Macrolepiota procera (Scop.) Singer and 230 soil/substrate samples, which were collected in foraging seasons 2015–2019 from 22 different locations in Slovakia. Total mercury content was determined by cold-vapor AAS analyzer AMA 254. The level of contamination and environmental risks were assessed by contamination factor (Cf), index of geoaccumulation (Igeo), and potential environmental risk index (PER). Bioaccumulation factor (BAF) was calculated for individual anatomical parts of M. procera. Mercury content in the soil/substrate samples varied between 0.02 and 0.89 mg kg−1 DW, and in mushroom samples between 0.03 and 2.83 mg kg−1 DW (stems), and between 0.04 and 6.29 mg kg−1 DW (caps). The obtained results were compared with the provisional tolerable weekly intake for Hg defined by WHO to determine a health risk resulting from regular and long-term consumption of M. procera.Wild-growing edible mushrooms are valuable food with a high content of proteins, fibers, antioxidants, and they are characterized by their specific taste and flavor. However, from an ecotoxicological point of view, they are a risk commodity because of their extremely high bioaccumulative capacity to accumulate the risk elements and contaminants from the environment. In the present study, we examined mercury (Hg) contamination in 230 fruiting bodies of  Nowadays, mushrooms are considered valuable foods, not only because of their attractive sensory characteristics and their culinary features but also for their nutritional properties . From anThe accumulation of risk elements by mushrooms is a complex process affected by both environmental and intrinsic factors . The mycMacrolepiota procera, known under the common name Parasol Mushroom, formerly called Lepiota procera, prefers light and warm places, especially calcareous and sandy soils that are well-drained in forests, meadows, and gardens [Macrolepiota species commonly collected by locals for their essential micro- and macroelements [ gardens ,13. It i gardens . It is p gardens . Parasol gardens . The speelements .M. procera and some other species are mercury bioaccumulators [Mercury, unlike other risk metals, can remain in the atmosphere and soil for the long-term and it can be distributed or migrate over long distances . Sourcesmulators . After emulators .M. procera and soil/substrate samples. Ecological risks of Hg were evaluated by calculating contamination factor, index of geoaccumulation, potential environmental risk index, as well as the bioaccumulation factor. Third, to assess the health risk resulting from the consumption of the studied mushroom by applying provisional tolerable weekly intake.The present study aims to determine the level of mercury contamination of This study was carried out in Slovakia in 22 different locations .Directly after the sampling, all mushroom samples (n = 230) were cleaned up from any organic and inorganic debris and the bottom part of the stem was cut off. After that, they were divided into two parts: cap and stem. The individual cap and stem samples were sliced into pieces using a ceramic knife and dried to a constant weight at 45 °C in a laboratory dry heat-oven with forced air circulation  for 22 h. The dried samples were pulverized in the rotary homogenizer  and stored in polyethylene bags until further analysis. Soil/substrate samples (n = 230) were collected together with mushroom samples at the same location from a depth of approximately 0.10 m. Under the laboratory conditions, the samples were air-dried at room temperature for 3 weeks. Afterward, they were sieved through a mesh sieve (2 mm) and stored in paper bags until the analysis.−6 mg kg−1 DW and the limit of quantification at 4.45 × 10−6 mg kg−1 DW. Two Certified Reference Materials (CRM) from the Institute for Reference Materials and Measurements were used to check the accuracy and precision of the analytical method. The recovery value for the loam soil (ERM-CC141), varied between 0.06 and 0.07 mg kg−1 DW, and for the Mussel tissue (ERM-CE278k), it varied between 0.05 and 0.06 mg kg−1 DW.Total mercury content was determined by cold-vapor AAS analyzer AMA 254 , in all types of dried and homogenized samples ). The limit of detection for Hg was set at 1.5 × 10To assess the level of ecological load of the monitored localities, we evaluated the Hg content in the soil/substrate using the following parameters:−1) [Contamination factor (ribed by  was usedmg kg−1) . The conEcological risk assessment is assessed by the potential ecological risk index . PER expresses the amount of potential ecological risk factor for the given risk element (Eri) :(2)PERHgigh risk ,23.geo) was calculated:Cn is the Hg content in the soil samples, 1.5 is the constant that is used due to potential variations in the baseline data  and Bi is the background value of Hg (0.06 mg kg−1) [geo values are divided into seven categories [geo ≤ 0); uncontaminated (0 < Igeo < 1); uncontaminated or slightly contaminated (1 ≤ Igeo < 2); slightly contaminated (2 ≤ Igeo < 3); moderately contaminated (3 ≤ Igeo < 4); strongly contaminated (4 ≤ Igeo < 5); very strongly contaminated (Igeo ≥ 5).To quantify the level of contamination on the sampling localities the geo-accumulation index (Img kg−1) . The Igetegories : backgroM. procera. It was calculated as follows:HgC is the measured mercury content mushroom samples and sC is the measured mercury content in soil/substrate. BAF < 1 indicates excluders, BAF > 1 indicates accumulators [c/s) was evaluated to compare the level of Hg translocation within the fruiting body.Bioaccumulation factor (BAF) was calculated to assess the level of transition and accumulation of Hg from soil/substrate to the above-ground parts (fruiting body) of mulators . The capHg is the measured content of Hg in the biological sample (mg kg−1 of fresh weight (FW) in mushrooms). If the detected value was greater than 100%, the consumption of mushroom samples from the area is potentially hazardous.The percentage of the provisional tolerable weekly intake (PTWI) assesses the health risk from exposure to each toxic element. The tolerable weekly intake per person weighing 70 kg for Hg is 0.28 mg per person per week . Taking p < 0.05 [p-value.All statistical operations were performed using R studio version 1.2.5033 . Spearmap < 0.05 . The KruSoil/substrate represents the main source of nutrients as well as risk elements  for the mycelium and mushroom’s fruiting body. When compared with the fruiting bodies, the soil/substrate contains mercury in minor contents. Mercury content in soil/substrate and mushroom samples (stem and cap) is shown in Hodruša-Hámre, Jedľové Kostoľany, Krajné, Sitnianska lehôtka, Snina, Šachtička, Štiavnik and Zbyňov belong to the 1st environmental region quality (regions with unisturbed environment), while Bojná, Divina, Duchonka, Kráľovce, Limbach, Lozorno, Nemečky, Orovnica, Pezinská Baba, Pitelová, Skýcov, Solčany and Tesáre) belong to the 2nd environmental region quality (regions with moderately disturbed environment).−1 [−1 DW (−1 DW) and the lowest in Lozorno (0.04 ± 0.02 mg kg−1 DW). In Slovakia, the background Hg content in the soil is 0.06 mg kg−1 . The ave1 [−1 DW . The hig−1 DW, while the highest average value of Hg content was detected in Macrolepiota procera (n = 8) and it was 0.13 mg kg−1. In another study, the content of total mercury in the underlying substrate ranged between 0.05 and 0.27 mg kg−1 (n = 33), while the average value of Hg content in the substrate in the case of mushroom samples with the highest content of mercury (Macrolepiota procera (Scop.) Singer, n = 5) was around 0.13 mg kg−1 ± 61.7 (mean ± RSD) ranging between 0.06 and 0.22 mg kg−1. The data of mercury content in the soil/substrate showed that none of the soil/substrate samples exceeded the mercury limit the soil/substrate [M. procera) were between 0.03 ± 0.02 and 0.09 ± 0.06 mg kg−1 DW . According to Falandysz and Gucia [−1 DW (means ranged between 0.02 and 0.36 mg kg−1 DW). More recently, Mleczek [−1 DW (it ranged between 0.05 and 0.08) in the soil corresponding to M. procera from Polish forests.When comparing our obtained results with the background value for mercury in soil/substrates, only samples from Snina exceeded the limit. We assume that a higher content of mercury was recorded in this locality due to chemical industries and landfills for industrial and hazardous waste located nearby. Árvay et al.  studied ubstrate . Falandyubstrate  determinkg−1 n = , while t Mleczek  measuredf, PER, and Igeo are shown in f = 2.86). The index of geoaccumulation was evaluated as uncontaminated for 5% of sampling sites, almost 73% as uncontaminated or slightly contaminated, 18% as slightly, and 5% as very strongly contaminated by Hg. The highest value of the geoaccumulation index of geoaccumulation value was found in Pitelová (Igeo = 11.4). Šefčík et al. [To assess soil pollution, contamination factor, the index of geoaccumulation, and the potential ecological risk index were calculated for each sampling point. The values of Ck et al.  used thek et al. .p = 3 × 10−14). This test  and the minimum in Hodruša-Hámre (0.69 ± 0.36). Regarding mushroom caps, Hg content varied between 0.04 and 6.29 mg kg−1 DW. The highest recorded mercury average value was 4.14 ± 1.12 mg kg−1 DW (Pitelová) and the lowest 0.89 ± 0.30 mg kg−1 DW (Kráľovce). The threshold for Hg in edible mushrooms (both cap and stem) is 0.75 mg kg−1 FW. When compared with our detected mercury contents, all analyzed samples (both stems and caps) did not exceed the limit.The element accumulation by wild-growing mushrooms has been the subject of numerous scientific papers around the world. Depending on the collection site, a higher, lower, or significantly differentiated ability of mushrooms to accumulate some toxic elements were reported. However, the efficiency of the element accumulation does not always depend on their content in a soil/substrate, but the element contents in such cases depend on mushroom species, genus, or the families to which they belong . In thisM. procera from Central Slovakia (Banská Bystrica) and they detected the average content of mercury in stems 1.40 (0.12–1.75) mg kg−1 DW. The highest mercury content was measured in M. procera cap, and the average value was 1.98 (between 0.41 and 3.20 mg kg−1 DW). Falandysz et al. [−1 DW) and stems (between 2.80 ± 1.30 and 3.00 ± 2.00 mg kg−1 DW). The Parasol Mushroom was also characterized to have a great potential to bioaccumulate mercury from the soil as evidenced by great bioconcentration factors (BCFs), which was between 170 ± 160 and 130 ± 120 for caps, and 110 ± 97.0 and 89.0 ± 92.0 for stems. Moreover, Falandysz and Gucia [−1 DW and between 0.05 and 20.0 mg kg−1 DW in caps and stems of M. procera, respectively. The caps generally contained higher contents of mercury when compared to stems, and the cap to stem mercury content quotient ranged between 1.30 and 4.60. Mleczek et al. [M. procera was 4.23 mg kg−1. The highest Hg contents were found in M. procera and S. bovinus. The common ranges reported in the literature for the elements of the trace elements with detrimental health effect are <0.5–5, <0.5–10, <0.2–10, <1–5, <0.5–5, and <1–5 mg kg−1 for Ag, As, Ba, Cd, Hg, and Pb, respectively. Mleczek et al. [Macrolepiota procera, and associated soil collected from Polish forests between 1974 and 2019. The average detected mercury content in M. procera was 1.74 (between 0.62 and 2.80) mg kg−1 DW. The BCF values for M. procera ranged between 0.084 and 2.85 mg kg−1. A comparison of the content of the toxic metal was done and they found out that the selected species make a rather significant contribution to elements intake, and M. procera had a rather significant contribution to Hg intake. In general, our results  and Kráľovce is significantly lower, and Pitelová is significantly higher compared to all studied localities .Kruskal-Wallis test has shown that there was a statistically significant difference between the tested variables , where analysis  demonstranalysis  showed tp-value  at the significance level of 5%, using the asymptotic p-value . This teM. procera ranged between 3.40 and 46.9 (caps) and 2.50 to 33.0 (stems). These results are closely correlated with the findings of other authors, who state that species with a BAF value > 1 accumulate the highest contents of risk elements in tubes and spores and flash of the cap [M. procera caps varied between 16 ± 6 and 220 ± 110 , and between 7.60 ± 2.60 and 130 ± 96  for the stems [c/s) was evaluated to compare the level of Hg translocation within the fruiting body. The quotient varied between 1.21 (Kráľovce) to 2.10 (Bojná). Falandysz et al. [Mercury can be efficiently bioaccumulated by many mushrooms . The bio the cap . In somehe stems ,39. The M. procera caps varied between 1.30 and 7.00 mg kg−1 DW. The estimated intake of Hg resulting from the consumption of 300 or 500 g portions of caps was assessed as 39–210 and 65–350 μg, and this is equivalent to 0.65–3.5 and 1.1–5.8 μg kg−1 body mass (BM) (a 76.0 kg adult). Taking into account that the provisionally tolerable weekly intake (PTWI) of mercury is 300 μg , and a reference dose of 0.30 μg kg−1 BM per day [M. procera caps for some areas might be of concern, especially if eaten by pickers frequently in the mushrooming season.In our study, %PTWI (using mean Hg content) ranged between 26% and 121% (for caps) and between 20.3 and 64.9% (for stems). The highest %PTWI for the cap was evaluated in Divina (105%) and Pitelová (121%). The recommended provisionally tolerable weekly intake from all of the analyzed samples was exceeded in 9% of the samples. Giannaccini et al.  analyzed per day ,42, mercM. procera and its corresponding soil/substrate from 22 different localities in Slovakia. The Parasol Mushroom is a species that is harvested in the wild for its unique taste. All analyzed mushroom samples of M. procera did not exceed the limit for Hg in edible mushrooms (0.75 mg kg−1 FW), while recommended provisionally tolerable weekly intake was exceeded in 9% of the analyzed samples. The level of ecological load of the monitored localities showed that the analyzed soils/substrates were polluted with mercury. The potential of Parasol Mushrooms to bioaccumulate certain metals, e.g., mercury in fruiting bodies can be very or extremely high. The contamination factor for the sampling sites was determined as low and moderate. The degree of the ecological risk of mercury was estimated as low risk (1 sampling site), moderate risk (16 sampling sites), considerable risk (4 sampling sites), and very high risk (1 sampling site). The majority of the studied soil samples showed moderate ecological risk and considerable ecological risk. Moreover, low (4.50%) and very high ecological risks (4.5%) were confirmed. The highest value contamination factor, geoaccumulation index, and PER were determined in Pitelová. This reaffirms that Pitelová belongs to the regions with a moderately disturbed environment in Slovakia. The current study validates that M. procera can uptake and accumulate Hg (and other elements) from its underlying soil/ substrate. Information provided by our study can bring a more comprehensive understanding of mercury transmission from the soil/substrate to the mushroom fruiting bodies. Concurrently, it can fill the vacancy and provide information about a significant detrimental impact on the environment, as well as consumers’ health resulting from long-term consumption of contaminated mushrooms.This study has been carried out to investigate the accumulation of mercury in edible wild-growing


Given the public health relevance of PSA‐based screening, various professional organizations have issued recommendations on the use of the PSA test to screen for prostate cancer in different age groups.Using a large commercial claims database, we aimed to determine the most recent rates of PSA testing for privately insured men age 30 to 64 in the context of screening recommendations.P = .11), age 30 to 34; −3.0% (P = .001), age 35‐39; −3.1% (P < .001), age 40 to 44; −2.4% (P = .001), age 45 to 49; −0.2% (P = .66), age 50 to 54; 0.0% (P = .997), age 55 to 59; and −3.3% (P = .054) from 2011 to 2013 and 1.2% (P = .045) from 2013 to 2017, age 60 to 64. PSA testing rate decreased from 2011 to 2017 for age groups between 35 and 49 by 13.4% to 16.9%.Data from employer plans were from MarketScan commercial claims database. Annual PSA testing rate was the proportion of men with ≥1 paid test(s) per 12 months of continuous enrollment. Men with diagnosis of any prostate‐related condition were excluded. Annual percent change (APC) in PSA test use was estimated using joinpoint regression analysis. In 2011 to 2017, annual testing rate encompassing 5.02 to 5.53 million men was approximately 1.4%, age 30 to 34; 3.4% to 4.1%, age 35 to 39; 11% to 13%, age 40 to 44; 18% to 21%, age 45 to 49; 31% to 33%, age 50 to 54; 35% to 37%, age 55 to 59; and 38% to 41%, age 60 to 64. APC for 2011 to 2017 was −0.5% (Based on these data, PSA testing rate has modestly decreased from 2011 to 2017. These results, however, should be considered in view of the limitation that MarketScan claims data may not be equated to actual PSA testing practices in the entire U.S. population age 30 to 64. Future research should be directed to understand why clinicians continue ordering PSA test for men younger than 50. In 2008, the U.S. Preventive Services Task Force (USPSTF) noted that there was insufficient evidence to screen men younger than 75.In the context of PSA‐based screening recommendations for prostate cancer and given its public health relevance, the objective of this study was to determine the more recent rate and trend for annual PSA testing in a sample of privately insured U.S. men age 30 to 64 in 2011 through 2017 using a large insurance claims database. This study is unique in that it is, to our knowledge, the only report of PSA testing rates and trends through 2017 that also includes men in younger age groups between 30 and 49. The unique observation made in our study for the most recent PSA testing trends is that from 2017 to 2018, PSA testing increased for the first time in recent years. Information gained from this study may be used to inform needed practice interventions for younger men (age 30‐49) who should not be routinely screened for prostate cancer using the PSA test according to practice guidelines issued by various professional organization.2We used claims data from large employer‐sponsored health plans obtained from IBM Watson Health's MarketScan databases from January 1, 2011 through April 30, 2019, encompassing 5.02 to 5.53 million men with continuous enrollment for each calendar year. These data represent a convenience sample of ~10% of the U.S. population, and they were captured using a web‐based tool provided by IBM called Treatment Pathways.2.1Annual rates for PSA test use in each calendar year were the proportion of included men with ≥1 PSA test(s) per 12 months continuous enrollment that were paid. Encounters in MarketScan databases reflect paid claims only, and unpaid claims are not captured.2.2P values related to statistical significance  for each APC estimate being different from zero.Annual percent change (APC) in PSA test use was estimated using joinpoint regression analysis, fitting trend data to identify the log‐linear model with the fewest number of inflection points.2.3We stratified MarketScan claims into age 30 to 34, 35 to 39, 40 to 44, 45 to 49, 50 to 54, 55 to 59, and 60 to 64. To study regional variation in PSA testing rates, we further stratified claims data into the 4 U.S. Census regions (https://www.census.gov/prod/1/gen/95statab/preface.pdf). These regions are Northeast (NE): CT, MA, ME, NH, RI, NJ, NY, PA, and VT; Midwest (MW): IA, IL, IN, KS, MI, MN, MO, OH, NE, ND, SD, and WI; South (S): AL, AR, DC, DE, FL, GA, KY, LA, MD, MS, NC, OK, SC, VA, TN, TX, and WV; and West (W): AK, AZ, CA, CO, HI, ID, MT, NM, NV, OR, UT, WA, and WY.2.4To ascertain that the trends observed for PSA testing rate were not due to changes in clinical laboratory testing in general, we determined testing trends for hemoglobin , a test not impacted by any changes in practice recommendations, as a measure of trend in laboratory testing.33.1The number of men included in each year from 2011 through 2017 ranged from 4.55 to 5.14 million after excluding claim encounters with diagnostic or non‐laboratory procedure codes relating to prostate related diseases or conditions, including codes related to elevated PSA and history of prostate cancer. Consequently, 92.6% to 93.1% of men remained for analysis after this exclusion. Table 3.2Annual rate of PSA testing ranged from approximately 1.4% in men age 30‐34 to 38%‐39% in men age 60‐64. Over the period from 2011 to 2017, PSA testing rate decreased by 2.7%, 16.5%, 16.9%, 13.4%, 2.1%, 1.3%, and 2.1%, respectively, for men age 30 to 34, 35 to 39, 40 to 44, 45 to 49, 50 to 54, 55 to 59, and 60 to 64. Table 3.3P = .001), 40 to 44 (P < .001) and 45 to 49 (P = .001). There were no significant trends for PSA testing rate in men age 30 to 34 (P = .11), 50 to 54 (P = .66), and 55 to 59 (P = .997) from 2011 to 2017. For men age 60 to 64, PSA testing rate trended downward from 2011 to 2013 (P = 0.054) and upward from 2013 to 2017 (P = .045). The trends of annual PSA testing rates for men ages 55 to 59 and 60 to 64 are shown in Figures P = .051) and −5.8% (P = .033), respectively; and from 2013 to 2017 it was +0.5% (P = .14) and +1.2% (P = .11), respectively. For the Northeastern region APC was −2.2% (P = .15) from 2011 to 2015; and it was +3.2% (P = .43) from 2015 to 2017. For the Midwestern region APC was +1.2% from 2011 to 2017 (P = .035).Except for PSA testing trend in men age 60 to 64, there was no inflection point in annual testing rate from 2011 to 2017. Annual testing showed significantly downward trends in men age 35 to 39 .None of the seven age groups or the eight age‐group/region combinations showed any inflection point from 2011 to 2017, and the slopes of the trend lines were not significantly different from zero ,The PSA testing rates we have obtained are comparable to those published in previous reports. We are not aware of any studies that have assessed PSA testing rates in men age 30 to 39 as reported here. Furthermore, to our knowledge, no published report has used nationally based claims or electronic data to assess PSA testing rate beyond 2015.P ≤ .001) for these age groups , showing no inflection points, downward trends  were not significant for men age 30 to 34, 50 to 54 and 55 to 59 (P = .11‐.997). For men age 60 to 64, even though PSA testing decreased from 2011 to 2013 , testing rate showed a modestly significant upward trend from 2013 to 2017 . Using the most recently released 100%‐sample database covering claims from January 1, 2012 to April 30, 2019, except for age groups 35 to 39 and 30 to 34  PSA testing rate increased from 2.7% for men age 40 to 44 to 10.7% for men age 60 to 64. These results are consistent with other studies up to 2015 based on the use of electronicWhen comparing PSA testing rates in 2011 to those in 2017 in each of the seven age groups, testing rate consistently decreased, ranging from 1.3% for men age 55 to 59 to 16.9% for those age 40 to 44. The greatest decreases of 13.4% to 16.9% were for the three age groups between 35 and 49. Although PSA testing rate decreased significantly , only about 7% were included in the study. As such, PSA testing rates and trends among uninsured men age 30 to 64 and those receiving government insurance (Medicaid and Medicare) cannot be deduced from these data. Nevertheless, regional distribution of the enrollees in this study reflected well the regional distribution for Americans as reported by the U.S. Census Bureau (data not shown). Temporal changes for specific laboratory tests may be impacted by changes in the utilization rate of laboratory tests in general. We used hemoglobin as a surrogate marker of laboratory test utilization, demonstrating that the testing rate was stable for all age groups and U.S. Census regions (P = .070‐.96). These limitations call for the need to access aggregate, nationally based electronic health data for men age < 65 to determine performed PSA testing rates.Our findings should be considered in view of several limitations of this study. The sample of U.S. men studied is restricted to those with private health insurance coverage. Also, we captured only paid claims data using non‐random MarketScan samples of the U.S. population.Our data show that the impact of the USPSTF recommendation against testingThe authors have stated explicitly that there are no conflicts of interest in connection with this article.Conceptualization, S.S., K.S., L.F., D.S.; Data curation, S.S., K.S., Methodology, S.S., K.S., L.F., D.S.; Investigation, S.S., K.S., L.F., D.S.; Formal Analysis, S.S., K.S.; Project administration, S.S.; Writing ‐ Original Draft, S.S.; Writing ‐ Review & Editing, S.S., K.S., L.F., D.S.; Visualization, S.S., K.S.; Supervision, S.S.; Validation, S.S., K.S.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Instituitional clearance and approval have been obtained prior to submission. Requirement for patient consent is not applicable for this study.


