different results, where people of different age categories (juveniles and adults) showed comparable knowledge of medicinal plants . Thus, iSuch results support that knowledge is concentrated in the older community members, since they indicated a higher amount of plant uses, and the youngest people had less knowledge of plant uses. Such lack of information may be due to young people’s lack of interest in useful plants species and may be influenced by increased technology. Furthermore, such useful knowledge, that is concentrated with the older members of the community, should be shared with other generations, as it could be lost if not passed on. Corroborating with our findings, ,97 highl-1. The preferentially used species were Strychnos pseudoquina and Hymenaea stigonocarpa. Our research showed that the older the participant was and the longer their residence time was, the more knowledge they had about useful plants. Such knowledge is concentrated in older community members. In this sense, encouraging knowledge socialization among the different generations is essential to ensure that beliefs, traditions, and culture are not lost over the years.This study significantly contributes to the value, socialization, and record of popular knowledge about useful plants, which was not being documented. This is the first record of popular uses of plant species in the Bananal rural community. This community revealed high species richness, with 152 useful plant species presenting values, including 136 medicinal plants. The Bananal community also presented 1,070 citations, with various use indications to treat health problems. Overall, the community showed high diversity of knowledge about the plant species, with a diversity index of 4.5 nats/indS1 Table2018.(DOCX)Click here for additional data file.


The objective of this systematic review protocol is to provide the methods for evaluating the effectiveness and safety of acupuncture on the treatment of myasthenia gravis (MG).We will search randomized controlled trials (RCTs) on this subject in 8 electronic databases and they are Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, PubMed, Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure (CNKI), the Wan-Fang Database, and Chinese Scientific Journal Database (VIP database). Other relevant literatures will be manually searched as a complement. Only RCTs related to acupuncture for MG will be included, without Language restrictions and limitation of publication types. The risk of bias and trial quality will be assessed by the Cochrane collaboration tool. The study inclusion, data extraction and quality assessment will be conducted independently by 2 reviewers. All data from the studies included will be analyzed by RevMan V.5.3 statistical software.This study will provide a high-quality synthesis of RCTs on the efficacy and safety of acupuncture as an adjuvant therapy in the treatment of MG.This systemic review will provide high quality evidence to evaluate acupuncture as adjuvant therapy in patients with MG.PROSPERO CRD42019133577. And mortality is up to approximately 40%. At present, the incidence and prevalence of MG are increasing, particularly in older individuals.,4 According to a large clinical study of unselected patients with MG in China, the most striking result is the high proportion of childhood cases mostly with purely ocular MG, which is different from clinical expression of caucasian with MG. Ocular weakness, the most common initial presentation of MG, occur in approximately 85% of patients. According to a large number of MG epidemiological studies, the prevalence of fatigue was 70%. Various clinical treatments for MG exist, including thymectomy, symptomatic, and immunosuppressive (IS) treatments, and immunomodulating therapies such as intravenous immunoglobulin (IVIg) and plasma exchange (PLEX).,9Myasthenia gravis (MG) is an autoimmune antibody-mediated disorder characterized by fluctuating fatigability and weakness affecting ocular, bulbar, and limb skeletal muscle groups.,10 Standards and possibilities for the diagnosis and treatment of myasthenia gravis show great variation within and between countries. In particular, orthodox therapy for effective symptom control often requires prolonged and even life-long IS treatment with high-dose steroids and add-on other IS agents.–14 Furthermore, despite there have been significant advances in the treatment of MG, an estimated 10% to 20% of patients with MG do not achieve an adequate response, are intolerant to conventional treatment. In addition, the adverse effects associated with these treatments are significant, such as diarrhea, nausea, vomiting, salivating, muscle twitching, and the treatments suffer from short effectiveness, difficult dosage control, strong dependence, and high cost. Implementing best-practice standards universally represents a major challenge.However, there is no internationally accepted standard of care, and no one treatment best for all patients because of heterogeneous of MG. Nowadays, an increasing number of patients with MG seek help from complementary and alternative medicine. Acupuncture, an important part of traditional Chinese medicine (TCM), has been used to treat MG diseases for a long time and obtained experimental evidence. According to our pre-search, many clinical trials, which were conducted to investigate the efficacy of acupuncture for patients with MG, indicated that acupuncture could relieve the patients’ symptoms. However, there has been no systematic evaluation of the safety and efficacy of acupuncture in the treatment of myasthenia gravis. Thus, we conducted a systematic review of acupuncture for MG focused on the clinical evidence according to the high-quality randomized-controlled clinical trials (RCTs).Complementary and alternative medicine (CAM) is increasingly used to treat MG, owing to its long-term efficacy and few side effects. Acupuncture is one of the most frequently used forms of complementary medicine.2This systematic review aims to analyze various RCTs, further summarize and critically evaluate the evidence for the effectiveness and safety of acupuncture treatment of MG.3This