Objective: The new adipokine, which is asprosin, affects glucose release from the liver to the blood, and thus, influences exercise metabolism. This is the first study assessing whether single anaerobic exercise affects asprosin secretion in women and men.Methods: 10 men and 10 women  performed a single 20-s bicycle sprint. Blood samples were collected before exercise and in the 3′, 15′, 30′, and 60′ of recovery, and 24 h after competition.Results: Only in women did asprosin (P = 0.001)  and irisin (P < 0.001)  concentrations increase. Leptin, however, decreased (P = 0.001) at 3′, 15′, and 30′ in women. There was an increase in interleukin-6 (P < 0.001) at 3′, 15′, 30′, and 60′ of recovery in men, at 15′, 30′, 60′, and 24 h of recovery in women, along with a simultaneous decrease in interleukin-1β (P < 0.001) at 15′, 30′, and 60′ of recovery in men, and at 15′ and 30′ of recovery in women . There was a positive correlation between asprosin and adiponectin and a negative one between asprosin and leptin. The increase in irisin concentration at 30′ of recovery was positively correlated with the increase in asprosin concentration and percentage fat content, while being negatively correlated with total and lean body mass (LBM).Conclusion: The single anaerobic effort induced an increase in asprosin and irisin secretion while reducing leptin secretion in women. Adipocytokine concentration changes are inter-related. Regardless of sex, anaerobic efforts induce anti-inflammatory effects. Via the G-protein/cAMP-protein kinase A pathway, it regulates the release of glucose from hepatocytes. Higher levels of asprosin were observed in obese subjects and in those with type 2 diabetes  was obtained for conducting research according to the presented methodology.The study included 10 men and 10 Caucasian women aged 21.64 ± 1.22 and 22.64 ± 1.49 years, respectively. All of them were non-smokers, and were physically active, but did not perform any sports disciplines. The participants’ physical activity  was assessed using the 7-day PAR questionnaire . None of2, 18.36 ± 2.99%, and 62.95 ± 7.29 kg and 59.75 ± 6.49 kg (P < 0.001), 21.50 ± 1.84 kg/m2 (P = 0.009), 24.20 ± 1.60% (P < 0.001), and 45.15 ± 4.12 kg (P < 0.001) for men and women, respectively.These were individuals with normal body mass (BM) and composition, and normal metabolic profile. BM, body mass index (BMI), percentage of body fat (%FAT), and lean body mass (LBM) significantly differed in both groups and totaled 77.13 ± 8.56 kg, 23.71 ± 1.58 kg/mBody height was measured using a Martin-type anthropometer to the nearest 1 mm (Poland). BM and body composition were determined using a multi-frequency , eight-electrode analyzer , based on bioelectrical impedance analysis (BIA).The participants’ state of health was confirmed by medical examination . The results of the analyses are presented in Table max and oxygen efficiency by measuring maximal oxygen consumption per minute – VO2max; and during the sprint: maximum (MAP) and mean (MP) anaerobic power of the lower limbs were measured (max was similar in both groups (P = 0.354) and was 201.0 ± 11.1 and 196.3 ± 10.1 b/min, in men and women, respectively. VO2max was significantly higher (P < 0.001) in men (55.61 ± 5.63 mL/kg), compared to women (44.75 ± 3.43 mL/kg). MAP and MP were also higher in the group of men than women and were as follows: 11.28 ± 0.82 and 8.59 ± 0.44 W/kg (P < 0.001) for MAP and: 9.41 ± 0.62 and 7.33 ± 0.49 W/kg (P < 0.001) for MP, respectively.In previous studies, in the graded test, the following were determined for each of the volunteers: maximum heart rate – HRmeasured . HRmax wEach participant performed a single 20-s bicycle sprint . Venous and capillary blood was collected before the exercise (0′) and five times during the recovery period: third minute (3′), 15th minute (15′), 30th minute (30′), 60th minute (60′), and 24 hours (24 h) following the test. Asprosin, irisin, adiponectin, leptin, glucose, and lactate were determined as well as interleukin concentration: IL-1β, IL-6, and IL-10. Hemoglobin and hematocrit were determined to calculate changes in plasma volume.P < 0.001), which corresponded to 0.075 × BM and 0.065 × BM. The test effort consisted in obtaining the maximum cadence of pedaling as quickly as possible and maintaining it for 20 s. The participants started the sprint after hearing the 3-2-1-GO! command and were vigorously encouraged. After 20 s, the load was removed and the subjects pedaled at 60 rpm for 3 min.Execution of the bicycle sprint has been previously described . First, The efforts were carried out under the supervision of a physician. The study was conducted from October to December in Poland, the external temperature was about 15°C to -5°C. Due to the possible impact of the time of day and ambient temperature on muscular power , laboratBlood plasma concentrations of adipocytokines: asprosin, irisin, adiponectin, leptin, and serum concentrations of interleukins: IL-1β, IL-6, and IL-10 were determined using the enzyme immunoassay (ELISA) method in accordance with the manufacturer’s manuals.The asprosin concentration was determined using the Human Asprosin ELISA KIT SK00229-06 . The concentration of irisin was determined using the Irisin/FNDC5 EK-067-16 . Adiponectin concentration was determined with the high-sensitivity Human Adiponectin Elisa RD191023100, whereas leptin concentration was measured with the Human Leptin Elisa RD191001100 test . The detection range for asprosin was 1–32 nmol/L, for irisin 0–100 ng/mL, for adiponectin 2–150 ng/mL, and for leptin 0–50 ng/mL. Intra-assay CV for asprosin was <8%, for adiponectin it totaled 4.4%, for leptin 7.6%, and the inter-assay CV for asprosin was <12%, for adiponectin it equaled 6.2%, while for leptin, it was 6.7%.IL-1β, IL-6, and IL-10 concentrations were determined using R&D Systems (United States). Test sensitivity for IL-1β was 0.057 pg/mL (HSLB00C), for IL-6, it totaled 0.039 pg/mL (HS600B), and for IL-10, it was 0.09 pg/mL (HS100C). Intra-assay CV for IL-1β equaled <10.2%, for IL-6 <7.8%, for IL-10 <9.4%, while the inter-assay CV for IL-1β was <10.4%, for IL-6 <7.2%, and for IL-10 <8.5%.via the colorimetric enzymatic method according to the manufacturer’s instructions using LC2389 and GL2623 , for which the absorbance and concentration dependence was maintained at 19.7 and 22.2 mmol/L, respectively.The concentrations of lactate and glucose were determined in the capillary blood plasma The hemoglobin concentration was determined with the cyanide-methemoglobin method , while hematocrit with the micro-hematocrit method in triplicate, and the mean was calculated. Knowing hemoglobin and hematocrit levels, which were determined in whole venous blood immediately after collection, plasma volume changes were calculated  and thenBecause the varied diet used by volunteers could affect the anaerobic power  and the t test for independent samples or the Mann–Whitney U test. The Friedman ANOVA test for multiple measurements and the Wilcoxon test were used to assess the effect of exercise on changes in biochemical markers. The results for men and women were compared with the Mann–Whitney U test. The Spearman correlation coefficient was calculated. For all variables, differences at the level of P < 0.05 were considered statistically significant. Data are presented as mean ± SD (standard deviation) or median and IQR (interquartile range). The calculations were performed using the Statistica 12 program .Data distribution was checked using the Shapiro–Wilk test. Significance of differences between sexes for single measurements was tested with the P < 0.01). In women, the highest lactate concentration was at 3′, and in men, 15′ after the exercise . In men, there were no changes in glucose levels caused by exercise  to baseline (0′), and then increased, attaining its highest value at 30′, which was significantly higher compared to the value at 3′ (P = 0.009) and 15′ (P = 0.017). Asprosin concentration at 60′ was still significantly higher compared to 3′ (P = 0.005) and 15′ (P = 0.047). In men, asprosin concentration did not change after the exercise (P = 0.312) (Table The asprosin concentration in women at 3′ after exercise was comparable (P < 0.001). In men, the concentration of irisin did not change (P = 0.187)  or men (P = 0.141) after exercise (Table The concentration of adiponectin did not change in women P = 0.53 or menP = 0.001) than at baseline. In men, the concentration of leptin following exercise did not change (P = 0.080)  by 0.58 ± 0.14 pg/mL at 15′, by 0.70 ± 0.14 pg/mL at 30′, by 0.78 ± 0.12 pg/mL at 60′, and by 0.25 ± 0.11 pg/mL 24 h after the exercise. In the group of men, the serum concentration of IL-6 was significantly higher (P < 0.001) than the baseline by 0.31 ± 0.12 pg/mL at 15′, by 0.49 ± 0.12 pg/mL at 30′, and by 0.48 ± 0.10 pg/mL at 60′ following exercise  by 0.34 ± 0.12 pg/mL and by 0.32 ± 0.11 pg/mL at 15′ and 30′, respectively. IL-1β concentration in men significantly reduced (P = 0.001) by 0.33 ± 0.11 pg/mL at 15′, by 0.27 ± 0.08 pg/mL at 30′, and by 0.22 ± 0.09 pg/mL 60′ after exercise  (Table In none of the groups were there any significant effects of the cycling sprint on the IL-10 concentration (5) Table .P < 0.001) than in men (1.27 ± 1.02 ng/mL). The concentrations of other biochemical markers before exercise were comparable in both groups (P > 0.05)  than in men 15′, 30′, and 60′ after the exercise (Table The concentration of irisin in the blood plasma of women was significantly higher (P < 0.05) in women for all measurements after the exercise  (Table Leptin concentration was significantly higher (P < 0.05) in women than in men. There were no significant differences between sexes in IL-1β and IL-10 concentrations , as well as the negative correlation between resting leptin concentration and BM , LBM  and a positive correlation of leptin concentration with %FAT . There was a simultaneous, negative correlation between leptin concentration and MAP  and MP .A positive correlation was found between the resting concentration of asprosin and BM , and a negative correlation between asprosin and leptin concentration    and negatively with IL-1β concentration , while IL-1β concentration showed a positive correlation with IL-10 concentration   and %FAT , and negatively with BM  and LBM   , which mIn our study, the exercise-induced increase in asprosin concentration during the 30′of recovery, as compared to the 3′ after exercise, was positively correlated with the increase in irisin concentration. Differences in irisin concentrations as a response to physical exercise may reflect the energy status of muscles during and after exercise. It has been proven that in less trained people, the concentration of irisin significantly increases during exercise, and in people training regularly, this increase is smaller . This isIt was found that aerobic efforts performed on a regular basis induce increased secretion of adiponectin . StrengtIn research concerning a group of men and women with normal body composition, there were significant individual differences in leptin reaction  to motor therapy of varying intensity . In turnOur research also points to the anti-inflammatory effect of anaerobic exercise. We have found that the bicycle-sprint sprout induced an increase in serum IL-6 concentration, at the same time associated with a decrease in pro-inflammatory IL-1β concentration. This effect of exercise was independent of gender, and the correlation between IL-6 and IL-1β concentration was negative, which in this case, demonstrates the anti-inflammatory function of IL-6 . IncreasIn healthy, young individuals, a single anaerobic exercise increases asprosin and irisin secretion and reduces leptin secretion in women. Changes in the concentration of adipocytokines are inter-related. Regardless of sex, anaerobic efforts induce anti-inflammatory