Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia.The B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms.Five of the seven B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats.This study showed that the The online version of this article (doi:10.1186/s12866-015-0377-9) contains supplementary material, which is available to authorized users. Bacillus cereus s. l. species are Gram-positive, facultative anaerobic, spore-forming rods. As soil-associated ubiquitous organisms, B. cereus s. l. are commonly found in food products where they can cause spoilage [Bacillus cereus on dairy farms has been extensively studied, particularly in Scandinavia and the Netherlands [The spoilage . In addispoilage . The incherlands ,4.B. cereus s. l. is complex and has been regularly updated by new findings. To date, seven members have been described: Bacillus cereus sensu stricto, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus anthracis and most recently Bacillus cytotoxicus [. These strains share a highly conserved genome, and their 16S rRNA gene sequences show high levels of similarity [B. cereus s. l. strains [B. cereus s. l. species on the basis of genetic closeness [B. cereus group strains, on the basis of various phenotypical traits. B. mycoides and B. pseudomycoides exhibit a typical rhizoid growth whereas the rest of the species have round to irregular colonies [B. mycoides strains are able to grow at 8°C but not at 40°C. B. weihenstephanensis strains are psychrotolerant and can thus grow at temperatures as low as 7°C but not higher than 43°C [B. cytotoxicus strains have a growth temperature range of 20°C to 50°C [B. thuringiensis strain produce parasporal toxin crystals with insecticidal properties [. Finally, B. anthracis strains are non-hemolytic on Sheep Blood Agar (SBA) plates [.The taxonomy of otoxicus . These s strains . These floseness ,9. Howevcolonies . B. mycohan 43°C . On the operties . Finally) plates .Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium commonly found in the environment and in animal intestinal tracks. It is also a human pathogen and has been identified as the second most common bacterial source of foodborne illness in the United States [C. perfringens strains are classified into toxigenic types A, B, C, D and E, based on the combination of four different toxins that they can produce . Moreover, the C. perfringens strains can also produce a diversity of other toxins, such as the beta2 enterotoxin and Clostridium perfringens enterotoxin (CPE).d States . C. perfB. cereus s. l. isolates was evaluated, in terms of taxonomy and toxin production potential at different temperatures in non-agitated reconstituted milk and broth. Molecular ecology of the B. cereus s. l. population was investigated using PFGE. C. perfringens isolates were subtyped using a toxin-gene-targeted multiplex PCR method. This study provided extensive data about the diversity of the on-farm populations of Bacillus cereus s. l. and C. perfringens in Victoria, Australia. The results regarding the growth conditions and toxin production potential of these pathogens provides information on the potential associated public health risk, and for the design of tailored bacterial control methods on the Victorian dairy farms.The isolates in this study were obtained from samples from various sources  collected on a mixed group of bovine, caprine and ovine dairy farms in Victoria, Australia . The divB. cereus s.l. isolates were taken from each sample to yield a total of 50 isolates from 27 positive samples .A total of 50 isolates were obtained from soil, faeces, feed (grain), raw milk and milk filter samples  collected on seven dairy farms. Where possible, two B. anthracis (non-hemolytic) or B. cytotoxicus (minimum growth temperature of 20°C) species were among the isolate pool.All the isolates exhibited hemolytic activity on SBA plates and were able to grow at a temperature of 10°C, indicating that no B. cereus s. s., B. thuringiensis, or B. pseudomycoides. One of these exhibited a rhizoid colony morphology, and was classified as B. pseudomycoides. Of the remaining 13 isolates, three produced bipyramidal or round toxin crystals on sporulation, and were identified as B. thuringiensis  showing rhizoid growth, and B. weihenstephanensis, with round to irregular colonies (n = 2).Another five isolates were incapable of growth at 40°C, implicating them as either B. cereus s.s or B. thuringiensis. One of these isolates produced toxin crystals and was designated as B. thuringiensis; the other 30 isolates were identified as B. cereus s. s.The remaining 31 isolates were capable of growth at both 8°C and 40°C, exhibited a round to irregular colony morphology, and were implicated as either B. cereus s. l. species identified in this study  is shown in Figure B. weihenstephanensis isolate reached exponential and stationary phase in the shortest times, with an average cell density of 5.55 ± 0.2 log(CFU/ml) measured on day 4. This was followed by the B. mycoides isolate, with an average cell density of 4.83 ± 0.09 log(CFU/ml), also measured on day 4. The B. cereus s. s. isolate reached numbers of 5.75 ± 0.17 log(CFU/ml) by day 6, at which time the representative isolates of both other species has reached stationary growth phase. Neither the B. thuringiensis nor B. pseudomycoides representative isolates grew at 8°C.The growth profile of one representative strain from each B. weihenstephanensis isolate also grew to higher cell densities on days 2, 3 and 4 at 10°C, when compared with the other 4 species. All representative isolates, with the exception of the B. pseudomycoides isolate, reached stationary stage after approximately 5 days at 10°C; this took approximately 7 days for the B. pseudomycoides isolate.The B. weihenstephanensis. Cell growth plateaued after 3 days  or 4 days (for the B. pseudomycoides isolate).At 12°C, highest cell densities after 2 days were observed for B. cereus s. s.; four B. thuringiensis; three B. mycoides; two B. weihenstephanensis; and one B. pseudomycoides. B. cereus s. s. was identified on all 7 farms, B. thuringiensis on 4 farms, and B. mycoides on Farms B and C. Both B. weihenstephanensis strains were recovered from Farm F, and the B. pseudomycoides isolate was from Farm B.PFGE subtyping was utilised to understand the population diversity in samples, across each farm environment, and comparatively across all farms Figure . Two isoB. cereus s. s. isolates Bc13-013 (isolated from soil) and Bc13-012 ; both samples were from Farm F, taken on the first visit (December 2013). The other pair of isolates indistinguishable by PFGE were Bc13-009 and Bc14-016, isolated from soil samples collected from Farm D in December 2013 and again in March 2014.Population analysis of the 46 remaining isolates yielded 44 distinct pulsotypes; two of these pulsotypes were identified in multiple samples (each of these strains were identified in two separate samples). A pulsotype was shared by B. mycoides isolates clustered together in this group, together with one B. weihenstephanensis isolate. The rest of the isolates were B. cereus isolates. Eighty-six percent of members of this grouping were psychrotrophic and weakly hemolytic; only Bc14-020 (showing the lowest similarity among clade 1 isolates) did not grow at 8°C, and was also strogly hemolytic. Clade 2 included 22 isolates: one B. pseudomycoides, one B. weihenstephanensis and 20 B. cereus s. s. isolates. Finally, clade 3 comprised 18 isolates, along with the B. cereus reference strain ATCC 10876. All the B. thuringiensis strains clustered in this last group. The remaining isolates were B. cereus s. s.. This group exhibited a broader phenotypic diversity, with 50% of strains capable of growth at 8°C. Similarly, 50% of strains displayed weakly hemolytic phenotypes.No pulsotypes identified in this study were shared across multiple Farms, and a diversity of between three and nine unique pulsotypes were identified on individual farms. The PFGE profiles clustered in 3 major groups: Clades 1, 2 and 3. Clade 1 comprised 7 isolates; all the B. weihenstephanensis representative isolate (Bc14-026) was the only isolate not to produce detectable Nhe after 24 h in TSBYE at this temperature. The B. mycoides Bc14-009 isolate was the only isolate to produce detectable Hbl enterotoxin under these conditions, after 48 h. With the exception of B. weihenstephanensis, all isolates produced higher amounts of Nhe in TSBYE at 25°C, when compared with production in 10% RSM. No detectable enterotoxin was produced by the B. pseudomycoides isolate in 10% RSM at 25°C, in contrast to strong Nhe production measured in TSBYE.Production of enterotoxins Nhe and Hbl was greatest at 25°C, for all 5 species tested Table . All fivB. cereus s. s. Bc14-005, all strains produced less enterotoxin at 37°C, in comparison with that measured at 25°C. B. thuringiensis was the only species with detectable Hbl toxin production at 37°C. No enterotoxin production was noted for B. weihenstephanensis in either TSBYE or 10% RSM at 37°C; interestingly, although all other representative isolates produced enterotoxin in TSBYE at this temperature, only the B. cereus s. s. and B. thuringiensis isolates produced detectable toxin in 10% RSM at 37°C.With the exception of B pseudomycoides nor B. weihenstephanensis produced detectable Nhe or Hbl at this temperature; production of Nhe by representative isolates of the other three species was low, and no Hbl production was detected at 12°C.Overall, toxin production was lowest at 12°C. Neither Clostridium perfringens enterotoxin and the beta2 toxin were not detected in any of the isolates. Thus, out of the 26 isolates, 77% were identified as type A and 23% as type D. Type A strains were present on every farm (A to G), whereas type D strains were found only on Farms D and F, which are caprine farms. On Farm D, the type D strains were isolated from feed (grain) and raw milk samples only. On Farm F, the type D strains were isolated from soil, milk and milk filter samples only. No type D strain was isolated from the animal faeces on these farms.Among the 26 isolates, two toxin types were detected - type A and type D Figure ; all isoB. cereus s. l., along with C. perfringens, may cause illness in both humans and animals. These bacteria are commonly found in many environments, including soil. As such, there is potential for both infection of animals and entry in food chains through contamination of crops, raw milk, or food animals themselves. These organisms are of particular importance in food safety, as their spores may survive thermal treatments, such as pasteurization [Species of rization . CharactB. cereus s. l. isolates collected was B. cereus s. s., which comprised 78% of the population. Species diversity across different farm environments was varied; Farms A and E showed the lowest diversity, where only B. cereus s. s. was identified; all other farms harboured either two or three species.The most common species identified among B. weihenstephanensis possessed the highest growth rates at all 3 temperatures examined . This characteristic is reflected in the psychrotolerant nature of the organisms, which is known to grow at temperatures as low as 5°C [B. pseudomycoides isolate showed the slowest growth rates at 10°C and 12°C, and did not grow at 8°C. Taken together, these results suggest that toxigenic B. weihenstephanensis isolates may be of higher risk in cases of contaminated refrigerated food products that have experienced temperature adulteration; conversely, the comparative risk may be lower in the case of B. pseudomycoides. Although this data provides an indication of the growth performance of these isolates at low temperature, the duration of lag phase, or the exact maximum growth rate, would require additional bacterial counts in the early growth phase.Comparative analysis of the growth profiles of representative isolates for each of the 5 species at low temperatures suggested w as 5°C ,17. The B. cereus s. l. isolates. Of the 23 samples where 2 isolates were taken, only 4 contained the same pulsotype (i.e. the pair of isolates from the other 19 samples possessed different pulsotypes). This indicated a high diversity of B. cereus s. l. among samples. Indeed, it is probable the actual population diversity of B. cererus s. l. in a proportion of the positive samples would have been found to be greater than was identified in this study, had more isolates been taken from those samples. That withstanding, this analysis identified 46 distinct isolates from the original pool of 50.PFGE analysis was