protocol of this systematic review has been registered on PROSPERO, the registration number is CRD42019133577. The protocol will be strictly developed under the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocols (PRISMA-P).3.13.1.1We will only include RCTs of acupuncture treating MG without publication or language restriction. And quasirandomized RCTs will be ruled out, such as case report, and the study without sufficient information about the randomized method or process.3.1.2 China guidelines for the diagnosis and treatment of myasthenia gravis. The other diagnostic criteria with comparable definitions were also used.We will include patients with any sex, age, nationality, and education background, who are diagnosed with myasthenia gravis. Myasthenia gravis diagnoses are on the grounds of the internationally recognized criteria, such as the diagnosis criteria settled by the Myasthenia Gravis Foundation of America (MGFA),3.1.33.1.3.1Clinical trials with all types of acupuncture intervention including regular acupuncture, electroacupuncture, fire needling, scalp needling, auricular acupuncture, catgut embedding acupuncture, and intradermal needling will be included. While we will exclude other forms of stimulation including laser acupuncture, cupping, moxibustion, bleeding therapy, acupotomy, pharmaco acupuncture, or point injection.3.1.3.2Eligible control group will include the studies that are interfered with no treatment, sham acupuncture, placebo or Western drug. Additionally, studies compare acupuncture plus the concomitant of other treatment with that treatment will also meet the control group including criteria.3.1.43.1.4.1We will consider the following outcomes measuring the extent of MG and muscle weakness:(1)Quantitative Myasthenia Gravis (QMG) scores.(2) The MG clinical absolute and relative scores are a 60-point scale evaluate ptosis, eyelid fatigue, eye movement in the horizontal direction, right and left arm held outstretched at 90°, flexion of the knee and hip at 90°, facial muscles, chewing and swallowing, and respiratory muscle function.MG clinical absolute and relative scores or other validated scales.(3)Clinical absolute score before and after treatment.(4)Clinical relative score and effective rate: The clinical relative score = (absolute score before treatment – absolute score after treatment)/absolute score before treatment 100%.3.1.4.2The secondary outcomes included:(1)Relapse rate after follow-up.(2)Adverse events.(3)Acetylcholine receptor antibody (AchRAb): The concentration of AchRAb in Serum.(4)Quality of life. Such as MG activities of daily living (ADL) profile (MG-ADL)-a simple 8-question survey of MG symptoms.3.2Two reviewers independently searched the following databases: PubMed, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Library, Chinese Biomedical Literature Database, China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP) Journals Database, Wan fang data Information Site without language restriction. The search time was from inception to September 30th of 2019. The reference lists of retrieved trials and previous systematic reviews will be searched for citation of potentially eligible trials. We will contact the author of articles if there were any question about trials.http://www.who.int/trialsearch) the US National Institutes of Health Ongoing Trials Register  and meta Register of Controlled Trials  for any unpublished or relevant ongoing trials. The following search terms will be used: myasthenia gravis, acupuncture, manual acupuncture, electroacupuncture, fire needling, auricular acupuncture, ear acupuncture, dermal needle, abdominal acupuncture, pyonex and plum blossom needle, controlled clinical trial, randomized, placebo, randomly. The search strategy for PubMed is shown in Table In addition, we will search the WHO International Clinical Trials Registry Platform , Acupuncture Research (1985–2019), China Journal of Traditional Chinese Medicine and pharmacy (1985–2019). Meanwhile, relevant conference papers will be manually retrieved. At the same time, we will also search the WHO International Clinical Trials Registry Platform (ICTRP) to contain new trails pertaining to the theme.3.33.3.1After electronic searches in the databases, 2 reviewers (SS and JXY) will screen the titles and abstracts respectively to exclude: the duplicates; the studies in which the participants in the experimental group did not receive acupuncture treatment as the primary intervention; the studies that were not RCTs with parallel design; and the studies in which the participants did not meet the criteria of MG. Then our reviewers will screen the full text articles that cannot be obviously screened by titles and abstracts only. And reviewers will analysis consideration to identify the included studies and assess the study eligibility. Reasons for the exclusion of studies will be recorded. In addition, all reviewers will have a group discussion on the consistency of all the studies included, exclude and eliminate those are not up to the theme topic till final team consensus arrived. The selection process is fully elucidated in the following PRISMA flow diagram Fig. .3.3.2An electronic form will be established via Excel to extract substantial contents, and then filled by 2 reviewers independently. Information was extracted from the qualified articles by using a standardized data extraction form as follows: general information: first author, the year of publication, and country; participants: sample size, sex, age, and disease duration; methodological characteristics: study design, MG severity, and diagnostic criteria; details of intervention: type of intervention, duration of treatment, and follow-up time; and outcome measures. Disagreements will be solved by group discussion or consult seniors. However, if we fail to reach the consensus, the authors of trials will be contacted for further details and verification.3.3.3Two independent authors assess the risk of bias of each included article according to the Cochrane Handbook for Systematic Reviews of Interventions. The methodological quality will be evaluated from the following domains: generation of random