Chronic excessive alcohol consumption  could induce serious liver injury. In this study, effects of lemon juice on chronic alcohol-induced liver injury in mice were evaluated. The serum biochemical profiles and hepatic lipid peroxidation levels, triacylglycerol (TG) contents, antioxidant enzyme activities, and histopathological changes were examined for evaluating the hepatoprotective effects of lemon juice in mice. In addition, the in vitro antioxidant capacities of lemon juice were determined. The results showed that lemon juice significantly inhibited alcohol-induced increase of alanine transaminase (ALT), aspartate transaminase (AST), hepatic TG, and lipid peroxidation levels in a dose-dependent manner. Histopathological changes induced by alcohol were also remarkably improved by lemon juice treatment. These findings suggest that lemon juice has protective effects on alcohol-induced liver injury in mice. The protective effects might be related to the antioxidant capacity of lemon juice because lemon juice showed in vitro antioxidant capacity.  Alcohol abuse and alcoholism could lead to serious health and socioeconomic problems worldwide. Chronic excessive alcohol consumption  could lead to several illnesses, such as gastrointestinal damage, pancreatitis, alcoholic liver disease, neurologic disorders, diabetes mellitus, and cancer , 2. Amon2−•), hydroxyl radical (OH•), and hydrogen peroxide (H2O2). The ROS can react with most cellular macromolecules and subsequently cause cellular damage [Chronic alcohol consumption can generate abundant reactive oxygen species (ROS), including superoxide anion radical , such as hesperidin, eriocitrin, naringin, neohesperidin, rutin quercetin, chlorogenic acid, luteolin, and kaempferol . The in The compounds 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tri(2-pyridyl)- S-triazine (TPTZ), quercetin, gallic acid, and Folin–Ciocalteu's phenol reagent were purchased from Sigma-Aldrich . Assay kits for the determination of SOD, lipid peroxidation, CAT, and TG contents were purchased from Nanjing Jiancheng Bioengineering Institute . Other chemicals were of analytical grade.g for 10 min, and the supernatant was obtained. The supernatant was used for the measurement of antioxidant capacity, total phenolic contents (TPC), and total flavonoid contents (TFC) and for animal experiments. Moreover, in animal experiments, the original supernatant and the diluted supernatant  were used as the high, medium, and low dose, respectively. The lemon juice was freshly prepared before gavage every time.Lemon was obtained from markets in Guangzhou, China. The fruit was cleaned with deionized water. The edible portion was weighed precisely and mixed with deionized water , and the mixture was ground into a homogenate with a homogenizer. Then, the homogenate was centrifuged at 5,000g for 10 min and the serum was collected. The obtained serums were stored at −22°C before determination. A piece of tissue was taken from liver and fixed in 4% paraformaldehyde, and then the remaining liver tissue was stored at −22°C until use.Male C57BL/6 mice (20–25 g) were employed in this study. Thirty mice were randomly divided into 5 groups, each group containing 6 mice. They were maintained in a SPF laboratory animal room, which kept a 12 h light/dark cycle at 22 ± 0.5°C with 40%–60% relative humidity. The animal study was performed according to the “Principles of Laboratory Animal Care” and approved by the Institutional Animal Ethics Committee of Sun Yat-sen University. The model group was treated daily with ethanol and distilled water (10 mL/kg) at the same time; the lemon juice treatment groups were treated daily with different concentrations (high dose 1 : 1 (m/v), medium dose 1 : 5, and low dose 1 : 10) of lemon juice (10 mL/kg) and ethanol simultaneously; the control group was treated daily with isometric distilled water. The model group and the lemon juice treatment groups were given ethanol according to the following ways: 35% ethanol (v/v) at a dose of 3 g/kg body weight for 7 days, 40% ethanol (v/v) at a dose of 4 g/kg body weight for the next 7 days, and 52% ethanol (v/v) at a dose of 5 g/kg body weight on the 15th day . All theThe levels of ALT, AST, and TG in serum were determined by a Hitachi-7180 automated biochemistry analyzer  with the corresponding reagent kit.The levels of TG, SOD, and CAT in liver tissue were measured using the commercial detection kits according to the manufacturer's instructions.The levels of lipid peroxidation in liver tissue were measured by thiobarbituric acid (TBA) method using the commercial detection kits according to the manufacturer's instructions. The reference standard was malondialdehyde (MDA), and the results were expressed as nmol MDA equivalent/mg prot.μm thickness, and stained with hematoxylin-eosin (H&E) for evaluation of histopathological changes. The histopathological changes of stained liver slices were observed under a bright-field microscope.The liver tissue fixed in 4% paraformaldehyde was embedded in paraffin, sectioned into 5 μL of the diluted sample was added to 3 mL of the FRAP reagent and the mixture was measured after 4 min at 593 nm. The standard curve was established using FeSO4 solution, and the results were expressed as μmol Fe(II)/g dry weight of lemon.The FRAP assay was performed based on the method described in the literature . In brie•+ stock solution was prepared from 2.45 mmol/L potassium persulfate and 7 mmol/L ABTS solution in a volume ratio of 1 : 1 and then placed in the dark for 16 h at room temperature. The ABTS•+ working solution was prepared by diluting the stock solution, and the absorbance of ABTS•+ working solution was 0.710 ± 0.05 at 734 nm. 100 μL of the diluted sample was mixed with 3.8 mL ABTS•+ working solution, and the absorbance of the mixture was measured at 734 nm after 6 min, and the percent of inhibition of absorbance at 734 nm was calculated. The reference standard was Trolox, and the results were expressed as μmol Trolox/g dry weight of lemon.The TEAC assay was carried out according to the procedure in the literature . BrieflyTPC were measured according to the literature . BrieflyTFC were measured according to the literature . In briep < 0.05 was regarded as significant.Statistical analysis was carried out by one-way analysis of variance (ANOVA) with post hoc LSD test using SPSS 13.0 software. p < 0.05) elevation of alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum of the model group compared with that of the control group. The administration of low and medium concentration of lemon juice slightly prevented the elevation of serum level of AST, while a high dose of lemon juice significantly (p < 0.05) decreased it. At the same time, the prevention of the elevation of serum levels of ALT was observed significantly (p < 0.05) in medium and high concentration of lemon juice group and displayed a dose-effect relationship.As shown in p < 0.05) increased in the model group compared with that in the control group (p < 0.05). In addition, hepatic TG content was significantly (p < 0.05) increased in model group compared with that in the control group (p < 0.05) reduced the accumulation of hepatic TG in a dose-dependent manner.Triacylglycerol (TG) content in serum was significantly (ol group . Adminisol group . Adminisp < 0.05) increase in the lipid peroxidation level of the model group. The administration of lemon juice significantly (p < 0.05) decreased the level of lipid peroxidation in a dose-dependent manner.The lipid peroxidation levels in liver tissue are shown in p < 0.05) in the model group compared with that in the control group. The CAT level in the liver decreased only slightly (p > 0.05) in the model group compared with the control group in this study. However, treatment with lemon juice significantly (p < 0.05) decreased the levels of SOD and CAT compared with those of the model group. In addition, all the biochemical parameters are summarized in Histopathology assessment of the liver was carried out for all groups . There wμmol Fe(II)/g dry weight (DW) and 19.88 ± 0.66 μmol Trolox/g DW, respectively. The total phenolic contents (TPC) and total flavonoid contents (TFC) of lemon were 6.21 ± 0.28 mg GAE/g DW and 0.30 ± 0.03 mg QE/g DW, respectively.The in vitro antioxidant activities of lemon were evaluated using ferric-reducing antioxidant power (FRAP) and Trolox equivalent antioxidant capacity (TEAC) assays. The FRAP and TEAC values were 50.82 ± 2.70 Alcohol use disorder causes substantial diseases, and the liver is the most adversely affected organ. In the present study, the effects of lemon juice on chronic alcohol-induced liver injury in mice were investigated. Ethanol induced impairment of liver in mice was evidenced by increased AST and ALT levels. Treatment with lemon juice lowered the increased levels of AST and ALT in serum. The return of the activities of aminotransferases (AST or ALT) in serum to normal indicates the regeneration of hepatocytes and the healing of hepatic parenchyma; therefore, lemon juice had a protective effect on alcohol-induced liver injury. The results were in agreement with previous reports that showed lemon possessing a hepatoprotective effect on liver injury induced by carbon tetrachloride and acute exercise , 13. In Various factors and mechanisms are associated with the pathological progress of alcohol-induced liver injury, and oxidative stress was one of them . ROS is β-oxidation and induce steatosis further [Liver steatosis is the earliest disease of the liver on account of chronic ethanol consumption, with the characteristic of fat accumulation. It is generally accepted that, in the development of hepatic steatosis, ethanol exposure increases the ratio of reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide in hepatocytes, which disturb mitochondrial fatty acid  further . In thisThe antioxidant enzymes, such as SOD and CAT, represent the defense response system to excessive ROS. SOD catalyzes the dismutation of two superoxide anions to hydrogen peroxide and oxygen, and then CAT degrades two hydrogen peroxide molecules to water and oxygen . SOD is Lemon contains numerous beneficial bioactive compositions, including phenolic compounds (mainly flavonoids), vitamins, carotenoids, essential oils, minerals, and dietary fiber . The hepIn this study, lemon juice revealed a protective effect on chronic alcohol-induced liver injury. Due to the fact that lemon contains a variety of bioactive ingredients, the hepatoprotective effect might be the result of joint action of multiple mechanisms, and it is difficult to clarify the specific mechanism of effect. The medium in vitro antioxidant capacities of lemon and reduced in vivo MDA levels indicated that lemon might reduce the oxidative stress induced by ethanol, thus exerting hepatoprotective effects. This study has found that lemon juice has a strong hepatoprotective effect, which provides valuable information for the general public to reduce harm of alcohol consumption. In the future, active components in lemon juice should be separated and identified, and the mechanism of action of the purified compound should be explored, including the action on the small intestine.Chronic alcohol consumption could induce liver injury. Lemon juice is readily available as a widely consumed beverage. In this study, we found that treatment with lemon juice exerted hepatoprotective effects on alcohol-induced liver injury in mice through decreasing the levels of serum ALT and AST as well as hepatic TG and lipid peroxidation. In addition, the in vitro antioxidant experiment of lemon showed that lemon had medium in vitro antioxidant capacities. Therefore, we speculate that the hepatoprotective effects might be related to the antioxidant capacities of lemon juice. The results showed that lemon juice might be a potential dietary supplement for the prevention and treatment of liver injury related to chronic alcohol consumption.