effects.However, further research is required on the influence of physical efforts of varying intensity on the secretion of asprosin and other adipocytokines/myokines. These trials should involve more subjects, including obese individuals.MW conceived the project and procured the project funding. MW, JS, MM, and ZS contributed to the collection of the data and reagents. MK developed and analyzed diets. MW performed the statistical analysis and interpretation of results. MW drafted the first version of the manuscript. ZS conducted substantive consultations. All authors contributed to revising the manuscript and gave their final approval of the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coconut cake is an abundant and good potential edible protein source. However, until now it has not been extensively used in the food industry. To promote its usage, the characterization, nutrition value and antioxidant activity of coconut cake protein fractions  were studied. Results revealed that all the albumin, globulin, glutelin-1 and glutelin-2 fractions showed a high nutrition value. The prolamine, glutelin-1 and glutelin-2 all exhibited good radical scavenging activity and reducing power, and the globulin and prolamine showed high ion chelating ability (89.14–80.38%). Moreover, all the fractions except glutelin-2 could effectively protect DNA against oxidative damage. Several peptides containing five to eight amino acids with antioxidant activity were also identified by LC-MS/MS from the globulin and glutelin-2 fractions. The results demonstrated that the coconut cake protein fractions have potential usages in functional foods. Reactive oxygen species (ROS) and free radicals are very unstable and react rapidly with other groups or substances in the body, leading to cell or tissue injury . RecentlCoconut cake, with a global annual output of 200 million tons, is the main byproduct of the coconut milk and oil industry, and contains approximately 10–16% protein . Apart fFollowing sequential fractionation, coconut cake albumin, globulin, prolamine, gultelin-1 and glutelin-2 fractions were separately obtained. Their contents were 8.84 ± 1.16, 54.35 ± 3.69, 0.92 ± 0.30, 20.49 ± 1.11 and 6.14 ± 1.42 g/100 g, respectively. Obviously, globulin was the predominant fraction, in accordance with the report of Angelia et al. [Size heterogeneity among the fractions was revealed by the electrophoretic separations A–D. The As shown in −1), the prolamine, glutelin-1 and glutelin-2 had a higher ABTS radical scavenging activity  form of pBR322 plasmid DNA will be transformed to its open–circular (OC) form after oxidative damage, and some antioxidants can protect the SC from degradation . As showactivity . A previreaction .The reported results in 2O2 induced damage [50: 22.16 mg·mL−1) [As shown in d damage ,18. Moremg·mL−1) . This almg·mL−1) , which id-ribose and plasmid pBR322 DNA were purchased from Shanghai Yeyuan Biotech. Co., Ltd., Shanghai, China. Low molecular weight protein markers were purchased from Shanghai DINGUO Biotech. Co., Ltd., Shanghai, China. Other reagents were all of analytical grade.Coconut cake was purchased from Nanye Coconut Food Co., Haikou, China. BHT, ABTS ), 2-Deoxy-n-hexane , dried, ground and passed through a sieve of 0.2 mm mesh to obtain defatted coconut cake (DCC). Different solvents, including 0.4 mol·L−1 NaCl, 70% (v/v) 2-propanol (IPA), 50% (v/v) glacial acetic acid and 0.1 mol·L−1 NaOH, were used in sequence to extract the five fractions, and protocol was summarized in −1) after 8 h at 4 °C. After centrifugation at 20,000× g for 30 min, the supernatant was collected and dialyzed against deionized water at 4 °C for 48 h, and then lyophilized in a freeze dryer . Extraction with each solvent was carried out in triplicate, and all the resulting fractions were stored at −20 °C. Protein concentration of each fraction was determined by the Bradford method [Prior the sequential fractionation described by Kwon et al. , the cocd method . Coconutb (97.4 kDa), BSA (66.2 kDa), ovalbumin (45.0 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (20.1 kDa) and lysozyme (14.4 kDa).SDS-PAGE was performed according to the method of Laemmli  on 7.5% 4 U/g),and gently shaken at 37 °C for 48 h and then centrifuged at 10,000 × g 30 min. The supernatant and the precipitate were collected, respectively. The precipitate was suspended in 10 mL of 0.1 M sodium phosphate buffer (pH 8.0) and 2.5 mg trypsin (1 × 105 U/g) was added. The mixture was gently shaken at 23 °C for 16 h. The digested mixture was then centrifuged and trichloroacetic acid (TCA) was added to the supernatant to reach a concentration of 8 M in the solution. The supernatant previously obtained from pepsin digestion was also treated in a similar manner. Precipitated proteins were removed by centrifugation at 10,000 × g 25 min. The TCA-soluble nitrogen content of the supernatant was determined by Branford method [Amino acid composition (expressed as g/100 g protein) of each CCP fraction was determined by the method of Wang et al.  with an d method . IVPD wa−1) was mixed with 2 mL of ABTS ) solution (7 mM), and then incubated in the dark at 30 °C for 6 min. Then, absorbance at 734 nm was recorded. BHT (100 µg·mL−1) was used as the control. The activity was calculated as follows:C was the absorbance of the control (distilled water replaced samples), AB was the absorbance of blank (without ABTS) and AS was the absorbance of the mixture containing samples.Following the method of Arts et al. , 20 µL o−1), 100 µL FeSO4-EDTA (10 mM), 100 µL 2-deoxyribose (10 mM), 1.4 mL phosphate buffer  and 100 µL H2O2 (10 mM). After incubation at 37 °C for 1 h, the reaction solution was mixed with 1.0 mL trichloroacetic acid (28 g·L−1) and 1.0 mL thiobarbituric acid (10 g·L−1), and then incubated at 100 °C for 20 min. The absorbance was measured at 532 nm. The activity was determined as follows:B was the absorbance of blank (without 2-deoxyribose).Following the method of Ren et al. , the reaSuperoxide radical-scavenging activity was measured using the pyrogallol assay as described by Gao et al. . 0.1 mL −1) and FeCl2  was reacted with ferrozine  for 30 min. Then the absorbance was read at 562 nm, and chelating activity was calculated as follows:B was the absorbance of the blank (without ferrozine).Following the method of Jeong et al. , the mix−1) was mixed with 0.2 M phosphate buffer (pH 6.6) and potassium ferricyanide (0.01 g·mL−1). The mixture was first incubated at 50 °C for 20 min. An aliquot (2.5 mL) of TCA (0.1 g·mL−1) was added to the mixture, followed by centrifugation at 3000× g for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 2.5 mL of ferric chloride (1 g·mL−1), and the absorbance at 700 nm was read after 10 min. BHT (100 µg·mL−1) was used as the positive control. Increased absorbance at 700 nm of the reaction mixture indicated increased reducing power.Reducing power was determined by the method of Yen et al. . Aliquot−1) was added to 11 µL of the reaction mixture, which contained 6 mM H2O2, 1.5 mM FeSO4 and 0.5 µg of plasmid pBR322 DNA. The mixture was incubated at 37 °C for 1 h [A 5 µL of sample solution  and Thermo LTQ linear ion trap mass spectrometer . The acquired MS/MS data were interpreted using the bioinformatics search engine Mascot version 2.1.0 .The CCP fractions with high antioxidation and potential antihypertension were subjected to LC-MS/MS analysis. Aliquot of sample solution (25 µL) was mixed with 5 µL of 0.2 M NHData were subjected to analysis of variance and Duncan value with a confidence interval of 95% was calculated to compare means.The albumin, globulin, prolamine, glutelin-1 and glutelin-2 protein fractions were successfully obtained by the sequential extraction method from coconut cake. All these fractions except albumin exhibited higher radical-scavenging activity and ion chelating ability. These fractions except glutelin-2 can also protect DNA from oxidative damage. These results indicate that these fractions can be used as natural antioxidant or food ingredient for some food such as meat patties to prolong shelf life of product. However, whether these fractions have antioxidant properties in vivo will need to be determined through further work.


The advantages of the synthesized fluorescent probe (compound 1) were the low detection limit (4 × 10−6 mol·L−1), good selectivity and high sensitivity which had been demonstrated through UV-vis, fluorescent titration experiments. Besides cytotoxicity test of compounds (1 and 2) was studied and the results indicated that compounds (1 and 2) showed almost no cytotoxicityat at a concentration of 150 μg·mL−1. The interacted mechanism was the thiolysis reaction of dinitrophenyl ether which had been confirmed by fluorescence and HRMS titration experiment. In addition, probe 1 can also detect HS− selectively by naked eye in pure DMSO solvent.A series of chemsensors ( Endogenous concentration of H2S is related to some diseases such as Alzheimer’s disease, Down syndrome, liver cirrhosis and diabetes17. What’s more, the regulation of H2S levels is also a potential drug development strategy19 and the importance of accurate detection of H2S cannot be over-emphasized. Therefore, it presents significant research related to track and quantify H2S inside living cells being crucial in order to understand the biological and pathological roles of H2S. Recently, some methods to determine H2S concentration in biological sample have been developed including the methylene blue, the monobromobimane (MBB), gas chromatography (GC), the sulphide ion selective electrodes (ISE) and fluorescent analysis23. Among these methods, fluorescent analysis has attracted great attention due to the high sensitivity and selectivity for the detection of H2S in many fields such as environment area, pharmacy area and so on31.In the past decade, we has seen a boost of research interest in hydrogen sulfide , (n-C4H9)4NAcO (AcO−), (n-C4H9)4NH2PO4 (H2PO4−), (n-C4H9)4NF (F−), (n-C4H9)4NCl (Cl−), (n-C4H9)4NBr (Br−), (n-C4H9)4NI (I−)) and amino acids (Glutathione (GSH), Cysteine (Cys), Homocysteine (Hcy)). Besides, four compounds designed and synthesized could exhibit the probes with strong electron-withdrawing groups located in 2,4-positions of fluorobenzene have strong binding ability for HS− detection which provides a good idea for the design of probe in future.In the present work, a series of “OFF-ON” probes based on coumarin derivatives to detect HS− Fig. . The res2 was synthesized according to the route shown in Fig. 2 was obtained by the standing method. The suitable single light yellow crystal was obtained by volatilizing ethyl acetate containing compound 2 at room temperature. The details of the crystallographic determination, selected bond lengths and angles were given in Table Compound 2 suitable for X-ray crystal analysis was obtained and the structure was also confirmed . In the crystal packing of compound 2  were recorded after addition of amino acids  and various anions  through UV-vis titration experiments in pure DMSO solution and aqueous solution  respectively. The data of UV-vis titration experiments manifested only compounds  displayed different binding abilities with the above anions and amino acids. The free 1 showed a main absorption at 320 nm, as the HS− increases in pure DMSO solution of probe 1, the absorbance at 320 nm was decreased gradually, along with the simultaneous emergence of a new absorption at 470 nm. In this process, two isosbestic points noted at 330 nm and 348 nm suggesting a clear chemical reaction. Based on the well-establish thiolysis reaction of dinitrophenyl ether, the new absorption at 470 nm could be attributed to coumarin derivative, which was also supported by fluorescence and HRMS titration experiment. Furthermore, the UV-vis spectra of probe 1 with HS− in aqueous solution were also performed  to pure DMSO