sequence, allocation concealment, blinding of participants and personnel, blinding of outcome assessments, incomplete outcome data, selective reporting, and other bias. Description of each aspect of risk of bias in each pooled study will be conducted in order to provide the rationale for the risk judgment. The risks will be classified into 3 levels: low, high, or unclear with a graphical presentation.3.3.4I2 statistic will be used to examine the heterogeneity between trial and control results. A fixed effects model (I2 < 50%) or a random effects model (I2 > 50%) will be used depending on the value of I2. The value of P < .05 was considered statistically significant. Dichotomous outcomes were calculated by the risk ratio (RR) with 95% confidence interval (CI), whereas continuous outcomes will be summarized using standardized mean difference (SMD) with 95% CI. Publication bias will be checked graphically by using the funnel plot, and approximately symmetric shows no existence of publication bias.Statistical analysis will be performed by Cochrane Collaboration Review Manager Software (RevMan 5.3). Standard chi-squared test and 3.3.5If any data information is not sufficient in included trials, we will try to contact the first or corresponding author by email or phone, requesting for adequate information and details of the studies included to retrieve missing or insufficient trial data. However, if the author is not available or sufficient information cannot be obtained, we will have a group discussion and analysis based on the current information. Meanwhile, the potential impact of missing data will be taken into account and relative discussion will be presented in the result section.3.3.6α = 0.1) and I2 value, respectively, to assess the heterogeneity. If I2 ≤ 50%, the studies included will indicate no presence of meaningful heterogeneity, and then a fixed-effect model will be employed to evaluate the effect sizes. While, if I2 > 50%, indicating the heterogeneity among studies are statistical significant, and a random-effect model will be adopted.Based on the guidelines in Cochrane Handbook for systematic review, we will perform a standardized chi-squared test .3.3.10After removing the low-quality studies, we will perform a sensitivity analysis to evaluate the robustness of the results according to following aspects: sample size, missing data, and methodologically quality. The sensitivity analysis is conducted to exclude the inappropriate trials without random generation.3.3.11The Grading of Recommendations Assessment, Development and Evaluation (GRADE) guidelines will be employed to evaluate the quality of evidence for primary outcomes. The strength of evidence will be graded it into very low, low, moderate, or high level.3.3.12Formal ethical approval is not required in this protocol. We will collect and analyze data based on published studies, and since there is no patients involved in this study, individual privacy will not be under concerns. The results of this review will be disseminated to peer-reviewed journals or submit to related conferences.4MG brings a significant adverse impact on patients’ daily activities, including quality of life, general and psychological health. Pharmacological methods have been associated with a slight increase in incidence of adverse effects and surgery is not appropriate for each patient. Acupuncture therapy, a suggested intervention in China that may have value to treat MG, has been shown to improve some symptoms of myasthenia gravis, such as eye weakness and fatigue. But no high-quality synthesis of the evidence exists. To our knowledge, there is no related systematic review published in English. Therefore, a high quality systematic review is needed. And we show the process of performing this study in Fig. This study may have limitations that might limit its ability to generate conclusions based on high confidence. Specifically, there may be significant heterogeneity in the forms of acupuncture therapies used and the qualities of methodology. There will also most likely be differences in outcomes measured and tools used.LC, ZHM, WYP conceived this study and developed the first frame of this manuscript. JXY, SS, drafted the manuscript. LB, JXY performed data collection; SS revised the manuscript. All authors contributed to the editing of the final manuscript and approved the final version.


Flubendazole, originally developed to treat infections with intestinal nematodes, has been shown to be efficacious in animal models of filarial infections. For treatment of filarial nematodes, systemic exposure is needed. For this purpose, an orally bioavailable amorphous solid dispersion (ASD) formulation of flubendazole was developed. As this formulation results in improved systemic absorption, the pharmacokinetic and toxicological profile of the flubendazole ASD formulation have been assessed to ensure human safety before clinical trials could be initiated.Safety pharmacology, toxicity and genotoxicity studies have been conducted with the flubendazole ASD formulation.in vivo toxicity studies with the ASD formulation, high systemic exposure to flubendazole and its main metabolites was reached. Flubendazole, up to high peak plasma concentrations, does not induce Cmax related effects in CNS or cardiovascular system. In repeated dose toxicity studies in rats and dogs, flubendazole-induced changes were observed in haematological, lymphoid and gastrointestinal systems and in testes. In dogs, the liver was an additional target organ. Upon treatment cessation, at least partial recovery was observed for these changes in dogs. In rats, the No Observed Adverse Effect Level (NOAEL) was 5 mg (as base)/kg body weight/day (mg eq./kg/day) in males and 2.5 mg eq./kg/day in females. In dogs, the NOAEL was lower than 20 mg eq./kg/day. Regarding genotoxicity, flubendazole was negative in the Ames test, but positive in the in vivo micronucleus test.In animals, flubendazole has good oral bioavailability from an ASD formulation ranging from 15% in dogs, 27% in rats to more than 100% in jirds. In Based on these results, in combination with previously described genotoxicity and