P. vivax in Qatar, and its ability to produce gametocytes, compared to parasites in main sites of imported cases, the Indian subcontinent (india) and East Africa (Sudan and Ethiopia). High diversity was seen among imported P. vivax in Qatar, comparable to parasites in the Indian subcontinent and East Africa. Limited genetic differentiation was seen among imported P. vivax, which overlapped with parasites in India, but differentiated from that in Sudan and Ethiopia. Parasite density among imported cases, ranged widely between 26.25–7985934.1 Pv18S rRNA copies/µl blood, with a high prevalence of infections carried gametocytes detectable by qRT-PCR. Parasitaemia was a stronger predictor for P. vivax gametocytes density . The extensive diversity of imported P. vivax and its ability to produce gametocytes represent a major threat for re-introduction of malaria in Qatar. The genetic relatedness between P. vivax reported in Qatar and those in India suggest that elimination strategy should target flow and dispersal of imported malaria into the region.Malaria control program in the Arabian Peninsula, backed by adequate logistical support, has interrupted transmission with exception of limited sites in Saudi Arabia and sporadic outbreaks in Oman. However, sustained influx of imported malaria represents a direct threat to the above success. Here we examined the extent of genetic diversity among imported  The escalating volume of international travel among people in endemic areas, driven by desire to improve quality of life and political unrest, has increased the risk of malaria importation into non-endemic areas, including Europe2.Global concerted and collaborative efforts, backed by WHO, have resulted in remarkable reduction in the burden of malaria, and disease elimination in many areas. Nonetheless, imported malaria represents a significant threat to the above achievements3, and active case detection to define existing malaria foci. Consequently, this has brought local transmission to a halt in many countries in the region, including Bahrain, Kuwait and the United Arab Emirates (UAE), and more recently Oman3. However, some sites in Yemen and southern Saudi Arabia remain malarious, with a high prevalence of drug-resistant Plasmodium falciparum4. The success of control programs in the region has prompted the health authorities in GCC countries to shift strategies towards malaria elimination and prevention of re-introduction in receptive areas5.Local malaria transmission in the Gulf Cooperation Council (GCC) countries has been interrupted as a result of sustained vector control7. The risk of malaria resurgence depends on the combined effect of receptivity and vulnerability. Receptivity is a function of the presence of local vectors and environmental conditions that favour malaria transmission, while vulnerability reflects the probability of importation of malaria parasites into a country8. The region is highly vulnerable to re-introduction of malaria, given the high volume of immigration from malaria endemic regions. This has been implicated in the persistence of high rates of imported malaria in many GCC countries9. For example, while a significant reduction in locally acquired malaria cases was observed between 2000 and 2014 in Saudi Arabia, imported cases remained consistent averaging between 250 and 830 cases per year10. Imported malaria is also common in Qatar, Bahrain, Kuwait and the UAE13, and has been implicated in repeated malaria outbreaks in Oman14. In Qatar, malaria transmission has been interrupted since 197015, however, a constant influx of imported malaria by the large number of immigrant workers from the Indian Subcontinent, and Sub-Saharan Africa, poses a risk for re-introduction16. Receptivity of the country to a re-introduction of malaria is evident by the presence of two potential vectors, Anopheles stephensi and A. multicolour17. The proportion of all imported malaria reported amongst travellers in Qatar has increased over the past decade, with P. vivax being the main prevalent parasite11.However, the above success is vulnerable to a possible resurgence of local transmission caused by imported parasites via asymptomatic travellers from malaria-endemic areasP. falciparum and P. vivax infections to produce gametocytes is well documented19. Currently there is no clear information on the actual magnitude of asymptomatic parasite reservoir in areas where malaria has been eliminated in the GCC countries and its population structure. Thus, the present study examined the source of imported P. vivax into the transmission free setting of Qatar, assessed its genetic complexity and its ability to produce gametocytes and transmit malaria. In addition, we compared the extent of diversity and genetic relatedness of imported parasites into Qatar with that in three countries of origin of immigrant malaria into Qatar, in Africa (Sudan and Ethiopia) and the Indian Subcontinent (India). Such knowledge would allow regional and national control programs to develop targeted policies to reduce the size of infectious reservoir, define the source of outbreaks and limit the risk of re-introduction of malaria.The impact of imported malaria, often as asymptomatic infection, on local epidemiology and local transmission is not well defined in Qatar and surrounding regions. However the ability of asymptomatic P. vivax cases (n = 318 [70.9%]), while P. falciparum (n = 118 [26.3%]) and mixed infection, with P. falciparum and P. vivax, was less frequent (n = 12 [2.7%])  were found to be positive. Species-specific PCR revealed a high prevalence of P. vivax cases were young males (93.7%), mean age 32 years, from the Indian Subcontinent (n = 264 [83%]); including, India (n = 148 [46.5%]), Pakistan  and Nepal . However, a small proportion of imported P. vivax cases were from Africa (n = 53 [16.7%]), including Sudan , and other African countries . On the contrary, the vast majority of imported P. falciparum cases were from Africa , with a fewer number of patients from the Indian Subcontinent  and others (n = 7 [6%])  were successfully examined by qPCR and qRT-PCR to quantify total parasitaemia and gametocyte density, respectively. Total parasite density estimated as transcript of Pv18s rRNA ranged between 26.2 and 7985934.1 copies/µl of blood (median = 1029.0).Ninety-five imported P. vivax isolates examined by RT-qPCR, 94 (98.9%) had detectable transcripts of Pvs25, indicative of the presence of gametocytes. The copy number of Pvs25 transcript, among the examined isolates ranged from 0.33 to −78441.24 copies of Pvs25/µl of blood (median = 300.3).Out of 95 Pv18srRNA and Pvs25 transcripts , suggesting an association between parasitaemia and gametocyte density , Ethiopia (n = 87) and India (n = 40). There was a high degree of polymorphism in all microsatellites, with number of alleles per locus varied from 8 for MS1 to 38 for MS8 among imported parasites in Qatar, 6 for MS7 to 22 for MS20 among parasites in Sudan, 4 for MS7 to 22 for MS8 among parasites in Ethiopia and 6 for MS5 to 19 for MS8 among Indian isolates  similar to that seen in endemic sites in Sudan (HE = 0.726), Ethiopia (HE = 0.684) and India (HE = 0.797) , Ethiopia (n = 34), Sudan (n = 33) and India (n = 30)  in Sudan each shared a distinct genotype.The predominant allele at each locus in each isolate was used to construct multi-locus genotypes (haplotypes). Each SA). This determines whether there is an association between alleles on different loci, and if the above high levels of diversity observed in P. vivax populations could be explained by frequent genetic exchange.The multi-locus genotype (haplotypes) data was used to estimate the standard index of association  compared to parasites from Ethiopia (0.0367) and India (0.0265) (Table Limited linkage disequilibrium (LD) was seen among the imported cases in Qatar Iiopia 0.067 and InP. vivax in Qatar, the isolates were divided into three populations  based on the nationality of patients. Pairwise analysis showed a close relationship between parasites from Pakistan and India (FST = 0.0008), compared to that seen between parasites from Sudan and India (FST = 0.0037) or Sudan and Pakistan (FST = 0.0011). Nonetheless, imported P. vivax from the three countries can be considered as one population. Similar close genetic relatedness was seen between imported P. vivax in Qatar, treated as one population, and isolates collected in India (FST = 0.0594). However, imported P. vivax in Qatar was slightly diverged from local parasite in Sudan (FST = 0.1435) and Ethiopia (FST = 0.1896).To assess the genetic relatedness among imported P. vivax population was distanced from the imported parasites in Qatar, while the Sudanese and Ethiopian parasites were slightly overlapped  Fig. . The Ethped Fig. . AnalysiP. vivax in Qatar and the East African isolates (Sudan and Ethiopia) when K = 2. However, when K = 3 there was some overlap between Sudanese isolates and imported cases in Qatar in agreement with the FST analysis. However, when K = 4, a clear overlap between the imported parasites to Qatar and parasite in Sudan and Ethiopia , Sudan (60.6%), and Pakistan (50.5%). Similar, high MOI was seen among local P. vivax in Sudan (44.70%), Ethiopia (67%) and India (62.5%).MOI defined as presence of more than one allele in any locus, was high among imported P. vivax from India (1.6), Sudan (0.7), and Pakistan (1.2) compared to that seen among endemic parasites in Sudan (2.4), Ethiopia (2.00) and India (2.00).Moreover, the minimum number of clones (MNC), estimated as the minimum number of alleles per locus, across all loci, was relatively low among imported 10 and sporadic outbreaks in Oman14 represent the final stage in the battle to eliminate endemic malaria in the region. However, the above success is threatened by the high influx of imported malaria by young immigrants from endemic countries in the Indian subcontinent  and East Africa (Sudan).The GCC countries have achieved great success in the fight against malaria. Four out of the six states accomplished complete interruption of malaria transmission and a malaria-free status, while limited sites in southwest Saudi ArabiaP. vivax malaria into GCC countries while Africa is the main origin of P. falciparum malaria. For some decades, the oil-rich region has been a popular destination for temporary labour workers, seeking employment opportunities. The high migratory turnover of immigrants from malarious areas is accompanied by increased reports of imported malaria in GCC countries9, reflecting a high vulnerability for re-introduction of malaria in areas where the disease has been eliminated. Though, receptivity, defined as the presence of the Anopheles vector, is limited as the result of current efficient vector control programs20. Nonetheless, autochthonous malaria cases were seen near livestock farms and building sites, where a large number of immigrants work, and environmental conditions favour vector habitats14. Analysis of a recent malaria outbreak in Oman identified Anopheles culicifacies as the vector involved14. Therefore, to minimize vulnerability, to re-introduction of malaria, a stringent surveillance system, based on highly sensitive molecular tools, should be structured to ensure timely detection and management of outbreaks seeded by imported malaria.The findings of the present study are in line with previous reports, demonstrating that the Indian Subcontinent is the most likely source for imported P. vivax in Qatar. Out of 95 P. vivax isolates successfully examined by RT-PCR, 83 (87.37%) harboured gametocytes evident by detectable transcripts of Pvs25 gene. This accords well with the findings of the high prevalence of low-density gametocyte carriage among P. vivax infection in many endemic sites21. P. vivax isolates examined in the present study were obtained from clinical cases. However, imported malaria often exists as asymptomatic low-level parasitaemia detectable only by PCR22 that may easily be missed by traditional microscopy and serological surveillance