utilised to examine population diversity among Two Farm F isolates, Bc13-013 isolated from soil and Bc13-012 isolates from faeces, were indistinguishable by PFGE. Contaminated soil may lead to contamination of the feeding pasture, which in turn may lead to passage to the animal intestinal tract, this may be transient, or lead to colonization of the gut. Either scenario may account for this observation. In the case of the other two non-distinguishable profiles (Bc13-009 and Bc14-016), these were identified in soil samples collected from Farm D in December 2013 and again in March 2014. Both samples were collected from the same pasture field, although not collected at the same area of the field. This suggests a persistent pattern of colonization by this pulsotype in this particular field. Although this pulsotype was not identified in faecal samples from animals on the farm, further insights into its colonization across the farm, and in the herd, would necessitate more extensive sampling. Nonetheless, taken together, both instances where an individual pulsotype was identified in multiple samples highlights the contamination risk associated with resident strains in the farm environment; such bacteria will conceivably be found at higher frequency around the farm environment and in faecal shedding of the herd, and thus at higher risk of contamination of milk, and entry to the dairy food chain.B. cereus s. l. isolates, with no shared PFGE profiles observed between farms; this observation should be contextualised in that the complete population diversity is likely more complex than this study identified. Farms had a minimum of between three and nine pulsotypes among samples taken, as determined by PFGE analysis. B. cereus s. s. dominated all farms, comprising a minimum of 63% of species in the case of Farm C, up to 100% of all species identified (Farms A and E). This suggests this species is the dominant member of these B. cereus s. l. populations. There was no apparent correlation with farm herd type  to a higher or lower diversity of isolate pulsotypes or number of species. Since isolation of the organisms does not involve an enrichment step, this does not introduce the possibility of certain isolates out-competing others in the enrichment process, nonetheless possible competition between strains on the Mannitol Egg Yolk Polymyxin Agar (MYPA) medium used for isolation, or indeed the medium itself, may affect their isolation. Certain B. cereus s. l. isolates may produce bactericidal toxins that inhibit other isolates [Further examination of the remaining 42 PFGE profiles indicated a diverse population, not only around individual farm environments, but across the farm environments at large. Each farm had its own distinct community of isolates ,19, and B. cereus s. l. isolates is central to the manifestation of clinical illness. The emetic form of illness is attributed, in a large part, to the heat-stable cereulide toxin [B. cereus s. l., one representative isolate for each of the species identified on farms was grown in either 10% RSM or TSBYE medium at various temperatures . Interesting, all isolates showed a trend of reduced toxin production in milk, when compared with TSBYE; the extent of which varied depending on the combination of temperature and time measured (24 h or 48 h). This may indicate a reduced risk of toxin production in milk and/or dairy products, relative to other food matrices, and thus for certain forms of B. cereus s. l.-induced illness as a result of ingestion of contaminated foodstuffs. Of the five species, the B. weihenstephanensis isolate produced the lowest detected Nhe toxin level under all conditions tested, when compared with other isolates. Production of Hbl toxin was lower than that of Nhe under all experimental conditions; indeed Hbl production was only detected for the B. mycoides and B. thuringiensis isolates. Contrary to this, all isolates produced Nhe. This suggests that all of these isolates may illicit diarrheal disease in human hosts under favourable conditions. Although production of enterotoxins was greatest at 25°C, production of Nhe was identified at 37°C for all isolates, with the exception of B. weihenstephanensis. This again implies the capacity of these isolates to cause diarrheal illness in humans, as this reflects the temperature conditions in vivo. That withstanding, based on the observation that no enterotoxin was detected in 10% RSM at 37°C, the food vector involved in the infection may play some role in the disease outcome. This may be reflected in the low prevalence of dairy products among foods linked to B.cereus s. l. food-poisoning outbreaks [B. cereus s. s. and B. thuringiensis isolates showed highest levels of toxin production across the range of conditions  tested.Enterotoxin production by de toxin ; in casede toxin , it is gde toxin . To undeutbreaks ,24; howeC. perfringens isolates: type A, which may cause foodborne illness in humans [C. perfringens type D strains are predominantly associated with goats and sheep, rather than cattle [C. perfringens enterotoxin gene (cpe), or the beta2 toxin gene (cpb2), as determined by the PCR screening method (data not shown) [Two toxin types were identified among the n humans , howevern humans ; and typn humans . Type D n cattle . None ofn cattle . Type A t shown) . Beta2 tt shown) ,29, suggBacillus cereus group of bacteria and Clostridium perfringens on farm populations in Victoria. First of all, both species were isolated from all seven farms and from the same types of samples. On each farm, the B. cereus s. l. population was found to be diverse and distinct to each localised environment. Enterotoxin production in milk was limited compared to broth in the same conditions, suggesting that enterotoxin production by B. cereus s. l. may be reduced for dairy products. Type A isolates were dominant among the C. perfringens population; however the presence of type D isolates on two of the three caprine farms may be a potential concern with regards to the risk of enterotoxemia in goats.This study provides insights to the diversity of the B. cereus s. l., and AS 5013.16-2006 for C. perfringens). This comprised 50 B. cereus s. l. and 26 C. perfringens isolates. Where possible, two presumptive-positive isolates were taken from each positive sample. Where both rhizoid and non-rhizoid colonies were identified in a positive sample, one of each was taken for further analysis.Isolates were obtained from soil, faeces, feed (grain), milk and milk filter samples from 7 dairy farms across Victoria, Australia . Sampling was performed on 2 separate occasions, in December 2013 and March 2014. Isolation from samples was performed as per Australian standard methods  agar plates and incubated overnight at 30°C. For each isolate, one colony was picked from the BHI plate with an inoculating loop and mixed into 5 ml of BHI broth. After overnight incubation at 30°C the cultures reached a concentration of approximately 10Trypticase Soy agar plates with 5% of Sheep Blood were divided in 6 equal sections and inoculated with 24 h BHI broth culture suspensions by touching the surface of the agar with a 1 μl loop. Plates were incubated for 24 h at 35°C and examined for hemolytic activity. Hemolytic strains were identified by a translucent halo of 2 to 4 mm of hemolysis.Analysis of toxin production was carried out based on the FDA standard method, which minimised sequential culturing, and utilises media without curing agents, to reduce the chance of plasmid curing . Nutrien8-109 CFU/ml. After serial dilutions, 3 ml cell suspensions with an approximate concentration of 102 CFU/ml were set in 12-well plates. Sterile BHI broth was used as a negative control and a blank to standardise the data.Isolates were streaked onto BHI agar plates and incubated overnight at 30°C. For each isolate, one colony was picked from the BHI plate with an inoculating loop and mixed into 5 ml of BHI broth. After overnight incubation at 30°C the cultures reached a concentration of approximately 10The plates were incubated at 8°C, 10°C and 12°C over a period of 14 days. Growth was monitored by taking optical density measurements at 600 nm with a plate-reader spectrophotometer. Growth curves for each strain and temperature were then plotted, using standard curves generated for each isolate to equate absorbance readings to log(CFU/ml) (data not shown). All growth curves and standard curves were measured in triplicate, with each replicate performed on separate occasions.B. cereus s. l. isolates. Test isolates were streaked on BHI agar plates and incubated for 14–18 h at 30°C. Cell suspensions were then prepared in 2 ml TE buffer , to a final cell optical density of 1.00 at 600 nm, as measured on a spectrophotometer. 400 μl of the cell suspension was mixed with 20 μl of a lysozyme solution (20 mg/ml) and incubated for 2 h at 56°C. Following 1% PFGE-grade agarose  was prepared with TE buffer and equilibriated at 55°C. 20 μl of a Proteinase K solution (20 mg/ml) was added to the cell suspensions, followed immediately by 400 μl of the 1% SKG agarose and gently mixed. The plugs were then cast in disposable plug molds . After 10 to 15 min at room temperature the plugs were transferred in a Cell Lysis Buffer  containing 25 μl Proteinase K solution (20 mg/ml). The plugs were lysed overnight in a 56°C shaker water bath at 160 rpm. Prior to plug washing, the water bath temperature was reduced to 50°C and the wash solutions were pre-warmed before use. To wash the plugs, the lysis buffer was drained from the tube, and replaced with 5 ml sterile molecular grade water. The tube was then replaced in the water bath at 50°C, and shaken at 160 rpm for 10–15 min. One more wash with molecular grade water was repeated as before, followed by four washes with TE buffer. Plugs were then transferred to 1.5 ml centrifuge tubes filled with 1 ml sterile TE buffer and stored at 4°C until used.An in-house method was developed for PFGE subtyping of SbfI or NotI endonucleases . For both enzymes, 40U was incubated into a 200 μl reaction mix at 37°C for 2 h. After restriction, the plugs were equilibrated for 10–15 min in 0.5X Tris Borate EDTA buffer (40 mM Tris–HCl: 45 mM boric acid: 1 mM EDTA ) at room temperature.For PFGE analysis, DNA plugs were restricted using either Saccharomyces cerevisiae CHEF DNA Size Marker , already restricted, and Salmonella ser. Braenderup H9812 chromosome, prepared according to the Standard Operating procedure for PusleNet PFGE for Salmonella [XbaI restriction endonuclease , were used simultaneously as DNA-size standards for analysis. Gels were observed after staining 30 min in a 300 ml 1% GelRed  solution and de-staining for 40 min in a 300 ml distilled water bath. Cluster analysis was performed with BioNumerics v6.5 software . Similarity matrixes were produced using the Dice coefficient for the two experiments (digestion with NotI and SbfI), then a combined analysis was performed using the unweighted pair group method with arithmetic mean (UPGMA), with optimisation and tolerance settings of 1%.DNA fragments were separated on a 1% SeaKem Gold agarose gel using a CHEF-Mapper  with the following program: auto algorithm; low molecular weight = 20 kb; high molecular weight = 700 kb; select default values except where noted by pressing “enter”; run time = 21 h; initial switch time = 5 s; final switch time = 80 s; tank temperature = 14°C. The lmonella  and restBacillus cereus s. l. species isolated in this study: B. cereus s. s. (Bc014-005), B. pseudomycoides (Bc14-006), B mycoides (Bc14-009), B. weihenstephanensis (Bc14-026), and B. thuringiensis (Bc13-016). Toxin production was measured at three temperatures: 12°C, measured at 48 h; and 25°C and 37°C, both measured after 24 h and 48 h. Toxin production was assayed in two test media: 10% reconstituted skim milk (RSM) or tryptic soy broth with yeast extract (TSBYE), separately for each representative isolate. Overnight cultures grown at 30°C were diluted to a concentration of approximately 106 CFU/ml in the test medium. A final concentration of 104 CFU/ml in 10 ml of the test medium was used for each test. The tubes were incubated without agitation. The Nhe and Hbl toxins were detected using the Duopath Cereus Enterotoxins detection kit . For 10% RSM cultures, 100 μl was added to 900 μl sterile molecular grade water; for TSBYE broth cultures, 100 μl of sample was added to 100 μl 10% RSM in 800 μl sterile molecular grade water. A volume of 150 μl was the applied to the toxin test chip and results were assessed after 30 min, according to the product datasheet. The intensity of the line was scored as – (no line), + (faint line), ++ (clear line), +++ (thick clear line), to achieve a semi-quantitative measure of toxin production.The production of the enterotoxins Nhe and Hbl was investigated with five isolates, representing each of the DNA extraction was achieved by picking up half a colony from fresh BHI cultures and mixing it in 200 μl molecular biology grade sterile water. After heating at 95°C for 15 min, the samples were centrifuged for 10 s at 14,000 rpm. The PCR was performed with a FTS-960 Thermal Sequencer , following the protocol described by Kalender et al. . Represe