reproductive toxicity data and the outcome of the preclinical efficacy studies, it was concluded that no flubendazole treatment regimen can be selected that would provide efficacy in humans at safe exposure. in vivo toxicology studies, increased systemic exposure does not induce Cmax-related effects in CNS and cardiovascular systems. Increased exposure upon repeated dosing results in changes in haematological, lymphoid and gastrointestinal systems and in testes. In dogs, the liver was an additional target organ. These changes were at least partially reversible. Flubendazole is negative in the Ames test but positive in the in vivo micronucleus test. Because of the carcinogenic risk associated with this positive effect in the in vivo micronucleus test, exposure duration in patients should not exceed one day. Flubendazole-induced toxicity and associated risk is monitorable and controllable in patients if stringent precautions are applied in view of testicular toxicity and previously described teratogenicity. Considering both, treatment regimen needed for efficacy and outcome of toxicity and genotoxicity studies, it was concluded that the risk/benefit associated with the use of orally bioavailable flubendazole for the treatment of onchocerciasis in the field does not support further development.This article describes pharmacokinetic profiles and results of safety pharmacology, toxicity and genotoxicity studies with an oral ASD formulation of flubendazole with improved bioavailability. Flubendazole administered as ASD formulation has good oral bioavailability in animals ranging from 15% to more than 100%. In  Onchocerca volvulus, which spreads through bites from infected black flies. The presence of larvae in the skin causes several symptoms, including intolerable itching. Larvae can migrate to the eye and cause decreased vision and blindness. Adult worms live in nodules in the skin, can survive for 10–15 years and produce thousands of larvae per day. Current treatment of onchocerciasis relies on three drugs, albendazole, ivermectine and diethylcarbamazine, agents working primarily against microfilarial stages. In tropical medicine, there is a need for a macrofilaricidal drug that safely kills adult filarial worms. [Onchocerciasis is a neglected tropical disease caused by the parasitic worm species l worms. In vivo activity of flubendazole against a variety of filariid species has been reported in animals and man after parenteral administration indicating systemic exposure is needed for the treatment of onchocerciasis. For this purpose, an orally bioavailable amorphous solid dispersion (ASD) formulation was developed. ASD is an approach to formulate poorly water-soluble drugs in the amorphous form, for the enhancement of dissolution rate and bioavailability.Flubendazole, originally discovered and developed by researcher Dr. Paul Janssen and his team, was first approved for human use in 1980 to treat soil transmitted helminths (STH), also known as intestinal worms. This methylcarbamate benzimidazole anthelmintic has been shown to have a marked macrofilaricidal effect on many filarial species.  Oral fluBecause of the significantly higher systemic exposure anticipated from the ASD formulation, the safe evaluation of this formulation in clinical trials could not be supported by nonclinical safety studies performed for the marketed oral flubendazole formulation. In the nonclinical safety studies performed in support of flubendazole for STH, systemic exposure was very low. The set of nonclinical studies performed for the safety evaluation of the ASD formulation and their design including number of animals used, were based on international standards described in guidelines of the International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH guidelines promote safe and ethical development of pharmaceuticals and reducing the use of animals in accordance with the 3R (reduce/refine/replace) principles is part of their objectives.in vivo micronucleus test [in vivo micronucleus test in rats as a solution/suspension in polyethylene glycol 400 (PEG400) and hydroxypropyl-β-cyclodextrin. An Ames test was performed with flubendazole. The results of the evaluation of flubendazole and its main metabolites in the Ames test and the in vitro micronucleus test have been described [With the ASD formulation, two-week repeated dose toxicity studies followed by 1-month recovery were performed in rats and dogs as well as a cardiovascular safety study in dogs and a CNS safety study in rats. Flubendazole amorphous solid dispersion had been tested in an eus test . FlubendThis paper describes the results of these preclinical safety studies and their impact/implications for the potential use of this new orally bioavailable ASD formulation in humans, for the conduct of clinical trials and finally for the risk/benefit associated with the use of orally bioavailable flubendazole for the treatment of onchocerciasis in the field.After intravenous administration, flubendazole (FBZ) showed a low plasma clearance in rodents (rats and jirds) and high clearance in dogs , a mediuhttps://doi.org/10.5061/dryad.5vv774m [Data available from the Dryad digital repository: When compared to vehicle dosing, a single oral administration of flubendazole at a dose of 20, 50 or 120 mg eq./kg to telemetered male beagle dogs did not exert any adverse effect on health status, body weight, electrocardiogram, body temperature, arterial blood pressure and respiratory parameters. Concentrations of flubendazole and the two metabolites R-FBZ and H-FBZ, measured approximately 6.5 hours after dosing at 20, 50 or 120 mg eq./kg, indicated exposure to flubendazole and both metabolites in all five dogs .Under the experimental conditions of the study, a single oral administration of flubendazole at a dose of 5, 15 or 60 mg eq./kg to male Sprague-Dawley rats did not adversely affect body weight, rectal temperature or neuro-behavioral parameters investigated in the modified Irwin test. The concentrations of flubendazole and the two metabolites were measured after completion of the modified Irwin test sessions, by treating the 6 rats previously treated with the