methods. An additional feature of asymptomatic infection, of particular relevance to prevention of re-introduction in areas where malaria has been eliminated, is its extended longevity. Asymptomatic P. falciparum infection can persist for several years, and discovered only when an individual develops clinical malaria23. Clinical presentations of P. falciparum malaria among immigrants have been found to delay by as long as 8 years after patients have left malaria-endemic areas24. Thus, asymptomatic parasite carriers can sustain long-term threat to malaria elimination programs.The vulnerability to re-introduction of local transmission is emphasised by the high prevalence of gametocytes carriage among imported 25. Amplification-based techniques are sensitive enough to detect and quantify gametocytes as low as 0.02–10 gametocytes per microliter26. Such low gametocytes densities are unlikely to be detected by microscopy and can make significant contributions to transmission27. In addition, parasites can increase gametocyte conversion rate in response to clues for transmission opportunity28. For example, the reappearance of Anopheles mosquitoes following the long dry period in areas of seasonal transmission can promote surge in gametocyte densities among asymptomatic parasite carriers26. This may explain the success of imported asymptomatic malaria to raise regular outbreaks in receptive sites, with low mosquito density in some GCC countries such as Oman14 and southern Europe29. Thus, molecular surveillance of migrants from endemic areas, in GCC countries and other receptive areas, can estimate the risk of re-introduction and allow targeted control measures to interrupt potential transmission.The presence of gametocyte among low-density asymptomatic parasite carriers is well documentedP. vivax parasites into Qatar [average HE = 0.782], similar to that in endemic sites where the majority of imported cases originate, in India [average HE = 0.797] and East Africa . This is consistent with previous reports on diversity of P. vivax in the Indian Subcontinent30 and East Africa31. The high level of diversity seen among imported P. vivax in Qatar reflects a constant flow of importation of diverse strains from multiple origins. This hypothesis is supported by the close genetic relatedness of imported P. vivax in Qatar and local parasite in India as shown by FST and PCoA analysis  and enhance the parasite diversity, as there is a direct relationship between the expected level of diversity and Ne32. In addition, the appearance of drug resistance in countries of origin such as India33, Pakistan34 and Ethiopia35 can augment the ability of the parasite to establish and evolve in the face of current control measures, if local transmission arises.High level of diversity was seen among imported P. vivax is higher than that seen amongst P. falciparum reported by immigrants in Qatar at the same time of the present study36. This is in line with data from different endemic areas that examined sympatric P. vivax and P. falciparum populations37. This has been attributed to the fact that the global P. vivax population is older, efficient in transmission, very diverse and less structured than P. falciparum38. This is conistsnt with the findings of low level of genetic differentiation between imported P. vivax in Qatar and local parasites in India (FST = 0.0594), and moderate differentiation between imported parasites in Qatar and that in East Africa  and Alkhor Hospital; Hamad General Hospital in Al-Doha city and Alkhor Hospital in the north area are the two main hospitals within HMC that assigned to admit patients with malaria, were examined microscopically for malaria between January 2013 and October 2016. All cases were diagnosed using conventional microscopic examination of Giemsa stained thick (100 fields) and thin blood (1000 RBCs). A total of 448 (76.8%) were found to be positive for the malaria parasites.All subjects were interviewed using a structured questionnaire to collect demographic information, including age, gender, nationality, history of travel and treatment. Patients were given treatment as per the Ministry of Public Health, Qatar.P. vivax infection, in Qatar. In addition, we also used 137 confirmed infected samples from Sudan, collected in Whatman qualitative filter paper, grade 3 between 2013–2014, 87 confirmed infected samples from Ethiopia collected in Whatman qualitative filter paper, grade 3 between 2010–2013 and 40 confirmed P. vivax samples from India collected between 2014–2016.The present study focused on analysis of 265 isolates obtained from patients with 35. Samples were collected after obtaining a written informed consent from the patients or guardians. All experiments in this study were performed in accordance with relevant guidelines and regulations of the above institutes.Ethical approvals were obtained from the Institutional Review Board of WCM-Q and HMC (Protocol no. 14-00097), ethical committee of the Postgraduate Institute of Medical Education and Research, Chandigarh, India (PGI/IEC/2014/88), Research Review Committee of Institute of Endemic Diseases, University of Khartoum (certificate research number 9/2016), and the Ethiopian isolates were reviewed and approved by the respective Ethical Boards of the Addis Ababa University College of Natural Sciences, Ethiopia (RERC/002/05/2013) and National Research Ethics Review Committee of Ethiopia (Ref.no. 3.10/580/06)P. vivax isolates in Qatar and parasites collected in India, using QIAamp DNA Blood Mini Kit as described by the manufacturer . DNA was isolated from blood collected on filter paper  from Sudan and Ethiopia using Chelex extraction method40.DNA was extracted from 200 µl of venous blood from the imported P. vivax isolate obtained from imported cases in Qatar using SV Total RNA Isolation kit . Conventional PCR was used to confirm the absence of co-extracted genomic DNA, and then RNA samples were converted to cDNA using the High Capacity cDNA Reverse Transcription Kit .RNA was extracted from 100 µl blood of P. vivax was confirmed using species-specific PCR as described by41. Total parasitaemia was quantified as copy number of the 18S rRNA transcripts in reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays based on TaqMan probe chemistry, primers and probes sequences are provided in Table 419. Pv18s rRNA copy numbers were estimated using in-plate standard curve generated from a 10-fold serial dilution  of a purified PCR product. Gametocytes were detected and quantified using RT-qPCR, targeting transcripts of Pvs25 gene, as described elsewhere21, with the use of modified probes and primers sequences (Table Species identification of 2 (1.5 mM), dNTPs (50 µM of each dNTPs) and 10 pmole of each primer. PCR conditions were, 94 °C/3 min, 35 cycles at 94 °C/30 s, 59.8 °C/30 s and 72 °C/45 s, and a final extension step at 72 °C/5 min42.We examined 9 polymorphic single copy tri-nucleotide repeats microsatellites , distributed across the whole parasite genome. In 25 μl premix containing Taq polymerase (2U), MgCl43.PCR products were analysed using ABI 3130XL Genetic analyser  with relative to an internal reference Genescan 500 LIZ . GeneMapper v4.0  for allele calling and quantification of peak height44. The minimum number of alleles across the 9 loci in each sample was calculated and this index value represented the minimum number of clones (MNC) (genotypes) per isolate. Then the average MNC for all isolates in each region was calculated44.The multiplicity of infection (MOI) refers to presence of more than one allele per isolate at least in one locus. To avoid overestimation of MOI, multiple alleles per locus were scored if electrophoretic peaks corresponding to minor alleles were ≥33% the height of the predominant allele45. This included determining the number of alleles per locus, and expected heterozygosity (HE). These two parameters were used to assess the level of polymorphism at each locus and determine diversity. HE was calculated using the formula for ‘unbiased heterozygosity’ also termed haploid genetic diversity, HE = [n/(n − 1)][1 − ∑p2] where n is the number of isolates and p the frequency of each different allele at a locus46.Genetic diversity parameters were calculated for the entire dataset using GenAlex v6.5P. vivax populations in different regions comprised a single panmictic population with a high degree of genetic exchange. Multilocus linkage disequilibrium  of the alleles at pairs of loci was estimated as the standard index of association (ISA) using the program LIAN version 3.547. The software tests for independent assortment of alleles by determining the number of loci at which each pair of MLGs differs, and from the distribution of mismatch values a variance VD  is calculated, which is then compared with the variance expected for LE, termed Ve. The null hypothesis that VD = Ve is tested by a Monte Carlo simulation and a parametric method and the results provide 95% confidence limits, which are denoted LMC and LPARA, respectively. If there is limited or no association between alleles at different loci, indicating panmixia, a value close to zero is obtained, whereas if association is detected, a value significantly greater than 0 is obtained, indicating non-panmixia47. The variance of pairwise difference (VD) between the data and that predicated for panmixia14 and L (LMC & LPARA), were calculated in order to test the hypothesis of panmixia. Therefore, when the VD value exceeds the L value, LD is indicted and the null hypothesis of panmixia is discarded. When the VD is less than L, LE is indicated and the null hypothesis of panmixia is accepted47.The predominant allele in each of the examined 9 microsatellites was used to construct multi-locus haplotypes to determine whether the FST) using Gene pop web interface48. Molecular variance (AMOVA) was calculated to estimate the variation within and between population and Principal component analysis (PCoA) was used to visualise the relationship between MLGs using GenAlEx 6.545.The genetic association between population pairs was evaluated by Wright’s fixation index , consisting of a burn-in period of 10,000 iterations followed by 100,000 Markov Chain Monte Carlo (MCMC) iterations, assuming a mixture model and correlated allele frequencies. K was defined according to49 by calculating the rate of change of K, ΔK using STRUCTURE HARVESTER v0.6.9450. Additional analysis of structure results was conducted using CLUMMP51 and DISTRUCT52. CLUMMP aligns the cluster assignment across replicate analyses while DISTRUCT performs a graphical display of the aligned cluster assignments.Structure software was used to elucidate the genetic structure and to detect the most likely number of clusters (K)