Our latest study reported the grim status of hypertension in rural China with the prevalence of hypertension reached 51.1 %. However, we lack the latest data about the prevalence and epidemiological features of dyslipidemia among hypertensive residents in rural China.A cross-sectional survey was conducted from July 2012 to August 2013 through a cluster multistage sampling to a resident group of 4048 individuals  with hypertension, age ≥ 35 years, in the rural Northeast China. Serum lipids level were proposed by National Cholesterol Education Program Adult Treatment Panel III.Of the hypertension residents without antihypertension treatment, 34.5 % had borderline high total cholesterol, 19.2 % had high total cholesterol, 11.4 % had low high-density lipoprotein cholesterol and 37.4 % had high non HDL-C. The population with borderline high, high, and very high low-density lipoprotein cholesterol was 20.9, 6.7 and 2.3 %, respectively. In addition, 14.3 % had borderline high triglycerides, 17.4 % had high TG and 2.4 % had very high TG. The awareness rate of dyslipidemia among the study population was 5.9 %. After adjusting for independent variables, fasting plasma glucose, body mass index, Han nationality, current drinking and smoking, higher annual income and classification of blood pressure were risk factors for dyslipidemia while moderate physical activity was protective factor for dyslipidemia. On the contrary, gender and current drinking decrease the risk of HDL-C.The prevalence of dyslipidemia was dramatically high and dyslipidemia screening was in-need in all diagnosed hypertensive individuals. With development of economy and improvement of living standards, more and more people surfer from atherosclerotic cardiovascular diseases (ASCVD) which constitute the leading cause of death . FurtherThe prevalence and epidemiologic characters of hypertension in China had been thoroughly studied in resent years and reached the conclusion that hypertension became prevalent not only in urban but also in rural areas . There wLiaoning Province is located in Northeast China. Our previous study, used a cluster multistage sampling method, was also conducted in Liaoning province from October 2004 to June 2006 . From JaData were collected during a single visit to the clinic by cardiologists and trained nurses using a standard questionnaire in a face-to-face interview. Before the survey was performed, we invited all eligible investigators to attend an organized training session. The training included the purpose of this study, how to administer the questionnaire, the standard method of measurement, the importance of standardization and the study procedures. A strict test was administered after this training, and only those who scored perfectly on the test were accepted as investigators in this study. During data collection, our inspectors had further instructions and support.Data regarding the demographic characteristics, lifestyle risk factors, dietary habits, family income and family history of chronic diseases were obtained during the interview using the standardized questionnaire. The study was guided by a central steering committee with a subcommittee for quality control. Educational level was assessed as completion of primary school or less, middle school or high school and higher. Self-reported sleep duration  was obtained from the questionnaire. The responses were categorized into four groups: ≤7, 7–8, 8–9 and >9 h/d. Family income was classified as ≤5000, 5000–20,000 and >20,000 CNY/year.According to American Heart Association protocol, blood pressure (BP) was measured three times at 2-min intervals after at least 5 min of rest using a standardized automatic electronic sphygmomanometer , which had been validated according to the British Hypertension Society protocol . The parWeight and height were measured to the nearest 0.1 kg and 0.1 cm, respectively, with the participants wearing light-weight clothing and without shoes. Waist circumference (WC) was measured at the umbilicus using a non-elastic tape (to the nearest 0.1 cm), with the participants standing at the end of normal expiration. Body mass index (BMI) was calculated as the weight in kilograms divided by the square root of the height in meters.Fasting blood samples were collected in the morning after at least 12 h of fasting. Blood samples were obtained from an antecubital vein into Vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA). Fasting plasma glucose (FPG), total cholesterol (TC), LDL-C, HDL-C, TG and other routine blood biochemical indexes were analyzed enzymatically using an autoanalyzer. All laboratory equipment was calibrated, and blinded duplicate samples were used for these analyses.2 and <30 kg/m2 were diagnosed as overweight, and those with ≥30 kg/m2 as obese.Hypertension was considered present if any of the following conditions were met: systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or reported use of a medication for hypertension and the classification of BP in response to JNC8. Stage 1 hypertension Clinic blood pressure is 140/90 mmHg or higher. Stage 2 hypertension Clinic blood pressure is 160/100 mmHg or higher. Stage 3 hypertension hypertension Clinic blood pressure is 180 mmHg or higher . TC, LDLPhysical activity included occupational and leisure-time physical activity. A detailed description of the methods for assessing physical activity has been presented elsewhere . Occupatt-test, ANOVA, non-parametric test or the χ2-test as appropriate. Multivariate logistic regression analyses were used to identify independent factors of dyslipidemia among hypertensive adults with odds ratios (ORs) and corresponding 95 % confidence intervals (CIs) calculated. All the statistical analyses were performed using SPSS version 17.0 software, and P values less than 0.05 were considered to be statistically significant [Descriptive statistics were calculated for all the variables, including continuous variables  and categorical variables (reported as numbers and percentages). Differences between different dyslipidemia groups were evaluated using Student’s P < 0.001), but current smoking and drinking, SBP, DBP and WC were significantly higher in men than in women (P < 0.001).Characteristics of the hypertensive residents enrolled in this study, as stratified by gender, are shown in Table P < 0.001). The age-specific prevalence of borderline high TC for women increased with increased age (P < 0.001) while high TC increased with increased age for both genders (P < 0.001). The prevalence of low HDL-C was 11.4 %. The prevalence of low HDL-C was significantly higher in men than in women . The prevalence of borderline high, high and very high LDL-C was 20.9, 6.7, and 2.3 %, respectively. The prevalence of borderline high, high and very high LDL-C was slightly higher in women than in men (P < 0.05). In addition, the prevalence of borderline high, high and very high TG was 14.3, 17.4 and 2.4 %, respectively. Among the high and very high group, the age-specific prevalence of men decrease from 35 years old (P < 0.001); however, it increased in the group of women in the borderline high and high group (P < 0.001). Women had higher prevalence of non HDL-C than men(40.4 % vs. 30.7 %). The age-specific prevalence gradually decrease from 35 years old in the group of men while on the contrary significantly increase in women. The prevalence of serum cholesterol and triglycerides by cardiovascular co-morbidities are presented in Table Table P < 0.001). Besides, the awareness and control rate among those with antihypertension treatment is statically higher than those without it .Figure P = 0.255). Besides, we also see a trend between HDL-C and the stage of BP in men and TC and the stage of BP in women .The main finding of this study was that during the past ten years, in rural areas of China, the prevalence of dyslipidemia in hypertensive was still high, and some kind of it even worse. In the present study, we found that the prevalence of isolated hypercholesterolemia (TC ≥ 240 mg/dL and TG <200 mg/dL), low HDL-C concentration, increased LDL-C concentration (LDL-C ≥ 160 mg/dL), isolated hypertriglyceridemia (TG ≥ 200 mg/dL) was 12.5, 11.4, 9.0 and 19.8 %, respectively. The prevalence of mixed hyperlipidemia (TC ≥ 200 mg/dL plus TG ≥200 mg/dL), severe hyperlipidemia (TC ≥ 300 mg/dL and/or TG ≥ 500 mg/dL), normotriglyceridemic hypoalphalipoproteinemia (HDL-C <40 mg/dL and TG <200 mg/dL) was 13.7, 4.1 and 6.2 %, respectively. Furthermore, 68.7 % of adult hypertensive had at least one type of dyslipidemia  in rural China. Despite the high prevalence of dyslipidemia in rural Northeast China, the awareness for those without antihypertension treatment was only 5.9 %. However, for those received antihypertension treatment, the awareness rate increased to 19.0 %. Surprisingly, the prevalence of dyslipidemia among those with antihypertension treatment were significantly higher than those without antihypertension treatment . The possible reason might be those hypertensive with antihypertension treatment paid more attention to their health. They were more willingly to complete the chronic diseases related health check and take measurement to intervene.Our study found that dyslipidemia was prevalent in rural hypertensive Chinese compared with those living in other areas of China. Luo JY and colleagues reported that the prevalence of dyslipidemia in the Northwestern China was 52.72 % . One recP < 0.001, 10.7 % vs. 2.5 %, P <0.001). The mean values of TG, TC, LDL-C and HDL-C in those hypertensives with antihypertension treatment are higher. Besides, the prevalence of different kind of dyslipidemia among those with antihypertension treatment are statically higher . The possible reason might be: First, in our study, those with antihypertension treatment had higher value of age, SBP, DBP and BMI than those without treatment . Many previous studies had proved that dyslipidemia was relevant to older age, hypertension and overweight/obesity [Interestingly, data from our study revealed that even though the awareness and treatment rates in hypertensive with antihypertension treatment are higher than those without antihypertension treatment (19.0 % vs. 5.9 %, /obesity , 23. Sec/obesity .In our study, after adjusted for independent variables, we found that fasting plasma glucose and BMI was the common risk factors of dyslipidemia. As previous study had confirmed diabetes and overweight/obesity increased the risk of dyslipidemia , 23. BesOur study has several limitations. The major limitation is the cross-sectional design which reflects only associations between dyslipidemia and risk factors, but unable to observe prospectively. Besides, in this study, the sample contains only hypertensive, lacking normotensive for comparison. The prevalence of dyslipidemia was based on a single assessment of blood samples, which may introduce error. In addition, although the researchers had been trained according to a standardized protocol of measurements, measurements at a single visit might lead to incorrect values for the anthropometric indexes. And In this study, the sample contained only hypertensives, lacking normotensives for comparison. Finally, we did not evaluate the medication of antihypertension which might be crucial for the evaluation of the characters of dyslipidemia.In summary, our population-based study updated the latest prevalence of dyslipidemia in rural Northeast China and revealed a higher prevalence of dyslipidemia in rural areas. Besides, the apparently lower awareness, treatment rates need our attention. In addition to some of the uncontrollable risk factors associated with dyslipidemia, including age, gender, ethnic, we should pay attention to the risk factors that can be intervened, like recommendation of controlling of blood glucose, quite smoking and drinking. Besides, we should strengthen the dyslipidemia screening for residents with higher risk like women and Han nationality.