Dimocarpus longan with a novel black D. longan that underwent a thermal ageing process. Pericarp, aril, and seed of both D. longan were macerated in 95% v/v ethanol. Their chemical compositions were investigated using a Folin–Ciocalteu assay, aluminum chloride assay, and high-performance liquid chromatography. Antioxidant activities were evaluated in terms of radical scavenging and iron (III) reduction capacity. An enzyme inhibition assay was used to evaluate the hyaluronidase inhibition. Inflammatory cytokine secretion was evaluated with an enzyme-linked immunosorbent assay. After being exposed to a heating and ageing procedure, gallic acid and ellagic acid content were increased tenfold, while the corilagin content was doubled. Black D. longan seed extract was the most potent anti-hyaluronidase and antioxidant with the strongest free radical scavenging and reduction power, while black D. longan aril extract resulted in the highest inhibition of inflammatory cytokine secretion. Black D. longan contained more biologically active compounds and possessed more potent biological activities than conventional dried D. longan. Therefore, thermal ageing treatment is suggested for producing black D. longan, for which seed extract is suggested as a cosmeceutical active ingredient and aril extract for anti-inflammation.This study is the first to compare the chemical compositions and biological activities of a conventional dried  Dimocarpus longan Lour., a subtropical evergreen plant in the family of Sapindaceae, is widely known as longan. D. longan is cultivated in several countries in East Asia and South-East Asia as well as Australia and some subtropical regions in the US1. China and Thailand are the largest areas of commercial D. longan cultivation2. The succulent and edible aril with delicious flavor and health benefits has led to the increased popularity of D. longan1. Since the aril of D. longan contains several polyphenols, flavonoids, organic acids, and polysaccharides, it possesses various beneficial biological activities, including antioxidant, antiglycation, anticancer, immunomodulatory, prebiotic, anti-osteoporotic, anxiolytic, and memory-enhancing effects3. A decoction of dried aril has been taken as a tonic for insomnia and neurasthenic neurosis treatment since ancient times4. Not only does the aril of D. longan, which is the only edible portion, have reported beneficial effects on health, but the pericarp has also been reported to contain abundant polyphenols, flavonoids, and polysaccharides, which possess antioxidant, anti-tyrosinase, and anti-hyperglycemic activities1. Furthermore, D. longan seed, which is a waste from the food and canning industry, contains antioxidative polyphenols and possesses anti-tyrosinase, antibacterial, and anti-fungal activity7. D. longan seed has also been administered to counteract heavy sweating, whereas the ground kernel has been used for the treatment of various conditions according to their chemical components such as saponin, tannin, and fat8. Although D. longan has been reported to contain a variety of biologically active components and have the potential to be used for the treatment of various conditions, the fruit of D. longan has a short storage life since its pericarp rapidly turns brown and hardens at ambient temperature9. The highly perishable nature and limited shelf life of D. longan fruit not only decreases its marketing value after deterioration but also burdens both domestic distribution and export to foreign countries10. Although a low temperature (1–5 °C) can protect D. longan from pathological degradation, the fruit deteriorates quickly after being removed from cold storage3. Moreover, the over-supply of off-seasonal D. longan production recently led to increasing production costs and a decrease in selling prices10. Sopadang et al. highlighted the issues in the D. longan supply chain and recommended adding value to D. longan and creating a range of D. longan products as improvement alternatives to enhance D. longan supply chain management efficiency10.D. longan are widely consumed and can be distributed worldwide. Generally, dried foods can be kept for a long period, but their sensory and nutritional characteristics are often lost along with the water removal during the drying processes11. Production of intermediate moisture food (IMF) is another technique to overcome this problem since the properties of IMF are close to fresh foods while extending shelf life12. A reduction of the moisture content and a water activity below 0.6 do not support microbial growth and lead to shelf-stable products13. Various types of food have been preserved as IMF, such as meat and several fruits, e.g., grapes, tomatoes, peaches, prunes, apricots, and strawberries14. However, some antimicrobial compounds and additives are required in the production of IMF for antimicrobial properties , along with the agents for water activity reduction and plasticizing, e.g., glycols and sorbitol13.Dried flesh and the canned product of Allium sativum L.), a processed garlic produced by thermal treatment of raw garlic at high temperature and high relative humidity for 60–90 days without using additives16. During the production process, raw garlic also undergoes the Maillard reaction, which occurs between amine groups and carbonyl compounds, finally resulting in brownish melanoidin17. Melanoidin has been shown to have a number of biological actions, including antioxidant, antibacterial, anti-inflammatory, hypoglycemic, hypotensive, and antitumor effects; prevention of obesity; lowering of serum lipopolysaccharide levels; and modulation of the composition of the gut microbiota18. Additionally, inhibition of oxidation and angiotensin I converting enzyme were enhanced in black garlic comparing to raw garlic16.Besides IMF, the heating and ageing process can also prolong the shelf life without refrigeration. A well-known food that undergoes this process is black garlic , followed by dried D. longan aril (17.6% w/w), dried D. longan pericarp (13.8% w/w), black D. longan aril (11.0% w/w), black D. longan seed (6.6% w/w), and dried D. longan seed (3.6% w/w). It was highlighted that black D. longan yielded higher extract content compared to dried D. longan in aril and seed but not in pericarp. The explanation is that the pericarp of dried D. longan lost more water content than that of black D. longan, which underwent a heating and ageing process in conditions that kept the relative humidity constant at 75%. Therefore, the mass of initial pericarp material of dried D. longan was lower and led to a higher extract yield since the yield was calculated from the initial pericarp material used in the extraction process.The external appearance of dried Dimocarpus longan extracts were investigated for contents of total phenolic compounds, total flavonoid, gallic acid, corilagin, and ellagic acid. Gallic acid and corilagin are natural polyphenolic compounds which belong to hydrolysable tannin, whereas ellagic acid belongs to a flavonoid group20. Among different parts of dried D. longan, pericarp extracts contained the significantly highest total phenolic content (p < 0.05) and the highest total flavonoids as shown in Fig. D. longan compared to the D. longan aril1. The total phenolic content of pericarp, seed, and aril extracts from dried D. longan, which were 967.6 ± 31.5, 739.3 ± 62.3, and 229.5 ± 2.6 µg GAE per g extracts, were found to be in agreement with a previous study, which reported that the total phenolic content of D. longan was in the range of 22.09–132.47 mg gallic acid equivalent (GAE/100 g), which was equivalent to 220.9–1324.7 µg GAE per g extract1. Interestingly, the dramatic increase in total phenolic content was observed in black D. longan seed extract. The ethanolic extract from black D. longan seed contained as much as 1827.1 ± 73.1 µg GAE per g extracts, which was much higher than previously reported1. On the other hand, there was no significant difference between the total phenolic content of dried and black D. longan extract from pericarp and aril (p > 0.05).D. longan seed extract also contained the significantly highest flavonoid contents (p < 0.05). Among various dried D. longan extracts, the pericarp contained the significantly highest flavonoid content of 2.8 ± 2.4 µg QE per g extract as shown in Fig. D. longan pericarp was 3.12 ± 0.76 mg/kg, which was equivalent to 3.12 ± 0.76 µg/g extract21. D. longan pericarp has been reported to contain slightly higher content of flavonoids than D. longan seed and aril22. Obviously, the flavonoid content of black D. longan seed extract, which was as high as 13.6 ± 2.5 µg QE per g extract, was dramatically enhanced and was about four times higher than previously reported.In addition to the total phenolic content, black D. longan did not affect the phenolic and flavonoid contents of D. longan pericarp and aril, the total phenolic and flavonoid contents of D. longan seed were obviously enhanced after the production process of black D. longan. The likely explanation might be the formation of biological compounds, which were not originally present in the D. longan seed, during the thermal ageing process such as resistant starch24. During the temperature/time-controlled incubation, the starch inclusion complexes were generated by interaction with other components in the seeds24.Although the thermal ageing process of D. longan extract. The peak of gallic acid, corilagin, and ellagic acid in the HPLC chromatograms were detected at around 3.7, 9.9, and 19.2 min, respectively. The results agreed with the previous studies, which identified these compounds as major polyphenolic components of D. longan pericarp and seed27.As shown in Fig. D. longan extract and dried D. longan extracts. The amounts of each compound in D. longan extracts are shown in Fig. D. longan seed extract contained the significantly highest quantities of polyphenolic compounds and flavonoid contents (p < 0.05).In the present study, the contents of these polyphenolic components were investigated in the context of a comparison between black D. longan fruit, seeds contained the significantly highest content of gallic acid, corilagin, and ellagic acid (p < 0.05) with 5.3 ± 0.0, 8.9 ± 0.1, and 1.9 ± 0.2 mg/g extract, respectively. Interestingly, the these phenolic and flavonoid contents were significantly enhanced after the production process of black D. longan (p < 0.05). The gallic acid, corilagin, and ellagic acid content of black D. longan seed extract were as high as 53.6 ± 0.9, 19.8 ± 2.9, and 24.5 ± 0.7 mg/g extract, respectively. After being exposed to a heating and ageing procedure, the amount of gallic acid and ellagic acid of D. longan were increased by around 10-fold, while the quantity of corilagin was doubled. The reason might be the liberation of free polyphenolic compounds and flavonoids from the bound forms  or the decline in enzymatic oxidation involving in the antioxidant compounds in the raw fruit28. The results of black D. longan were in accordance with those of black garlic, as the total phenolic and total flavonoid contents of the garlic subjected to the thermal processing steps were significantly higher than those of fresh garlic28. The previous study reported that the phenolic content was increased by about 4–10-fold in the black garlic cloves compared with the fresh garlic23.Among various parts of dried D. longan fruit, different methods used in the drying process also affected