Stenotrophomonas maltophilia  is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections. S. maltophilia is inherently resistant to most of the available antimicrobial agents. Spread of resistant strains has been attributed, in part, to class I integrons. In vitro susceptibility studies have shown trimethoprim-sulfamethoxazole and new floroquinolones as two important agents with activity against these organisms.S. maltophilia were isolated from clinical samples such as respiratory discharges, sputum, and catheter and hospital environments. These isolates were also subjected to susceptibility testing and polymerase chain reaction for four groups of genes including int encoding integron elements, sulI and sulII encoding trimethoprim-sulfamethoxazole resistance and smqnr encoding quinolone resistance.150 isolates of sulI and sulII genes.The rate of resistance to trimethoprim-sulfamethoxazole was up to 27 (18%) and the highest resistance to quinolone family belonged to ofloxacin (20%) and the lowest rate was for gatifloxacin (16%). The results showed that 14% of isolates contained integron elements concomitantly with S. maltophilia to co-trimoxazole and fluoroquinolones and detection of integron elements between isolates in this study showed that this rate corresponded to other data obtained from other studies.Resistance rate of  Stenotrophomonas maltophilia is an aerobic, nonfermentative, gram-negative, catalase-positive and oxidase-negative bacterium. S. maltophilia is ubiquitous in aqueous environments including water, urine, or respiratory secretions, soil and plants 1. This bacterium causes nosocomial infections in immuno-compromised patients and frequently colonizes breathing tubes such as endotracheal or tracheostomy tubes, the respiratory tract and urinary catheters. Infection easily commences by the presence of prosthetic material , and the most effective treatment is removal of the prosthetic devices. Therefore, growth of S. maltophilia isolated from respiratory or urinary specimens in microbiological media is difficult to interpret and not a proof of infection. However, isolation of S. maltophilia from sterile body regions  usually represents true infection. In immunocompetent individuals, S. maltophilia is a relatively unusual cause of pneumonia, urinary tract infection, or blood stream infection. S. maltophilia is naturally resistant to many antibiotics  and often difficult to eradicate. Although resistance has been increasing, many strains of S. maltophilia are sensitive to co-trimoxazole and ticarcillin 2. It is not usually sensitive to piperacillin, and sensitivity to ceftazidime is variable. S. maltophilia is resistant to many β-lactams, β-lactamase inhibitors, and aminoglycosides 4.5. Because of low resistance levels (∼5%), trimethoprim-sulfamethoxazole (TMP/SMX) has remained the choice of antimicrobial therapy against S. maltophilia infections worldwide. Although there are a few surveillance studies for Stenotrophomonas infections, resistance to TMP/SMX appears to be emerging, and recent in vitro modeling studies have shown that combination therapies by TMP/SMX plus ciprofloxacin or TMP/SMX plus tobramycin exhibit a greater killing capacity than TMP/SMX alone 6. In addition, S. maltophilia can acquire antimicrobial resistance through integrons, transposons, and plasmids. Class 1 integrons have been characterized from S. maltophilia strains isolated in Argentina and Taiwan, which indicates that they contribute to TMP/SMX resistance through the sulI gene carried as part of the 3′ end of the class 1 integron 7. Sul genes have been reported to contain class 1 integrons and insertion element common region (ISCR) elements that are responsible for high rate of resistance to TMP-SMX in S. maltophilia9.A recent survey has indicated that newer fluoroquinolones demonstrated good efficacy against these bacteria. The most active antimicrobials were levofloxacin and gatifloxacin in which resistance rates were reported to be 6.5 and 14.1%, respectively S. maltophilia has identified a novel family of resistance genes (smqnr) encoding proteins containing pentapeptide repeats, which confer low-level resistance to quinolones 10. Qnr gene exists in several bacterial genera and is located in S. maltophilia chromosome, designated as smqnr which encoded a protein that contributes to intrinsic resistance to quinolones 9. This gene could be plasmid-borne and results in high resistance to quinolones in wild type and mutant bacteria 12.Recent analysis of S. maltophilia genome sequence (R551-3) also identified a novel family of genes (smqnr) that encode proteins with homology to the Qnr quinolone protection proteins found in the Enterobacteriaceae 5. These Smqnr proteins  in which pentapeptide repeats could be found, confer low-level quinolone resistance by protecting DNA gyrase and topoisomerases. In Enterobacteriaceae members, Smqnr proteins are usually located in association with other resistance determinants on large plasmids 13. The clinical importance of plasmid-mediated quinolone resistance is uncertain, although it is postulated that it may help stabilize or select for mutations in the Quinolone Resistance-Determining Region (QRDR) of DNA gyrase and topoisomerase, which then confers high-level quinolone resistance 13. This study could determine the relation between dissemination and increasing antibiotic resistance of S. maltophilia isolated from hospital and patient’s samples.Examination of K279a and another S. maltophilia antibiotic resistance. In one typical study, 3% of 895 isolates were resistant to co-trimoxazole.Some studies have been performed in Iran that focused on S. maltophilia were collected from different clinical settings in Tehran, Iran and clinical samples like respiratory samples, ventilator associated pneumonia, discharges of patients, surgery devices and catheters. Body fluids were inoculated to BACTEC media. BACTEC system was pereferred for better performance in growth and identification of microbial agents because detection procedure was based on on-time and precise computed mechanism.During a two year period between 2012 to 2014, 150 isolates of °C.Susceptibility testing was performed by disc diffusion methods (Mast diagnostics) and E-test (AB Bio-disk) for trimethoprim-sulfamethoxazole, ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin on Muller Hinton agar as described by Clinical and Laboratory Standards Institute. Resistant strains to trimethoprim-sulfamethoxazole and quinolones were stored at −70ml containing 2 μl template DNA, 5 μl 10× concentrated PCR buffer, 1 μl of each appropriate primer, 10 μl dNTPs, 1 μl Taq DNA polymerase, and 21 μl sterilized distilled water 10. Primer designation and sequences are shown in Extraction and purification of DNA from bacterial colonies and plasmids was accomplished by commercial extraction kits from the isolates (QIAmp mini kit from Qiagen). Polymerase Chain Reaction (PCR) was carried out in 50 μg/ml, whereas the sensitive controls possessed TMP/SMX MICs ranging from 0.5 to 2 μg/ml. MIC for ciprofloxacin resistant strains was >4 μg/ml and for gatifloxacin resistant strains, moxifloxacin resistant strains and ofloxacin resistant strains of S. maltophilia was >8 μg/ml  and this rate for quinolone family was: ciprofloxacin 27 (18%), gatifloxacin 24 (16%), moxifloxacin 25 (17%), and ofloxacin 30 (20%). The TMP/SMX-resistant isolates possessed MICs >32 >8 μg/ml .int I F, int IR (5′ conserved region) with DNA bands of 510 bp  isolates contained smqnr genes  and Salmonella enteric8. The data presented in this study showed that sulII gene may spread by Enterobacteriaceae origins among S. maltophilia strains. These data suggest that microbiology laboratories need to carefully monitor S. maltophilia strains which show resistance to TMP/SMX, because they have the potential to increase by means of mobile genetic elements.Out of these SXT resistant isolates, 27% of them carried et al showed that 91% of strains were susceptible to SXT and MIC 14. Based on a study in 2001, rates of resistance to SXT ranged from 2% in Canada and Latin America to 10% in Europe 15. In another study performed in Saudi Arabia in 2006, two resistant cases to SXT were reported. Both isolates were resistant to TMP-SXT (MIC >8/152 μg/ml by MicroScan system and MIC >32 μg/ml by E-test strip). The two isolates were also resistant to gentamicin (MIC >8 μg/ml), both meropenem and imipenem (MIC >16 μg/ml) and ciprofloxacin (MIC >4 μg/ml). They were sensitive to ceftazidime (MIC <2 μg/ml) and ticarcillin-clavulanate (MIC=16/2 μg/ml). The sensitivities to amikacin, chloramphenicol, tetracycline, levofloxacin, aztreonam and piperacillin-tazobactam were variable between the two isolates 2. In a study performed in England in 2005, it was indicated that none of S. maltophilia isolates from salad was resistant to trimethoprim-sulfamethoxazole, ciprofloxacin, but these isolates concomitantly were resistant to chloramphenicol; 7 (78%) to piperacillin/tazobactam; 5 (56%) to ceftazidime, and 2 (22%) to gentamicin. But it should be considered that the number of samples  in the mentioned study was so small and cannot be compared with this study because our study was about clinical samples and there were many specimens 16.Betrieu S. maltophilia developes and many isolates should be considered for testing in laboratory. The most significant study ever performed on susceptibility of S. maltophilia was a study in 1999 in Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario K7L 3N6, Canada entitled “Multiple Antibiotic Resistance in Stenotrophomonas maltophilia: Involvement of a Multidrug Efflux System”.It can be concluded that as the time passes, the rate of resistance of first line effective antibiotics to 4. There are some studies performed in Iran which focused on S. maltophilia isolates and its antibiotic resistance. In a study in 2011 among a total of 12922 blood specimens, 2300 specimens had a positive blood culture (17.7%); the specimens were collected early at hospitalization and as a result, blood samples were collected before initiation of any treatment. Not considering fungal growth, 21 microorganisms were recognized, with S. maltophilia being the most common one . There were 95 sensitive and 5 resistant species in both the disk diffusion method and E-test for co-trimoxazole 25.In this study, the mechanisms of resistance and percentage of susceptibilities to antibiotics were indicated


Equisetum arvense L. extract and the mechanisms involved in the in vitro effects on endothelial vascular cells exposed to hyperosmotic stress were evaluated.  The antimicrobial activity of the Equisetum arvense L., caffeic acid, and cathechin, were quantified.  Antimicrobial activity was evaluated by disk diffusion method and minimum inhibitory concentration (MIC) determination, and oxidative stress, inflammation, and apoptosis, in pretreatment with Equisetum arvense L. exhibited antibacterial effects only on pathogenic gram-positive cocci. The modulatory activity of Equisetum arvense L. on endothelial cells exposed to hypertonic medium was different and depended on the concentration used. Low concentrations of tested compounds exerted antioxidant effect and diminished the activity of caspase-8 and also increased IκB expression while in high doses, Equisetum arvense L. was prooxidant, induced apoptosis, and decreased IL-6 secretion.  The results have shown that Equisetum arvense L. has antibacterial effects on gram-positive cocci and, administered in low dose, may be a new therapeutic approach for diseases associated with hypertonic conditions or oxidative stress and apoptosis. These experimental findings suggest that  Worldwide, research in the past decades has shown an increased interest in the phytochemical products and plant extracts, due to frequent use in the prevention and treatment of some diseases. Several studies have demonstrated that the antioxidants found in plants are of major interest to medicine, owing to the fact that they protect the organism against oxidative stress, generated in the context of some diseases: atherosclerosis, ischemic cardiac disease, cancer, Alzheimer disease, Parkinson disease, aging, and even in infectious diseases . ExperimEquisetum arvense L (field horsetail) is a perennial fern from the Equisetaceae family. It has a yellowish nonphotosynthetic spore-bearing fertile stem, produced in early spring. The green, photosynthetic, heavily branched sterile stems are produced in late spring and persist to late autumn. The sterile stem is the medicinal product of the plant (Equiseti herba) mentioned in European Pharmacopeia (Ph. Eur. 8) [Equisetum arvense L. are flavonoids, phenolic acids, alkaloids, phytosterols, tannins, and triterpenoids [ Eur. 8) . The mosrpenoids .Equisetum arvense L. extract or tea with natural extract, such as antioxidant, anti-inflammatory, antibacterial, antifungal, vasorelaxant, neuro and cardio protectors [Several studies have described different biological effects of otectors , 9, and otectors , 10.Cardiovascular diseases are a leading health problem worldwide . 