Whether HbA1c is a predictor of end-stage renal disease (ESRD) in type 2 diabetes patients remains unclear. This study evaluated relationship between HbA1c and ESRD in Chinese patients with type 2 diabetes.Patients aged ≥ 30 years who were free of ESRD (n = 51 681) were included from National Diabetes Care Management Program from 2002–2003. Extended Cox proportional hazard model with competing risk of death served to evaluate association between HbA1c level and ESRD.A total of 2613 (5.06%) people developed ESRD during a follow-up period of 8.1 years. Overall incidence rate of ESRD was 6.26 per 1000 person-years. Patients with high levels of HbA1c had a high incidence rate of ESRD, from 4.29 for HbA1c of  6.0%–6.9% to 10.33 for HbA1c ≥ 10.0% per 1000 person-years. Patients with HbA1c < 6.0% particularly had a slightly higher ESRD incidence (4.34 per 1000 person-years) than those with HbA1c  of 6.0%–6.9%. A J-shaped relationship between HbA1c level and ESRD risk was observed. After adjustment, patients with HbA1c < 6.0% and ≥ 10.0% exhibited an increased risk of ESRD  compared with those with HbA1c of 6.0%–6.9%.Diabetes care has focused on preventing hyperglycemia, but not hypoglycemia. Our study revealed that HbA1c level ≥ 7.0% was linked with increased ESRD risk in type 2 diabetes patients, and that HbA1c < 6.0% also had the potential to increase ESRD risk. Our study provides epidemiological evidence that appropriate glycemic control is essential for diabetes care to meet HbA1c targets and improve outcomes without increasing the risk to this population. Clinicians need to pay attention to HbA1c results on diabetic nephropathy. Diabetes has become one of the most common causes of end-stage renal disease (ESRD) in numerous countries, and a 44.6%, 44.5%, and 43.7% incidence of ESRD in patients is caused by diabetes in Japan, Taiwan, and the United States, respectively . BecauseHyperglycemia is the most crucial factor in the progression of microvascular complications of diabetes, including nephropathy. The American Diabetes Association (ADA) recommended that target HbA1c should be below or around 7.0% . A longiPrevious studies have evaluated the associations between intervention targeting strict glycemic control and substantially clinical renal outcomes, such as ESRD requiring dialysis therapy in type 2 diabetes patients –11. HoweWhether extreme levels of HbA1c increase the risk of ESRD incidence in the Han Chinese population has not been reported. The effect of HbA1c on ESRD among type 2 diabetes patients warrants further investigation. Therefore, we estimated the incidence of ESRD according to HbA1c levels, and evaluated whether a J- or U-shaped relationship between HbA1c levels and ESRD risk exists in a large cohort of ethnic Chinese patients with type 2 diabetes enrolled in the National Diabetes Care Management Program (NDCMP).Our study population from a national cohort of Taiwan Diabetes Study comprised 62 656 diabetic patients enrolled in the NDCMP from 2002–2003 in Taiwan. The date of entry into the NDCMP was defined as the index date. The NDCMP provided a set of integrated examinations and performance measures for patients. At the beginning of entering the NDCMP, patients underwent a series of medical tests for blood, urine, and body measurements; and patients were required to complete a standardized and computerized questionnaire administered by a case management nurse to record the previous status of their medication and lifestyle behaviors. This nationally managed care for diabetes patients was established by the Bureau of National Health Insurance (BNHI) in November 2001. Its goal is to integrate related departments, expertise, and hospitals at diverse levels to provide continual, approachable, and high-quality care for diabetes patients. All patients diagnosed with diabetes ; code 250) based on ADA criteria were invited to participate in the NDCMP. In this study, patients with type 1 (ICD-9-CM codes: 250.x1/x3) or gestational (ICD-9-CM codes: 648.83) diabetes (n  =  2072), aged less than 30 years (n  =  651), and those missing diabetes-related variables information (n  =  7091) were excluded. We also excluded patients with a diagnosis of ESRD at baseline or incidence ESRD cases within 1 year of the index date to eliminate cause-and-effect (n  =  317), along with patients who died or withdrew from the National Health Insurance (NHI) program within the first year after participating in the NDCMP (n  =  844). Finally, 51 681 participants were included for further analysis. This study was approved by the Ethical Review Board of China Medical University Hospital. Patient records and information were anonymized and de-identified prior to analysis.2), diabetic ketoacidosis (DKA) , hyperosmolar non-ketoacidosis (HHNK) , severe hypoglycemia , and neuropathy . Regarding DKA, HHNK, severe hypoglycemia, and neuropathy, we used data collected in the NDCMP, which used the standardized and computerized questionnaire to record whether patients had ever occurred each disease event. Renal function was evaluated using glomerular filtration rate (GFR); estimated GFR (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation: eGFR (mL/min/1.73 m2)  =  141×min α × max −1.209 × 0.993Age×1.018  ×1.159 (if black), where Scr is serum creatinine, κ is 0.7 for women and 0.9 for men, α is -0.329 for women and -0.411 for men, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1 [The key independent variable was baseline HbA1c. According to the clinical criteria of HbA1c, the participants were grouped into 6 categories: < 6.0%, 6.0%–6.9%, 7.0%–7.9%, 8.0%–8.9%, 9.0%–9.9%, and≥10.0%. Covariates at baseline consisted of sociodemographic factors, lifestyle behaviors, biochemical variables, diabetes-related variables, and comorbidities including age, sex, smoking status , alcohol consumption , fasting plasma glucose (FPG), total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum creatinine, duration of diabetes, type of antidiabetes medication , antihypertensive medication , obesity (body mass index≥27 kg/mBaseline comorbidities included hypertension (ICD-9-CM codes: 401–405), hyperlipidemia (ICD-9-CM code: 272), cerebral vascular accident , coronary artery disease , congestive heart failure , and cancer . A diabetic patient was defined as having one of these comorbidities by using at least one inpatient or three ambulatory claims data which were collected for 1-year period prior to cohort entry. Data were from the National Health Insurance Research Database (NHIRD), which is affiliated with the Taiwan NHI program initiated on March 1, 1995 and maintained by the National Health Research Institutes. In 2007, more than 98% of Taiwan’s population of 23 million were covered by the NHI program [Our outcome of interest was ESRD defined by requiring dialysis at least 3 months. These incident ESRD cases (ICD-9-CM code: 585) were identified from the NHIRD Registry for Catastrophic Illness Patient Database excluding patients with acute kidney failure. The physician diagnosed ESRD by performing a physical examination and tests to check renal function. Tests may include blood tests, urine tests, renal imaging, and kidney biopsy. Patients with ESRD approved for catastrophic illness registration cards are those who have received hemodialysis or peritoneal dialysis for over 3 months. Insured ESRD patients with catastrophic illness registration cards are not required to pay medical expenses when they seek health care for ESRD-related conditions.P < .05.The baseline period was 2002–2003. Participants were followed until the development of ESRD, death, or the end of 2011. The baseline demographic and clinical characteristics of the participants in the NDCMP were examined according to HbA1c levels. Continuous variables were reported as the mean±standard deviation (SD), and categorical variables were tallied as a number and percentage. Bivariate statistical methods  were used to explore data features. Incident ESRD density rates per HbA1c level were estimated, and Kaplan-Meier cumulative incidence curves were derived. The extended Cox proportional hazards model using the Lunn-McNeil approach was applied as a modified Cox proportional hazards model that considered competing risks and allowed for multivariable adjustment . The extThe demographic and clinical characteristics of the 51 681 NDCMP participants are summarized according to baseline HbA1c levels in P < .001 and 95% CI: 3.80–5.14, P < .001, respectively) than were patients with HbA1c levels of 6.0%–6.9% after considering age, sex, diabetes duration, lifestyle behaviors, eGFR, medications, and comorbidities. In a sensitivity analysis, we observed similar results after excluding patients with DKA, HHNK, severe hypoglycemia, CVA, CAD, and CHF; the adjusted HRs and their 95% CIs were 2.26 (1.76–2.92), 1.94 (1.59–2.37), 2.33 (1.90–2.85), 2.98 (2.42–3.66), and 4.99 (4.15–6.01) in HbA1c < 6.0%, 7.0%–7.9%, 8.0%–8.9%, 9.0%–9.9%, and≥10.0%, respectively, compared with that in HbA1c 6.0%–6.9%. Furthermore, to reduce the random error of one HbA1c measurement, we used the mean value of HbA1c measurements obtained within 1 year as the predictor of ESRD risk (n  =  31 747). We also observed a J-shaped relationship between HbA1c and ESRD risk; that is, the adjusted HRs and their 95% CIs were 2.20 (1.70–2.85), 1.80 (1.47–2.19), 2.10 (1.72–2.56), 2.66 (2.16–3.29), and 4.32 (3.53–5.28) in HbA1c < 6.0%, 7.0%–7.9%, 8.0%–8.9%, 9.0%–9.9%, and≥10.0%, respectively, compared with that in HbA1c 6.0%–6.9%. P < .0001).2), and types of antidiabetes medication . We still observed a J-shaped relationship between HbA1c and ESRD risk across these different stratification subgroups. Comparing HbA1c level ≥10.0% with 6.0%–6.9%, the ESRD risk consistently increased across various strata . There were similar trends in comparing HbA1c level < 6.0% with 6.0%–6.9%, except age≥65 years and eGFR≥60 ml/min/1.73m² subgroups. Significant interaction between HbA1c and sex (P < .001); interaction between HbA1c and age (P < .001); interaction between HbA1c and eGFR (P < .001); and interaction between HbA1c and types of antidiabetes medication (P < .001) on ESRD risk were observed.P < .05).We used NDCMP data and nationwide NHI claims data to estimate the incidence of ESRD from 2002 to 2011, and evaluate the association between baseline HbA1c and incident ESRD in patients with type 2 diabetes. A total of 2613 patients were diagnosed with new-onset ESRD during follow up. The lowest incidence of ESRD was 4.29 per 1000 person-years in the groups with HbA1c levels of 6.0%–6.9%; the highest ESRD incidence was 10.33 per 1000 person-years in the HbA1c of ≥ 10.0% group. A J-shaped relationship between HbA1c and ESRD risk was observed in the entire sample. Moreover, interaction between HbA1c and types of antidiabetes medication on ESRD risk was significant (P  =  .017). The risks of ESRD in HbA1c 6.5%–7.49% and≥ 7.5% were significantly increased compared with that in HbA1c < 6.5%, respectively. Because of the limited sample size and hospital-based study design, the authors did not evaluate the effect of a more extreme cutoff point of HbA1c on ESRD risk. Our study revealed that HbA1c < 6.0% or≥7.0% increased the risk of incident ESRD in the entire sample of the Han Chinese population, with a greater