their bioactive compounds. The thermal ageing process was hence proposed for the enhancement of bioactive compounds in D. longan.Apart from the findings showing differing content of the biologically active component in various parts of D. longan extracts were investigated by two assays with different mechanisms of action. The ABTS assays measure the electron transfer reaction and represent the radical scavenging activity of the tested samples, while the FRAP assay is concerned with the ion reduction process, which represents the ability of the tested compound to convert ferric ions (Fe3+) to ferrous ions (Fe2+)29. The ferric reducing antioxidant power (EC1) and Trolox equivalent antioxidant capacity (TEAC) values of dried and black D. longan extracts are shown in Fig. The antioxidant activities of dried and black D. longan extracts were not significantly different from those for the dried D. longan extracts, except in the aril. The dried D. longan aril extract had no antioxidant activity, whereas the black D. longan aril extract possessed some antioxidant activity with a TEAC value of 4.1 ± 1.4 µg Trolox/mg extract. A probable explanation lies in the greater Maillard reaction, which occurs in the aril as compared with the others. As D. longan aril is composed of glucose, fructose, and various types of amino acids, such as γ-aminobutyric acid, it tends to undergo Maillard reactions, which are the chemical reactions between an amino acid and a reducing sugar that occur in the presence of heat30. These non‐enzymatic browning reactions gave black D. longan a darker color and resulted in the formation of some antioxidant compounds28.The TEAC values of black D. longan pericarp and seed extracts possessed the same radical scavenging activity as those from dried D. longan. A likely explanation might be the degradation of some oxidative compounds during the heating process, although some free polyphenolic compounds and flavonoids were liberated from the bound forms23. Interestingly, the TEAC values of pericarp and seed extracts from both dried and black D. longan were comparable to ascorbic acid, gallic acid, and corilagin (p > 0.05). Ellagic acid was remarked as the most potent radical scavenger (TEAC = 23.4 ± 0.3 µg Trolox/mg), followed by ascorbic acid (TEAC = 12.3 ± 0.0 µg Trolox/mg), gallic acid (TEAC = 12.8 ± 0.2 µg Trolox/mg), and corilagin (TEAC = 12.7 ± 0.1 µg Trolox/mg). Thereby, ellagic acid was found to be the main compound responsible for the free radical scavenging activity of D. longan extracts together with gallic acid and corilagin31. Although the previous study reported that among various polyphenolic compounds, tannins demonstrated the strongest ABTS·+ radical scavenging activity32, in the present study it was observed that ellagic acid, which belongs to a flavonoid group, was more potent than gallic acid and corilagin, which belongs to hydrolysable tannin1. Furthermore, D. longan extracts from both pericarp and seed could therefore be considered as natural extracts with potent radical scavenging activity.On the other hand, black D. longan extracts also possessed a reduction ability as shown in Fig. D. longan extracts was in accordance with their phenolic and flavonoid contents. Gallic acid possessed the significantly highest EC1 value of 237.0 ± 1.6 mM FeSO4/mg, which was comparable to that of ascorbic acid (238.3 ± 0.2 mM FeSO4/mg), followed by corilagin (226.2 ± 2.9 mM FeSO4/mg) and ellagic acid (192.3 ± 0.7 mM FeSO4/mg). However, both phenolic compounds and flavonoids were responsible for their reduction capacity33. The black D. longan seed extract, which contained the highest total phenolic, total flavonoid, gallic acid, corilagin, and ellagic acid contents, thus possessed the significantly highest reduction ability with an EC1 value of 150.0 ± 1.0 mM FeSO4/mg extract (p < 0.05). Consequently, the black D. longan seed extract was suggested as the most potent antioxidant extract with the strongest free radical scavenging and reduction ability. Because of its potent antioxidant effect, black D. longan seed has been proposed as a natural antioxidant source for use in food and cosmetic products. Since the portions of pericarp and seed account for 30% of the whole fruit dry weight34, the utilization of these by-products would not only reduce the agricultural waste product but also increase its value.Aside from radical scavenging activity, 35, of dried and black D. longan extracts were investigated. RAW 264.7 macrophage cells were used in the present study since they can secret these cytokines after the stimulation of LPS. The RAW 264.7 macrophage cell viability after treatment with dried and black D. longan extracts is shown in Table D. longan extracts since the cell viability was more than 100%. Dexamethasone, corilagin, gallic acid, and ellagic acid were also found to be safe for RAW 264.7 macrophage cells.The inhibitory activities against the secretion of IL-6 and TNF-α, which are key players involved in the age-related inflammatory processD. longan extracts are shown in Fig. 37. Among various parts of D. longan fruit, aril of both dried and black D. longan was predominant in IL-6 and TNF-α inhibition. Gallic acid was suggested to be the main compound responsible for both IL-6 and TNF-α inhibitory activities. In contrast, corilagin was responsible only for TNF-α inhibition. Although D. longan extracts and their major chemical components exhibited only low to moderate anti-inflammatory activities compared to dexamethasone, a corticosteroid used in the treatment of inflammation, they could be consumed as natural anti-inflammatory supplements with no steroidal side effects.The IL-6 and TNF-α inhibitory activities of dried and black 38. Hyaluronan plays a pivotal role in the maintenance of the elastoviscosity of liquid connective tissues and controls the water transportation related to the tissue hydration39. The degradation of hyaluronan resulting in the production of breakdown products, including lower molecular mass polymers. These breakdown products of hyaluronan exhibited distinct biological properties from the larger precursor molecules40. The hyaluronan depolymerization occurs in tissue injury and initiates the inflammatory response38. Additionally, hyaluronan is known as a lubricant and shock-absorber in joints and connective tissues41. Its degradation hence leads to the deterioration of the viscoelastic properties of the synovial fluid42.Hyaluronidase, a homologous enzyme that hydrolyzes or depolymerizes hyaluronan, plays an important role in the modulating activity of many pathological processesD. longan extracts are shown in Fig. D. longan extracts exhibited low anti-hyaluronidase activity, the inhibitory effect of black D. longan seed was significantly enhanced compared to the dried D. longan seed extract. Since the anti-hyaluronidase activity of black D. longan seed extract (18.4 ± 2.0%) was the most significantly potent (p < 0.05), black D. longan seed extract could be suggested to have anti-hyaluronidase activity in addition to its antioxidant activities.The inhibitory activities against hyaluronidase of dried and black D. longan was successfully developed after undergoing a heating and ageing procedure at a controlled temperature of 70 °C and a relative humidity of 75%. A novel black D. longan contained a larger quantity of biologically active compounds and possessed more potent biological activities than a conventional dried D. longan. The ethanolic extract from the seed of black D. longan contained the most significantly abundant of biologically active compounds, including total phenolic, total flavonoid, gallic acid, corilagin, and ellagic acid content (p < 0.05). Furthermore, it possessed the most significantly potent antioxidant and anti-hyaluronidase activities (p < 0.05). Since oxidative stress is known to be related to ageing and skin wrinkles, black D. longan seed extract with a potent antioxidant activity (p < 0.05) was suggested for further topical use as a cosmeceutical ingredient for anti-skin ageing. On the other hand, degradation of hyaluronan in the skin resulted in the loss of the skin’s natural moisturizing factor, while the loss of hyaluronan from the synovial fluid in joint resulted in joint pain and several conditions. Therefore, black D. longan seed extract, which significantly inhibited hyaluronidase activity (p < 0.05), is suggested for both topical use for anti-skin ageing and joint pain relief. On the other hand, the aril of D. longan, which possessed the significantly highest anti-inflammatory activities, is suggested as a natural edible anti-inflammatory agent.In conclusion, black l-Ascorbic acid, aluminum chloride (AlCl3), 2,2′-azino-bis3-ethylbenzothiazoline-6-sulfonic acid (ABTS), calcium chloride (CaCl2), corilagin, dexamethasone, disodium phosphate (Na2HPO4), ferric chloride (FeCl3), ferrous chloride (FeCl2), ferrous sulfate (FeSO4), formic acid, Folin–Ciocalteu reagent, 4,4′,5,5′,6,6′-hexahydroxy-diphenic acid 2,6,2′,6′-dilactone (ellagic acid), hyaluronic acid, hydrochloric acid (HCl), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), bovine testicular hyaluronidase (E.C.3.2.1.3.5), potassium acetate (CH3COOK), potassium persulphate (K2S2O8), sodium acetate (C2H3NaO2), sodium carbonate (Na2CO3), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH2PO4), sodium phosphate (Na3PO4), 2,4,6-tripyridyl-striazine (TPTZ), and 3,4,5-trihydroxybenzoic acid  were analytical grade and purchased from Sigma-Aldrich . Amphotericin B, Dulbecco's modified Eagle's medium, high glucose (DME/HIGH), l-glutamine, penicillin/streptomycin, trypan blue, and secondary antibody conjugated with HRP were bought from Invitrogen . Lipopolysaccharides (LPS) were bought from Cell Signaling Technology . GlutaMAX-I supplement was bought from Thermo Fisher Scientific, Inc. . Newborn bovine calf serum , fetal bovine serum (FBS) , and bovine serum albumin (BSA)  were bought from Gibco . Analytical grade acetic acid, ethanol, and dimethyl sulfoxide (DMSO) were purchased from Labscan, Ltd. . HPLC-grade acetonitrile was purchased from Labscan, Ltd. .D. longan fruits were collected from Chiang Mai Province in Northern Thailand in January 2020 by the gardeners according to WHO Guidelines on Good Agricultural and Collection Practices (GACP) for Medicinal Plants43. The permission to collect D. longan fruits were obtained from the farm owner. The plant materials were identified and authenticated by Ms. Wannaree Charoensup, a botanist at the Herbarium of Faculty of Pharmacy, Chiang Mai University. A voucher specimen number 0023268 of D. longan has been deposited in an Herbarium, Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University. The preparation of conventional dried and black D. longan was performed by the Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, Thailand.The D. longan was obtained after the whole fruit of fresh D. longan was incubated in an oven set at the temperature of 50 °C until dryness (moisture content below 16–18%). The sample of dried D. longan was kept in a sealed plastic bag to prevent contact with air and humidity in the room temperature until further experiments.Conventional dried D. longan was obtained after the whole fruit of dried D. longan was incubated for 20 days at a controlled temperature of 70 °C and 75% relative humidity. The sample of black D. longan was kept in a sealed plastic bag and placed in a desiccator chamber cabinet to prevent contact with air