vehicle with a dose of 60 mg eq./kg. Toxicokinetic parameters were calculated .https://doi.org/10.5061/dryad.5vv774m [Data from safety pharmacology studies available from the Dryad digital repository: Flubendazole dosed orally to male and female rats for 14 or 15 consecutive days resulted in premature sacrifice of all high dose animals  on Days 7, 8 or 11, due to poor general condition. Flubendazole-related mucosal atrophy in the small and large intestines and bone marrow atrophy were considered to be the main contributors to the moribund condition of these rats.The primary effects associated with flubendazole administration involved the haematological and gastrointestinal systems. Additional effects were seen in the testes and thymus.At the low dose , no flubendazole-related adverse effects were observed.At the mid dose, males (15 mg eq./kg/day) exhibited a flubendazole-related decrease in body weight gain from Day 8 onwards, which correlated with decreased food consumption. Tables  Slight tBased on the information above, the No Observed Adverse Effect Level (NOAEL) in this rat study was considered to be the low dose, i.e. 5 mg eq./kg/day for males and 2.5 mg eq./kg/day for females. The exposures were higher in female compared to male rats. The sex related difference for flubendazole is probably due to the differential expression of various sex-dependent forms of cytochrome P450s (CYP450) mediated by hormonal regulation, enzymes which are involved in the metabolism of flubendazole. In general, this gender-related difference in CYP450 expression and metabolism is more striking in the rat than in any other species, Plasma levels of flubendazole and both metabolites, R-FBZ and H-FBZ, were measured and toxicokinetic parameters at the end of dosing are shown in Flubendazole dosed orally to male and female dogs for 14 or 15 consecutive days resulted in the premature sacrifice of one dog at 100 mg eq./kg/day, based on poor general condition. Flubendazole-related liver enzyme activity increase and associated hepatocellular necrosis were considered the main cause of morbidity. The primary effects associated with flubendazole administration involved the haematological and gastrointestinal systems. Additional effects were seen in the liver, testes, epididymides, thymus, lymph nodes and bone marrow.Gastrointestinal clinical signs  were observed at 40 and 100 mg eq./kg/day. Dogs dosed at 20 or 40 mg eq./kg/day minimally lost weight during the dosing period (-2% to -3%) while a more pronounced body weight loss was noted in dogs dosed at 100 mg eq./kg/day .  At all dThere was complete recovery for clinical signs, body weights, food consumption and haematology parameters at 40 and 100 mg eq./kg/day, and liver enzyme activities and microscopic findings at 40 mg eq./kg/day. There was partial recovery for alanine aminotransferase activity and spleen weight in females at 100 mg eq./kg/day and microscopic findings in the testes/epididymides and the perivascular inflammatory infiltrate in the liver at 100 mg eq./kg/day. Based on the information above, a NOAEL for dogs after 14 days of treatment could not be determined.Plasma levels of flubendazole and both metabolites, reduced flubendazole and hydrolyzed flubendazole were measured and plasma toxicokinetic is shown in No biological relevant and/or dose-related increases in the number of revertant colonies were observed with any of the tester strains up to the precipitating concentration of 2000 μg/plate either in the presence or absence of an exogenous metabolic activation system (S9-mix). These results indicate that flubendazole was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of S9 mix. In the peripheral blood micronucleus test, a slight reduction in red blood cell proliferation was observed in male rats dosed with flubendazole at 15 and 50 mg/kg/day. In female rats, a pronounced reduction in red blood cell proliferation was observed at 15 and 50 mg/kg/day. Administration of flubendazole did not result in a biologically relevant increase in micronucleated reticulocytes in male or female rats at any of the dose levels tested.  In the bBased on the results of this study, it is concluded that flubendazole induces structural and/or numerical chromosome aberrations in erythrocytes of rat bone marrow. There were no biologically relevant increases in micronuclei in male rats up to the 15 mg/kg/day dose, however, the lowest dose to cause biologically relevant induction of micronuclei in female rats was 5 mg/kg/day.Flubendazole as currently formulated for the treatment of gastrointestinal parasites has poor systemic availability when given orally. Due to its poor bioavailability and solubility, the original oral formulation induces limited systemic or gastrointestinal toxicities and is negative in the bone marrow micronucleus tests.  FlubendaIn vivo oral toxicity studies with the ASD formulation described above, clearly show relevant systemic exposure to flubendazole and its main metabolites, reduced flubendazole and hydrolyzed flubendazole. (Tables max in plasma) following treatment with the marketed flubendazole formulation at the usual dosage (100 mg once or twice a day for 3 days). [in vivo filarial efficacy studies described by MP Hübner et al., 100% efficacy could not be achieved with oral treatment. Highest efficacy achieved with oral treatment with the ASD formulation was 90% in the Litomosoides jird model at 15 mg eq./kg/day for 10 days [max of 50 ng/ml and an AUC0-24h of 1100 ng.h/ml, both on day 5 . Notwitle jirds ). The phle jirds . These dmax at 50 mg eq./kg/day b.i.d. in the 2-week repeated dose toxicity study in male dogs on day 1 was 1660 ng/ml. These data indicate that flubendazole up to high peak plasma concentrations, does not induce Cmax related effects in the central nervous system or cardiovascular system.In the CNS safety study in rats and the cardiovascular safety study in dogs, a single dose up to 60 mg eq./kg in the rat and 120 mg eq./kg in the dog, did not result in relevant CNS findings in the rat or cardiovascular findings in the dog. In the rat, the dose of 60 mg eq./day was associated with a peak exposure of 4330 ng/ml.  