solution of probe 1, only Cys induced similar changes in the UV-vis spectra compared with HS−, which exhibited Cys also interacted with compound 1 . However, the additions of the above amino acids and anions to aqueous solution induced almost no spectra changes of compound 1. The result indicated that compound 1 showed different binding abilities for HS− and Cys in pure DMSO solution and almost no binding abilities with above amino acids and anions in aqueous solution. Therefore, compound 1 could be used as a sensor to detect HS− in aqueous solution.The additions of amino acids  and other anions , which indicated that compound 1 can be used for the detection of HS− as a colorimetric sensor.Subsequently, the colorimetric sensing capabilities of compound ons Fig. . Obvious33 of compound 1 (4.0 × 10−6 mol·L−1) was implemented through fluorescence titration . Therefore, the detection limit of compound 1 for HS− was determined to be 4.0 × 10−6 mol·L−1.Moreover, the detection limition Fig. . Further2 to various anions  were tested. Upon the addition of increasing amounts of HS−  to DMSO solution of compound 2 a new absorption peak appeared gradually at 485 nm. While, the additions of other anions  to compound 2, almost no spectra changes was observed.Next, the UV-vis titration experiments of compound 1–4) in DMSO solvent were also investigated with addition of various amino acids and anions. Just as Fig. 1 exhibited at about 385 nm. Upon the addition of increasing amounts of HS−, the fluorescence intensity increased obviously at about 392 nm. Similar fluorescence response of compound 1 was observed upon the addition of Cys  compared with HS−. Furthermore, the interactions of probe 1 with amino acids  and other anions  were also investigated. The additions of H2PO4−, AcO− and F−  induced the appearance of fluorescence emission bands centered at about 386 nm, however, nominal changes were induced in the presence of GSH, Hcy, F−, Cl−, Br−, I−.The photo-physical responses of four compounds  and various anions  were also examined in aqueous solution. From Fig. 1 showed very weak fluorescence at the absence of anions. After HS− was added, double emission peaks appeared and the fluorescent intensity increased gradually. Results demonstrated that compound 1 showed strong binding ability for HS−. However, weak spectral responses that even could be ignored were induced with addition of amino acids  and H2PO4−, AcO−, F−, Cl−, Br−, I−.Besides, the fluorescence spectral responses of compound 2, the fluorescence intensity noted at 434 nm increasing rapidly by titration of HS− . No obvious responses of compound 2 were observed with titration of other anions  and Cys. For compounds (3 and 4), Similar experiments were carried out, however, no significant spectral responses were observed with the addition of other anions  into DMSO solution of two compounds (3 and 4) which indicated the weak binding abilities of compounds (3 and 4) and other anions  and Cys could be ignored.For compound 1 and 2) interacted with amino acids and various anions as the ratio of 1:1 or 1:2. The UV-vis spectral data was used to calculate the binding constants by non-linear least square method35, and the binding constants were listed in the Table 1 and 2) with amino acids and various anions followed the order of HS− ≫ H2PO4−, Cys, AcO−, F−, Br−,Cl−, I−, Hcy and GSH. In general, both compound 1 and compound 2 showed the strongest binding ability for HS− among amino acids and anions. Besides, a theoretical basis and these binding constants were necessary for the optimization of sensor.The job-pot curves suggested two compounds 1 and  interact1 and 2) with two electron-withdrawing groups on the fluorobenzene ring were stronger than that of compounds (3 and 4) which had one electron-withdrawing group on the fluorobenzene ring. In addition, the anion binding ability of compound 1 was stronger than that of compound 2 due to the electron-withdrawing ability of nitro group was greater than the trifluoromethyl group36. The above results indicated that strong electron-withdrawing groups located in 2, 4-positions of fluorobenzene provides a easily site for intermolecular HS− attack. Besides, for HS−, the binding ability trend of compounds (1–4) followed the order of 1 > 2 ≫ 3, 4 also could be confirmed by comprehensive analysis of UV-vis, fluorescence titration.The anion binding abilities of compounds  was also carried by performing MS-HRMS after the addition of 2 equiv. NaHS. The probe 1 itself exhibited a dominant peak at m/z = 341.0551(M-H)− . However, the above peak vanished and a new peak at m/z = 199.0364 (M + Na)+ appeared which was the ion-peak of 7-hydroxy-4-methylcoumarin  was 1:2. The above results indicated the possible interacted mechanism was thiolysis reaction of dinitrophenyl ether in vitro was conducted using MTT assay. Owning to Glutathione peroxidase (GPx1) being an important selenoprotein and not found in human breast cancer cells (MCF-7) according to scientific research, therefore, the special cell lines (MCF-7 cell) were selected by us46. The MTT assay results indicated that compound 1 and 2 showed very low cototoxicity over a concentration range of 0–150 μg·mL−1 especial probe 2  ( <150 μg·mL−1). In agreement with the binding constants, compounds (1 and 2) showed a high binding capacity and low cytotoxicity, especial probe 1, which indicated that compounds (1 and 2) have a potential application to H2S detection in cells.For further biological application point of view, a quantitative cytotoxicity study of two compounds (n-C4H9)4NCl, (n-C4H9)4NBr, (n-C4H9)4NI, (n-C4H9)4NAcO and (n-C4H9)4NH2PO4) and amino acides  were purchased from Aladdin Chemistry Co. Ltd . Dimethyl sulfoxide (DMSO) was distilled in vacuum after being dried with CaH2. 1H NMR spectra were recorded using an Unity Plus-400-MHz spectrometer. HRMS was obtaioned with a Mariner apparatus. Absorption spectra were obtained on UV-vis spectrophotometer . Fluorescence emmission spectra were taken on a Cary Eclipse Fluorescence Spectrophotometer . The binding constant (Ks) was obtained by non-linear least squares calculation method for data fitting.Most staring materials were obtained commercially, all reagents and solvents were of analytical grade and used as received without further purification unless otherwise stated. Sodium hydrolfide and all anions in the form of tetrabutylammonium salts  at 490 nm wavelength measurement. Cell viability (expressed in%) was calculated considering 100% growth at the absence of fluorescence probe, and the viability of other groups were calculated by comparing the optical density reading with the control. The IC50 was defined as the compound concentrations required for 80% inhibition of cell growth.The cells that were at logarithmic growth phase were seeded in a 96-well plate at a density of 2.0 × 101–4) were synthesized according to the route shown in Fig. The compounds –4 were s47. The experimental details were described in supplementary material.7-Hydroxy-4-methylcoumarin was synthesized according to the literature2CO3  were dissolved in DMF(40 mL) solution with stirring. The above mixture were heated for 4 h at 90 °C at N2 atmosphere and then cooled to room temperature. The reaction was poured into ice-water (400 mL) and extracted with ethyl acetate (3 × 25 mL), dried with MgSO4 overnight. Filtration, purification with column chromatography (CH3COOCH2CH3:CH2Cl2 = 1:40). Stood overnight a yellow precipitate was obtained. Yield: 68%. m.p. 187.9–189.8 °C. 1H NMR  δ 8.94 , 8.51 , 7.92 , 7.40 , 7.28 , 6.43 , 2.47  . Elemental analysis: Calc. for C16H10N2O7: C, 56.15; H, 2.94; N, 8.18. Found: C, 56.21; H, 2.94; N, 8.16. MS-HRMS (m/z): 341.0551(M-H)- . IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1288 cm−1 .7-Hydroxy-4-methyl coumarin  and 2,4-dinitrofluorobenzene , K2–4) were also synthesized according to the above similar procedure.Compounds  δ 8.56 , 8.51 , 7.91 , 7.39–7.30 , 7.23 , 6.42 , 2.46  . Elemental analysis: Calc. for C17H10FNO5: C, 55.90; H, 2.76; N, 3.83. Found: C, 55.83; H, 2.76; N, 3.84. MS-HRMS (m/z): 366.0589(M + H)+ . IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1274 cm−1 .Suitable single light yellow crystal for X-ray crystal structure analysis was obtained. Yield: 74%. m.p. 139.3–142.1 °C. 1H NMR  δ 8.16 , 7.86–7.76 , 7.51 , 7.39 , 7.09–7.01 , 6.35 , 2.43  . Elemental analysis: Calc. for C16H11NO5: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.72; H, 3.72; N, 4.72. MS-HRMS (m/z): 320.0550(M + Na)+ . IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1273 cm−1 .Yield: 75%. m.p. 140.9–143.6 °C. 1H NMR  δ 8.35–8.27 , 7.88 , 7.28 , 7.19 , 6.40 , 2.46  . Elemental analysis: Calc. for C16H11NO5: C, 64.65; H, 3.73; N, 4.71. Found: C, 64.82; H, 3.74; N, 4.70. MS-HRMS (m/z): 320.0533 (M + Na)+ . IR: C-O-C (Diphenyl oxide Phenyl ether Biphenyloxide): 1267 cm-1 .Yield: 78%. m.p. 165.5–167.3°C. 2 with dimensions of 0.45 nm × 0.32 nm × 0.23 nm was mounted on a glass fiber. X-ray single-crystal diffraction data was collected on a Rigaku saturn CCD area detector at 293 K with Mo-Ka radiation (λ = 0.71073 Å). The structure was solved by direct methods and refined on F2 by full-matrix least squares methods with SHELXL-9748.A light yellow crystal of compound 2S successively with OFF-ON” fluorescence response. The fluorescence probes, especially compound 1, exhibited remarkable response to H2S against other anions and amino acids in pure DMSO solvent. Otherwise, the probe 1 also showed strong binding ability for HS− in HEPES buffer solution. The interacted mechanism of host-guest was the thiolysis reaction of dinitrophenyl ether. In addition, compound (1 and 2) showed highly sensitivity and low cytotoxicity to MCF-7 cells and probe 1 can also detect HS− selectively by naked eye in pure DMSO solvent. The results of our efforts highlight that the probes, especially compound 1, hold a potential chemical tool for the detection of H2S.In conclusion, we developed a series of fluorescence probes based on coumarin derivatives for the detection of HSupplementary Information


Our study investigates whether Native Thiol, Total Thiol and disulphide levels measured in serum of patients with prostate cancer and prostatitis and of healthy subjects, have any role in differential diagnosis.Patients followed up for histopathologically verified diagnosis of prostate cancer and prostatitis in 2016-2017 at the Medicalpark Gaziantep Hospital Urology Clinic were included in the study. Native Thiol (NT), Total Thiol (TT), Dynamic Disulphide (DD) levels in serum were measured by a novel automated method.NT, TT, DD, NT / TT ratios, DD / TT ratio and DD / NT ratio were measured as 118.4 ± 36.8μmoL / L, 150.3 ± 45.3μmoL / L, 15.9 ± 7μmoL / L, 78.8 ± 7μmoL / L, 10.5 ± 3.5μmoL / L, 13.8 ± 5.8μmoL / L respectively in patients with prostate cancer; as 116.4 ± 40.5μmoL / L, 147.5 ± 50.1μmoL / L, 15.5 ± 8.7μmoL / L, 79.7 ± 9μmoL / L, 10.1 ± 4.5μmoL / L, 13.5 ± 7.2μmoL / L in patients with prostatitis and as 144.1 ± 21.2μmoL / L, 191 ± 32.3μmoL / L, 23.4 ± 10.1μmoL / L, 76.1 ± 98.3μmoL / L, 11.9 ± 4.1μmoL / L, 16.4 ± 6.9μmoL / L in healthy subjects. Significant difference was detected between groups of NT, TT and DD levels . No significant difference was detected in terms of the NT / TT, DD / TT and DD / NT rates .Serum NT, TT, DD levels in patients with prostatitis and prostate cancer were found significantly lower compared to the control group. This indicates that just as inflammation, prostate cancer also increases oxidative stress on tissues. Prostate cancer is the second most prevalent type of cancer in men and is the second most prevalent cause of death from cancer in men. Past studies have shown the important role of age, genetic predisposition, androgen hormones, diet-related factors, inflammation and oxidative stress in the development of this disease . It was It was reported