variation of this effect in subgroups based on eGFR level and types of antidiabetic medication.A study in South Korea was performed to assess the appropriate HbA1c level for minimizing ESRD incidence in diabetic patients by using the Seoul National University Bundang Hospital database . The autP  for trend: < .001, HR: 1.04, 95% CI: 1.03–1.06 for every 1% increment in HbA1c).Our study observed a J-shaped relationship between HbA1c level and ESRD, in contrast, a population-based cohort study in Canada conducted by Shurraw et al did not observe an overall relationship between HbA1c level and ESRD . They foIn previous RCTs exploring the effect of intensive glucose control intervention on DN, 2 RCTs have demonstrated that intervention targeting intensive glucose control can reduce the risk of new-onset micro- and macroalbuminuria in type 2 diabetes patients , 9, but DN is a complex disease, and multiple mechanisms contribute to DN development. Hyperglycemia plays a pivotal role in DN development, which affects mesangial cells and glomerular injury. Mesangial cells are of critical importance to kidney function in maintaining glomerular capillary structure and regulating glomerular filtration . PreviouThe current study has several strengths. First, we used nationwide data with a large sample size, and all patients diagnosed with diabetes were invited to participate in the NDCMP, which provides a set of integrated examinations and performance measures for participants. Second, the NHI program in Taiwan provides continuing universal coverage for the entire population, which avoids selection bias. Third, using NHI datasets eliminated the need to minimize the number of cohort participants lost to follow up. In addition, we easily obtained a large sample of geographically dispersed patients. Fourth, we defined the new-onset ESRD cases based on the Registry for Catastrophic Illness Patients Database, which resulted in the high ascertainment validity of the ESRD cases. This could minimize ESRD misclassification bias. Finally, we controlled for numerous crucial clinical and demographic factors, such as laboratory markers, types of antidiabetes medication, antihypertensive medication, and diabetes-related comorbidities, which could minimize the effect of potential confounders.Nevertheless, three limitations deserve to be mentioned. First, we did not conduct repeated measurements of HbA1c for all the study participants within 1 year of the follow-up period to reduce the random error of once HbA1c measurement. Using approximately 60% of the participants, the mean value of HbA1c measurements obtained within 1 year is a significant predictor of ESRD risk, demonstrating a J-shaped relationship between HbA1c and ESRD risk. Second, we also did not acquire the HbA1c measurement for the later follow-up period; thus, we could not examine the effect of time-varying HbA1c on ESRD risk. Finally, some patients  with CKD stage 5 in baseline assessments were included. Prior studies have provided evidence that HbA1c levels were false low –31 due tIn conclusion, our data suggest that a low or high HbA1c value is linked to increased ESRD risk in the population of Chinese patients with type 2 diabetes in Taiwan. Our findings are consistent with the literature indicating that HbA1c level plays a crucial role in ESRD development. Diabetes care has focused on preventing hyperglycemia, but has ignored hypoglycemia. Because a low level HbA1c has the potential risk of causing ESRD to develop, appropriate glycemic control is essential for minimizing ESRD risk, which should be emphasized in diabetes care. Clinicians need to pay attention to HbA1c results on DN.tcli@mail.cmu.edu.tw.All relevant data are within the paper. Data are from the Taiwan Diabetes Study whose authors may be contacted at


The inclusion complexes were formed by physical mixing, kneading, coevaporation, and lyophilisation methods. Inclusion complexes were characterized by FTIR, DSC, XRD, NMR, and mass spectral studies. Inclusion complexes prepared using kneading, and lyophilisation techniques in the molar ratio 1 : 5 with β-cyclodextrin were used for formulating orodispersible tablets by direct compression with different superdisintegrants like croscarmellose sodium, crospovidone, sodium starch glycolate, and low substituted hydroxypropyl cellulose in varying concentrations. The directly compressible powder was evaluated for precompression parameters, and the prepared orodispersible tablets were evaluated for postcompression parameters. Drug-excipient compatibility studies showed no interaction, and characterization proved the formation of inclusion complex. In vitro disintegration time was found to be within 3 minutes, and all the formulations showed complete drug release of 100% within 20 minutes. The optimized formulation was found to be stable after 6 months and showed no significant change in drug content. This work proved β-cyclodextrins to be effective solubilizing agent in improving the solubility of poorly water soluble drugs.The aim of this study was to investigate the use of inclusion complexation technique employing  Cyclodextrins consist of six, seven, and eight D-glucose units, respectively, attached by α-1, 4-linkages. Cyclodextrins consist of -linked α-D-glucopyranose units and contain a somewhat lipophilic central cavity and a hydrophilic outer surface. Due to the chair conformation of the glucopyranose units, the cyclodextrins are shaped like a truncated cone [Any drug from a given dosage form to be absorbed must be present in the form of solution at the site of absorption. Low aqueous solubility is one of the major problems encountered during formulation development of new chemical entities especially in the process of generic product development. More than 40% of new chemical entities developed in pharmaceutical industry are practically insoluble in water. Various techniques are used for the enhancement of the solubility of poorly soluble drugs including physical and chemical modifications of drug like particle size reduction, crystal engineering, salt formation, solid dispersion, use of surfactant, hydrotropy, cosolvency, use of surfactants, and complexation . Inclusited cone . In aqueβ-cyclodextrins.For the inclusion complex formation, different methods were employed such as physical mixture, kneading, coevaporation, and lyophilisation techniques. The concept of mouth dissolving drug delivery system emerged from the desire to provide patient with more conventional means of taking their medication. It is difficult for many patients to swallow tablets. Hence, they do not comply with prescription, which results in high incidence of noncompliance and ineffective therapy. A mouth-dissolving drug delivery system, in most cases, is a tablet that dissolves or disintegrants in the oral cavity without the need of water or chewing . Candesaβ-Cyclodextrin (β-CD) was obtained from S D Fine-Chem. Ltd. . Crospovidone, croscarmellose sodium, and low substituted hydroxypropyl cellulose were obtained from Yarrow Chem. Products . Aspartame and aerosil was obtained from Himedia Laboratories Pvt. Ltd. . Mannitol was obtained from Thermo Fischer Scientific India Pvt. Ltd. . Sodium starch glycolate was obtained from E. Merck Ltd. . Methanol was obtained from RFCL Ltd. . All other ingredients used were of analytical grade.Candesartan cilexetil was a gift sample from Hetero Drugs Pvt. Ltd. (Hyderabad). β-CD was prepared by mixing candesartan cilexetil with β-CD in a mortar and pestle. Mixing was continued for one hour with constant trituration and then passed through sieve no. 100. The resulting sample was stored in a desiccator until further use. The physical mixture was prepared in 1 : 1, 1 : 3, and 1 : 5 molar ratios.The physical mixture of candesartan cilexetil with β-CD was taken in a mortar, and 10 mL of 50% ethanol was added and triturated to get slurry. Then, slowly drug was incorporated into the slurry, and trituration was further continued for one hour. Slurry was then air-dried at 25°C for 24 hours, pulverized, and passed through sieve no. 100. The resulting sample was stored in a desiccator until further use. Candesartan cilexetil complexes with β-CD were prepared in 1 : 1, 1 : 3, and 1 : 5 molar ratios.β-CD and candesartan cilexetil in 10 mL of 50% aqueous ethanol. The solution was stirred till a clear solution was obtained, and the resulting solution was evaporated under vacuum at a temperature of 45°C. The solid residue was further dried completely at 45°C for 48 h. The dried complex was pulverized into a fine powder and sieved through sieve no. 100. The resulting sample was stored in a desiccator until further use.Inclusion complex was prepared by dissolving 1 : 1, 1 : 3, and 1 : 5 molar ratios of β-CD in molar quantities of 1 : 1, 1 : 3, and 1 : 5 were taken separately in 20 mL of water and mixed thoroughly using a magnetic stirrer. The resultant solution was frozen in a deep freezer at −20°C for about 12 h. The frozen mixture was then freeze-dried in the freeze-dryer for 8 h at −50°C under vacuum. The resultant product was stored in a desiccator.Candesartan cilexetil and β-CD such as 0, 2, 4, 6, 8, and 10 milimoles/liter. All the solutions were shaken in rotary shaker for 72 hours. The solutions were filtered, and their absorbance was measured at 255.5 nm [Kc = apparent stability constant and S0 = intercept.Phase solubility studies were carried out according to the method reported by Higuchi and Connors. An excess of candesartan cilexetil was added to 10 mL portions of distilled water, each containing variable amount of β-CD inclusion complex was accurately weighed and transferred to 100 mL volumetric flask. To this, 50 mL of 5% PEG 400 solution and 50 mL of distilled water were added. The resulting solution was diluted suitably, and drug content was estimated spectrophotometrically  at 232 nm using distilled water as blank. The drug content was calculated using calibration curve.100 mg of drug β-cyclodextrin, and complexes were recorded by using Philips Holland-PW 1710 scanner with filter Cu radiation over the interval 5–60°/2θ. The operation data were as follows: voltage 35 kV, current 20 mA, filter Cu, and scanning speed 1°/min.The XRD patterns of drug, 1H nuclear magnetic resonance spectra were recorded on AMX400 NMR spectrometer. Chemical shifts (δ) are reported in parts per million downfield from internal reference standard tetramethylsilane [ylsilane . Mass spylsilane .In vitro dissolution of candesartan inclusion complexes were studied in USP type II dissolution apparatus using 900 mL of distilled water as dissolution medium at 75 rpm and at a temperature of 37 ± 0.5°C. Complex equivalent to 8 mg of candesartan was used in each test. 5 mL of sample of dissolution medium was withdrawn at suitable time intervals, diluted suitably, and analyzed by measuring the absorbance at 255.5 nm. The volume withdrawn at each time interval was replaced with fresh quantity of dissolution medium to maintain sink conditions. The amount of candesartan released was calculated and plotted against time and compared with pure drug.