and humidity at room temperature until further experiments.Black D. longan fruit was ground into fine powder using a 20-in. herbal medicine grinder tub with a powerful motor . Dried D. longan powder was subsequently macerated in 95% v/v ethanol for three cycles of 24 h. The proportion of plant material to solvent was 1:5 by weight. The same protocol of maceration was used for each part of dried and black D. longan. The extracting solvent from three cycles was combined and removed using a rotary evaporator . All extracts were stored in the refrigerator  was mixed with 180 μL of Folin–Ciocalteu reagent (10% w/v). After incubation at ambient temperature for 4 min, 80 μL of sodium carbonate solution  was added. After the resulting mixtures were incubated for 2 h. they were measured for an absorbance at 750 nm using multimode detector . The results were presented in the form of gallic acid equivalent values (GAE) representing an amount of gallic acid (µg) per g of the D. longan extracts. GAE was calculated following Eq.  was mixed with 80 μL of AlCl3 aqueous solution  and 100 μL of CH3COOK aqueous solution . After the resulting mixtures were incubated for 30 min in the dark, they were measured for an absorbance at 415 nm using a multimode detector . Quercetin was applied as a standard compound to construct a calibration curve. Finally, the results were presented as quercetin equivalent (QE) values, which represented a µg of quercetin per g of the D. longan extracts. QE was calculated following Eq. , ellagic acid (5–100 µg/mL), and corilagin (2–80 µg/mL) solution were used for the construction of standard curves for quantitative determination. Subsequently, the content of gallic acid, corilagin, and ellagic acid was then calculated using the following Eqs. . A gradient mobile phase system composed of two phases was used, including phase A (0.05% formic acid in acetonitrile) and phase B (0.05% formic acid aqueous solution). The program was set for gradient elution of 10% A (0–8 min), 20% A (8–28 min), 30% A (28–30 min), and 10% A (30–35 min), eluting the sample at a flow rate of 1.0 mL/min. The UV detector was set at 280 nm with a Eurospher II 100-5 C18 . All samples, standard solution, and mobile phase were filtrated through a 0.45 mm Millipore filter, type GV  before injection into the HPLC system. The injected volume was set at 20 μL. The sample of + of D. longan extracts, gallic acid, corilagin, and ellagic acid were evaluated using the ABTS assay and reported in terms of Trolox equivalent antioxidant capacity (TEAC), which represents the quantity of Trolox that equivalent to 1 mg of the D. longan extracts46. Firstly, 20 μL of the ethanolic solution of D. longan extracts (1 mg/mL) was mixed with the mixture of 72 μL ABTS solution  and 108 μL potassium persulfate solution , which had been previously mixed and incubated in the dark for 16 h at ambient temperature. After the resulting mixtures were incubated for 5 min, they were measured for an absorbance at 750 nm using multimode detector . TEAC values were calculated following Eq.  was mixed with 150 μL acetate buffer pH 3.6 (0.3 M), 15 μL TPTZ solution  in HCl , and freshly prepared 15 μL FeCl3 . After incubation at ambient temperature for 4 min, 80 μL of sodium carbonate solution  was added. After the resulting mixtures were incubated for 5 min, they were measured for an absorbance at 595 nm using multimode detector . The ferric reducing/antioxidant power of each D. longan extract was expressed in the form of equivalent concentration (EC1), representing the ferric-TPTZ reduction capacity, which is equivalent to 1 mg of the D. longan extract. The EC1 values were calculated following Eq. , penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL) were seeded in wells of 24 well-plates and incubated in a CO2 incubator  for 24 h. Thereafter, 1 µL of the D. longan extracts or dexamethasone (100 µg/mL) was added and further incubated in a CO2 incubator . Three replicates per sample were performed. After 24 h of the extract treatment, 250 μL of LPS in DMEM (4 µg/mL) was treated in each well and incubated in a CO2 incubator  for another 24. After that, the treated cells along with the supernatant were divided into two parts. The medium (500 μL) was collected for cytokine dosages, while the attached cells were subjected to the viability assay using MTT dye. The collected medium was then centrifuged for 10 min at 13,500×g, and the supernatant was investigated for cytokine secretion using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s protocol (R&D Systems). The remain medium, which was left over in the wells, was investigated for cell viability using a 3--2,5-diphenyltetrazolium bromide (MTT) assay. To reduce variation due to cell density differences, secretion of IL-6 and TNF-α from RAW 264.7 cells was normalized to MTT levels48. RAW 264.7 cells without lipopolysaccharide treatment served as a negative control, while 100% of cytokine secretion was from the positive control of RAW 264.7 cells treated with LPS. The inhibitory activities of each sample were calculated following Eq. (Murine monocyte-macrophage (RAW 264.7) cells  treated with LPS were used to investigate the effect of D. longan extracts, gallic acid, corilagin, and ellagic acid was evaluated by measuring a product from the cleavage of sodium hyaluronate by hyaluronidase49. Prior to the experiment, the enzyme activity of hyaluronidase was determined. More than 90% enzyme activity was used in the anti-hyaluronidase activity determination. Firstly, 20 μL of the ethanolic solution of D. longan extracts (1 mg/mL) was mixed with hyaluronidase (15 unit/mL). After incubation at 37 °C for 10 min, hyaluronic acid solution (0.03 % w/v) in phosphate buffer pH 5.35 was added and further incubated for 45 min. Immediately after the addition of acid bovine serum albumin, the extracts were measured for an absorbance at 600 nm using a multimode detector . The hyaluronidase inhibitory activity was calculated according to Eq.  followed by Tukey's post-hoc tests using the SPSS software . A value of p < 0.05 was accepted as significant.


Apis mellifera L.) venom (HBV) has been determined by HPLC and FTIR-ATR spectroscopy, and tested for antibacterial activity, as well as efficiency with regard to conventional antibiotics. The investigated HBV was of high quality with melittin and total protein contents of 70.10 ± 7.01%, and 84.44 ± 3.12 g/100 g, respectively. The purity of HBV was confirmed by FTIR-ATR spectral profiling, which revealed a unique pattern of absorption bands that are characteristic of its major fractions. In addition, HBV showed a broad spectrum of activity against all three tested biomasses of potentially pathogenic Gram-positive and Gram-negative bacteria with MIC values ranging between 12.5 and 200 µg/mL, and MBC between 12.5 and 400 µg/mL. When compared to conventional antibiotics, HBV (400 µg) showed up to 27.8% efficiency of tetracycline (30 µg), 52.2% erythromycin (15 µg), 21.2% ciprofloxacin (5 µg), and 34.6% of ampicillin-sulbactam (20 µg). The overall results demonstrate the therapeutic potential of the analyzed HBV.The incidence of antibiotic resistance in pathogenic bacteria has become an alarming clinical and social problem. Therefore, the demand for alternative antimicrobial compounds has increased. In this study, a chemical profile of honey bee ( Since the discovery of antibiotics, their uncritical application and overuse have led to the development of antimicrobial drug resistance in numerous bacterial pathogens. Such bacteria are becoming a serious clinical and social problem throughout the world. Thus, new effective antibacterial agents with a novel mode of action need to be developed . AntimicApis mellifera L.) venom (HBV), secreted in honey bee workers’ venom glands as a protection mechanism, has long been used to treat an array of medical conditions [Natural products such as venoms from different animals  are an important source of such new bioactive peptides ,5 and prnditions ,8. It isnditions ,10,11,12nditions ,14,15,16nditions ,18.The major compound and the principal bioactive peptide with antimicrobial activity in HBV is mellitin, which comprises up to 60% of the dry HBV weight . The antStaphylococcus aureus, Listeria innocua, and Bacillus cereus), and Gram-negative  bacteria by determining the minimum inhibitory (MIC) and bactericidal (MBC) concentrations, and compare it with the conventional antibiotics. In addition, we aimed to determine the quality and purity of the HBV sample prior to microbiological assay by performing a detailed chemical profiling using classical, chromatographic and spectroscopic analytical methods.Therefore, the objective of this study was to evaluate the antibacterial activity of honey bee venom against three different biomass quantities  of selected potentially pathogenic Gram-positive  are not only the most abundant and the most studied compounds of honey bee venom ; they ar−1 (with an absorption maximum at 3288 cm−1), which is assigned to the N–H stretching vibrations (amide A band) of the peptide and protein secondary structures [−1 (amide B). The latter may also indicate an overtone of the amide II band [−1 and 2928 cm−1 correspond to CH2 asymmetric stretching vibrations, and they are followed by the lower intensity analyte signals at 2873 cm−1 and 2855 cm−1 that are attributed to CH2 symmetric stretchings.The results of the spectral analysis revealed a characteristic FTIR-ATR spectrum of honey bee venom powder distinguished by a unique pattern of absorption bands primarily arising from molecular vibrations related to the structure of honey bee venom’s major fractions—peptides and proteins . Given tructures ,39, whil II band . Medium −1 (fingerprint region) is populated by a series of absorption bands that are unique to the peptide/protein secondary structure. The most prominent vibrations in this region, occurring at 1645 cm−1 and 1537 cm−1, are assigned to the amide I (C=O stretching) and amide II (N–H bending and C–N stretching vibrations) bands, respectively. This is in compliance with the report on the basic spectral features of the bee venom provided by Park et al. [−1 are likely to result from the protein side chain COO−. A broad medium intensity absorption occurs between 1290 and 1240 cm−1 due to the amide III band, which comprises 30% of C–N stretching, 30% of N–H bending, 10% of C–O stretching, and 10% of O=C–N bending vibrations, while the rest belongs to other vibrations [−1 can be attributed to the C–H in-plane deformation vibration of various aromatic structures in the bee venom. A weak analyte signal occurs nearby at 805 cm−1 due to the symmetric CNC stretching vibration of proteins (enzymes). Amide IV (primarily O=C–N bending) and amide V (N–H bending) bands have been observed at 660 cm−1 and 730 cm−1, respectively.The spectral region between 1700 and 700 cmk et al. . Other abrations . A mediu−1 region) in the dried form of HBV compared to a liquid form of genuine bee venom, and an overlapping of the H–O–H deformation of water with the amide I band at 1645 cm−1. This was followed by higher absorption intensities of the analyte signals of other peptide/protein-based venom components observed in dried HBV. Given that no analyte signals other than those specific for HBV proteins were observed, these results confirmed the purity of dried bee venom used for the microbiological assay.The antibacterial activity of HBV was determined by evaluating minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations against different biomass quantities  of selected Gram-positive and Gram-negative bacteria. The MIC values were calculated as the concentrations that inhibited more than 95%, and MBC more than 99%, of bacterial growth. The results are summarized in Both MIC and MBC values correlate with the initial biomass of the pathogen, where, with an increase in bacterial density, higher concentrations of HBV were needed for bacteriostatic and bactericidal effects. At lower and medium initial biomass levels, similar HBV concentrations  suppressed the growth or completely inhibited  all tested pathogens. When biomass was extremely high (8 log CFU/mL), high HBV concentrations were needed to inhibit (25.0–200.0 µg/mL) or to completely kill (50.0–400.0 µg/mL) all tested bacterial strains.E. coli at 5 µM, and different Bacillus species at 0.3–0.4 µM, while Ebbensgaard et al. [E. coli at 16 µg/mL, and both S. aureus and L. monocytogenes at 2–4 µg/mL. On the contrary, Ebbensgaard et al. [Salmonella enterica (32–64 µg/mL).In the studies of Oren and Shai , Han et d et al.  showed td et al.  determinS. aureus, and from 1800 to 3800 µg/mL for E. coli, while Han et al. [S. aureus. Such differences can be attributed to the extraction method and additional venom purification, resulting in different HBV purity and mellitin content values. For example, while Hegazi et al. [Contrary to our study, Hegazi et al.  reportedn et al. , on the i et al.  obtainedi et al.  further E. coli was 0.25 µg/mL and for S. aureus 0.06 µg/mL. On the contrary, Al-Ani et al. [S. aureus (10–60 µg/mL) and E. coli (60–200 µg/mL). Such differences could be explained by different bacterial species and strains, and different initial biomass used. Although our results are in line with previous studies as the lowest MIC value in our study was 12.5 μg/mL, to increase the resolution, especially at the lower range, HBV concentrations < 12.5 μg/mL should be tested.Similarly to our study, Han et al.  investigi et al.  detectedL. innocua, S. enterica subsp. enterica and E. coli treated with HBV could only be detected after 48h; thus, the true HBV killing potential could be determined only after prolonged incubation. L. innocua, S. enterica subsp. enterica and E. coli are known for forming biofilms [Furthermore, in the case when bacterial growth was detected in our study only in the control wells after 24 h, the incubation was prolonged, and MIC and MBC values were determined after 48 h. The growth of biofilms ,44, whicbiofilms .B. cereus, most likely due to its ability to sporulate [Generally, the MBC values for all tested pathogens were either the same or double when compared to MIC values, except for porulate  and survAntibacterial activity of HBV (100–400 µg) and conventional antibiotics (tetracycline (TE 30 µg) and erythromycin (E 15 µg) for Gram-positive bacteria, and ciprofloxacin (CIP 5 µg) and ampicillin-sulbactam (SAM 10 µg + 10 µg) for Gram-negative bacteria) was determined by the disc diffusion method. The sensitivity of all pathogens to HBV was achieved at 100 µg for Gram-negative and 300 µg for Gram-positive bacteria. Increased concentrations of HBV, up to 400 µg, were applied in order to achieve saturation. The results are summarized in S. aureus; in addition, 25.5 ± 1.7% TE efficiency and 47.8 ± 1.0% E efficiency towards B. cereus, and 19.9 ± 0.2% TE efficiency and 21.4 ± 0.3% E efficiency towards L. innocua, were achieved.All pathogens were sensitive to conventional antibiotics and their sensitivity to HBV increased with the HBV concentration. Only minor differences were observed between 300 and 400 µg of HBV, indicating that saturation in HBV concentration was achieved. At HBV concentrations of 300 µg, 29.1 ± 0.4% TE efficiency and 33.4 ± 0.1% E efficiency were reached when applied to E. coli was more sensitive to HBV than S. enterica. More specifically, at HBV concentrations of 100 µg, 15.3 ± 0.3% CIP and 25.0 ± 0.0% SAM efficiency when applied to E. coli, and 13.8 ± 1.8% CIP and 22.6 ± 2.7% SAM towards S. enterica, was reached.At HBV concentrations of 100 µg, S. aureus was the most sensitive to the HBV. In general, Gram-positive pathogens were found to be more sensitive to HBV than Gram-negative, [The disc diffusion method showed that egative, ,47,48. Degative, ,35,49. Tegative, .Furthermore, the antimicrobial activity of HBV at the highest tested concentration of 400 µg showed a considerable efficiency of conventional antibiotics. For example, HBV showed up to 27.8% TE and 52.2% E efficiency when applied to Gram-positive bacteria, and up to 21.2% CIP and 34.6% SAM in Gram-negative bacteria. Some studies have revealed that the antimicrobial activity of HBV increases when applied in combination with conventional antibiotics ,34, wherApis mellifera carnica Pollman, 1879) colonies (pooled sample) by an electro-stimulation method. An internal bee venom collector, placed at the top of the 10-frame Langstroth hives under a dome, was powered by a 12-volt battery. Honey bees were stimulated with electric impulses (12 V) for 10 s followed by 10 s pauses. During the electro-stimulation, the bee workers stung the glass plates through a plastic foil. Venom was left to dry at 27 °C and a relative humidity of 50% for 35 min and collected by scraping the dried HBV (powder) from the glass slide of the venom collector using a sharp scraper. Dried HBV was stored in the dark and dry place at 4 °C prior to further analyses.Honey bee venom was obtained directly from the primary producer with an apiary situated in Bjelovar-Bilogora County (Croatia) during 2018. HBV was collected from 100 Carniolan honey bee , and the result was expressed in percentage (%). The standards were supplied by Sigma-Aldrich. The analysis was performed in triplicate and the results are expressed as mean values with standard deviation.A modified version of the reversed-phase chromatography method of Rybak-Chmielewska and Szczęsna  was appl−3 HCl (volumetric standard) and used for titration of the flask content after distillation in a Kjeltec system. At the endpoint of the titration, the color of the solution became pale pink. The volume of hydrochloric acid for the titration of the sample was recorded. The analysis was performed in triplicate and the results were expressed as mean values with standard deviation. Nitrogen content (%) was calculated according to the Equation (1):a is the volume of HCl spent for titration of the sample (mL), b is the volume of HCl spent for titration of blank (mL), c is the concentration (mol/L), f is the acid factor, and m is the sample weight (g).Protein (nitrogen) content was determined according to the standard ISO 1871:2009   spectroscopy in order to provide an insight into the total chemical composition and ensure the purity of investigated HBV sample.−1) using a nominal resolution of 4 cm−1 (at 23 ± 2 °C). Two replicate spectra of the sample (32 scans per spectrum) were recorded using different aliquots. Raw spectral data were stored and pre-analyzed using the Agilent Resolutions Pro version 5.3.0 software package , and further spectral data analysis and processing were carried out using Origin version 8.1 (Origin Lab Corporation). An assignation of molecular vibrations observed in the bee venom spectrum was performed using electronic spectral libraries  in combination with venom-relating spectral data from the available scientific literature.Dried HBV sample (powder) was analyzed by Fourier transform infrared spectroscopy (FTIR) coupled with the Attenuated Total Reflectance (ATR) recording technique to provide an insight into its total chemical composition. Infrared (IR) spectra of the bee venom sample were acquired using the Cary 660 Fourier FTIR spectrometer  and the Golden Gate single-reflection diamond ATR accessory (Specac). The sample was analyzed as obtained (it was only pulverized into a finer powder with a porcelain mortar). Approximately 50 mg of bee venom powder was used to acquire the spectra of a thin uniform layer of the sample. This was achieved by pressuring the bee venom powder on a diamond ATR plate using a self-leveling sapphire anvil. Absorption spectra were recorded in a mid-infrared region , the spectra of analyzed bee venom powder were compared with a spectrum of genuine bee venom sample collected directly from a 24-day old worker bee (forager) in a liquid form using a 10 µL microcapillary tube sting stimulation .S. aureus subsp. aureus (DSM 20231), B. cereus (DSM 6791) and S. enterica subsp. enterica (DSM 14221) from the German Collection of Microorganisms and Cell Cultures, and L. innocua (ATCC 3309) and E. coli (ATCC 25922) from American Type Culture Collection. All isolates were stored as glycerol (25%) culture at −20 °C in the Department of Microbiology, University of Zagreb Faculty of Agriculture.The bacterial strains used in this study were 8 CFU/mL) was achieved. Cell suspensions were then diluted in sterile saline solution (0.85%) to obtain target biomass.The minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of HBV were determined by broth microdilution according to the Clinical and Laboratory Standards Institute reference method M27-A3  in 96-wet is the absorbance of MH + HBV + bacterial suspension measured after 24 or 48 h, A0 the absorbance of MH + HBV + bacterial suspension measured at the beginning of the experiment (0 h), Act the absorbance of MH + bacterial suspension measured after 24 or 48 h, and Ac0 the absorbance of MH + bacterial suspension measured at the beginning of the experiment (0 h).HBV was dissolved in distilled water and then filtered through a membrane filter  to obtain a working solution of HBV (1000 µg/mL). HBV was serially diluted two-fold in Mueller–Hinton broth in microtiter plates, to test the HBV concentration range 12.5–800 µg/mL. All tested pathogens  were incubated with two-fold serial dilutions of HBV, at 37 °C for 24 or 48 h under constant agitation . The experiment was performed in triplicates. The bacterial growth was measured by the spectrophotometric assay as turbidity at 660 nm wavelength . The bacterial growth inhibition was calculated according to Equation (2):8 CFU/mL). A sterile cotton swab was used for spreading diluted cell suspension on Mueller–Hinton  agar plates. Sterile blank paper discs  were then placed on the MH agar’s surface, and HBV  was added to each disc. Three replicates were made for each treatment. The results were presented as a mean inhibition zone, including the diameter of the disc, in mm.The antibacterial effects of HBV and conventional antibiotics were tested by using disc diffusion assays ,56. PureAntibiogram disks including tetracycline  and erythromycin  for Gram-positive isolates, and ciprofloxacin  and ampicillin-sulbactam  for Gram-negative isolates, were added. The plates were incubated at 37 °C for 24 h, and the zones of inhibition were measured. The results were calculated as mean inhibition zone in mm, and as a percentage (%) of antimicrobial activity compared to the antimicrobial activity of tetracycline and erythromycin for Gram-positive isolates, and ciprofloxacin and ampicillin-sulbactam for Gram-negative isolates.p < 0.01 were considered significant. All statistical analyses were performed in R environment version 3.0.2 [All data were expressed as mean values ± standard deviation of the mean (SD). Statistical differences between groups were determined using Analysis of Variance (ANOVA) followed by the post-hoc Tukey HSD test. Differences with on 3.0.2 .Staphylococcus aureus, Listeria innocua, Bacillus cereus, Escherichia coli and Salmonella enterica) was observed, irrespective of the bacterial biomass applied . The MIC values ranged between 12.5 and 200 µg/mL , and MBC values ranged between 12.5 and 400 µg/mL . When compared to conventional antibiotics, HBV (400 µg) showed up to 27.8% efficiency of tetracycline (30 µg), 52.2% erythromycin (15 µg), 21.2% ciprofloxacin (5 µg), and 34.6% of ampicillin-sulbactam (20 µg). The results presented in this study revealed above-average high-quality properties of investigated honey bee venom, and indicate that HBV is a good source of alternative antimicrobial compounds.The chemical composition, purity, and antibacterial activity of honey bee venom (HBV) obtained directly from the primary producer were investigated in this study. The analyzed HBV showed above-average high-quality properties with melittin and total protein contents of 70.10 ± 7.01%, and 84.44 ± 3.12 g/100 g, respectively. The results of the spectral analysis revealed a characteristic FTIR-ATR spectrum of HBV powder distinguished by a unique pattern of absorption bands primarily arising from molecular vibrations related to the major fractions of HBV—peptides and proteins. Besides high melittin and protein content, the broad spectrum of antibacterial activity of the investigated HBV against all tested potentially pathogenic Gram-positive and Gram-negative bacteria (