31% of An increased extracellular osmolarity has many negative effects on the endothelium: promotes water flux out of the cells, triggers cell shrinkage and intracellular dehydration , increasκB/Rel family of transcription factors, and stimulate the releasing of proinflammatory cytokines [α) by the activated macrophages, T-lymphocytes, smooth muscle cells, and endothelial cells. Reactive oxygen species (ROS) are highly reactive molecules which cause, on one hand, lipid peroxidation, DNA damage, and protein oxidation [κB.In order to equalize the intracellular and extracellular pressure, the cells develop an adaptive response to osmotic stress by induction of genes responsible for synthesis and transports of “compatible osmolytes.” Many of them, such as heat shock protein, antioxidant enzymes, and adhesion molecules, are required for preserving the protein structure and function  and for ytokines  such as xidation  and, on Staphylococcus aureus is known as an etiologic agent for many skin and mucous infections, especially those caused by methicillin-resistant S. aureus (MRSA) at immunocompromised patients or postoperative [E. coli is frequently described as a cause of chronic and recurrent urinary infection [C. albicans is the most pathogenic species in humans, isolated from biofilms formed on implanted medical devices [The study of the cytotoxicity and antimicrobial activity of plant extracts is important for their clinical application in medical practice. Bacterial infections, especially those that form biofilm or are associated with antibiotic failure, require the finding of agents with complementary action or synergic effect with antibiotherapy. perative . E. colinfection  while C. devices  or is in devices . Several devices –27.Equisetum arvense L. extract for its antimicrobial activity and for anti-inflammatory, antioxidant, and antiapoptotic properties on endothelial cell cultures. The plant used comes from nonpolluted areas of Bihor County, the harvest time being the months of April and May, when we obtained the highest values of phenolic compound concentration. The effect against gram positive, gram negative, and Candida albicans by disc diffusion test and minimum inhibitory concentration (MIC) was evaluated. Oxidative/antioxidative status and inflammatory status in addition with caspase-3 and 8 activities and their interaction with two doses of Equisetum arvense L. extract, on osmotic stress in vitro, were investigated.Starting from the previously obtained results, we aimed to test Equisetum arvense L. collected in May 2015 (Bihor County) was dried at 40°C, for 96 h, and used for preparation of lyophilized extracts. The extraction of 10 g of sterile stems was made with 100 ml 70% aqueous ethanol for 20 minutes. After centrifugation , the extract was evaporated to dryness and then was transferred to a sample via lyophilized extract. Shimadzu SCL-POA reversed-phase high-performance liquid chromatography (HPLC) system consisted of a LC-10ADVP pump equipped with SPD-10AVP Diode Array detector was used to assay compositions of phenolic compounds. The column Kintex 5u RPC 18 and mobile phase composed of 0.05% formic acid and 0.05% formic acid-acetonitrile (50 : 50 v/v) were used. The antioxidant activity was evaluated by CUPRAC assay, DPPH method, and FRAPS method. Total polyphenol content was determined with the Folin–Ciocalteu method.In our previous research, the plant collection and sample preparation were described . Thus, tα were obtained from R&D Systems . NF-κB, phospho-NF-κB p65 (Ser 536), (93H1), IκBα, and phospho-IκBα (Ser 32/36) were purchased from Cell Signaling Technology Inc., Danvers, USA.2-Thiobarbituric acid and Bradford reagent were obtained from Merck KGaA , while absolute ethanol and n-butanol were purchased from Chimopar . ELISA tests for evaluation of caspase-3, caspase-8, IL-6, and TNF-Equisetum arvense L. was evaluated by disk diffusion method using the standard methodology [Antimicrobial activity of hodology  and by dStaphylococcus aureus (ATCC 29213), Streptococcus pneumoniae (ATCC 49619), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 90029)  and human clinical isolates—Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and group G beta-hemolytic streptococcus.In our study, we used the following test organisms: reference microbial strains—Escherichia coli, and Pseudomonas aeruginosa isolates, Mueller-Hinton Agar (Oxoid) was used and for streptococcal strains, Mueller-Hinton 2 agar + 5% sheep blood (BioMerieux). Candida albicans was plated onto Sabouraud Gentamicin Chloramphenicol 2 agar (BioMerieux).For staphylococci, Candida albicans, equivalent to a 0.5 McFarland standard. Each strain was plated onto the appropriate culture media with a sterile cotton swab, and plates were dried for 10–15 minutes. Sterile filter paper discs of 6 mm diameter (HiMedia Laboratories) saturated with 20 μl of Equisetum arvense L. extract were placed onto inoculated plates. After overnight incubation, at 37°C, inhibition zone diameters were measured in millimeters. Filter papers impregnated with distilled water (20 μl) were used as negative controls, and penicillin , vancomycin discs , ofloxacin discs , meropenem discs , and fluconazole  were used as positive controls. Each test was performed in triplicate and mean values were selected.Inoculums were prepared by direct colony suspension in salina from 20–24 h growth from Columbia agar + 5% sheep blood (Biomeriex) for bacterial strains and from Sabouraud Gentamicin Chloramphenicol 2 agar (BioMerieux) for Staphylococcus aureus (ATCC 29213), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes group A (ATCC®12384™ Culti-Loops™) and human clinical isolates—group G beta-hemolytic streptococcus and Staphylococcus aureus. For staphylococci isolates, Mueller-Hinton Broth (Oxoid) was used and for streptococcal strains, Mueler-Hinton Broth plus 5% sheep blood (BioMerieux). We used 75 × 12 mm sterile capped tubes, in two rows for each microbial strain to cover the range of Equisetum arvense L. The first tube, an inoculated tube, controlled the adequacy of the broth to support the growth of the organism, while the second tube was used to check the sterility (an uninoculated tube).The MIC is defined as the lowest concentration of a drug that will inhibit the visible growth of an organism after overnight incubation. We used Broth dilution MIC's—macrodilution for the following test organisms: reference microbial strain—Equisetum arvense L. extract, five dilutions were prepared as follows: 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, and 6.25 mg/ml. Inoculums were prepared by direct colony suspension in salina from 20–24 hours grown from Columbia gar supplemented with 5% sheep blood (BioMerieux), equivalent to a 0.5 McFarland standard. 0.1 ml inoculum for each tube was used and all the tubes were incubated at 37°C overnight.From 200 mg/ml μg/ml, and amphotericin 100 μg/ml  in a humidified CO2 incubator at 37°C. Cell cultures in the 23rd to 26th passages were used. The analysis of surface markers was performed by flow cytometry  and used different monoclonal antibodies .Commercial human umbilical vein endothelial cells (HUVEC) were bought from the European Collection of Cell Cultures  and multiplied in RPMI medium, supplemented with 10% fetal calf serum (FCS), gentamicin 50 4/cm2 in cell culture Petri dishes (TPP) were settled for 24 h in medium then exposed for 24 h to either Equisetum arvense L. extract (25 or 2.5 μg/ml), caffeic acid (10 or 1 μg/ml), and cathechin (10 or 1 μg/ml), as positive controls; afterwards, the cells were washed 3 times with PBS and incubated for 24 h either in normotonic (137 mmol/l) or hypertonic conditions (200 mmol/l). Cells were collected by scraping and treated with a lysis buffer containing IGEPAL-Nonidet 1% (Sigma) and 1% protease inhibitor complex (Sigma) in PBS for 1 h, on ice. The protein content was determined by the Bradford method . All the experiments were performed in triplicate.Cells seeded at a density of 10Equisetum arvense L. extract, the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method was employed. Human umbilical vein endothelial cells (HUVECs), seeded at a density of 104/well in ELISA 96-well microtitration flat bottom plaques , were allowed to settle for 24 h in medium, then treated with different concentrations of extract in medium ranging from 6.5 to 5000 μg/ml, for 24 h. Afterwards, the viability assays were performed. The viability testes were done using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay as indicated by the producer. The optical density values were read at absorbance of 490 nm using an ELISA plate reader  for MTS. All the experiments were conducted in triplicate. Untreated cultures exposed to medium were used as controls.To explore the cytotoxicity of Lipid peroxides were determined using the fluorimetric method with 2-thiobarbituric acid (TBA) described by Conti et al.  and wereα levels and apoptosis by evaluation of caspase-3 and caspase-8 activities. In addition, the expressions of total NF-κB protein and the active form (phospho-NF-κB), IκB, and phospho-IκB were evaluated by Western blot in cells exposed to low doses of the three compounds used for treatment. IL-6, TNF-α, the human active caspase-3 immunoassay kits, and caspase-8 colorimetric assay kit were purchased from R&D Systems Inc. (USA). Cell extracts were treated as indicated by the producer, readings were done at 450 nm with correction wavelength set at 540 nm, using an ELISA plate reader .Inflammation was quantified by measurement of IL-6 and TNF-μg protein lysates/lane were separated by electrophoresis on SDS PAGE gels and transferred to polyvinylidene difluoride membranes, using Bio-Rad Mini-PROTEAN system (Bio-Rad). Blots were blocked and then incubated with antibodies against NF-κB, pNF-κB, IκB, and pIκB, then further washed, and incubated with corresponding secondary HRP-linked antibodies (1 : 1500) (Santa Cruz Biotechnology). Proteins were detected using Supersignal West Femto Chemiluminescent substrate  and a Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software (Bio-Rad). GAPDH  was used as a protein loading control .For Western blotting, 20 http://www.graphpad.com). A p value < 0.05 was considered statistically significant.The statistical significance of the difference between treated and control groups was evaluated with two-way ANOVA, followed by Dunnett's multiple test using GraphPad Prism version 4.00 for Windows  and Staphylococcus epidermidis, the last one being a methicilin-resistant strain. Our extract was the most effective against Streptococcus pneumoniae and against the clinical isolate of Streptococcus pyogenes. No antimicrobial activity was found on Candida albicans or on gram-negative bacteria. In MIC determinations, the lowest concentration of Equisetum arvense L. extract, at which there was visible growth, was 25 mg/ml for Staphylococcus aureus (ATCC 29213) and human isolate Staphylococcus aureus and 12.5 mg/ml for streptococcal strains: Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes group A, and group G beta-hemolytic Streptococcus. Subcultures on Columbia agar + 5% sheep blood (BioMérieux) from tubes with 25 mg/ml Equisetum arvense L. extract for staphylococci and 12.5 mg/ml for streptococcal were positive. These values represent the MICs. Subcultures from tubes with 50 mg/ml for staphylococci and 25 mg/ml for streptococcal were negative; these values represent the minimum bactericidal concentrations (MBCs).One of the goals of our study was to determine the antimicrobial activity of the Surface marker analysis performed by flow cytometry showed the presence of ICAM-1, CD105, CD29, and CD73 thus suggesting that the cells used in experiment exhibit endothelial functional parameters.Equisetum arvense L. extract tested on HUVECs did not lead to any significant changes in cell viability even when high doses (5000 μg/ml) were used (The toxicity of the ere used .p < 0.0001) in the cells exposed to high concentrations of saline solutions  and in the hypertonic solutions (p < 0.001). The caffeic acid and cathechin significantly decreased the lipid peroxide levels compared to the extract (p < 0.001) . The best protection of endothelial cells against hyperosmotic stress was exerted by the low concentration of cathechin (p < 0.001)  . When us< 0.001) .