that oxidative stress resulted from the disruption of the balance of antioxidants and reactive oxygen radicals and that this caused various systemic diseases. Over-production of reactive oxygen types (ROT) causes damage in proteins and lipids. Oxidative damage is irreversible in serum and tissue proteins and significant changes occur in the structure and activity of proteins and biomolecules. Oxidative modifications in DNA and proteins can impact certain cellular functions, resulting in cell damage, death or mutation and carcinogenesis formation .Thiols are essential and strong anti-oxidant molecules in the sulfhydryl group, consisting of hydrogen atom and sulfur atom bonded to a carbon atom . The disWhen oxidative stress occurs, it has been noted than reduced thiol concentration increases and disulphide values increase in correlation . StudiesProstate Specific Antigen (PSA) is a critical marker in prostate cancer diagnosis. Serum total prostate specific antigen (t PSA) levels together with abnormal digital rectal examination were the most prevalent methods used in prostate biopsy indication in recent years . IncreasProstatitis is a disease that is observed at a rate of 8.2% 2.2-9.7%) in men. Acute bacterial prostatitis (ABP) is a pyogenic urinary system infection of the urinary system. It is observed at a rate of 5% among general prostatitis  is added for reduction of disulphide bonds into thiol groups again. The sum of residual thiol and reducted thiol groups encloses the total thiol. The remaining NaBH4 and DTNB ) are removed by formaldehyde. In this way, the amount of native and reduced thiol groups are defined separately. The difference between the TT and the NT is divided by two to calculate the quantity of the DD bonds. Also, we calculate the NT, TT, DD / TT, NT / TT and DD / NT percent ratio .Measurement of serum t PSA levels were conducted automatically with the electro-chemiluminescent method using the Hitachi Modular Analytics E 170 device .The patients were classified into risk groups according to Gleason score and t PSA values. Split into risk groups was as follows: low risk prostate cancer: T1-T2a stage and Gleason score ≤ 6 and t PSA ≤ 10, moderate risk prostate cancer: T2b stage and / or Gleason score = ≤ 7 and 10 ≤ t PSA ≤ 20 and high-risk prostate cancer: ≥ T2c stage or Gleason score 8-10 or t PSA > 20 .Statistical analyses were performed using the SPSS for Windows 15.0 package software. Compliance of the variables to normal distribution was examined using visual (histogram and probability graphs) and analytic methods (Kolmogorov-Smirnov / Shapiro-Wilk tests). In the Kolmogorov-Smirnov test, cases with p value greater than 0.05 were accepted as normal distribution. Differences between prostate cancer, prostatitis and the control groups in terms of NT, TT and DD were compared using the unilateral ANOVA test, as these variables showed normal distribution. The homogeneity of the variations was evaluated using the Levene test. Cases where the p value was lower than 0.05 were evaluated as statistically significant results. In cases with significant difference between the groups, post-hoc pair comparisons were performed using the Tukey's Test.NT / TT, DD / TT and DD / NT ratios were detected to not show normal distribution. The differences between these variables between prostate cancer, prostatitis and control groups were compared using the Kruskal-Wallis test. Pair comparisons were performed using the Mann-Whitney U test and evaluated using the Bonferroni correction. Total type-1 error level was used as 5% for statistical significance.A total of 80 subjects were included in the study, consisting of 30 (37.5%) prostatitis patients, 25 (31.3%) prostate cancer patients and 25 (31.3%) healthy subjects. Patients diagnosed with prostatitis had a mean age of 60.5 ± 12.8 (range 31-83). Patients diagnosed with prostate cancer had a mean age of 70.6 ± 6 (range 58-82). The age difference between the two groups was evaluated using the Student t test as they had normal distribution. Statistically significant difference was detected between the two groups in terms of age (p = 0.001). Patients diagnosed with prostatitis consisted of younger patients.Total Prostate Specific Antigen was detected as 139 ± 257.8 (range 4-1200) in patients with prostate cancer and as 51.3 ± 112 (range 3.9-405) in patients with prostatitis. Statistically significant difference was detected (p = 0.020) between patients with prostate cancer and prostatitis and tPSA value was detected to be higher in prostate cancer .Prostate cancer patient group NT was detected as 118.4 ± 36.8 (range 68.1-201.3) μmoL / L, TT 150.3 ± 45.3 (range 87.2-243.9) μmoL / L, DD 15.9 ±7 (range 4.4-31) μmoL / L, NT / TT rate 78.8 ± 7 (range 67.1-91), DD / TT rate 10.5 ± 3.5 (range 4.5-16.4) and DD / NT rate 13.8 ± 5.8 (range 4.9-24.4).In the prostatitis patient group NT was detected as 116.4 ± 40.5 (range 53.5-281.3) μmoL / L, TT 147.5 ± 50.1 (range 61-326.8) μmoL / L, DD 15.5 ± 8.7 (range 2.7-32.3) μmoL / L, NT / TT rate 79.7 ± 9 (range 66.7-94.7), DD / TT rate 10.1 ± 4 .5 (range 2.6-16.6) and DD /NT rate 13.5 ± 7.2 (range 2.7-24.9).In the control group NT was detected as 144.1 ± 21.2 (range 106.9-194.2) μmoL / L, TT 191 ± 32.3 (range 133.3-280.4) μmoL / L, DD 23.4 ± 10.1 (range 5-43.1) μmoL / L, NT / TT rate 76.1 ± 98.3 (range 66.2-92.8), DD / TT rate 11.9 ± 4.1 (range 3.5-16.8) and DD / NT rate 16.4 ± 6.9 (range 3.8-25.5).NT, TT and DD levels were detected to have normal distribution. Significant difference was detected between the three groups in NT levels (p = 0.008) . SignifiStatistical difference was detected in terms of TT levels between the prostate cancer, prostatitis and control groups (p = 0.001). Significant difference was not detected between prostate cancer and prostatitis in the Tukey post-hoc analysis (p = 0.970). Significant difference was detected between the prostate cancer group and the control group (p = 0.004). Significant difference was detected between the prostatitis group and the control group (p = 0.001) .Statistical difference was detected in terms of DD levels between the prostate cancer, prostatitis and control groups (p = 0.002). Significant difference was not detected between prostate cancer and prostatitis in the Tukey post-hoc analysis (p = 0.986). Significant difference was detected between the prostate cancer group and the control group (p = 0.009). Significant difference was detected between the prostatitis group and the control group (p = 0.004) .NT / TT, DD / TT and DD / NT ratios were detected to not show normal distribution. No significant difference was detected between the three groups in terms of the NT / TT, DD / TT and DD / NT rates .When the patients with prostate cancer were separated into risk groups according to t PSA and Gleason score, 2 (8%) had low risk, 8 (32%) had moderate risk and 15 (60%) had high risk. Significant difference was not detected between NT, TT and DD levels within the risk groups . Significant difference was also not detected between NT / TT, DD / TT and DD / NT rates within the risk groups .In our study we considered that serum NT, TT and DD levels could be a good marker in differentiating patients with high PSA values and normal patients. In this study we aimed to show the change in thiol/disulphide values in two different diseases with acute and chronic progression occurring on the same tissue.Many studies have been conducted on oxidative stress in urological patients regarding prostate cancer, benign prostatic hyperplasia or prostate inflammation –14. SomeMany recent studies have shown disorder in plasma thiol / disulphide homeostasis in the enteropathogenesis of diabetes mellitus, cardiovascular diseases, cancer, rheumatoid arthritis, chronic kidney disease, Parkinson disease and liver diseases –21. TherStudies have shown that oxidative stress biomarkers such as thiobarbituric acid reactive substances, total oxidative status, malondialdehyde, plasma nitrite / nitrate levels, lipid peroxide activities increased in prostate cancer patients compared to the control group . Some stPSA is the most commonly used marker in prostate cancer diagnosis. PSA value, tumor volume and Gleason score are the most important prognostic factors in the course of prostate cancer. High PSA values were present in both groups in our study. PSA level increases rapidly in the event of acute bacterial prostatitis. However, blood values such as CRP, ESR and White Blood Cell also rise.Prostate cancer is a chronic disease (except for high-risk prostate cancer), PSA values usually rise slowly and can also be detected at extremely high values depending on tumor aggressiveness. In this study, we aimed to show the change in thiol / disulphide values in two different diseases with acute and chronic progression occurring on the same tissue. According to our findings, NT, TT and disulphide values were found to be significantly different in both prostatitis and the prostate cancer compared to the control group. However, a statistically significant difference was not observed when both groups were compared. This shows that both cancer and inflammation trigger a similar oxidative stress on the tissue. Although oxidative stress markers rise suddenly in acute events, this increase takes place gradually in chronic processes. However, this process continues in cancer patients unless treated. In inflammation, the oxidative process returns to normal after the causative infectious event is remedied. Thiols, which are an anti-oxidant structure in the serum, may have decreased upon exposure to severe oxidation because anti-oxidant defense weakens or oxidation increases in prostate cancer or prostatitis patients. Oxidation products more progressed than disulphides were formed as thiols were subject to severe oxidation. As these are usually products of irreversible thiol oxidation, it is considered that disulphides are also low .Earlier studies have not shown the relation between tPSA level and thiol / disulphide values. In our study tPSA values and thiol/disulphide values were compared in both groups. However, although tPSA values were high in both groups, it was detected to be statistically higher in the prostate cancer group. However, no difference was detected between both groups in Thiol / Disulphide ratios. High tPSA values are a direct indicator of prostate tissue damage.The main limitations of the present study are its retrospective and non-randomized nature. In addition, the number of patients involved is small. These results need to be supported by prospective, randomized studies, and comprehensive patient series.Calculating thiol / disulphide values, which is a new marker, is an easy, inexpensive and reliable method. Serum NT, TT, DD levels in patients with prostatitis and prostate cancer were found significantly lower compared to the control group. This shows that just as inflammation, prostate cancer also increases oxidative stress on tissues. We consider that NT, TT, DD levels measures in serum can be used in the differential diagnosis of these pathologies.Approval was obtained for this research with the decision of the SANKO University Faculty of Medical Local Ethics Committee dated 29.03.2018 / numbered 2018 / 04 and the study was conducted in compliance with the Helsinki Declaration Rules. All patients participating in the study were informed about the study and their written consent was obtained. The data supporting the results reported in the article are applicable.The authors declare that they have no competing interests.We have no funding body in this study. MS did physical examinations and acquired the data of patients. HÇ studied on design and biochemical parameters. MY performed the statistical analysis and interpretation of data drafting of the manuscript. NO worked on critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.