In the dIn the 2-week repeated dose toxicity studies in rats and dogs, main flubendazole-related changes were observed in haematological and lymphoid systems, gastrointestinal system and testes. In the dog, the liver was an additional target organ of toxicity of flubendazole. . From 3.46 mg eq./kg/day  onward, flubendazole markedly reduced embryonic development by GD 12.5. On GD 20, 80% of fetuses showed malformations. [Results of an explorative oral embryofetal developmental toxicity study in the rat have been published by M. Longo et al.  They alsin vitro. This aneugenicity is the consequence of the binding of flubendazole to the tubulin protein, inhibiting polymerization of tubulin and consequently causing mitotic spindle disruption. Spindle poisons all have the potential to induce polyploidy and aneugenicity. [in vitro MNT, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. Like flubendazole itself, both metabolites are negative in the Ames test. [in vivo micronucleus test with the ASD formulation of flubendazole published by Tweats et al. also showed evidence of induced aneugenicity. [in vivo micronucleus test with an aqueous solution/suspension of flubendazole in demineralised water containing polyethylene glycol 400, hydroxypropyl-β-cyclodextrin and HCl, described in this article. In this study, flubendazole induced structural and/or numerical chromosome aberrations in erythrocytes of rat bone marrow from a dose of 5 mg/kg/day onwards, at similar exposure levels as with the ASD formulation. . The study design was in general compliance with the following animal health and welfare guidelines: Guide for the care and use of laboratory animals, 2011; Decree n° 2013–118 relating to the protection of animals used in scientific experiments described in the Journal Officiel de la République Française on 01 February 2013; Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The Test Facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.Both rat and dog repeated dose toxicity studies were carried out by Charles River Laboratories Den Bosch B.V.  according to their internal Standard Operating Procedures. The protocols were reviewed and agreed by the Animal Welfare Officer and the Ethical Committee (DEC 14–59) as required by the Dutch Act on Animal Experimentation (February 1997).The rat in vivo micronucleus study was performed in an AAALAC-approved laboratory of Johnson & Johnson In Belgium. Johnson & Johnson vivarium facilities meet inspection agency standards, and all animals are treated humanely and cared for in accordance with the European  and BelgFlubendazole has a molecular weight of 313.28, and a molecular formula of C16H12FN3O3.For the Ames assay, flubendazole was prepared as a solution in dimethyl sulfoxide (DMSO).in vivo pharmacokinetic studies with intravenous administration, a solution of flubendazole was made in polyethylene glycol 400 (PEG400) with 20% of hydroxypropyl-β-cyclodextrin pH = 4.2 for the rat. For the intravenous pharmacokinetic studies in the jird and the dog, a solution of flubendazole was made in 10 v/v % polyglycol/PEG400 ratio 1–1 and 35 v/v % hydroxypropyl-β-cyclodextrin at pH = 4. For oral pharmacokinetic studies and two-week repeated dose toxicity studies and safety pharmacology studies, the test article/drug product was an amorphous solid dispersion (ASD) powder with a potency of 94.8 mg active ingredient flubendazole per g ASD powder. Because the active ingredient flubendazole only constituted 9.48% of the ASD powder, dose levels were expressed as mg eq./kg body weight meaning mg base/kg body weight. To calculate the amount of ASD powder equivalent to the mg base, conversion factor was as follows: 1 mg base = 10.5 mg drug product. The test article was prepared as a suspension containing 0.5% w/v Methocel A4M (Sigma-Aldrich) in Elix water.For For the rat in vivo micronucleus test, an aqueous solution/suspension in demineralised water containing 10% polyethylene glycol 400, 20% hydroxypropyl-β-cyclodextrin and HCl to pH 1.5± 0.1 was prepared.max), the time to reach the Cmax (tmax), the half-life (t1/2), the area under the curve from time zero to infinity (AUC0-inf), the clearance (CL) and the volume of distribution (at steady state Vdss or Vd)The pharmacokinetic studies were performed after single intravenous administration to determine key parameters such as plasma clearance, volume of distribution and half-life, and after single oral administration to determine exposure of flubendazole and its metabolites. The toxicokinetics of flubendazole and its metabolites, which is the determination of the exposure within the toxicology studies were also performed. In both studies, the plasma samples were analyzed individually for flubendazole, H-FBZ and R-FBZ using a qualified LC-MS/MS method. After preparation and addition of the internal standard, samples were precipitated with acetonitrile, mixed and centrifuged. The supernatant was evaporated to dryness under nitrogen flow at 50°C and reconstituted with a mixture of 0.1% formic acid and acetonitrile . The extract was injected onto an Acquity UPLC BEH C18 column  . The chromatographic system consisted of a Shimadzu SIL30ACMP autosampler and Shimadzu LC30 pumps . The mobile phase was a mixture of 1% formic acid and acetonitrile with a flow rate of 0.6 ml/min and a 2.5-minute gradient going from 20 to 60% acetonitrile followed by a 1-minute step gradient to 98% acetonitrile. Mass spectrometric detection was performed on an API4000 triple quadrupole mass spectrometer  with Turbo Ion Spray ionization operated in positive ion mode. Flubendazole, hydrolyzed flubendazole (H-FBZ) and reduced flubendazole (R-FBZ) were quantified against calibration samples and quality control samples, prepared in the same matrix as the study samples, by means of a qualified analytical method with the lowest limit of quantitation of 0.2, 0.4 and 0.2 ng/ml respectively and an upper limit of quantitation of 3000 ng/ml for all three analytes across the different studies. Flubendazole was administered intravenously by bolus at 1 and 2 mg/kg in male jirds and male rats, respectively or by a short infusion (15 minutes) in male dogs at 0.5 mg/kg. Flubendazole was administered orally by gavage at 20 mg eq./kg in male jirds and male rats and at 35 mg eq./kg in male dogs. The jird and the rat were not fasted. The dogs were fasted overnight. 