LOW) in mL/min/g were related to reference perfusion measurements (PREF) through regression by PLOW = 1.04 PREF − 0.08 . The Morphological and physiological assessment of coronary artery disease (CAD) is necessary for proper stratification of CAD risk. The objective was to evaluate a low-dose cardiac CT technique that combines morphological and physiological assessment of CAD. The low-dose technique was evaluated in twelve swine, where three of the twelve had coronary balloon stenosis. The technique consisted of rest perfusion measurement combined with angiography followed by stress perfusion measurement, where the ratio of stress to rest was used to derive coronary flow reserve (CFR). The technique only required two volume scans for perfusion measurement in mL/min/g; hence, four volume scans were acquired in total; two for rest with angiography and two for stress. All rest, stress, and CFR measurements were compared to a previously validated reference technique that employed 20 consecutive volume scans for rest perfusion measurement combined with angiography, and stress perfusion measurement, respectively. The 32 cm diameter volumetric CT dose index ( Computed tomography angiography (CTA) is a powerful tool for coronary artery disease (CAD) risk stratification. Nevertheless, CTA only assesses the morphological severity of segmental CAD and cannot define the physiological severity of concurrent multi-vessel, diffuse, and microvascular disease, or myocardial scar. Hence, additional physiological assessment with single-photon emission computed tomography, stress echocardiography, cardiac magnetic resonance, static positron emission tomography (PET), or static CT is recommended for better stratification of patient risk. However, such modalities only provide metrics of relative perfusion or scar extent; hence, they cannot determine the true severity of CAD –8.Fortunately, the spatial distribution of absolute stress perfusion in mL/min/g and coronary flow reserve (CFR), defined as the ratio of stress to rest perfusion, can be used to overcome these limitations by enabling localization and delineation of focal, diffuse, and microvascular disease, as well as scar , 9. WhilThere is a major unmet clinical need for an accurate, low-dose CT technique capable of combined morphological and physiological assessment of CAD. Enabled by wide-detector CT, our prior research has sought to provide a solution. First, we developed a new dynamic CT perfusion technique, validating that global perfusion measurement was feasible in an idealized phantom model of the heart using only two volume scans, with an ultrasonic flow probe as the reference standard . Next, wAVE) within the entire myocardium, modelled as a single compartment, is proportional to the rate of contrast mass entry over the measurement time , normalized by the average blood pool contrast concentration  and total myocardial tissue mass , assuming measurements are made prior to contrast outflow. Of importance, dMC/dt and Cin are not directly measured but are instead linearly related to the tissue and blood enhancement in Hounsfield Units (HU) by the same constant. As this constant cancels in ratio in Eq. AVE is also proportional to the average change in contrast concentration, represented by the change in enhancement, ΔHUAVE, in the entire myocardium over the measurement time. Given this derivation, only two whole-heart volume scans, V1 and V2 shown in Fig. C/dt is calculated by summating all voxel values (in HU) within the myocardium of both the V1 and V2 volume scans, determining their difference, then dividing by time . Similarly, ΔHUAVE is calculated by averaging all voxel values (in HU) within the myocardium of both the V1 and V2 volume scans and determining their difference. Cin is defined as the central enhancement of blood pool in the aortic root in HU averaged between V1 and V2. Finally, MT is defined as the density of myocardial tissue  multiplThe study, approved by the Institutional Animal Care and Use Committee at the University of California Irvine, was performed on twelve male Yorkshire swine (41 ± 11 kg) between February and December of 2017. Of the twelve animals, three had sub-occlusive balloon stenosis in the left anterior descending (LAD) coronary artery. Of additional note, the stress data from eight of the twelve animals (including the three with stenosis) were used in a prior study . Each anAnesthesia was induced with Telazol (4.4 mg/kg), Ketamine (2.2 mg/kg), and Xylazine (2.2 mg/kg), and was maintained with 1.5–2.5% Isoflurane . Sheaths were placed  in both femoral veins for intravenous adenosine and contrast administration. In three of the animals, an extra sheath was placed in the right carotid artery to pass a Judkins Right (JR) catheter  into the left coronary ostium. A pressure wire  was then advanced into the distal LAD. A balloon was passed over the wire into the mid LAD and was used to generate several levels of sub-occlusive stenosis with FFR  severities of 0.7–0.9 under maximal vasodilation . Beta blockers were not administered for heart rate control and nitroglycerin was not administered during CTA.For rest perfusion conditions, contrast  was injected  followed by a saline chaser (0.5 mL/kg) at the same rate. Twenty consecutive whole-heart volume scans were then acquired during diastole using prospective ECG-gating. All scanning was performed in CINE mode at 100 kVp and 200 mA, and all volumes were acquired as full projections. For stress perfusion conditions, maximum vasodilation was induced for two minutes prior to imaging, and was maintained during acquisition, where contrast injection and volume scan acquisition remained the same as above. All volume scans had a 0.35 s rotation time and 320 × 0.5 mm collimation for a total of 16 cm of z-axis coverage. The 32 cm diameter volumetric CT dose index  were calculated as the integrated change in HU within the compartment between corresponding volume scans (dMC/dt) normalized by the blood pool contrast concentration  and myocardial tissue mass . The average compartmental perfusion was then combined with the average and per-voxel changes in HU (ΔHUAVE and ΔHU) between volume scans to yield voxel-by-voxel rest perfusion, stress perfusion, and CFR measurements. Finally, Vitrea was used for accurate semi-automatic extraction of the LAD, LCx, and RCA centerlines from the V2REST CTA volume scan  [For each acquisition, all volume scans were first reconstructed from full projection data at 75% of the R-R interval using AIDR 3D reconstruction   , 27, andnka, MN) –20, 28. Using Shapiro–Wilk testing, all rest and stress perfusion measurements were first verified to be normally distributed. The measurement variance within each animal was then assessed as compared to the measurement variance between each animal, resulting in an intra-cluster correlation of 0.49, indicating minimal correlation between intra-animal measurements; hence, all measurements were assumed to be independent for the remainder of analyses. Low-dose perfusion measurements in the LAD, LCx, and RCA were quantitatively compared to corresponding reference standard perfusion measurements through regression, Bland–Altman, root-mean-square-error (RMSE: accuracy as compared to the reference standard), root-mean-square deviation (RMSD: precision as compared to the regression fit), and Lin’s concordance correlation coefficient (CCC). Student’s T-tests were also performed to compare low-dose perfusion and CFR measurements to corresponding reference standard perfusion and CFR measurements. All data are reported with 95% confidence intervals. All other data are reported as mean ± standard deviation. P-values less than 0.05 indicate significant differences. Statistical software was used for analyses .The average rest and stress heart rates of the swine were 83 ± 8 and 99 ± 7 beats per minute, respectively, while the average rest and stress mean arterial pressures of the swine were 72 ± 10 mmHg and 66 ± 10 mmHg, respectively. The time delay between V1 and V2 acquisition ranged from 4.10 to 8.93 s with an average of 5.69 ± 1.35 s. The average data processing time per swine for all acquisitions combined was approximately one hour, with most of the time spent on semi-automatic segmentation of the myocardium and coronary centerlines. The average low-dose rest and stress perfusion in all three coronary arteries combined was 0.50 ± 0.22 and 1.93 ± 0.84 mL/min/g, respectively, while corresponding reference standard rest and stress perfusion was 0.58 ± 0.21 and 1.92 ± 0.73 mL/min/g, respectively. The average CFR in all three coronary arteries combined was 3.07 ± 1.66, while corresponding reference standard CFR was 3.37 ± 1.67. The average low-dose stress perfusion in the LAD with and without stenosis was 1.20 ± 0.32 and 2.07 ± 0.83 mL/min/g, respectively, while corresponding reference standard stress perfusion was 1.40 ± 0.40 and 2.04 ± 0.73 mL/min/g, respectively. The average CFR in the LAD with and without stenosis was 1.80 ± 0.81 and 3.11 ± 1.17, respectively, while corresponding reference standard CFR was 2.19 ± 0.69 and 3.46 ± 0.85, respectively. All other low-dose perfusion and CFR measurements in the LAD, LCx, and RCA individually as compared to corresponding reference standard perfusion and CFR measurements are shown in Table LOW) and reference standard perfusion (PREF) measurements in all three coronary arteries combined under rest and stress perfusion conditions were related through regression by PLOW = 1.04 PREF −0.08, with a Pearson's correlation of r = 0.94, a concordance correlation of ρ = 0.94, a RMSE of 0.32 mL/min/g, and a RMSD of 0.32 mL/min/g, as shown in Fig. LOW = 1.04 PREF −0.07, with a Pearson's correlation of r = 0.90, a concordance correlation of ρ = 0.89, a root-mean-square-error of 0.36 mL/min/g, and a root-mean-square deviation of 0.36 mL/min/g, as shown in Fig. LOW = 0.60 PREF −0.16, with a Pearson’s correlation of r = 0.56, a concordance correlation of ρ = 0.53, a