We conducted a single-arm prospective phase II study to determine the efficacy and safety of the first-line treatment of advanced pancreatic cancer with nab-paclitaxel and S-1 followed by S-1 maintenance therapy.2. S-1 at 120 mg/day (for surface area ≥ 1.5 m2), 100 mg/day (for surface area between 1.25–1.5 m2), and 80 mg/day (for surface area < 1.25 m2) were given two times daily on days 1–14 every 3 weeks. Patients who achieved response and stable disease after 6 cycles were given S-1 maintenance treatment in the same schedule until disease progression or unacceptable toxicity developed. The primary endpoint was objective response rate (ORR), and the secondary endpoints were disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Between 01/2015 and 07/2017, 32 patients were enrolled.Nab-paclitaxel was administered intravenously on days 1 and 8 at 120 mg/mN = 32) was 53.1%, and the DCR was 87.5%. In the 30 evaluable patients, the ORR and DCR were 56.7 and 93.3%, respectively. The median follow-up time was 18 (range 12–36) months, the median PFS was 6.2 (range 4.4–8) months, and the median OS was 13.6 (range 8.7–18.5) months. The incidence of grade 3/4 neutropenia was 27.6%. Other grade 3 adverse events included 1 (3.1%) hand–foot syndrome, 1 (3.1%) rash and 2 (6.3%) diarrheas.The ORR in the intention-to-treat (ITT) population (Nab-paclitaxel and S-1 regimen has presented encouraging ORR, OS, and manageable toxicities as first-line therapy for advanced pancreatic cancer. Pancreatic cancer is one of the most fatal malignant disease worldwide with an increasing incidence. It is the fourth and seventh leading cause of cancer-related mortality in the world  and ChinGemcitabine has been approved as the standard chemotherapy for advanced pancreatic cancer since 1996, but the efficacy is extremely limited with a response rate of 6–8% and median survival of 5.5–7 months. Gemcitabine-based combination either with 5-FU or oxaliplatin and irinotecan failed to gain overall survival benefit compared to gemcitabine alone , 5. GemcS-1, a new oral fluoropyrimidine derivative, has been approved for advanced pancreatic cancer in Japan , 8 and iThe study was approved by the Ethical Committee of Cancer Institute and Hospital, Chinese Academy of Medical Sciences, No.14–102/892.Patients aged ≥ 18 years with histologically or cytologically confirmed pancreatic cancer were enrolled. Patients with locally advanced or metastatic disease, performance status 0–1, presence of at least one measurable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST 1.1), no prior systemic chemotherapy, normal blood routine, normal liver and kidney functions, and roughly normal electrocardiograph index were recruited.2. S-1 at 120 mg/day for surface area ≥ 1.5 m2, 100 mg/day for surface area between 1.25 and 1.5 m2, and 80 mg/day for surface area < 1.25 m2 were given 2 times daily on days 1–14 every 3 weeks. S-1 maintenance treatment was given to patients who achieved response and stable disease after 6 cycles of therapy until disease progression or unacceptable toxicity developed. Routine blood work, liver and kidney function tests, and electrocardiogram examination were performed before treatment. Doses were reduced by 20–25% in the next cycle for any grade 4 hematologic toxicity and grade 3 non-hematologic toxicity (with the exception of nausea and hair loss).Patients with locally advanced or metastatic pancreatic cancer were treated with nab-paclitaxel and S-1 as first-line therapy. Nab-paclitaxel was administered intravenously on days 1 and 8 at 120 mg/mAll patients were monitored by imaging examination every 2 cycles to evaluate the efficacy according to RECIST 1.1  for compThe primary endpoint was objective response rate (ORR), and the second endpoints included disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety.χ2 test used an α = 0.05.SPSS 17.0 software was used for statistical analysis. Survival was analyzed using Kaplan–Meier method and log-rank test. Cox proportional hazard regression model was used to examine both univariate and multivariate associations with survival. The 32 patients were enrolled between 01/2015 and 07/2017. The median age was 53 years (range 37–70 years), including 21 males and 11 females. All patients had performance status of 0–1. The most common metastatic site was liver, accounting for 71.9%. The baseline characteristics are described in Table N = 32) was 53.1% (Table 32 patients received a median 6 range 1–6) cycles. One patient withdrew from the study after 1 cycle of treatment due to abdominal pain. One carcinoma in pancreatic head with involvement of duodenum and hepatic metastasis withdrew and received palliative radiotherapy after 1 cycle of treatment for duodenal obstruction. The ORR in the intention-to-treat (ITT) population (–6 cyclesIn 29 patients (90.6%) with elevated carbohydrate antigen 19-9 (CA19-9) at baseline, 15 (52.3%) achieved ≥ 50% decline from baseline.19 patients (59%) completed 6 cycles of planned treatment. 13 patients received S-1 maintenance treatment, in which 11 patients were treated for 6 cycles, while the other 2 patients had response but started maintenance treatment after 4 cycles due to patients’ preference or adverse event. The median time of maintenance treatment was 3 (range 1–6) months.11 patients received gemcitabine plus oxaliplatin as the second-line treatment after failure of first-line chemotherapy, 2 PR (18.1%), 2 SD (18.1%), and 7 PD (63.6%) were reported. Irinotecan-based two drug combination regimen was given as the third-line chemotherapy for four patients, one had PR and three had PD.The median follow-up time was 18 range 12–36) months, median PFS was 6.2 (range 4.4–8) months, and median OS was 13.6 (range 8.7–18.5) months, and 12 patients survived as of the last follow-up , primary lesion (pancreatic head vs. tail), number of metastatic sites (1 vs. more), gender , age (≥ 60 vs. < 60), ≥ 50% decline from baseline CA19-9 (yes vs. no), with only liver metastasis (yes vs. no) by Cox regression model. No significant correlation was observed between the efficacy of the combination regimen and above clinical factors in the treatment.As shown in Table Nab-paclitaxel and S-1 act on the M and S phases of the cell cycle, respectively. Masaya  found thP = 0.0008), and has never been surpassed so far.In this study, nab-paclitaxel plus S-1 followed by S-1 maintenance therapy was proved an encouraging outcome as the first line treatment for advanced pancreatic cancer. The ORR and DCR reached 53.1 and 87.5%, higher than those of FOLFIRINOX, gemcitabine plus nab-paclitaxel (AG) and gemcitabine plus S-1(GS) in phase III trials. In the MPACT study , 861 patP = 0.000015) and the PFS of 5.5 vs. 3.7 months (P = 0.000024), in AG and gemcitabine group in MPACT trial. The PFS was 6.4 vs. 3.3 months (P < 0.0001), and the OS was 11.1 vs. 6.8 months (P < 0.001) in the FOLFIRINOX and GEM group, respectively [The survival in this study also seemed pretty encouraging. The PFS was 6.2 months and the OS exceeded 12 months in contrast to the median OS of 8.5 vs. 6.7 months (GS is another widely used combination regimen in Asia. In the phase III study (GEST) reported by Oka et al. , the ORRThe high response rate of nab-paclitaxel and S-1in our study is supported by another phase II trial recently reported by Shi et al. , in whicIn this study, the combination of nab-paclitaxel and S-1 indicated favorable safety profile. The most frequent grade 3/4 adverse effects were neutropenia with an incidence of 27.6% and most of the other adverse events were limited to grade 1/2. Only one patient developed neutropenic febrile. FOLFIRINOX brought survival benefit compared to gemcitabine but led to significantly increased adverse events with an incidence of grade 3/4 neutropenia of 45.7%, fatigue of 23.6%, vomiting of 14.5% and diarrhea of 12.7%, compared to 37% incidence of grade 3/4 neutropenia in MPACT trial, which is higher than the combination of nab-paclitaxel and S-1 in our study.Findings of this study demonstrated an encouraging efficacy and safety profile of nab-paclitaxel plus S-1 therapy. Further prospective randomized controlled phase III studies are needed to buttress these data. The high antitumor activity may provide a new option in the neoadjuvant setting for patients with locally advanced pancreatic cancer.


Artemisia afra (Asteraceae), a prominent feature of South African folk medicine, is used for the treatment of a number of diseases, including malaria. In search of transmission-blocking compounds aimed against Plasmodium parasites, the current study endeavoured to isolate and identify gametocytocidal compounds from A. afra.Optimal adoption of the malaria transmission-blocking strategy is currently limited by lack of safe and efficacious drugs. This has sparked the exploration of different sources of drugs in search of transmission-blocking agents. While plant species have been extensively investigated in search of malaria chemotherapeutic agents, comparatively less effort has been channelled towards exploring them in search of transmission-blocking drugs. Plasmodium falciparum (NF54 strain), using a parasite lactate dehydrogenase assay. Chemical structures of isolated compounds were elucidated using UPLC-MS/MS and NMR data analysis.A bioassay-guided isolation approach was adopted wherein a combination of solvent–solvent partitioning and gravity column chromatography was used. Collected fractions were continuously screened in vitro for their ability to inhibit the viability of primarily late-stage gametocytes of 50 < 10 μg/ml; ~ 10 μM) against both gametocytes and intra-erythrocytic asexual P. falciparum parasites. Interestingly, 1α,4α-dihydroxybishopsolicepolide was significantly more potent against late-stage gametocytes than to early-stage gametocytes and intra-erythrocytic asexual P. falciparum parasites. Additionally, both isolated compounds were not overly cytotoxic against HepG2 cells in vitro.Two guaianolide sesquiterpene lactones, 1α,4α-dihydroxybishopsolicepolide and yomogiartemin, were isolated and shown to be active  contains supplementary material, which is available to authorized users. Artemisia annua (Asteraceae) [Plasmodium falciparum parasites but, unfortunately, most have limited transmission-blocking activity [With over 1000 genera, 13 tribes and 20,000 species, the Asteraceae is one of the most diverse and largest flowering plant families in the world [eraceae) . Artemiseraceae) . ACT priactivity .Plasmodium parasites have been identified as a priority target for transmission-blocking efforts [Plasmodium parasites [Plasmodium parasites [With the shift in global malaria management programmes from control to eradication of the disease, the transmissible, asymptomatic, intra-erythrocytic sexual gametocyte stages of  efforts . However efforts . While Aarasites , 9. The arasites . This liarasites –16. One arasites .Plasmodium parasites, both in vitro and in vivo [Vernonia amygdalina (Asteraceae) has in vivo activity against Plasmodium berghei gametocytes with pronounced potency on microgametocytes compared to macrogametocytes [Parthenium hysterophorus (Asteraceae) and Tanacetum parthenium (Asteraceae), respectively, are active against mature stage-V gametocytes of P. falciparum [Plasmodium transmission-blocking drugs, and one species which warrants exploration is Artemisia afra (Asteraceae) [Despite the limited interrogation of plant species in search of transmission-blocking compounds, crude extracts of plants and plant-derived compounds have encouragingly been shown to be potent against late-stage gametocytes of  in vivo –21. Aste in vivo , 18, 22.eraceae) .Artemisia afra is a commonly used indigenous medicinal plant species in South Africa for the treatment of colds, coughs and fever [A. afra crude extract against the transmissible late-stage gametocytes of chloroquine-sensitive P. falciparum parasites was reported. However, the identity of the gametocytocidal compounds in A. afra was not determined [A. afra. The agents, a recent study investigated and provided fractionation steps for isolation procedure were primarily guided by in vitro activity against late-stage gametocytes of P. falciparum as measured using the parasite lactate dehydrogenase (pLDH) assay [nd fever , 23. Itsnd fever –26. In stermined . The preH) assay , 27.A. afra were collected from the Manie van der Schijff Botanical Garden at the University of Pretoria in July 2015. Artemisia afra leaves were collected from the same plant used for an earlier study [A. afra was subjected to solvent–solvent partitioning using five solvent systems of varying polarity in sequential order. The crude extract was dissolved in distilled water and extracted three times with hexane. After separation, the water was extracted separately with chloroform, ethyl acetate and methanol. The organic solvents were kept separate and evaporated to dryness under a stream of air.Leaves of er study . Voucherer study . Plant mer study . Dried cP. falciparum (NF54 strain) was cultured as described by Verlinden et al. [Plasmodium parasites generated through d-sorbitol (5% w/v) synchronization. Gametocytogenesis initiation, induction and gametocyte culturing was done as described by Reader et al. [The chloroquine-sensitive n et al.  and homor et al. .A. afra and isolated compounds  on the viability of both intra-erythrocytic asexual parasites and gametocytes stages of P. falciparum were assessed using the pLDH assay [P. falciparum (1% parasitaemia) was incubated for 72 h after which pLDH activity was quantified spectrophotometrically [The effect of dried fractions of DH assay , 27 withtrically . Both meA. afra as well as that of the active compounds isolated  in the study. Cytotoxicity assays were