α were quantified  . In hypertonic medium, the pattern is different; the three tested substances had a stronger anti-inflammatory effect (p < 0.001). The lower doses of the three studied compounds had no proinflammatory effects in normal conditions, but caffeic acid and cathechin were proinflammatory under hypertonic conditions. The low dose of Equisetum arvense L. extract had no significant effect on the IL-6 levels in hypertonic medium (p > 0.05). In hypertonic conditions, we registered an increased concentration of IL-6 in the medium compared to the normotonic situation (p < 0.01). The administration of the high dose of Equisetum arvense L. and caffeic acid had no significant effect on the IL-6 levels found in the growth medium (p > 0.05)  .μg/ml of cathechin reduced significantly IL-6 secretion in the medium. The lower doses of Equisetum arvense L., caffeic acid, and cathechin had no effect on IL-6 secretion found in the growth media . The lower doses of the three compounds did not influence significantly the TNF-α secretion in both experimental conditions  complex, and IκB is an inhibitor which sequesters NFκB dimers in the cytoplasm in an inactive state. In normotonic medium, the treatment with low dose of Equisetum arvense L. extract (p < 0.05) and low doses of caffeic acid (p < 0.001) and cathechin (p < 0.001) diminished the expression of NF-κB compared to controls, in parallel with increasing of IκB expression, especially after cathechin administration (p < 0.05)  .κB level decreased compared to normotonic medium (p < 0.001) and increased only after caffeic acid treatment (p < 0.01). In hypertonic medium, all natural compounds used in the experiment increased the expression of IκB, mostly cathechin in low dose (p < 0.001). The best inhibitory effect on IκB activation was noticed in cells exposed to cathechin (p < 0.001), then after Equisetum arvense L. (p < 0.01) and caffeic acid (p < 0.01) treatments.In hypertonic conditions, NF-κB represents a modest part of NF-κB protein, both in normotonic and in hypertonic medium, as illustrated in κB protein expression (p < 0.001), effect amplified by cathechin administration (p < 0.001) which exerted an inhibitory effect on pIκB (p < 0.001). Equisetum arvense L. and caffeic acid had also a strong inhibitor effect on activation of IκB in hypertonic medium while in normotonic conditions, the same compounds enhanced its expression .The active form of NF-p < 0.01) while in hypertonic medium, the same compound did not influence the enzyme activity (p < 0.01) in hypertonic conditions while the normotonic medium did not change the enzyme activity   but, in lower doses, decreased the caspase-8 activity, especially Equisetum arvense L. and cathechin, and thus the induction of apoptosis in the same conditions (p < 0.001)  . Higher  Figures .Equisetum arvense L. extract exhibited an antibacterial effect on Streptococcus pneumoniae and the clinical isolate of Streptococcus pyogenes.In previous studies, it has been demonstrated that plants, fruits, and vegetables, due to their phytochemicals composition, have antioxidant, antimicrobial, and anti-inflammatory actions and are used in medicine and nutrition including the cosmetic industry –33. In oin vitro antimicrobial activity Equisetum arvense L. extract, but with some conflicting results. Milovanović et al. [Candida albicans while in our study, we did not obtain any inhibitor effect on Candida albicans strain. Our results are in agreement with previous findings reported by Uslu et al. [Equisetum arvense L. extract at a concentration of 50 mg/mL was effective against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Candida albicans, Candida tropicalis, and Candida glabrata, microorganisms involved in oral infections [Equisetum arvense L. extract on Staphylococcus aureus and gram-negative bacteria are also described, both in the aqueous and alcoholic extract, with better response in the case of the alcoholic extract [Equisetum arvense L. was enough for its strong antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis, and fungi: Aspergillus niger or Candida albicans. The effects were explained by a composition rich in the essential oil of the plant extract which inactivated the microbial adhesion proteins and transport proteins and induced the rupture of the microbial membrane [Candida albicans due to trapping free radicals directly or scavenging them through reactions with antioxidant enzymes [Studies over the past decade attested the ć et al.  describeu et al.  and Čanau et al. . De Olivfections . Protect extract , 39. Rad extract  stated tmembrane . In addi enzymes .Staphylococcus aureus and Escherichia coli [However, other studies showed no effect of the aqueous extract on hia coli , 43. Onehia coli  or diffehia coli . From a hia coli . In simihia coli .in vitro strong antioxidant activity of the extracts obtained from the sterile stems of Equisetum arvense L. Using the FRAP technique, the value determined was 84.160 μmol Trolox equivalent/g DW (TE), using CUPRAC technique, 49.2 μmol Trolox equivalent/g DW (TE), and using DPPH method, 87.3% [In our extract, the total content of polyphenols and flavonoids was high, as it resulted from findings performed with spectrophotometric techniques . Using Hd, 87.3% .Osmotic stress is involved as a pathogenetic mechanism in many cardiovascular diseases and is associated with the efflux of water from the cells and shrinking of cells. In this process, the activity of water channels (aquaporins) and electrolyte transporters, and also the accumulation of osmolytes within the cells  for prot+-K+-2Cl− cotransporter, Na+/H+ exchanger, and the Cl−/HCO3− exchanger which lead to intracellular accumulation of ions and increasing of cell volume [myo-inositol transporter [It is known that under severe hypertonic conditions or when the adaptation mechanisms are outdated, the cells enter to apoptosis and die . Moreovel volume . In the l volume . In thisnsporter . In addinsporter .α, IL-1β, IL-6, and IL-8 secretion from different cells [κB p65 phosphorylation at serine 536 and consequently the induction of inflammatory NF-κB p65-responsive genes were involved [κBα. This is an inhibitor which can mask the nuclear localization of NFκB and sequester NFκB dimers in an inactive state in cytoplasm. IκB phosphorylated is then ubiquitinated and degraded by the proteasome, freeing NF-κB to enter the nucleus and induce expression of inflammatory genes [κB was obtained when cells were exposed to hypertonic medium alone or in association with cathechin administration, suggesting that cathechin addition increased both the total NF-κB protein and its activation. The active form of IκB protein diminished in cells exposed to Equisetum arvense L., caffeic acid, and cathechin in parallel with increasing of IL-6 levels and NF-κB activation, especially after cathechin administration.Cytoskeletal remodeling, antioxidant response, and unfolded protein response are other important mechanisms of osmoadaptation. In addition, hyperosmotic stress was found to directly induce TNF-nt cells . In thesinvolved . Moreovery genes . A highe2 and generation of prostaglandins including arachidonic acid is one of the mechanisms involved in the production of redox misbalancing. Phospholipase A2 activates the NADPH oxidase and increases the superoxide anion formation [In the experiment, the caspase activities were evaluated only at 24 h after incubation in the hypertonic environment, when the whole adaptation process took place. However, at this time, both oxidative stress and inflammation induced by hypertonicity were maintained at an increasing rate compared to normotonic conditions. In general, oxidative stress is caused by the intracellular accumulation of reactive oxygen species (ROS) or a disturbance of the cellular redox state and can cause intracellular proteins, lipids, and DNA damage. ROS have also been implicated as major mediators of stress-induced signaling as a response to various kinds of environmental stress including osmotic stress. In hyperosmotic stress, the activation of membrane-associated phospholipase Aormation . In addiormation .κB activation and diminished pIκB in cells and did not influence the caspase-3 activity. The high doses of the three substances had the opposite effect on inflammation and oxidative stress. Thus, Equisetum arvense L., cathechin, and caffeic acid exerted prooxidant effect and reduced the IL-6 secretion in cells. In addition, caffeic acid and cathechin in high concentrations induced the secretion of TNF-α in the endothelial cells in parallel with prooxidant and proapoptotic effects suggesting the cellular toxicity of high doses of polyphenols.In our study, the low doses of the three compounds reduced the lipid peroxidation and protected the cells against stress induced by hypertonic conditions and diminished the activity of caspase-8 and consequently the apoptosis triggered by extrinsic mechanism. In the same conditions, caffeic acid and cathechin, in low concentrations, increased IL-6 secretion and NF-α), oxidative stress, and angiotensin II and after vascular injury [α complex. This complex initiates the IL-6 signaling [The different effect on IL-6 secretion can be explained by double functions of IL-6 in cardiovascular systems. IL-6 is secreted in different cells of cardiovascular structures including macrophages, fibroblasts, and endothelial cells as a response to the exposure to cytokines (IL-1, TNF-r injury . IL-6 caignaling  and actiignaling .Equisetum arvense L. extract. The anti-inflammatory activity of the extract demonstrated by Do Monte et al. [Our results confirmed the findings of Bessa Pereira et al.  regardine et al.  in woundEquisetum arvense L. extract exhibited antibacterial effects on pathogenic gram-positive cocci but had no effect on gram-negative bacteria and Candida albicans. The three compounds tested exert the antioxidant activity and diminished the activity of caspase-8 only in low concentrations, protecting the cells exposed to hyperosmotic stress. In high doses, Equisetum arvense L., cathechin, and caffeic acid had prooxidant effects, induced apoptosis, and decreased IL-6 secretion.Equisetum arvense L. extract on endothelial cells exposed to hypertonic medium are different and depend on the concentration used. These experimental findings suggest that Equisetum arvense L. administered in low doses may be a new therapeutic approach to decrease the enhanced oxidative stress and apoptosis associated with hypertonic conditions. In addition, Equisetum arvense L. extract exhibits antimicrobial activity against gram-positive cocci. However, further more complex studies on different cell lines with multiple doses or exposure times are necessary to prove its clinical potency.The modulatory effects of