β-cyclodextrin complex, mannitol, low substituted hydroxy propyl cellulose, crospovidone, croscarmellose sodium, sodium starch glycolate, aspartame, aerosol, and talc were weighed and passed through no. 60 mesh separately. Then, the ingredients were mixed thoroughly and compressed into tablets using 6 mm punches in rotary tablet press . Formulations of candesartan cilexetil-β-cyclodextrin complex orodispersible tablets by direct compression method are shown in Candesartan-The prepared granules were evaluated for angle of repose, bulk density, tapped density, Hausner's ratio, and Carr's compressibility index , 11.Twenty tablets were selected at random and individually weighed, and the average weight was calculated. The uniformity of weight was determined according to pharmacopoeial specifications.2, which was determined using Monsanto hardness tester. Preweighed sample of tablets was placed in the friabilator (Roche Friabilator) and operated for 100 revolutions. Tablets were dusted and reweighed. Hardness is the tensile strength of tablets expressed in kg/cmWinitial = initial weight of 20 tablets and Wfinal = final weight of 20 tablets.Percentage friability was calculated by using the formulaFive tablets from each formulation were selected randomly, crushed and mixed. From the mixture powder equivalent to 10 mg of candesartan, cilexetil was weighed and dissolved in 100 mL of 5% PEG 400 in distilled water. The resulting solution was filtered through Whatman filter paper no. 41, diluted suitably, and the absorbance of the resulting solution was measured spectrophotometrically at 232 nm using distilled water as blank. Experiments were carried out thrice, and average percentage drug content was considered.Drug content was estimated by the formulaIn vitro dispersion time was measured by dropping tablets in a 10 mL measuring cylinder containing 6 mL of buffer solution simulating saliva fluid (pH 7.4). The time for the tablet to completely disintegrate into fine particles was noted. Six tablets from each formulation were randomly, selected and In vitro dispersion time was noted. The disintegration time for all formulations was carried out using tablet disintegration test apparatus. Six tablets were placed individually in each tube of disintegration test apparatus, and discs were placed. The water was maintained at a temperature of 37°  ± 2°C, and time taken for the entire tablet to disintegrate completely was noted.R, is determined using following equation:Wb and Wa are the weights of tablet before and after water absorption, respectively.A tablet is placed on piece of tissue paper that was folded twice and kept in a petri dish  containing 6 mL of water, and the time for complete wetting is measured. The wetted tablet is then weighed, and the water absorption ratio, In vitro drug release studies were carried out using 900 mL of distilled water as dissolution medium in USP dissolution apparatus type II rotating paddle apparatus. The rotating speed was maintained at 75 rpm, and temperature was maintained at 37°C ± 0.5°C. 5 mL of solution was withdrawn at regular predetermined time intervals, and same volume of sample was replaced with fresh dissolution medium. The samples were diluted suitable and the absorbances of the resulting solution were measured at 255.5 nm using UV/Visible spectrophotometer . All the experiments were carried out in triplicate.in vitro disintegration time at intervals of 2 months [The selected formulations were closely packed in aluminum foils and then stored at 40°C ± 2°C/75% RH ± 5% in stability chamber for 6 months and evaluated for their physical appearance, drug content, percent friability, and 2 months . in vitro dissolution study, and complexes prepared by kneading method and freeze-drying method in the molar ratio of 1 : 5 were used for further study. The drug content of all the candesartan cilexetil-β cyclodextrin complexes was found to be 98.4%–99.9%. All the complexes were subjected to β-cyclodextrin and its physical mixture to detect any sign of interaction which would be reflected by a change in the position or disappearance of any characteristic peaks of the compound. The IR spectra of the pure candesartan cilexetil ; such endothermic peak signifies that candesartan cilexetil used was in pure crystalline state. β-Cyclodextrins peaks are distinct in β-cyclodextrin was in pure crystalline state. DSC thermogram of the physical mixture of candesartan cilexetil with β-cyclodextrin  calculated according to equation were found to be 0.9867 and 0.04848 M−1. When the complex is first order with respect to ligand and first or higher order with respect to substrate, then AL-type phase solubility profile is obtained. The phase solubility diagram was classified as AL-type indicating the formation of a 1 : 1 candesartan cilexetil : β-CD inclusion complex. The lower values of the stability constants (Kc) suggest that the candesartan cilexetil : β-CD interaction is weak and also that the inclusion process is exothermic, defined as enthalpically driven and spontaneous [Analyzing the phase solubility profiles, it was observed that the increase of candesartan cilexetil solubility in the system was due to due to molecular interaction with β-CD . Candesantaneous . θ diffraction angles of 9.97°, 17.04°, 18.74°, 19.40°, 21.63°, 23.94°, 25.16°, 27.96°, and 29.28°. X-ray diffraction pattern in β-cyclodextrin was in a crystalline state as it showed sharp distinct peaks at 2θ diffraction angles of 12.4° and 19.0° 2θ. X-ray diffraction pattern of inclusion complexes prepared by kneading and lyophilisation techniques (Figures θ diffraction angles of 12.4°, 19.0°, 17.04°, 18.74°, and 19.40° that are characteristic for both candesartan cilexetil and β-cyclodextrin. The complex formation led to the broadening of the existing peaks, appearance of a few new peaks, and shifting of certain peaks. This could be due to the conversion of crystalline to amorphous form during complexation [The XRD results were in good agreement with the thermal analysis data. X-ray diffraction patterns in  Figures  showed sβ-cyclodextrin inclusion complexes prepared by kneading method and lyophilisation technique. From m/z scale. Other important fragments obtained were at 299 and 266 on m/z scale. From m/z scale. Other important fragments obtained were at 299 and 148 on m/z scale. Extensive fragmentation seen in mass spectra may be attributed to large molecular weight of candesartan cilexetil and β-cyclodextrin. So based on the above, the molecular weight of the prepared complex was found to be 423.18 Dalton.Mass spectroscopic studies were carried out to find out the molecular weight of candesartan cilexetil-1H-NMR chemical shift for each proton of candesartan cilexetil and βCD was evaluated for the formation of inclusion complex between candesartan cilexetil and βCD at molar ratio 1 : 5 of candesartan cilexetil and βCD. The chemical shifts for different protons in candesartan cilexetil (β-cyclodextrins (β-cyclodextrins were found in inclusion complexes prepared by kneading method and lyophilisation technique (Figures β-cyclodextrins.The ilexetil  were seedextrins  were see Figures . Since tIn vitro release studies were carried out using USP type II tablet dissolution test apparatus paddle method at 75 rpm and at a temperature of 37 ± 0.5°C, using 900 mL of distilled water as dissolution medium. Aliquots of 5 mL were withdrawn at a regular interval of 15 minutes and analyzed spectrophotometrically at 255.5 nm. The in vitro dissolution profiles of all the 12 complexes prepared by four different methods, namely, physical mixing, kneading, coevaporation, and lyophilisation techniques in three molar ratios of 1 : 1, 1 : 3, and 1 : 5, indicated faster drug release from all the formulations, and maximum drug release of 93.96% and 101.2% was obtained from formulations C6 and C12 that are prepared by kneading method and lyophilisation technique at the end of 105 min when compared to complexes prepared by coevaporation and physical mixture in all the other ratios.β-cyclodextrin inclusion complexes prepared by kneading method and lyophilisation technique at molar ratio 1 : 5 were selected for formulating into orodispersible tablets. Each complex was formulation into 12 formulations using four superdisintegrants, each of which is in three different concentrations as shown in Tables Candesartan cilexetil-The precompression parameters for the all 24 formulations were carried out, and the results were shown in Tables The weights of all tablet formulations ranged between 137 and 161 mg . As the 2.The hardness of all formulations was determined, and the results were shown in The percentage friability of all the formulations was found to be not more than 0.7%, which is well within the limit of less than 1%. The results of friability indicated that the tablets were mechanically stable.The drug content studies for all 24 formulations were carried out by a validated method using 5% PEG 400 in distilled water and was found to be in the range of 98.14%–100.28% of candesartan cilexetil, and the results were shown in Wetting time of the formulations was determined, and all the formulations showed wetting time of 90 to 210 seconds. Water absorption ratio of all the formulations was calculated using the equation, and all the formulations showed good water absorption ratio from 72.58 to 81.42. Wetting time of the dosage form is related to contact angle. Lower wetting time implies a quicker disintegration time .In vitro dispersion time of all the formulations of orodispersible tablets was determined and was found to be 66 to 184 seconds. Formulations F12 and F24 showed good in vitro dispersion time of 94 and 66 seconds, respectively. In vitro disintegration time of formulations F1 to F24 was determined, and all the formulations showed disintegration time within three seconds. According to the European pharmacopoeia, the fast disintegrating or orodispersible tablets should disintegrate within 3 minutes without leaving any residue on the screen. in vitro drug release profiles. Formulations F1 to F12 were prepared using complexes prepared by kneading method, and formulations F13 to F24 were prepared using complexes prepared by lyophilisation method. Formulations containing low substituted hydroxyl propyl cellulose as superdisintegrant, namely, F1, F2, and F3, showed 97.8%, 100.01%, and 99.5%, respectively, at the end of 20 minutes, whereas formulations containing crospovidone as superdisintegrant, namely, F4, F5, and F6, showed 95.6%, 97.8%, and 98.9%, respectively, at the end of 20 minutes. Formulations containing croscarmellose sodium as superdisintegrant, namely, F7, F8, and F9, showed 97.7%, 98.8%, and 98.5%, respectively, at the end of 20 minutes, whereas formulations containing sodium starch glycolate as superdisintegrant, namely, F10, F11, and F12, showed 97.8%, 101.9%, and 101.2%, respectively, at the end of 20 minutes. Formulations containing low substituted hydroxyl propyl cellulose as superdisintegrant, namely, F13, F14, and F15, showed 95.6%, 96.7%, and 98.8%, respectively at the end of 20 minutes, whereas formulations containing crospovidone as superdisintegrant, namely, F16, F17, and F18, showed 97.8%, 99.9%, and 97.7%, respectively, at the end of 20 minutes. Formulations containing croscarmellose sodium as superdisintegrant, namely, F19, F20, and F21, showed 97.7%, 97.8%, and 98.8%, respectively, at the end of 20 minutes, whereas formulations containing sodium starch glycollate as superdisintegrant, namely, F22, F23, and