assessed on the HepG2 cell line  as previously described [The LDH leakage assay was used to examine the in vitro cytotoxicity of the dried chloroform fraction of escribed . EmetinePlasmodium parasites and HepG2 cell controls. Full dose–response assays  on Plasmodium parasites were carried out over a concentration range of 0.0003–40 μg/ml. Data were analysed on Microsoft Excel and sigmoidal dose–response curves were plotted using GraphPad Prism (v5). Unless otherwise stated, all data have been expressed as mean ± SEM . Statistical analysis was carried out on GraphPad Prism (v5) using unpaired Student-t test with a level of significance of P < 0.05.Assay data were normalized against untreated A. afra, gravity column chromatography was used. The dried chloroform fraction produced from the solvent–solvent partitioning step (and shown in vitro using the pLDH assay to be the most potent against late-stage gametocytes) was fractionated by primary column chromatography . The collected fractions from column 1 (F1–F24) were screened in vitro against late-stage gametocytes of P. falciparum using the pLDH assay, following which fractions F13 and F19 were subsequently prioritized for further purification. Fraction F13 was purified using secondary and tertiary columns , while F19 was purified using secondary column chromatography (designated column 4 with eluent of hexane: dichloromethane: acetone at ratio of 5:5:3). For column 1, all solvents used were technical grade, while for columns 2, 3 and 4 only analytical grade solvents from Merck were used. All column chromatography fractionation steps were carried out using silica gel 60 .To isolate and purify gametocytocidal compounds from 18 column  using 0.1% (v/v) formic acid in water (eluent A)  and 0.1% (v/v) formic acid in acetonitrile (eluent B) . Solvent gradient varied over time as follows; eluent A%: eluent B%—time; 97: 3–0.1 min; 80: 20–6 min; 70: 30–9 min; 70: 30–9.2 min; 0: 100–20 min; 0: 100–23.5 min; 97: 3–24 min; 97: 3–25 min. Flow rate was set at 0.4 ml/min at a pressure of 0–15,000 psi. Mass spectral data were acquired using electrospray ionization in positive mode (ESI +ve mode). The conditions were set as follows: 2.6 kV capillary voltage, 30 V sampling voltage, 4 V extraction cone voltage, 120 °C source temperature and 600 °C desolvation temperature. Scan mass range was set for 50–1200 m/z. MassLynx (Version 4.6) software was used for data acquisition and processing.To monitor separation of compounds during fractionation, thin layer chromatography (TLC)  and ultr1H, 13C and 13C DEPT-135) and 2D  NMR experimental spectra were recorded at room temperature, on a Bruker Avance III 400 spectrometer, operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. The isolated compounds from A. afra were dissolved in deuterated chloroform (CDCl3) . The chemical shifts from 1H NMR and 13C NMR spectra are reported in parts per million relative to the solvent residual peak (δH—7.26 and δC—77.0).1D  yielded four fractions, namely hexane (9.2 g), chloroform (8.2 g), ethyl acetate (1.4 g), and methanol (2.2 g) .From an initial 400 g of dried A. afra and the four dried fractions  on the viability of late stage gametocytes of P. falciparum was assessed using the pLDH assay . Methylene blue (10 μM) served as positive control drug inhibiting > 70% in vitro viability of late gametocyte stages. The hexane and methanol fractions were the least active (< 10% inhibition). By contrast, the chloroform fraction was potently active against late-stage gametocytes, inhibiting viability of these P. falciparum parasites at 56–99%, compared to 9–21% inhibition seen with the crude extract  purification of fraction F19 resulted in isolation of a white powder designated compound 2.Secondary column chromatography purification (column 2) of F13 yielded a crystalline compound of colourless to off-brown colour on visual inspection as the major compound in this fraction. The presence of an off-brown colour indicated presence of an impurity in the major compound, which necessitated a final column chromatography purification step (column 3) yielding clear ‘needle-like’ crystals, designated compound 1 and 2 against late-stage gametocytes ) compared to compound 2 (IC50 = 15.8 µM (5.3 µg/ml)) .In vitro full dose–response activity of compounds a et al.  has been1 and 2 were additionally screened in vitro against early-stage gametocytes (> 85% stage II–III) and intra-erythrocytic asexual P. falciparum parasites using the pLDH assay  difference between the activities of compounds 1 and 2 and that of the parent chloroform fraction of A. afra against asexual parasites of Plasmodium, compound 1 was significantly  more potent against late-stage gametocytes compared to both early-stage gametocytes and intra-erythrocytic asexual parasites of P. falciparum. By contrast, compound 2 was the most potent against early-stage gametocytes compared to compound 1 and the chloroform fraction .Motivated by the need for discovery of pan-reactive anti-malarial drugs, compounds 1 and 2 was interrogated using the LDH leakage assay against HepG2 cells and compared to that of the parent chloroform fraction of A. afra . The NMR assignments for the compound are shown in Table 1H, 13C and DEPT-135) and 2D  NMR experiments. The 1H NMR spectrum showed signals for two pairs of exomethylene protons at δH 5.13 , 4.86 , 6.32  and 5.86 , while signals for the 2,3 alkene protons resonated at δH 5.62  and 6.00 . Two singlets integrating for three protons each were observed at δH 2.14 and 1.32 for the acetyl methyl and vinyl methyl signals, respectively. Additionally, four methine signals appeared at δH 2.51 , 4.16 , 3.63–3.70  and 4.86–4.93 , as well as the signals for the methylene protons at δH 2.90  and 2.73 . The 13C NMR showed the presence of 17 carbon atoms with two signals at δC 169.9 and at δC 169.8 indicating the presence of two carbonyl groups. The signals appearing at δC 142.9, 140.6, 135.5, 134.4, 125.3, and 117.4 pointed to the presence of the six olefinic carbon atoms. The DEPT-135 showed the presence of two methyl, three methylene, six methine and six quaternary carbon atoms.The structures and identity of compounds 1 are shown Fig. 2 (H-13a and H13b) and C-12 confirming that the lactone moiety contains the classical exocyclic double bond seen in sesquiterpene lactones. A three-bond correlation between the acetate carbonyl and the proton at C-8 also confirmed that the acetate group is located at position C-8 . The NMR assignments for compound 2 are shown in Table 1H NMR spectrum exhibited doublets at δH 6.21  and δH 5.56 , which are characteristic of the exomethylene group. The three singlets upfield resonating at δH 1.10, 1.56 and 2.13, each integrating for three protons, are assigned to two methyl carbinol groups and one acetyl methyl group. The 1H NMR also revealed the presence of four oximethine protons appearing at δH 3.67 , 3.27 , 4.24 9 , and 5.25 , two methine protons resonating at δH 2.85  and 3.67–3.77 , and one methylene group appearing at δH 2.41  and 1.77 . The 13C NMR spectrum of compound 2 showed the presence of 17 carbon atoms, which indicated that the compound might be a sesquiterpene. The two signals appearing at δC 170.0 and 169.3 indicated the presence of two ester-functionalized carbonyl groups for the acetoxy and lactone, respectively. The carbon signals resonating at δC 137.4 and 121.5 indicated that the alkene is present in the structure. The DEPT-135 spectrum showed the presence of three CH3, two CH2, six CHs of which four are oxygen linked and six quaternary carbon atoms.Compound 2 are shown in Fig. H 6.21 (H-13a), 5.56 (H-13b) and δC 170.0. A three-bond correlation between oximethine proton appearing at δH 5.25 (H-8) and the carbonyl of the acetate group showing at δC 169.3 in the HMBC spectrum confirmed that the acetate group is located at position C-8 of the molecule  to C-1 and C-9 was observed, which suggests that this methyl group is situated at the carbinol carbon C-10. The relationship between H-2 and H-3 was established by the three-bond COSY correlation, which indicated that these protons are adjacent to each other  and 3.27 (H-3) on the 1H NMR; these chemical shift signals are consistent to those reported for epoxide protons found on a cyclopentane ring of a guaianolide sesquiterpene lactone [C 76.5 (C-1), 58.5 (C-2), 57.0 (C-3), and 70.0 (C-4)  parasites with a reported IC50 value of 8.7 µg/ml (27.2 µM) [50 value of 6.0 µg/ml (18.7 µM) measured in this study for this compound against asexual chloroquine-sensitive P. falciparum (NF54 strain) parasites. The work presented in this study was the first to provide a report on the gametocytocidal activity of these two compounds. Both compounds showed good activity against early- and late-stage gametocytes with IC50 values of < 10 µg/ml. Interestingly, 1α,4α-dihydroxybishopsolicepolide was significantly more potent against late-stage gametocytes than early-stage gametocytes and intra-erythrocytic asexual P. falciparum parasites. This is consistent with a previous report showing that the crude extract of A. afra was more active against late-stage gametocytes than early-stage gametocytes [Plasmodium, marking them as potential pan-reactive anti-malarial drug candidates. This pattern of in vitro pharmacological activity has been observed for the sesquiterpene lactone, artemisinin, which is known to exhibit pan-reactivity in vitro [50 values) across all three stages investigated [50 of > 12.5 µM, > 90% stage V) [The two compounds, 1α,4α-dihydroxybishopsolicepolide and yomogiartemin, were both found to be the principal components responsible for the gametocytocidal activity of 27.2 µM)  which coetocytes . The isoin vitro –43. Howestigated –44 althostage V) . Parthenstage V) , and parstage V) . Data pr.0 µg/ml .7 µM meastage V)  stronglystage V) .2+-ATPase (SERCA) pump, which has been shown to be targeted by two other sesquiterpene lactones, thapsigargin and artemisinin [Sesquiterpene lactones have been known to exert their therapeutic effect through a number of biological activities –50. Modeemisinin . Inhibitemisinin . Primaquemisinin . Drug-in50 values (< 10 µg/ml) against the asexual parasites of Plasmodium.Unfortunately, the same mechanism in which sesquiterpene lactones exert their therapeutic value may also be responsible for their cytotoxicity , 51. ThiA. afra to that of the two gametocytocidal compounds isolated in this study. The pure compounds were more selective towards the gametocyte stages while activities against asexual stages were identical to that of the chloroform fraction. However, activity against gametocytes was still in the same range as that observed for the crude extract of A. afra [A. afra, the activities of the two isolated compounds were not better than the crude extract despite the fact that concentration of the active compounds, on mass basis, was several orders of magnitude lower in the crude extract than in the pure compounds dosed. This pharmacological effect can partly be explained by the possible synergistic role played by other natural product compounds in A. afra. These could aid in the uptake of the active compounds, inhibit extrusion pumps, decrease metabolism or transport of the active compound leading to either synergistic or additive effects. Such effects have been observed for whole A. annua compared to pure artemisinin, with the former containing same concentration of the latter during the investigation. The whole dry plant was seen to be more active in vivo than the single pure compound, with the authors explaining that this was possibly due to improved bioavailability as well as synergism with other compounds, in particular the flavonoids [A. annua, may possibly potentiate artemisinin activity by inhibiting the enzyme thioredoxin reductase, which plays a vital role in the maintenance of redox balance in the intra-erythrocytic asexual parasites of Plasmodium [Plasmodium gametocytes to disruption in redox balance has been reported recently [Artemisia afra is a known producer of flavonoids [Plasmodium has been also reported in other studies [Plasmodium parasites.Synergy and additive effects in crude extracts of plants have been observed in other investigations, in some cases leading to greater activity in comparison to pure isolated compounds . This pr A. afra . Hence, avonoids . The autasmodium  making irecently . The autrecently . Artemisavonoids  includinavonoids  which stavonoids . It is t studies . There iPlants remain poorly investigated for their activity against the sexual transmittable gametocyte stages of the malaria parasite. The current study has clearly shown that a bioassay-guided strategy can be adopted fruitfully using phenotypic screening, which led to the isolation of two sesquiterpene compounds: 1α,4α-dihydroxybishopsolicepolide and yomogiartemin. Both of these have novel gametocytocidal activity, with negligible overt cytotoxicity. These compounds are unique starting points for their optimization to develop safe and efficacious pan-reactive transmission-blocking anti-malarial drugs.Additional file 1: Table S1. Inhibition of in vitro viability of late stage gametocytes of Plasmodium falciparum (NF54 strain) by crude extract and fractions of Artemisia afra.Table S2. Inhibition of Plasmodium falciparum late gametocyte stages by fractions from column 1. Table S3. IC50 values of Artemisia afra chloroform fraction, compounds 1 and 2 on intra-erythrocytic asexuals, early gametocytes and late stage gametocytes. Fig. S1. Base Peak Ion chromatograms from UPLC-MS analysis using ESI +ve mode for fractions A) F10, B) F11, C) F13 and D) F19. Fig. S2. Full dose-response curve plots for artemisinin (ART) and methylene blue (MB) against late-stage Plasmodium falciparum gametocytes.