root-mean-square-error of 0.21 mL/min/g, and a root-mean-square deviation of 0.18 mL/min/g, as shown in Fig. The low-dose perfusion  in the absence and presence of a physiologically significant LAD stenosis (FFR = 0.70) are also shown in Fig. 4For the three swine with LAD stenosis, nine low-dose cardiac CT acquisitions were performed: three per swine. Corresponding CFR versus stress perfusion measurements were then assessed in relation to previously reported physiological cutoff thresholds , and disLow-dose vessel-specific, rest perfusion, stress perfusion, and CFR measurement were in good agreement with corresponding reference standard measurement. Stress perfusion and CFR agreed with the physiological severity of induced LAD stenoses, i.e., stress perfusion and CFR decreased proportionally as FFR decreased, as expected with focal disease. The spatial distribution of CFR and stress perfusion also agreed with the induced flow conditions, where CFR and stress perfusion without a LAD stenosis remained high, while CFR and stress perfusion distal to a LAD stenosis with a FFR severity of 0.70 was markedly reduced. Furthermore, the total Stress dynamic CT perfusion has been shown to correlate well with invasive FFR . However. used a tiered approach, where patients with positive calcium scores underwent CTA, those with positive CTAs underwent CT perfusion testing, and those with significant perfusion defects underwent invasive catheterization. Such a testing strategy improved the accuracy of CAD diagnosis as compared to standard testing, reduced the rate of negative catheterization, and exposed patients with lower risk, i.e. negative calcium scores or negative CTA, to less radiation and contrast dose. Nevertheless, the mean radiation doses were 1.3, 3.5, and 10.6 mSv for calcium scoring, CTA, and stress CT perfusion, yielding a total mean radiation dose of 15.5 mSv for patients receiving all three tests [. proposed a protocol comprised of CTA followed by static stress CT perfusion, where FFR-CT was also derived from the CTA data. While the results showed improved diagnostic accuracy when incorporating FFR-CT and stress CT perfusion, again the mean radiation dose of CTA and static stress perfusion combined was 5.2 mSv [Nevertheless, other groups are pursuing alternative approaches to comprehensive CT-based cardiovascular diagnostics. Lubbers et alee tests . Alterna 5.2 mSv , withoutSTRESS exposure time is increased, there is also potential for cardiac output, ejection fraction, wall motion, and myocardial strain to be assessed [The low-dose cardiac CT technique was shown to enable accurate, vessel-specific rest perfusion, stress perfusion, and CFR measurement, with simultaneously acquired co-registered CTA data using only four volume scans and two contrast injections, respectively, where such a protocol had not been fully realized by our prior work –21. Hencassessed  by our tWhile the time between paired acquisitions was 15-min, given that repeat injections were made in each animal, small increases in the baseline blood pool enhancement over time were unavoidable. However, the use of prospective triggering at 140 HU above the baseline blood pool enhancement maintained accurate timing and perfusion measurement. The variable time delays between V1 and V2 for the low-dose acquisitions were also estimated from the reference standard acquisitions, which is clinically unrealistic. Consequently, each time delay can be determined with a diluted test bolus and single slice CINE scanning , with onThe swine also had a small effective diameter (23 cm) as compared to the average 34 cm effective diameter of patients with CAD , i.e., tOn average, the swine also had high heart rates. To avoid blunting of stress perfusion, beta-blockers were not given. Nitroglycerin was also avoided during CTA to prevent further exacerbations in heart rate. As a result, the images were motion degraded. That said, the low-dose technique derives perfusion using the integrated change in HU within the entire myocardium over time; thus, the impact of motion on the accuracy of perfusion measurement was not significant. Still, despite optimal CTA acquisition at the peak of the aortic enhancement , 37, theLastly, only segmental disease was assessed in the absence of infarct, i.e., multi-vessel disease, diffuse disease, microvascular disease, and myocardial scar were not evaluated. However, the technique can spatially resolve both absolute perfusion (in mL/min/g) and CFR on a voxel-by-voxel and vessel-specific basis, where such metrics overcome the normalization dependent limitations of static perfusion. Hence, detection of focal, gradient, and global perfusion deficits, as well as balanced ischemia and myocardial scar are still feasible. Nonetheless, the accuracy of voxel-by-voxel perfusion and CFR measurement depends on image noise. Fortunately, voxel binning can be used to suppress image noise while maintaining adequate spatial resolution. More importantly, minimum-cost-path myocardial assignment , 28 can The low-dose cardiac CT technique is a new method that enables accurate, vessel-specific rest perfusion, stress perfusion, and CFR measurement, with simultaneously acquired CTA data, using only four volume scans and two contrast injections, respectively. As a result, the total combined radiation dose of CT-based CAD workup has the potential to be reduced.


Alveolar osteitis (AO) is the most common painful post‐operative complication after tooth extraction. The common modalities used in the management of AO are lavage, placement of medicated dressings, analgesics, and antibiotics. The present study was undertaken to compare platelet‐rich fibrin (PRF) and zinc oxide eugenol (ZOE) for pain relief in AO.t test, and Mann‐Whitney U test.All cases meeting the eligibility criteria received two different treatment modalities over a span of 18 months. At the analysis stage, the final sample size comprised 70 patients, with 35 patients appropriated in each group. Group A patients received ZOE and Group B received PRF. Pain scores were measured on “1st, 3rd, 5th, and 7th” days based on a visual analogue scale (VAS) and compared in both groups of patients. The collected data were analyzed using the chi‐square test, In patients treated with ZOE dressing, the average VAS scores observed were 7.4 ± 1.5, 5.1 ± 1.1, 3.4 ± 0.9, and 2.1 ± 0.7, respectively, on the “1st, 3rd, 5th, and 7th” follow‐up days. In patients treated with PRF, the average VAS score observed were 4.1 ± 1.2, 2.6 ± 0.9, 1.7 ± 0.9, and 0.8 ± 0.8 respectively.Both ZOE and PRF were effective in pain control during the follow‐up period. However, the pain intensity measured as a pain score using VAS was, lower in the PRF group than in the ZOE group on all follow‐up days. Blum defined dry socket as “postoperative pain in and around the extraction site, which increases in severity at any time between 1 and 3 days after the extraction, accompanied by a partially or totally disintegrated blood clot within the alveolar socket, with or without halitosis” while, excluding other possible causes of pain in the region.A multifactorial etiology has been suggested for the dry socket which includes oral anaerobic microorganisms, anatomic location, traumatic extractions, immoderate use of irrigating agents at the surgical site, curettage of the extraction socket, mechanical dislodgement of blood clot, fibrinolysis of blood clot, smoking, oral contraceptives, immune suppression, female gender, and vasoconstrictors.The emergence of AO as a common postoperative sequela of extraction has prompted surgeons to explore various treatment options, focusing on optimal healing and control over pain. It is fair to argue that the primary aim in the management of AO should be pain control until normal reparative processes are initiated.Platelet‐rich fibrin (PRF) is a second‐generation platelet concentrate developed by Choukroun et al in 2001.The definitive management of dry sockets remains inconclusive. It has been felt that a meaningful study must be carried out to evaluate the relative effectiveness of two different modalities used for pain management in AO. The present study aimed to compare ZOE and PRF for pain relief in AO.21. Study designThe design of the study was single‐blinded prospective study.2. Sample selection procedureAll cases meeting the eligibility criteria received two different treatment modalities over 18 months. The methods of screening, grouping, interventions, follow‐up, and analysis are summarized in the flow diagram . σ = SD.5. Estimation of sample sizeIn this study,σ) = 0.495.Pooled SD of VAS score at seventh day (Mean différence in VAS score on seventh day between methods (Δ) = 0.33AgeGenderPain based on visual analog scale (VAS)6. Outcome measures2.1Intense throbbing pain in relation to the extraction socket often radiates in nature and tends to increase in severity for a period between 1‐ and 3‐days post‐surgery.Extraction socket denuded the blood clot with or without halitosis.The following features were clinically diagnostic of AO.Group A patients received ZOE and Group B received PRF as the treatment modality. Pain score was measured on “1st, 3rd, 5th, and 7th” days based on a VAS and compared in both groups.In group A patients, ZOE was used as an intra‐alveolar obtundent dressing . .Copious irrigation of the extraction socket was undertaken using a mixture of isotonic saline and povidone‐iodine solution, to clear the socket of any remnants of disintegrated blood clots and debris. A thin paste of ZOE was soaked in a gauze piece and placed in the extraction socket, adhering to aseptic measures. The dressings were changed on “1st, 3rd, 5th, and 7th” days.2.1.1Samples were collected in an autoclavable test tube without anticoagulant and immediately centrifuged at 3500 rpm for 15 minutes until a thick, viscous, fibrous gel was obtained  Figure . CentrifIn all group B patients, local anesthesia was administered using 2% lignocaine with adrenaline at a 1:200,000 concentration. Thorough irrigation of the extraction socket was performed using a mixture of isotonic saline and povidone‐iodine solution. PRF was then placed in the unhealed socket, and the surgical site was closed using a 3‐0 black silk suture. PRF‐treated wounds on seventh post‐operative day demonstrated satisfactory wound healing Figure .2.2t test, chi‐square test, and Mann‐Whitney U test, and a P value <.05, which was defined as the level of statistical significance. SPSS Inc. Released 2007. SPSS for Windows, version 16.0. Chicago. SPSS Inc.The data were entered into Microsoft Office Excel 2007 version and the data were analyzed using SPSS for Windows, Version 16.0.* Qualitative variables are expressed as means, frequencies, and percentages. Qualitative variables were analyzed using the 3The study was approved by the Institutional Ethics Committee, Government Dental College, Thiruvananthapuram, Kerala, India . Informed consent was obtained from all the participants.4The results are summarized in Tables In the present study, among 35 patients in group A, 8 (22.9%) were men and 27 (77.1%) were women. In group B, 9 (25.7%) patients were men and 26 (74.3%) were women. The incidence of AO was more common in females among the treatment groups. There was no significant difference in the number of male and female patients treated in either group. Hence the two study groups were homogenous in terms of sex.P < .01)  Figure .P < .01)  on day 5  on day 7  have been explained.