2h post dose, dogs were given their regular food. After intravenous administration, blood samples were collected at 0.05, 0.25, 0.5, 1, 3, 7 hours after dosing in male jirds (bolus), at 0.0117, 0.333, 1, 2, 4, 7 and 24 hours after dosing in male rats (bolus), at 0.125,0.25 (end of infusion), 0.33, 0.42, 0.50, 0.75, 1.25, 2.25, 4.25, 7.25 and 24.25 hours after the start of infusion in male dogs. After oral administration, blood samples were collected at 0.5, 1, 2, 4, 7 and 24 hours in male jirds, rats and dogs. Plasma concentrations of flubendazole and its 2 metabolites were determined. Several pharmacokinetic parameters were determined: the maximum concentrations  and central nervous system (CNS).Flubendazole was administered orally via gavage to five telemetered male beagle dogs at single doses of 0, 20, 50 or 120 mg eq./kg, at a dosing volume of 10 mL/kg, on Days 1, 4, 7 and 12, respectively (ascending dosing schedule). Each animal served as its own control, and a minimum wash-out period of 2 days between each dose was respected. Telemetry signals were recorded for at least 1.5 hours before, and 24 hours after dosing. The following parameters were studied: mortality, clinical observations, body weight, arterial blood pressure, heart rate, ECG quantitative parameters and rhythm, respiratory parameters, body temperature, bioanalysis and evaluation of exposure. Blood sampling for exposure was done at a single time-point, 6.5 hours after dosing, time point at which maximum plasma concentration was expected.max), their times of occurrence (Tmax), the time of the last quantifiable mean concentration (tlast) and the mean plasma concentrations at 24 hours post-dose (C24h) were the observed values. Areas under the mean plasma concentration time curves (AUC) were calculated. Cmax, Tmax and AUC0-24h are described in this paper. Mortality, behavioural observations in the cage, during handling and while in an open field, general clinical observations, body weight and rectal temperature were evaluated on Day 1 (day of single administration), Day 2 and Day 7.Flubendazole was administered orally via gavage to male Sprague-Dawley rats at single doses of 0, 5, 15 or 60 mg eq./kg, in order to evaluate the neurofunctional integrity of the rat , using a modified Irwin test procedure . MeasureSafety pharmacology studies in animals were carried out by Charles River Laboratories France Safety Assessment SAS , following internal Standard Operating Procedures and applicable ICH guidelines  and were GLP regulated , 21, 22.max), their times of occurrence (Tmax), the time of the last quantifiable mean concentration (tlast) and the mean plasma concentrations at 24 hours post dose (C24h) as the observed values. Areas under the mean plasma concentration-time curves (AUC) were calculated. Cmax and AUC0-24h are described in this paper. Samples for toxicokinetic analysis were taken on day 1 and day 14 of the dosing period from satellite animals for all flubendazole-dosed groups except the high dose groups and on day 1 and day 10 of the dosing period for the high dose groups. In the vehicle dose groups, samples were taken on day 3 and day 11 of the dosing period. Sampling time points were 30 minutes, 1, 2, 4, 7 and 24 hours after dosing for the flubendazole-dosed groups and 1 and 7 hours after dosing for the vehicle dosed groups. Animals of vehicle-dosed groups were sampled to confirm absence of exposure to flubendazole in these animals.Flubendazole was administered by oral gavage for 14 or 15 consecutive days to Sprague Dawley rats. Dose levels for males were 0, 5, 15 and 30 mg eq./kg/day; dose levels for females were 0, 2.5, 5 and 10 mg eq./kg/day. Dose levels in females were lower compared to males because exposure in females was at least twice the exposure in males for flubendazole after a similar single dose. Because of the higher exposure, more pronounced toxicity findings were observed in females when compared with males. A similar group of rats received the vehicle only, according to the same regimen and served as a vehicle control group. Initially, the study included a recovery period of one month to investigate the reversibility of any possible toxicological effect induced by repeated oral administration of flubendazole after treatment was stopped. For this purpose, extra rats for the vehicle and the high dose group  were added to the main toxicity study . However, all high dose animals including animals assigned for recovery, were killed in extremis on Day 7, 8 or 11 of the dosing period. Since there were no high dosed animals left to evaluate recovery after treatment was stopped, recovery animals of the control group were necropsied on Day 4 of Recovery. In this study, the following parameters were studied: mortality, clinical and ophthalmoscopic observations, body weight and weight gain, food consumption, haematology, coagulation, clinical biochemistry, urinalysis, organ weights, gross pathology and histopathology. The toxicokinetic parameters for flubendazole and the 2 metabolites, reduced flubendazole and hydrolyzed flubendazole were also determined in satellite animals . The toxicokinetic parameters determined were maximum mean plasma concentrations  at dosages of 0, 20, 40 or 100 mg eq./kg /day during 14 or 15 consecutive days. The high dose group received twice daily 50 mg eq./kg, with a 6-hour interval. A similar group of dogs received the vehicle only (once daily) and served as a vehicle control group. In addition, a recovery period of 1 month was added to investigate the reversibility of any possible toxic effect induced by repeated oral administration of flubendazole. For this purpose, 2 extra dogs/sex for the vehicle, mid and high dose group were added to the study.max), maximum plasma concentrations after the first daily dose (Cmax1) and after the second daily dose (Cmax2), their times of occurrence , the time of the last quantifiable concentration (tlast) and the plasma concentrations at 24 hours