The influence of Amphotericin B (AmB) dose and the addition of fluconazole (Flu) on the AmB + 5-flucytosine (5FC) regimen for cryptococcal meningitis (CM) treatment remain debatable.A retrospective study was conducted to compare 44 CM patients treated with AmB + 5FC and 78 CM patients treated with AmB + 5FC + Flu using the propensity score matching method. The effects of AmB dosage, AmB course and Flu addition on the cerebrospinal fluid (CSF) chemical profile recovery, adverse effects, and 90-day mortality were compared between the groups.P = 0.637) were observed between the AmB + 5FC group and the AmB + 5FC + Flu group. However, the incidence rates of hypokalemia (33.9%) and creatinine elevation (7.1%) in patients treated with an AmB dosage of 0.4–0.5 mg/kg/d were less than those  treated with an AmB dosage of 0.6–0.7 mg/kg/d . The 90-day cumulative survival rate was 70.1% for patients treated with AmB for <14 days and 96.4% for patients treated with AmB for ≥14 days (log-rank P < 0.001). Multivariate Cox proportional hazards models suggested the hazard ratio was 26.8 (95% CI: 3.9–183.2) for patients treated with AmB < 14 days than those treated with AmB ≥ 14 days (P = 0.001).No differences in adverse effects, the rate of the 14-day CSF chemical profile recovery and 90-day cumulative survival rate (91.2% vs. 87.5%, Treatment with AmB less than 14 days was associated with a higher 90-day mortality in CM patients. A relative lower dosage but prolonged course of AmB in the +5FC ± Flu regimen led to favorable trends of fewer adverse effects and comparable clinical efficacy. The current treatment using antifungal drugs against cryptococcosis involves three categories of drugs, namely, amphotericin B (AmB), triazoles, and 5-flucytosine (5FC). The standard protocol for the treatment of cryptococcal meningitis (CM) is divided into three phases: AmB (0.7–1 mg/kg/d) + 5FC (100 mg/kg/d) as an initial 2- to 4-week induction therapy, followed by fluconazole (Flu) 400–800 mg/d for 8 weeks as consolidation therapy and fluconazole 200 mg/d for at least 6 months as subsequent maintenance therapy .There are still some variations in this treatment recommendation. For example, the usage of AmB is usually associated with efficient fungal clearance and an improved survival rate in HIV-infected CM patients . HoweverCryptococcus infection , AmB dose (0.4–0.5 mg/kg/d vs. 0.6–0.7 mg/kg/d), and Flu supplementation on the clinical outcomes of CM patients treated with AmB + 5FC to delineate the optimal antifungal regimen for CM patients.Cryptococcus neoformans, (2) CSF India ink smear of centrifuged sediment positive for Cryptococcus, (3) histopathology compatible with Cryptococcus (5- to 10-μm encapsulated yeasts observed in brain tissue using silver and/or PAS stain), or (4) probable CM with the clinical syndrome of meningitis and positive cryptococcal antigen titer ≥1:4 or positive CrAg lateral flow assay in CSF even without microbiological or pathological documentation.The diagnosis of CM was defined as the presence of at least one of the following criteria: (1) culture of cerebrospinal fluid (CSF) positive for Cryptococcus counting procedure is explained briefly as follows: 1 ml of CSF was collected and centrifuged at a speed of 3000 rpm × 10–15 min. Then, the sediment (≈100 μl) was mixed with a small drop of India ink. A large coverslip was applied over 100 μl of mixture on the glass slide and pressed gently to obtain a thin mount. The slide was scanned under a low power field in reduced light using a microscope, then switched to a high power field (HPF) for counting if cryptococcal capsules were found. The Cryptococcus count (cells/HPF) = /10.The Hypokalemia was defined as serum potassium less than 3.0 mmol/L. Anemia was defined as a concentration of hemoglobin less than 110.0 g/L, and creatinine elevation was defined as a serum creatinine level higher than 110.0 mmol/L.A total of 427 CM patients treated at the First Affiliated Hospital, College of Medicine, Zhejiang University between January 2007 and December 2017 were eligible for this retrospective study. Of those, 124 (26.1%) had human immunodeficiency virus (HIV) infection, 81 (17.0%) had hepatitis B virus (HBV) infection, 41 (8.6%) had liver cirrhosis, 52 (10.9%) had diabetes, 19 (4.0%) had chronic kidney disease (CKD), 14 (2.9%) had chronic hematological disease (CHD), 10 (2.1%) had malignancies, 28 (5.9%) underwent solid organ transplantation (SOT), and 37 (7.8%) had autoimmune diseases. Overall, HIV-uninfected CM patients were predominant (73.9%). There were 27 patients treated with voriconazole (VCZ) ± 5FC as the induction therapy, 3 with mono-AmB, 3 with AmB + VCZ, 53 with AmB + 5FC, 54 with a liposomal amphotericin B (L-AmB)-based regimen, 108 with Flu ± 5FC, 151 with AmB + 5FC + Flu, 18 with AmB + Flu, 4 with AmB + Itraconazole (ITC) ± 5FC, and 6 with other regimens. Fifty-three patients treated with AmB + 5FC and 151 patients treated with AmB + 5FC + Flu were eligible for observation after the exclusion of patients treated with other antifungal regimens. Propensity score matching (PSM) for age, sex, body mass index (BMI), predisposing diseases, routine blood test results, liver function, and renal function was used to select patients according to a match ratio of 1:2. There were 44 patients treated with the induction treatment of AmB + 5FC, and 88 patients (including 10 repeated cases) treated with AmB + 5FC + Flu were selected for study. Finally, 44 patients who received induction treatment with AmB + 5FC and 78 patients who received initial treatment with AmB + 5FC + Flu were enrolled in the present study after exclusion of 10 repeated cases. The enrollment was performed according to the process indicated in Forty-four patients were initially treated with amphotericin B deoxycholate (AmB) + 5FC, and 78 were initially treated with AmB + 5FC + Flu. According to the patients’ tolerance, AmB that was administered to patients ranged from 30 to 60 mg/d (approximately 0.4–0.7 mg/kg/d) via peripheral intravenous (IV) in 5% dextrose in a total volume of 500 ml. One liter of saline was administered intravenously immediately prior to amphotericin administration to minimize renal toxicity. The oral 5FC dosage was 80–100 mg/kg/d, and the Flu dosage was from 400 to 800 mg/d via IV. The mean dose of AmB was 0.4–0.5 mg/kg in 56 (45.9%) patients and 0.6–0.7 mg/kg in 66 (54.1%) patients whose dosage of AmB was 0.4–0.7 mg/kg/d during induction treatment. There were 42 patents treated with AmB for less than 14 days. Of those, 4 patients died, and 38 patients had adverse effects. The AmB treatment duration was 6.0 (3.0–13.0) days for patients treated with AmB for less than 14 days and 29.5 (19.0–39.8) days for patients treated with AmB for more than 14 days.Basic medical information of the patients  was recorded and saved in the electronic medical records system (EMRS) of the hospital. The time to follow-up was calculated from the first day of antifungal treatment. Patients were followed up for 90 days.t-test or Mann-Whitney U test. Categorical variables were compared using the χ2 analysis or Fisher’s exact test. Patient survival was analyzed using the Kaplan–Meier method. CM-related death was defined as an “event.” Data for patients were censored at the date of the final visit , the date last known to be alive  or the date of death from a cause that was not known to be AIDS related. Patients were followed up until 10 weeks after initiation of therapy. These covariates were first analyzed using the univariate Cox model. Thereafter, covariates with P < 0.2 in the univariate model were selected for the multivariate Cox proportional hazards model using the forward stepwise (likelihood ratio) method. The missing data were excluded from the multivariate analysis. Covariates  were used for propensity matching with a matching tolerance of 0.1 according to the match ratio of 1:2. P < 0.05 (two-tailed) was considered significant. Data analyses were performed using SPSS version 24.0  and GraphPad Prism version 6.0 .Continuous normal variables are presented as the means ± standard deviations, and continuous abnormal variables are presented as the medians . Categorical variables are presented as the number of cases (percentage). Continuous variables were compared using the Student’s The study protocol was in accordance with the Declaration of Helsinki 1975 guidelines and was approved by the Ethics Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University  (No. 2017-688). Written informed consent was obtained from all the patients, and all data analyzed were anonymous.Cryptococcus culture positivity, 4 by CSF Cryptococcus culture positivity combined with either cryptococcal antigen titer ≥1:4 or CrAg lateral flow assay in CSF, 24 by India ink staining combined with either cryptococcal antigen titer ≥1:4 or CrAg lateral flow assay in CSF, and 19 patients by cryptococcal antigen titer ≥1:4 or positive CrAg lateral flow assay in CSF but without the finding of Cryptococcus.Cryptococcal meningitis was diagnosed in 55 individuals via the method of CSF India ink staining and culture after admission, 20 by sole CSF P = 0.126). Similarly, 36.4% of the patients were treated with AmB doses ≤ 0.5 mg/kg/d, and 63.6% were treated with AmB doses > 0.5 mg/kg/d in the AmB + 5FC group; 51.3% of the patients were treated with AmB doses ≤ 0.5 mg/kg/d, and 48.7% were treated with AmB doses > 0.5 mg/kg/d in the AmB + 5FC + Flu group (P = 0.112). The CSF cryptococcal fungal burden was 2 (0–5) cells/HPF in patients treated with AmB + 5FC group and 2 (0–4) cells/HPF in patients treated with AmB + 5FC + Flu (P = 0.497). There were 15 HIV-infected (12.3%) and 107 HIV-uninfected CM patients (87.7%) in our cohort. The demographic characteristics and laboratory test results are listed in Forty-four of the 53 patients who received induction treatment