Kawasaki disease is diagnosed when fever lasts for more than 5 days with the presence of four out of five of the following clinical features: bilateral conjunctival congestion, changes in the lips and oral cavity, polymorphous exanthem, changes in peripheral extremities, and acute nonpurulent cervical lymphadenopathy . The average age of onset is 2 years and 90% of patients are below 5 years of age. Boys are more affected than girls . This case report describes an adolescent female who was initially managed as having septic shock and subsequently found to have Kawasaki shock syndrome. A 12-year-old girl presented with high spiking fever (103°F) for 3 days with headache, body aches, and an itchy rash for one day. There was no bleeding from gums, skin, urine, or stools. She did not have any urinary or respiratory symptoms. There was no contact history of fever or rash. She had a past history of simple febrile convulsions. She did not give a history of any known allergies, atopy, or asthma.Examination revealed a febrile ill child with cervical lymphadenopathy and urticarial rash over the body which changed in the distribution with fever spikes . Her per3 with 85% neutrophils and platelet (Plt) count of 116000/mm3, C-Reactive Protein (CRP) of 161 mg/l, and Erythrocyte Sedimentation Rate (ESR) of 40 mm/1st hour. Summary of investigations is shown in Investigations on admission showed white blood cell (WBC) count of 5580/mmOn Day 4 of illness, child developed nonpurulent conjunctivitis with periorbital oedema. She was noted to have haemodynamic instability with hypotension and tachycardia. She received ICU care and needed two inotropes to maintain her haemodynamic stability for 3 days (Day 4–Day 6). As the inotropes were given for short duration, significant side effects were absent. Her urine output was normal throughout the illness. With the presence of fever, rash,and haemodynamic instability, viral myocarditis, toxic shock syndrome, and Steven Johnson syndrome were considered as possible diagnosis compared to atypical Kawasaki disease (KD) with the evolving clinical situation on Day 4 of illness.2D Echo on Day 8 of illness showed anteroseptal hypokinesia with dilated left anterior descending (LAD) artery with normal Left ventricular and right ventricular function and no pericardial effusion with a negative troponin I and CK-MB. LAD diameter was 2.3 mm with dilated area with a diameter of 3.5 mm. Alanine transaminase (ALT) and aspartate transaminase (AST) were 86 U/l and 115 U/l on Day 4 which persisted above the reference range till Day 16 of illness. Serum bilirubin was 12 mg/dl on Day 4. Ultrasound scan showed bilateral pleural effusions and moderate ascites without hepatosplenomegaly. Repeated blood cultures did not reveal any bacterial growth. Serology for measles, leptospirosis, rubella, Dengue, Epstein-Barr virus, and Polymerase Chain Reaction (PCR) for influenza was negative. ANA was negative. Hypoalbuminemia was noted from Day 4 of illness and persisted for two weeks. CRP was 289 mg/l on Day 5 of illness which came down to 9.2 mg/l on Day 16. Serum Ferritin was 694.5 ng/ml on Day 10 of illness. Intravenous immunoglobulin 2 g/kg over 24 hours was given on Day 8 of illness for the treatment of Kawasaki disease. Aspirin 80 mg/kg in four divided doses was started and continued. Fever settled on Day 10. Ascites and pleural effusions settled subsequently. Two months after the illness, child was asymptomatic and 2D Echo done two months later showed dilated LAD without aneurysm formation.Initial differential diagnoses in our patient are viral exanthema with myocarditis, systemic lupus erythematosus, toxic shock syndrome, Scarlet fever, systemic onset Juvenile Idiopathic Arthritis (s-JIA), Steven Johnson syndrome, and atypical KD.KD is an acute febrile illness diagnosed with fever for >5 days with 4 out of 5 of the following signs: bilateral conjunctival congestion, changes in the lips and oral cavity, polymorphous exanthema, changes in peripheral extremities, acute nonpurulent conjunctivitis, and cervical lymphadenopathy . KD is tOur patient had conjunctivitis, exanthema, and cervical lymphadenopathy which led to the diagnosis of atypical KD. Serious cardiac complications in KD include coronary artery aneurysms, decreased myocardial contractility, congestive heart failure (CHF), arrhythmias, and myocardial ischemia . Heart f Mycoplasma and Streptococcus [Toxic shock syndrome will have similar presentation with hypotension and elevated ALT and conjunctivitis but coronary artery dilation is unlikely in it . Pleuraltococcus , 7. Incrtococcus .Kawasaki shock syndrome (KSS) characterized by systolic hypotension or clinical signs of poor perfusion is found in 6% of KD patients . It was One study showed serum ferritin levels were significantly elevated in s-JIA patients compared with KD patients with a cut-off value of 369.6 ng/ml . It was


Garcinia pedunculata (GP) fruit in rats were investigated. The acute antihyperglycemic effect of different doses of GP was studied in normal male Wistar rats. Diabetes was induced by streptozotocin (STZ) injection in another cohort of male Wistar rats and they showed significantly higher blood glucose and glycated hemoglobin (HbA1c) levels, altered lipid profiles, and lower insulin levels compared to nondiabetic control animals. There were increased lipid peroxidation and reduced levels of cellular antioxidant enzymes in different tissues of diabetic rats. However, oral administration of GP extracts, especially the highest dose (1000 mg/kg), significantly ameliorated hyperglycemia (42%); elevated insulin levels (165%); decreased HbA1c (29.4%); restored lipid levels , liver and renal function markers, and lipid peroxidation ; and stimulated tissue antioxidant enzymes to near normalcy. Overall, the findings suggest that GP fruit is effective against hyperglycemia and could be used in the treatment of diabetes and its complications and other oxidative stress-mediated pathological conditions.The antihyperglycemic, antidiabetic, and antioxidant potentials of the methanolic extract of DM is atabolism , 3. HencSeveral antidiabetic drugs are available, but all of them have many adverse side effects such as lactic acidosis, hyperglycemia, diarrhea, or flatulence, which impose an economic burden –6. ThereGarcinia pedunculata (GP) is an indigenous medicinal plant that belongs to the family Clusiaceae and is commonly known as “Taikor” in Bangladesh and “Amlavetasa” in India. Availability is especially common in Sylhet district in Bangladesh and in Assam, Arunachal Pradesh, India. The mature GP fruit is greenish yellow and is consumed as a cooked vegetable, a raw fruit, or an ingredient for pickle preparation [β-carotene (45.00 mg/100 g), thiamine (0.03 mg/100 g), and riboflavin (0.02 mg/100 g) as well as minerals  [paration . It has paration . The fruparation , 11, antparation , anti-inparation , hypolipparation , hepatopparation , nephropparation . GP frui, resp.) . Recentl, resp.) , 14. In , resp.) , 19.In our previous study, we reported the potentials of GP fruit extracts in the regulation of blood glucose level and positive effects on pancreatic and liver functions as well as lipid profile in rats .Therefore, considering the traditional use of the fruit as an alternative medicine and its inherent phytoconstituents, we aim to evaluate the antihyperglycemic, antidiabetic, and antioxidant effects of a GP fruit extract in rats and also to identify its potential compounds using high-performance liquid chromatography (HPLC) analysis.Streptozotocin (STZ) and methanol were purchased from Sigma . Glibenclamide was purchased from Square Pharmaceuticals Ltd., Bangladesh. Ketamine was acquired from Popular Pharmaceuticals Ltd., Dhaka, Bangladesh. Glucose oxidase/peroxidase reactive strips were purchased from Abbott Diabetes Care, Inc., USA. All chemicals and reagents used in this experiment were of analytical grade.Fresh, mature GP fruits were collected from Sylhet district of Bangladesh in March 2016. A methanolic fruit extract (25%) was prepared according to a method described by Lanjhiyana et al.  with sliThe fine powder (200 g) was mixed with sufficient absolute methanol to dissolve it (800 mL) and was kept in a shaker  at 150 rpm and 30°C for 72 hours. The mixture was filtered with Whatman No. 1 filter paper, and the methanol solvent was completely evaporated using a rotary evaporator  under reduced pressure (100 psi) at a controlled temperature (40°C). The concentrated extract was preserved at −20°C for subsequent use.μm syringe filter . 20 μL of the filtrate was loaded onto the HPLC system  equipped with a UV detector . A Luna Phenomenex C18 100A  HPLC column was used. A linear gradient at a flow rate of 0.5 mL/min was employed throughout with a total analytical time of approximately 35 min.The phenolic compounds in GP extract were detected following previous methods , 23 with trans-cinnamic acids as well as flavonoids  were purchased from Sigma-Aldrich  and were used as reference standards.The binary mobile phase consisted of solvent A (ultrapure water with 0.1% phosphoric acid) and solvent B (pure methanol with 0.1% phosphoric acid). Elution from the column was achieved with the following gradient: 0 to 10 min of solvent B, increased from 35% to 55%; 10 to 25 min of solvent B, increased to 62%; 25 to 30 min of solvent B, increased to 85%; and the final composition was kept constant up to 35 min. All solvents were of HPLC grade. The detection wavelength was fixed between 200 and 450 nm, with specific monitoring conducted at 265 nm. The identification of phenolic and flavonoid compounds was performed by comparing the retention times of the analytes with the reference standards. Phenolics which included tannic, gallic, pyrogallol, vanillic, benzoic, andn = 48) weighing between 175 and 250 g (aged 16–18 weeks) were used in this study. The animals were maintained under controlled environmental conditions in an animal housing facility at the Department of Biochemistry and Molecular Biology, Jahangirnagar University, at an ambient temperature of 22 ± 3°C and relative humidity of 44% to 56% under a 12 h/12 h light/dark cycle. They were fed with a standard pellet rat diet along with water ad libitum. The study was conducted in accordance with the internationally established principles of the US guidelines . The experimental protocol was approved by the Biosafety, Biosecurity & Ethical Committee of Jahangirnagar University.Male Wistar rats  was performed to observe the acute antihyperglycemic effects of the GP fruit extracts using different sets of normal rats. Five groups of six rats each were used in this study. Briefly, group 1 served as a normal control; group 2 served as a glibenclamide control, orally treated once with glibenclamide at 10 mg/kg; and groups 3, 4, and 5 were orally treated once with fruit extracts .In overnight-fasted rats, fasting blood glucose level was measured; this time point was defined as 0 min. Then, different doses of the fruit extract  were administered orally to the appropriate groups of rats. Blood glucose levels were measured 30 min later. The animals were then given glucose solution (2 g/kg) orally, and their glucose tolerance was measured hourly. Blood glucose levels were estimated using glucose oxidase/peroxidase reactive strips.Group 1 . Animals receiving only a normal diet for 28 days. Group 2 (Diabetic Control). Diabetic animals receiving a normal diet for 28 days. Group 3 (Diabetic + Glibenclamide). Diabetic animals treated with glibenclamide at a dose of 10.0 mg/kg body weight for 28 days. Group 4 (Diabetic + GP 250). Diabetic animals treated with methanolic extract of GP at a dose of 250 mg/kg body weight for 28 days. Group 5 (Diabetic + GP 500). Diabetic animals treated with methanolic extract of GP at a dose of 500 mg/kg body weight for 28 days. Group 6 (Diabetic + GP 1000). Diabetic animals treated with methanolic extract of GP at a dose of 1000 mg/kg body weight for 28 days. A total of 48 male Wistar rats were acclimatized one week prior to the experiment and randomly divided into six groups (each group containing 8 rats) as follows: ad libitum during the experimental period. In addition, the rats' body weights and fasting blood glucose levels were measured on a weekly basis. The animals were also monitored for any behavioral changes during the study period.All animals received a standard laboratory diet and drinking waterAn OGTT was performed to investigate the effects of the GP fruit extracts on glucose-induced hyperglycemia in rats with STZ-induced diabetes. The blood glucose levels were measured in overnight-fasted rats at a time point designated as 0 min. The animals were then orally dosed with glucose solution (2 g/kg) using a gavage needle, and their glucose tolerance was measured hourly for 4 hours. Blood glucose levels were estimated by glucose oxidase/peroxidase reactive strips.At the end of the experimental period, all rats were fasted for 15 hours before being sacrificed by intraperitoneal injection of ketamine hydrochloride (500 mg/kg) . Blood sThe relative organ weight was calculated by dividing the individual weight of each organ by the final body weight of each rat according to the following formula : relativBlood samples (4 mL) were placed in dry test tubes and were allowed to coagulate at ambient temperature for 30 min. Serum was separated by centrifugation at 2000 rpm for 10 min.Immediately following collection, the tissue samples were washed with ice-cold phosphate-buffered saline (PBS). The samples were homogenized in phosphate buffer  to make approximately 10% w/v homogenates. The homogenates were centrifuged at 1700 rpm for 10 min, and the supernatant was collected and stored at −20°C for further biochemical analysis. Part of each tissue sample was preserved in formalin solution (10%) for histopathological examination.