post dose (C24h) were the observed values. Areas under the plasma concentration-time curves were calculated. Cmax and AUC0-24h are described in this paper. Samples for toxicokinetic analysis were taken from all flubendazole-dosed dogs on day 1 and day 14 of the dosing period and from vehicle dosed dogs on day 3 and day 13. Sampling time points were pre-dose, 30 minutes, 1, 2, 4, 7 and 24 hours after dosing in the vehicle, low and medium dose groups. In the high dose groups, dogs were sampled 30 minutes, 1, 2, 4, 6 (before second dose), 7, 9 and 24 hours after the first dose. Animals of vehicle-dosed groups were sampled to confirm absence of exposure to flubendazole in these animals.The following parameters were studied: mortality, clinical and ophthalmoscopic observations, ECG and heart rate, body weight and weight gain, food consumption, haematology, coagulation, clinical biochemistry, urinalysis, organ weights, gross pathology and histopathology. The toxicokinetic parameters for flubendazole and 2 metabolites, reduced flubendazole and hydrolyzed flubendazole were also determined. Maximum plasma concentrations  according to their internal Standard Operating Procedures. They were GLP regulated  and wereGenotoxicity assessments were performed to detect if flubendazole induces genetic damage.Flubendazole was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al.  and EschThe Ames test was performed by BioReliance Corporation, Rockville, MD 20850, United States of America, according to their internal Standard Operating Procedures and was GLP regulated .Flubendazole was administered orally via gavage to young male and female rats to assess its potential to induce structural and/or numerical chromosome aberrations in bone marrow and peripheral blood immature erythrocytes. Male and female rats (5 rats/sex/group) were dosed once daily with flubendazole at 0.15, 0.5, 1.5, 5, 15, 50 and 150 mg/kg body weight/day for two consecutive days prior to the collection of bone marrow and peripheral blood at 24 hours after the last administration. A similar group of rats (5 rats/sex/group) received the vehicle only, according to the same regimen and served as a vehicle control group.The number of micronucleated and nonmicronucleated reticulocytes (RETs), and the number of micronucleated and nonmicronucleated normochromatic erythrocytes (NCEs) in peripheral blood was recorded by the flow cytometer. Data acquisition was stopped when 20,000 RETs had been collected. The percentage of RETs was calculated  as an index for bone marrow toxicity. The percentage of micronucleated RETs  was calculated as a measure for induction of structural and/or numerical chromosome aberrations by the test item.Bone marrow smears were evaluated by fluorescence microscopy. Analysis of red blood cell proliferation was based on the proportion of polychromatic erythrocytes assessed by counting a total of 1000 erythrocytes  per rat. Analysis of red blood cells for micronuclei was based on a total of 2000 PCEs per rat and the number of micronucleated PCEs. At the same time, the number of micronucleated NCEs was also recorded in the fields containing these 2000 PCEs.max) and corresponding peak times (Tmax). The area under the plasma concentration time curve (AUC) were calculated. Cmax and AUC0-24h are described in this paper. Samples for toxicokinetic analysis were taken on day 1 of dosing from the satellite animals. Sampling time points were 30 minutes, 1, 2, 4, 7 and 24 hours after dosing.The toxicokinetic parameters for flubendazole and the 2 metabolites, reduced flubendazole and hydrolyzed flubendazole were also determined in satellite animals . The toxicokinetic parameters determined were peak plasma concentrations (CThis study was classified as a non-GLP study. This study was carried out based on the current OECD guideline 474 (1997)  The studIn the rat study, the following statistical methods were used to analyze the data: If the variables could be assumed to follow a normal distribution, the Dunnett test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the vehicle control groups for each sex. The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data could not be assumed to follow a normal distribution. The Fisher-exact test was applied to frequency data. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.In the dog study, no statistical analysis took place due to the limited number of animals in the study.in vivo micronucleus test, the significance of differences between each test item group (and positive control group) and the vehicle control group was assessed by the following statistical methods: For mortality and clinical observations, Fisher Exact Probability test was applied. For body weight/weight gain and bone marrow data of the micronucleus test, the Mann-Whitney U test was applied. For flow cytometric analysis data, data (the percentage of RETs and the percentage of micronucleated RETs), were checked for non-normality by applying the Shapiro-Wilk test. When data were not normally distributed, the percentage of RETs was square root transformed and the percentage of micronucleated RETs was log transformed and subsequently rechecked for non-normality using the Shapiro-Wilk test. When data were normally distributed, homogeneity of variances was assessed through the Levene’s test. When data were either not normally distributed or homogeneity of variances could be rejected, then Dunn’s test (with a closed multiple comparison procedure following Holm) was applied for comparison of the test article groups to the vehicle control group. Otherwise, Dunnett’s test was applied. The above procedure was adopted for the comparison of all dose groups versus vehicle control and for the comparison of vehicle control versus the positive control. In the last comparison, where only two groups are compared, Dunnett’s test reduces to a standard t-test and Dunn’s test reduces to a Wilcoxon-Mann-Whitney test. In addition, Jonckheere Terpstra trend tests for dose trends were performed. All analyses of flow cytometric analysis date were performed with SAS version 9.2. All tests were performed at a significance level of 5% or less.In the