with AmB + 5FC and 78 of the 151 who received induction treatment with AmB + 5FC + Flu were enrolled after PSM. Of those patients, 19 (43.2%) were treated with AmB for less than 14 days, and 25 (56.8%) were treated with AmB for ≥14 days in the AmB + 5FC group; 23 (29.5%) were treated with AmB for <14 days, and 55 (70.5%) were treated with AmB for ≥14 days in the AmB + 5FC + Flu group (P = 0.857), anemia , creatinine elevation , or chills  between the AmB + 5FC group and the AmB + 5FC + Flu group, respectively.The main adverse effects that were observed in our study were hypokalemia, anemia, renal damage, and chills. Of the patients, 54 (44.3%) presented with hypokalemia, 34 (27.9%) patients had anemia, 19 (15.6%) had creatinine elevation, and 3 (2.5%) patients had chills during treatment. No differences were found in the incidence of hypokalemia . The overall incidence of creatinine elevation was 7.1% (4/56) in patients treated with AmB doses 0.4–0.5 mg/kg/d and 22.7% (15/66) in patients treated with AmB doses of 0.6–0.7 mg/kg/d (P = 0.018). However, the incidence of anemia was 23.2% (13/56) in patients treated with AmB doses of 0.4–0.5 mg/kg/d and 36.4% (21/66) in patients treated with AmB doses of 0.6–0.7 mg/kg/d (P = 0.291).The overall incidence of hypokalemia was 33.9% (19/56) in patients treated with AmB doses of 0.4–0.5 mg/kg/d and 53.0% (35/66) in those treated with AmB doses of 0.6–0.7 mg/kg/d (P = 0.126). In the AmB + 5FC group, 17 patients discontinued AmB treatment because of adverse effects, and 2 patient discontinued AmB treatment because of death. Furthermore, in the AmB + 5FC + Flu group, 21 patients discontinued AmB treatment because of adverse effects, and 2 patients discontinued AmB treatment because of death. Among the patients who survived more than 14 days, the percentage who received AmB for <14 days was 40.5% (17/42) in the AmB + 5FC group and 27.6% (21/76) in the AmB + 5FC + Flu group (P = 0.153).There were 42 patents treated with AmB for less than 14 days, namely, 19 patients in the AmB + 5FC group and 23 patients in the AmB + 5FC + Flu group : 0.7 ± 1.3 vs. 0.6 ± 1.1, respectively, P = 0.684; chlorine (mmol/L): 4.4 ± 5.7 vs. 4.2 ± 7.0, respectively, P = 0.866).The CSF protein level in the AmB + 5FC group was 0.8 (0.6–1.2) g/L at admission and decreased to 0.6 (0.5–0.9) g/L after 2 weeks of antifungal treatment. In addition, the CSF protein level in the AmB + 5FC + Flu group was 0.8 (0.5–1.4) g/L at admission and decreased to 0.6 (0.4–0.9) g/L after 2 weeks of antifungal treatment. There were no significant differences in the protein level reduction between the two groups [AmB + 5FC group: −0.2 (−0.4–0) g/L vs. AmB + 5FC + Flu group: −0.2 (−0.6–0) g/L; Cryptococcus culture sterilization was 69.2% (18/26) in the AmB + 5FC group and 75.9% (22/29) in the AmB + 5FC + Flu group (P = 0.581) among those patients with data available.In addition, the 2-week CSF P = 0.637) (P = 0.744) (Ten patients (8.2%) died within 90 days; 4 patients died within 14 days, and 6 patients died in 15–90 days after antifungal treatment. Of those patients, there were 3 patients in the AmB + 5FC group and 7 patients in the AmB + 5FC + Flu group. The 90-day cumulative survival rate was 91.2% in patients treated with AmB + 5FC and 87.5% in patients treated with AmB + 5FC + Flu (= 0.637) . We furt= 0.744) .P < 0.001)  .vs. ≥14 days) were taken together for analysis. We found that the 90-day cumulative rate was 100% for patients treated with an AmB course ≥14 days along with an AmB dose of 0.4–0.5 mg/kg/d, 93.9% for patients treated with an AmB course ≥14 days along with an AmB dose of 0.6–0.7 mg/kg/d, 73.8% for patients treated with an AmB course <14 days along with an AmB dose of 0.6–0.7 mg/kg/d and 66.6% for patients treated with an AmB course <14 days along with an AmB dose of 0.4–0.5 mg/kg/d (log-rank P = 0.001)  and AmB course (<14 days = 0.001) .9/L; <10.0 and ≥10.0), hemoglobin , serum albumin , intracranial pressure (ICP) , CSF WBC count , CSF glucose , CSF chlorine , CSF total protein , CSF fungal count , induction treatment (AmB + 5FC vs. AmB + 5FC + Flu), AmB dose , and AmB course . In the unadjusted univariate model, our data suggested that a WBC count in peripheral blood ≥10 × 109/L (P = 0.050), CSF chlorine < 105.0 mmol/L (P = 0.005), CSF fungal count ≥20.0 capsules/HPF (P = 0.044), and an AmB course <14 days (P = 0.002) were associated with 90-day mortality after the diagnosis of CM. CSF WBC < 30.0 × 106/L (P = 0.161), CSF glucose < 1.5 mmol/L (P = 0.113), and CSF protein (P = 0.084) were close to 90-day mortality. However, sex, age, CRP, hemoglobin, serum albumin, ICP, AmB + 5 FC ± Flu, and AmB dose were not associated with an increase in CM-related mortality. In the multivariate Cox proportional hazard model, only the WBC count in peripheral blood, CSF chlorine < 105.0 mmol/L and an AmB course <14.0 days in anti-fungal treatment were still associated with the 90-day cumulative survival rate. The hazard ratios (HRs) were 25.1 (95% CI: 3.7–171.1) for patients with CSF chlorine < 105.0 mmol/L compared with that of patients with CSF chlorine ≥105.0 mmol/L (P = 0.001), 5.0 (1.1–23.3) for patients with a WBC count in peripheral blood ≥10.0 × 109/L compared with that of patients with a WBC count in peripheral blood <10.0 × 109/L (P = 0.042) and 24.4 (95% CI: 3.6–163.6) for patients treated with AmB < 14.0 days compared with those treated with AmB ≥ 14.0 days (P = 0.001) , age , C-reactive protein level (CRP) , WBC count (×10= 0.001) .Although AmB plus 5FC followed by a switch to Flu is the standard treatment for CM, there are some challenges to its implementation in clinical practice. For example, some patients cannot endure the side effects of AmB, which leads to course shortening or dose reduction in the application of AmB. In addition, the concentration of AmB in the CSF is only 1% of the serum concentration , whereasIn our present study, we compared the efficacy of AmB plus 5FC alone or supplemented with Flu in Chinese CM patients, and we found the following: (1) adding Flu to the AmB + 5FC regimen did not improve the recovery of the 2-week CSF chemical profile nor did it improve the 90-day survival rate of the patients; (2) treatment with AmB for less than 14 days increased the risk of death for CM patients, especially patients treated with an AmB dosage of 0.4–0.5 mg/kg/d; and (3) an AmB dosage of 0.4–0.5 mg/kg/d in the AmB + 5FC ± Flu regimen led to a lower incidence of adverse effects but could achieve clinical efficacy that was comparable to the results of a dosage of AmB 0.6–0.7 mg/kg/d.Cryptococcus infection. In some cases, Flu ± 5FC can be used as an alternative induction treatment for patients when AmB is unavailable or unendurable. Particularly, high-dose fluconazole plus 5FC was reported as an alternative treatment for HIV-infected CM patients  had a higher incidence of satisfactory outcomes and a higher rate of Cryptococcus sterilization in the CSF than did therapy with AmB + 5FC  was associated with increased mortality in CM patients. The hazard ratios were 27 times higher for patients treated with AmB for less than 14 days than for those who were treated with AmB for ≥14 days in patients treated with AmB + 5FC ± Flu. Our study results were consistent with previous recommendations that AmB + 5FC should be administered at least for 2 weeks . HoweverCryptococcus. Second, higher serum AmB concentration does not necessary relate to higher CSF concentration. The AmB concentration in CSF is only 1% of its concentration in serum  produced more safety and similar efficacy to prevent or treat fungal infections . The posin serum ; consequin serum . Third, in serum .Cryptococcus infection could be considered for CM patients. Notably, the 90-day mortality of 8.2% in our patients is significantly lower than that of approximately 35% in African CM patients  as induction treatment for patients , 32% in patients , and 15–patients , indicatpatients . Higher There were some limitations in our study. First, our study was a retrospective study, although we tried to reduce certain biases by using a PSM method. Therefore, a prospective study is needed to further address the role of AmB in treatment with AmB + 5FC ± Flu. Second, our data were based on a small sample size and a short follow-up duration. Consequently, a study based on a larger sample size and prolonged observation is needed in the future. Third, the influence of 5FC and Flu on the outcomes of patients was not discussed. Nevertheless, we and other researchers understand the potential effects of 5FC and Flu upon patient outcomes. For example, a study indicated that the addition of 5FC and a short course of AmB therapy to high-dose Flu therapy significantly enhanced early fungicidal activity and may be associated with favorable trends in survival .In summary, our study suggested that compared with induction treatment with AmB + 5FC, the addition of Flu to AmB + 5FC for induction treatment did not improve the clinical outcomes of Chinese CM patients. The short-term (less than 14 days) together with low dose (less than 0.7 mg/k/d) usage of AmB is associated with a higher mortality risk in patients. The use of low dosages of AmB (such as 0.5–0.7 mg/kg/d) and a prolonged course (more than 14 days) in an AmB + 5FC ± Flu regimen were safer and of comparable efficacy in Chinese CM patients.All datasets generated for this study are included in the manuscript and/or the supplementary files.The study protocol was in accordance with the Declaration of Helsinki 1975 guidelines and was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University  (No. 2017-688) and all data analyzed were anonymous.LX, JH, and BZ conceived and designed the study. RT, JW, XD, and CH collected the data. LX analyzed the data. YH followed up the study. LX and YC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.