γ-glutamyltransferase (GGT), total bilirubin (TB), total protein (TP), albumin (ALB), globulin (GLB), and albumin/globulin (A/G) ratio; parameters for kidney function such as creatinine, urea, and uric acid; and parameters for lipid profile including total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were determined following standard procedures using an automated chemistry analyzer . Another lipid profile parameter, serum low-density lipoprotein cholesterol (LDL-C) level, was estimated based on the following Friedewald formula [Biochemical parameters for liver function such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH),  formula :(1)LDL-CMalondialdehyde (MDA) levels were analyzed to detect lipid peroxidation (LPO) products in the liver, kidney, heart, and pancreas tissues of normal and treated rats. MDA is referred to as a thiobarbituric acid reactive substance (TBARS) and was measured with thiobarbituric acid (TBA) at 532 nm according to the method described by Ohkawa et al. . The levThe levels of endogenous antioxidant or antiperoxidative enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPx), were determined in the rats' liver, kidney, heart, and pancreas tissues using rat-specific SOD and GPx ELISA assay kits . To this end, the tissue homogenates were recentrifuged at 12,000 rpm for 10 min at 4°C using an Eppendorf 5415D centrifuge . The clean supernatants were used for analysis. The levels of SOD and GPx were expressed as pg/mL and mIU/mL, respectively.μm thick sections using a rotary microtome and subsequently stained with hematoxylin and eosin (H&E) dye [The tissue samples were fixed in 10% neutral formalin and processed by the paraffin embedding technique. The histopathological specimens were cut into 5 H&E) dye . Photomip value of less than 0.05 was considered statistically significant.All results were represented as the mean ± standard deviation (SD). Data were analyzed using Microsoft Excel 2007 , GraphPad Prism version 6 , and SPSS . All the data from the treatment groups  were compared with the results from the diabetic control group using a one-way ANOVA followed by Dunnett's multiple comparison test. A A total of eleven phenolic standards were used in this study in which seven were phenolic acids and four were flavonoid compounds. Only a flavonoid and catechin (13.59 mg/g) were detected in GP extract while some other peaks remained unidentified since there were no proper standards available .Blood glucose levels returned to baseline or even lower 1 hour after glucose administration in all animals. Animals treated with glibenclamide and fruit extracts showed marked decreases in blood glucose level compared with normal control animals, beginning only 1 hour after glucose administration. In particular, the rats given glibenclamide and GP at 500 and 1000 mg/kg doses showed significant reductions in blood glucose level compared with the normal control rats at each time point .Blood glucose levels were significantly higher in rats with STZ-induced diabetes than in normal control rats one hour after oral administration of glucose solution. However, the administration of glibenclamide and GP at three doses caused gradual decreases in the blood glucose levels compared with those of diabetic control rats. The effects were significant in rats treated with glibenclamide and GP extract at 1000 mg/kg as observed up to 4 hours after treatment .The effects of GP fruit extract on body weight and relative organ weights are summarized in p < 0.05).The effects of GP on serum insulin, HbA1c, and Hb levels are presented in A marked increase in circulating levels of TG, TC, and LDL-C and a decrease in HDL-C level were observed in diabetic control rats compared with normal control animals . TreatmeThe effects of GP extract and glibenclamide on the renal function were investigated by measuring serum creatinine, urea, and uric acid levels . There wThe effects of oral GP administration on liver function in diabetic rats were investigated by measuring serum ALT, AST, ALP, GGT, and LDH activity as well as TB, TP, ALB, and GLB levels and A/G ratio . Serum ABased on the investigation of oxidative stress biomarkers, there was a significant increase in LPO levels in STZ-induced diabetic control rats as evidenced by increases in liver, kidney, heart, and pancreas MDA levels compared with those of the normal control group. However, GP tended to confer a protective effect: the animals that were treated with fruit extracts showed a dose-dependent reduction in MDA levels compared with the diabetic controls. Significantly reduced LPO levels were observed in liver, kidney, heart, and pancreas tissues from the rats treated with 1000 mg/kg GP and with glibenclamide, and the rats treated with 500 mg/kg GP had significantly reduced LPO levels in both liver and heart tissues compared with diabetic control rats .The effects of GP supplementation on the activity of cellular antioxidant enzymes including SOD and GPx in the liver, kidney, heart, and pancreas of rats are shown in Normal control rats had normal livers whose hepatic lobules had a uniform pattern of polyhedral hepatocytes radiating from the central vein (CV) towards the periphery . In contNormal control rats showed normal renal parenchymal morphology with well-defined glomeruli and tubules . In contNormal control rats showed normal cardiac muscle fibers . HoweverNormal control rats showed normal islets of Langerhans (IL) . The rat in vivo. Weekly measurement of body weight of the animals gives us an overview of the changes in their body weight. Gradual weight loss is a common symptom in DM due to degradation of structural protein and abnormal carbohydrate metabolism [To our knowledge, this is the first study to report on the antihyperglycemic, antidiabetic, and antioxidant potentials of GPtabolism , 31. In tabolism . The relα-glucosidase and α-amylase enzymes by GP as reported by Sarma et al. [The OGTT measures the body's ability to utilize blood glucose. The GP fruit extract exhibited a dose-dependent effect on the glycemic status of rats. An OGTT study in normal rats and diabetic rats showed that administration of GP extract reduced blood glucose levels significantly, as we also observed in rats treated with a standard drug, glibenclamide. In the normal rats, administration of GP at 500 mg/kg and 1000 mg/kg doses significantly reduced blood glucose levels within 3 to 5 hours. Diabetic rats treated with 1000 mg/kg GP showed significantly reduced blood glucose levels within 2 to 4 hours of treatment. The hypoglycemic activity of GP might be the result of an improved insulin level, which is observed in the GP-treated rats. This suppression of hyperglycemia may result from the inhibition of a et al. .In the present study, we found a continuous increase in blood glucose in the diabetic control group. However, oral administration of GP fruit extract showed significant improvement in blood glucose levels. Our findings indicate that the 1000 mg/kg GP dose significantly reduced blood glucose within the third week of the experiment. This finding strongly supports the antidiabetic effects of GP. The antidiabetic role of GP may result from insulin-like action such as improving the uptake of cellular glucose or enhancing glycogenolysis.β-cells and consequently decreased the serum fasting insulin level in rats. However, oral administration of GP extract significantly increased serum insulin levels compared with those of the diabetic control group in a dose-dependent manner. The insulin levels in GP-treated diabetic rats were found to be in the normal physiological range for rats. This indicates that GP extract might have effects on both insulin secretion and insulin action. In this case, the phytochemicals of GP extract might act as stimulating agents for both insulin secretion and insulin action. Among the compounds present in GP, garcinol has been reported to enhance insulin action by increasing the expression of GLUT2 and GLUT4.Both insulin deficiency and resistance are responsible for the pathogenesis of DM. Hence, increasing insulin secretion and maintain its level within the normal physiological range are very important for antidiabetic therapy. In this study, administration of STZ caused damage to pancreatic Hb and HbA1c are closely associated with DM, and HbA1c is a diagnostic marker used to predict DM progression in individuals . HbA1c iα-amylase and α-glucosidase, inhibit glucose absorption in the intestine by sodium-dependent glucose transporter 1 (SGLT1), stimulate insulin secretion, and reduce hepatic glucose output. Polyphenols may also increase insulin-dependent glucose uptake, activate 5′ adenosine monophosphate-activated protein kinase (AMPK), modify the microbiome, and have anti-inflammatory effects. Detailed experimentation should be conducted to identify the exact mechanism.Catechin was detected in this study by HPLC analysis while significant amounts of some other polyphenols were also identified in some other studies , 18. It The most common lipid abnormalities in DM are hypertriglyceridemia and hypercholesterolemia . ElevateDiabetic nephropathy is one of the major complications of DM. High blood sugar from DM destroys the tiny blood vessels in the kidneys, leading to impaired renal function and diabetic nephropathy . In our During DM, insulin deficiency contributes to increased serum levels of transaminase enzymes because sufficient amounts of amino acids stimulate the occurrence of gluconeogenesis and ketogenesis . Increas2−, H2O2, and −OH). Excessive lipid peroxidation causes lipid degradation that impairs cell membrane function, resulting in tissue damage and leading to many pathological conditions and cytotoxicity in the body [Oxidative stress is considered a potential contributor to the development of DM as well as its complications. Oxidative stress develops in DM through increased generation of reactive oxygen species (ROS) and a decrease in ROS scavenging capacity . The excthe body , 50. In Cellular antioxidant enzymes are considered the first line of defense against free radicals . Free raHistopathological examination of different organs showed that STZ causes degenerative necrosis of hepatocytes, vacuolization of the hepatic lobules, degeneration of the kidney glomeruli with inflammatory infiltrates, massive separation of cardiac muscle, and necrotic damage to the myocytes of the heart tissue and islets of Langerhans of the pancreas in diabetic control group rats. Administration of different doses of GP extract restored the morphology of these tissues to normalcy. These histopathological findings further confirmed the potential of GP fruit extract against STZ-induced tissue damage.The findings of this study indicated that oral administration of GP in Wistar rats can ameliorate hyperglycemia, diabetes, and diabetic complications and protect against oxidative stress-induced damage. Although establishing the mechanisms behind these effects is far beyond the scope of this study, we can certainly mention that the phytoconstituents of GP might be responsible for these pharmacological properties. We can conclude that GP is a nontoxic source of natural antioxidants that could be used to treat diabetes and its complications. However, more detailed biochemical and molecular studies should be conducted to identify the main active ingredients in GP and their mechanistic roles as well.