F24, showed 98.9%, 100.9% and 101.1%, respectively at the end of 20 minutes. Formulations F12 and F24 prepared using cros povidone and sodium starch glycolate in the concentration of 8% showed lowest disintegration time of 74 and 68 seconds and highest drug release of 101.12 and 101.14 at the end of 20 minutes. The In vitro drug release study indicated faster and maximum drug release from formulations F12 and F24. The release profiles of formulations F12 and F24 were demonstrated in The prepared orodispersible tablets were evaluated for their 12 and F24 were kept at 40 ± 2°C with 75 ± 5% RH for a period of 6 months. The physical appearances, friability, disintegration time, and drug content were measured for these tablets at the end of 2, 4, and 6 months. The results showed that there was no significant difference between the initial and aged orodispersible tablets. The percentage drug content and friability were found to be 96.1 and 0.81% for formulation F12 and 96.3 and 0.88% for formulation F24 at the end of 6 months. This indicated that the physicochemical parameters, disintegration time, and percentage drug content were not affected by aging. The optimized formulations were physically and chemically stable for a period of 6 months at accelerated stability conditions. The accelerated stability studies of orodispersible tablets were performed as per the ICH guidelines to investigate whether the orodispersible tablets are affected during storage conditions. Optimized formulations Fβ-cyclodextrins in molar ratio of 1 : 5 were prepared and characterized. These complexes were formulated into orodispersible tablets using varying concentrations of superdisintegrants. Precompression and postcompression parameters were determined, and all the parameters met the requirements. All the tablet formulations showed In vitro disintegration time of less than 3 minutes and 100 percent drug releases within 20 minutes. By this study, it can be concluded that β-cyclodextrins can be used as a solubilizing agent to improve solubility of poorly water soluble drugs. The oral bioavailability of the drug could be improved by this methodology by more than two times due to improved aqueous solubility when compared to pure drug. This methodology of inclusion complexation can be further exploited for the successful delivery of poorly water soluble compounds.In the present study, complexes of candesartan cilexetil with


We assessed the incidence of infants born small-for-gestational-age (SGA) and large-for-gestational-age (LGA) in an Italian cohort over 20 years (1993–2013). Furthermore, we investigated maternal factors associated with SGA and LGA births.th year over a 20-year period, specifically examining data for 1993, 1998, 2003, 2008, and 2013. Infants with birthweight <10th percentile were defined as SGA, and those with birthweight >90th percentile as LGA. Data collected included newborn anthropometry, birth (multiple vs singleton), maternal anthropometry, previous miscarriage, gestational diabetes, hypertension, and smoking during pregnancy.A retrospective review of obstetric records was performed on infants born in Chieti  covering every 5p < 0.05), while greater pre-pregnancy BMI and gestational diabetes were risk factors for LGA births .There were a pooled total of 5896 live births recorded across the 5 selected years. The number of SGA (+60.6 %) and LGA (+90.2 %) births increased considerably between 1993 and 2013. However, there were no marked changes in the incidence of SGA or LGA births . Maternal factors associated with increased risk of SGA infants included hypertension, smoking, and previous miscarriage (all There was an increase in the number of SGA and LGA births in Chieti over the last two decades, but there was little change in incidence over time. Most maternal factors associated with increased odds of SGA and LGA births were modifiable, thus incidence could be reduced by targeted interventions. Birth size is an essential parameter to consider in the clinical evaluation of newborns. Either weight or length at birth represent the expression of growth in utero as a result of maternal, placental, and fetal factors, and have been historically used to identify newborns at potential risk of complications early in life . In addiThere is considerable variation in the prevalence of infants born SGA (4.6–15.3 % across Europe) . The varSize at birth has been reported to vary over generations –11. DuriAs a result, we aimed to assess the trends in SGA and LGA births over a 20-year period (1993–2013) in Chieti . In addition, we investigated the maternal risk factors associated with SGA and LGA births.Ethics approval for this study was not required, since i) it was an audit; ii) it was confined to anonymised and unidentifiable data that are routinely collected at the S.S. Annunziata Hospital (Chieti); and iii) study findings would not affect patient care.th year on all infants born in the Department of Obstetrics and Gynecology, S.S. Annunziata Hospital, between 1st January 1993 and 31st December 2013 . This is the largest maternity hospital in the province of Chieti, covering approximately half of all births.The province of Chieti  has a population of 397,395 (2012 census), mostly of Caucasian origin. Data were retrospectively collected for every 5http://www.inescharts.com/). According to the Italian Neonatal Study (INeS) reference charts [th and 90th percentile (−1.28 to 1.28 SDS); SGA, defined on a birthweight and/or length <10th percentile (<−1.28 SD); and LGA, defined on a birthweight and/or length >90th percentile (>1.28 SD). Classification of newborns from multiple pregnancies as SGA or LGA was based on specific percentile curves [A retrospective review of obstetric and delivery records was performed, including information on sex, gestational age, anthropometry (birthweight and length), and birth order. Note that when assessing birth order, miscarriages at less than 20 weeks of gestation were disregarded . Gestatie curves . Unless Maternal height, pre-pregnancy BMI, conception , birth (multiple vs singleton), and delivery  were recorded. Maternal factors, such as previous miscarriage, parity, chronic illnesses, gestational diabetes, hypertension (including pre-eclampsia) , and useThe factors associated with SGA or LGA birth were identified using stepwise linear regression analysis with significance levels of 0.4 and 0.2 required for a variable to enter and stay in the model, respectively . The various parameters included at model entry were: birth year, gestational diabetes, maternal hypertension, maternal ethnicity, maternal age, smoking during pregnancy, alcohol consumption during pregnancy, previous miscarriage, conception , birth (multiple vs singleton), sex, and birth order. Subsequent models were also run on the subgroup with maternal anthropometric data, with the addition of maternal height and BMI into the models. Odds ratios (OR) are provided with 95 % confidence intervals in brackets. The agreement in SGA or LGA classifications according to birthweight and birth length was evaluated using the Cohen’s kappa statistic. All statistical tests were two-tailed and significance was maintained at the 5 % level.There were a pooled total of 5896 live births recorded across the 5 specific years covered by this study, mostly of Caucasian ethnicity (97.5 %). Detailed pregnancy information and birthweight data were available for 5759 live births (97.7 %).Pregnancy outcomes for the whole cohort are reported in Table p = 0.028), were hypertensive (OR 2.93 (2.11–4.06); p < 0.0001), or had a previous miscarriage (OR 1.35 (1.08–1.68); p = 0.008).The odds of being born SGA were greater in the offspring of mothers who smoked tobacco during pregnancy (OR 1.49 (1.05–2.14); p = 0.028), and in those born to mothers with gestational diabetes (OR 1.80 (1.25–2.57); p = 0.001) or who did not smoke during pregnancy (OR 1.61 (1.04–2.48); p = 0.032).Conversely, the odds of being born LGA were greater in males (OR 1.21 (1.02–1.43); 2 increase in BMI there were higher odds of having a LGA baby (OR 1.10 (1.07–1.13); p < 0.0001) but lower odds of having a SGA baby (OR 0.94 (0.91–0.98); p = 0.002). The odds for every 1 cm increase in height followed an identical pattern: being for LGA 1.06  and for SGA 0.94 .Maternal anthropometric data were available for a subset of 3071 live births (52.1 %), and these data were re-analysed incorporating maternal pre-pregnancy BMI and maternal height into stepwise regression analysis. For every 1 kg/mData on birth length were available for 3792 infants 64.3 %), as this parameter was not routinely recorded until the last decade. As a result, birth length data were available for 10.9 % of births in 1993, 6.2 % in 1998, 70.2 % in 2003, 94.5 % in 2008, and 93.1 % in 2013. Maternal pathologies and pregnancy outcomes observed amongst these births are reported in Table .3 %, as This study shows that there was an increase in the number of SGA and LGA newborns in Chieti over a 20-year period, but this was a result of increased birth rates. The incidence of SGA and LGA births varied over time, and there were no marked changes observed. Maternal factors associated with increased odds of SGA babies included maternal hypertension, smoking in pregnancy and previous miscarriage, while greater pre-pregnancy BMI and gestational diabetes were risk factors for LGA births.In a recent prospective cohort data of 75,296 newborns from 12 European countries 1983–2006), the prevalence of SGA births varied from 4.6 % in Finland to 15.3 % in Portugal, with a rate of 9.1 % reported in  Italy (Lazio) –2006, th, 23, 24.In regards to LGA births, there has been a progressive increase in rates reported in several countries over the last decades , 13. HadThere are a number of maternal factor known to be associated with an increased risk of delivering a SGA or LGA infant , 18. ConA previous miscarriage represented an additional risk factor for SGA birth in Chieti. A spontaneous miscarriage, which occurs in approximately 15 % of clinically recognized pregnancies , has beeIn accordance with existing evidence , 38, we Infants can be defined as SGA or LGA based on birthweight, birth length, or both . In our th year, so that it is not possible to ascertain what the incidence of SGA and LGA births was in the intervening years. Nonetheless, our study covers comprehensive pregnancy information and birthweight data.The main limitation of our study was the lack of complete maternal and newborn data on the whole cohort. In particular, the reduced number of infants with maternal anthropometric and/or birth length data could possibly limit the applicability of specific findings across the whole population. In addition, we only provide annual data collected every 5In summary, the incidence of SGA and LGA births has not markedly changed in Chieti over the last two decades, even though numbers of babies born small and large have increased due to a growing population. However, most of the maternal factors observed to be associated with increased odds of SGA and LGA births are modifiable. Thus, it is important that interventions target women both before and during pregnancy to reduce SGA and LGA incidence, and consequently minimize potential short- and long-term adverse effects on offspring health.


This extract (0–100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL.The present study aims to evaluate the Leishmania spp. through the bite of female sandfly. This disease is characterized by chronic skin lesions and leaves permanent scars as deformation of the infected area , fetal calf serum (FCS), fetal bovine serum (FBS), RPMI-1640 medium with L-glutamine, and Griess reagent (A and B) were prepared from Sigma-Aldrich . All other chemicals and solvents had the highest commercially available purity. P. khinjuk were collected from rural regions of Kerman province, south east of Iran, from May to September 2013  were separately extracted by percolation method with 70% aqueous ethanol successively for 72 h at room temperature. The extracts were passed through filter paper  to remove plant debris. The extracts were finally concentrated in vacuum at 50°C using a rotary evaporator  and stored at −20°C until testing . P. khinjuk ethanolic extract was carried out to determine the presence of tannins, saponins, alkaloids, flavonoids, and glycosides as described elsewhere using the following reagents and chemicals [2 and H2SO4, and saponin with ability to produce suds.The preliminary phytochemical analysis of thehemicals : alkaloi L. tropica (MHOM/IR/2002/Mash2) and L. major (MRHO/IR/75/ER) were kindly prepared from Center for Research and Training in Skin Diseases and Leprosy, Tehran, Iran. The parasites were cultured at RPMI 1640 supplemented with penicillin (200 IU/mL), streptomycin (100 μg/mL), and 15% heat-inactivated FCS. Murine macrophage cells (J774-A1) were obtained from Pasteur Institute of Iran, Tehran, Iran. The cells were cultured and maintained at Dulbecco's modified eagle's medium (DMEM) supplemented with 10% FBS at 37°C in 5% CO2.Standard strains of P. khinjuk, J774-A1 cells (5 × 105) were cultivated with various concentrations of alcoholic extract (0 to 5000 μg/mL) in 96-well tissue culture plates at 37°C in 5% CO2 for 48 h. Cell viability was determined by colorimetric MTT assay and the results were displayed as the percentage of dead cells compared to macrophages treated with MA and nontreated macrophages (100% of viability) [In order to determine the cytotoxic effects ofability) . P. khinjuk ethanolic extract against promastigote forms of L. tropica, colorimetric cell viability MTT assay was used as described elsewhere [μL of the promastigotes (106 cells/mL) harvested from logarithmic growth phase was added to a 96-well tissue culture plate. Then, 100 μL of various concentrations of alcoholic extract and MA (0–100 μg/mL) was added to each well and incubated at 25 ± 1°C for 48 h. After the incubation, 10 μL of MTT solution (5 mg/mL) was added to each well and incubated at 25°C for 4 h. Promastigotes were cultured at complete medium with no drug as positive control and complete medium with no promastigotes and drugs as blank. The absorbance was measured for each well at 560 nm using an ELISA reader .To evaluate the antipromastigoteeffects oflsewhere . At firs P. khinjuk extract was performed using the methods described by Mahmoudvand et al. [2 cover slips were placed in the wells of 6-chamber slides . In the next step, 200 μL of macrophage (J774) cells (105/mL) was incubated at 37°C in 5% CO2 for 2 h. Then, 200 μL (106/mL) promastigotes in stationary phase was added to the murine macrophages so that proportion of Leishmania/macrophage was 10 : 1 and incubated again in a similar condition for 24 h. Free parasites were removed by washing with RPMI 1640 medium and the infected macrophages were treated with 50 μL of various concentrations of 0–100 μg/mL alcoholic extract at 37°C in 5% CO2 for 72 h. Finally, the dried slides were fixed with methanol, stained by Giemsa, and studied under a light microscope. Also, the macrophages containing amastigotes without extract and those with no parasite and extract were considered positive and negative controls, respectively. Activity of anti-intramacrophage amastigotes of the extracts was evaluated by counting the number of amastigotes in each macrophage by examining 100 macrophages (% amastigotes viability) in comparison with those obtained by positive control.In this study, antiamastigote activity ofd et al. . Briefly P. khinjuk extract against the promastigotes invasion of macrophages, as one of the most important pathogenic and biological criteria of Leishmania parasites, was investigated. In this assay, promastigotes of L. tropica were preincubated in P. khinjuk extract (5 μg/mL) for 2 h at room temperature. Then promastigotes were washed with RPMI-1640 medium and incubated with macrophage cells for 24 h. The macrophages were stained by Giemsa and studied by a light microscope, to evaluate the frequency of infection by counting 100 macrophages [The inhibitory effect ofrophages .μL of supernatants was collected 48 h after introducing the various concentrations of P. khinjuk extract  into the culture medium. The assay was done in triplicate wells in a 96-well tissue culture plate. To this, 60 μL of Griess reagent A and then 60 μL of Griess reagent B were added. The plates were read at 540 nm in an ELISA plate reader .In this study, nitric oxide (NO) release in supernatants of macrophage culture was measured by the Griess reaction for nitrites compared to the untreated macrophages. One hundred The experimental procedures carried out in this survey were in compliance with Guidelines of Kerman University of Medical Science  for the care and use of laboratory animals in line with Animal Ethics Committee (permit number 91/21).Thirty-two male BALB/c mice (6–8 weeks old) were obtained from Animal Breeding Stock Facility, Razi Institute of Iran, Karaj, Iran. The animals were housed in a colony room with a 12 : 12 h light/dark cycle at 21 ± 2°C and handled according to standard protocols for the use of laboratory animals . L. major (2 × 106 cells/mL) harvested from the stationary phase was subcutaneously injected into the base of the tail [To infect the male BALB/c mice, 0.1 mL of the promastigotes ofthe tail . Thirty-After 5 weeks when leishmanial lesions appeared, the treatment of infected mice was started. At first, the diameter of lesions was measured before the treatment. Then, lotion (5 g/kg ethanolic extract at the concentrations of 20 and 30%) and MA were applied for each tested subgroup daily for 30 days topically. The control subgroups received distilled water and ethanol topically.D and d) at right angles to each other, and the size (mm) was determined according to the formula S = (D + d)/2 [Before and after the treatment, the diameter of lesions was measured using vernier caliper in two diameters (D + d)/2 . negative: 0 parasite/10 fields, 1+: 1 parasite/10 fields, 2+: 1–10 parasites/10 fields, 3+: 10–100 parasites/10 fields, 4+: 1010–1000 parasites/10 fields, 5+ and more: more than 1000 parasites/10 fields.Microscopical examinations were performed by laboratory demonstration of the parasite load in the lesions by making stained smears at the end of the experimental period. Lesions were cleaned with ethanol and punctured at the margins with a sterile lancet and exudation material was smeared. The smears were dried in air, fixed by methanol, and stained with Giemsa to determine the load of parasites by light microscopy as follows:50 (cytotoxic concentration for 50% of cells) and IC50 (50% inhibitory concentrations) lineal regression was used. Selectivity index (SI), calculated based on the equation of CC50 for murine macrophage cells/IC50 for amastigote forms of L. tropica, was used to compare the toxicity and activity of ethanolic extract of P. khinjuk, as described by Weniger et al. [t-test. In addition, P < 0.05 was considered statistically significant.In this survey, all the tests were carried out in triplicate. To determine CCr et al. . Data an P. khinjuk demonstrated the presence of high amount of tannins, phenols, and glycosides and lacking of the alkaloids in this plant.In this study, the findings of primary phytochemical screening of the ethanolic extract of P. khinjuk alcoholic extract had remarkable antileishmanial activity against the promastigote forms of L. tropica based on a dose-dependent response (P < 0.05). IC50 value for the P. khinjuk extract and MA against promastigotes of L. tropica was 58.6 ± 3.15 μg/mL and 88.3 ± 4.05 μg/mL, respectively.The results revealed that P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of intramacrophage amastigotes as a dose-dependent response. The obtained IC50 values were 37.3 ± 2.51 μg/mL and 44.6 ± 2.51 μg/mL for the P. khinjuk extract and MA, respectively.The findings demonstrated that, similar to promastigote stage, L. tropica with no drugs were able to infect 84.3 ± 6.5 percent of macrophage cells. While promastigotes treated with the P. khinjuk had potency to infect only 38.6 ± 3.05 percent of the macrophages cells, thus, these findings revealed that infectivity of promastigotes of L. tropica significantly reduced (P < 0.05) with P. khinjuk extract preincubation.The results revealed that promastigote forms of P. khinjuk at concentration of 3.125 μg/mL triggered production of 14.3 ± 1.5 μM of nitric oxide compared to the untreated macrophages with value of 11.3 ± 0.5 μM (P > 0.05), whereas this extract at higher concentrations (≥6.25 μg/mL) decreased production of NO to ≤7 μM, compared to the untreated macrophages (P > 0.05).The findings of this assay demonstrated that the in vitro cytotoxic activity of alcoholic extract of P. khinjuk, it could be observed that P. khinjuk extract had no significant cytotoxicity against J774 cells. CC50 values for P. khinjuk extract and MA were 511.6 ± 7.15 μg/mL and 1225.6 ± 11.6, respectively. SI values for the alcoholic extract P. khinjuk and MA are 13.7 and 27.5, respectively.In the assessment ofIn vivo antileishmanial evaluation of the P. khinjuk alcoholic extract demonstrated that, in the infected mice treated with the extract concentration of 30%, the number of parasites significantly (P < 0.05) reduced with respect to MA, whereas 20% of the extract decreased the number of parasites intermediately. Control subgroups (distilled water and ethanol) had no decrease in the number of parasites  and consequently IC50 values demonstrated that P. khinjuk alcoholic extract significantly inhibited the growth rate of promastigote forms of L. tropica. In addition, this extract significantly decreased mean infection rate and subsequently the viability of amastigote forms in the macrophages compared with the control group. The findings exhibited that amastigote forms were more susceptible to P. khinjuk extract than promastigote forms. This difference in the susceptibility of promastigote and amastigote forms could be related to their structural, biochemical, and morphological features as previously demonstrated by other researchers [ in vivo assay, it was found that P. khinjuk extract at the concentration of 30% had potent suppression effects on CL in male BALB/c mice infected with L. major with 87.5% recovery, whereas P. khinjuk extract at the concentration of 20% displayed the suppression effects as intermediate. In line with our results, it has been proven that gum obtained P. atlantica controlled cutaneous leishmaniasis in mice infected with L. major [ Pistacia species, particularly P. khinjuk, have been demonstrated [ Pistacia species including the study conducted by Orhan et al., which proved that P. vera branch extract at the concentration of 4.8 μg/mL significantly (77.3%) inhibited the growth of L. donovani, whereas dry leaf extract (PV-DL) was active against Plasmodium falciparum (60.6% inhibition) [50 values of these extracts were determined as 2.3 μg/mL for the amastigotes of L. donovani grown at axenic culture and 3.65 μg/mL for P. falciparum.According to WHO, more than 80% of the world's population rely on traditional medicine for their primary healthcare needs. In the past decades, the advent of synthetic antimicrobial drugs has caused reluctance in plants as a rich resource for antimicrobial agents  However,earchers , 23. In L. major . These rL. major . Previouibition) . This re P. khinjuk extract demonstrated the presence of terpenoids, phenols, flavonoids, fatty acids, and sterols and lack of alkaloids in this plant. At present, individual activities of these compounds have been proven [ p-cymene, thymol, carvone, limonene, and terpinene [ P. khinjuk extract could be responsible for their antileishmanial effect though their exact mode of action is not clear. In the case of cytotoxic effects, the present findings showed that P. khinjuk alcoholic extract had no significant cytotoxic effects against J774 cells. Furthermore, SI value ≥ 10 of P. khinjuk alcoholic extract demonstrated its safety against the macrophage cells and specificity to the parasite [ P. khinjuk alcoholic extract is safe for mammalian cells.The preliminary phytochemical analysis ofn proven . Moreoveerpinene –30. Therparasite . Thus, i P. khinjuk alcoholic extract had potent antileishmanial activity and could control cutaneous leishmaniasis in mice infected with L. major. This result also provided the scientific evidence that natural plants could be used in the traditional medicine for the prevention and treatment of cutaneous leishmaniasis.In conclusion, the findings of the present study demonstrated that