In this study, we examined the anti-inflammatory effect of the new human histamine HE243 -flavone and IIIa–IIIc—chalcones) for human H3R was evaluated in the radioligand binding assay. The cytotoxicity on BV-2 cell viability was investigated with the MTS assay. Preliminary evaluation of anti-inflammatory properties was screened by the Griess assay in an in vitro neuroinflammation model of LPS-treated BV-2 cells. The expression and secretion of pro-inflammatory cytokines were evaluated by real-time qPCR and ELISA, respectively. The expression of microglial cell markers were determined by immunocytochemistry.The affinity of flavonoids  compound IIIc was the most active in reducing the level of nitrite in Griess assay. Additionally, IIIc treatment attenuated inflammatory process in murine microglia cells by down-regulating pro-inflammatory cytokines  at both the level of mRNA and protein level. Our immunocytochemistry studies revealed expression of microglial markers  in BV-2 cell line.Chalcone derivatives showed high affinity at human HThese results emphasize the importance of further research to accurately identify the anti-inflammatory mechanism of action of chalcones. Microglia are called the ‘third element’ of the central nervous system (CNS), along with neurons and astrocytes, since in 1918 Pio del Rio Hortega developed a method to stain microglia and distinguish them from the neighbouring cells of the CNS . Recentl1 receptors (H1R), histamine H2 receptors (H2R), histamine H3 receptors (H3R), and histamine H4 receptors (H4R). Among them, H1R, H2R, and H3R are observed in the brain, whereas the results about H4R are inconsistent + 380.07.Synthesis from 4-methoxybenzaldehyde . Yield 12% (49 mg), m.p. 174–177 °C, C25H31NO4 x HCl (MW = 445.98). 1H NMR  δ: 10.46 , 8.14 , 7.80 , 7.63 , 7.50 , 7.34 , 7.05 , 6.98 , 4.15 , 3.82 , 3.78 , 3.41 , 3.09—3.19 , 2.76—2.89 , 2.14—2.26 , 1.70—1.83 , 1.67 , 1.35 . 13C NMR  δ: 187.7, 162.6, 151.7, 149.5, 144.3, 131.4, 128.1, 124.4, 120.0, 114.9, 112.0, 111.2, 66.0, 56.3, 56.1, 53.8, 52.5, 23.7, 22.9, 21.9. LC/MS purity: 100%, tR = 4.71 s, (ESI) m/z [M + H]+ 410.30.Synthesis from 3,4-dimethoxybenzaldehyde . Yield 25% (110 mg), m.p. 173–176 °C, C26H33NO5 x HCl (MW = 476.00). 1H NMR  δ: 10.48 , 8.16 , 7.87 , 7.63 , 7.19 , 7.06 , 4.15 , 3.83 , 3.67 , 3.40 , 3.13 , 2.83 , 2.21 , 1.70—1.87 , 1.66 , 1.35 . 13C NMR  δ: 187.8, 162.7, 153.6, 144.3, 140.1, 131.5, 131.2, 130.9, 121.7, 115.0, 107.0, 66.0, 60.7, 56.7, 53.8, 52.5, 23.7, 22.8, 21.9. LC/MS purity: 100%, tR = 4.84 s, (ESI) m/z [M + H]+ 440.34.Synthesis from 3,4,5-trimethoxybenzaldehyde . Yield 20% (95 mg), m.p. 176 °C dec., C3 receptor as we described previously [3H]-N-α-methylhistamine was used as radioligand and (R)(-)-α-methylhistamine was used to define nonspecific binding. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software) and Ki values were estimated from the Cheng − Prusoff equation.Radioligand binding studies were conducted using CHO-K1 cells stably transfected with the human Heviously . All ass1R or Sf9 cells transiently expressing the H4R were performed as described previously [3H]Pyrilamine (H1R) and [3H]histamine (H4R) were used as radiolignads. Affinities were determined using at least five different concentrations of tested compounds in triplicate independent experiments.Radioligand binding studies, using CHO-K1 cells stably expressing the Heviously . [3H]Pyr2 at 37 °C. BV-2 cells were used for experiments at passages 3 to 11. In all experiments, the concentration of FBS was reduced to 5%.The murine microglial BV-2 cell line was purchased from the Interlab Cell Line Collection (ICLC) cell bank . The BV-2 cells were cultured in DMEM/F12 medium  containing 10% of heat-inactivated fetal bovine serum  in a humidified incubator containing 95% air and 5% CO4 cells/100 μl/well) were cultured in transparent 96-well plates  in DMEM/F12 supplemented with 5% FBS in the presence of dimethylsulfoxide  or increasing doses of tested compounds (0.78 × 10–6 – 50 × 10–6 M). Compound treatment was performed for 24 h. After the incubation time, cell viability was examined using an MTS-based [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H tetrazolium] CellTiter96® AQueous One Solution Cell Proliferation Assay  following the manufacturer’s protocol. Briefly, 20 μl of MTS solution was pipetted into each well containing 100 μl of culture or culture medium without cells (blank control), mixed well and incubated at 37 °C for 55 min. After the appropriate time, the absorbance was measured at 490 nm using the EnSpire microplate reader . IC50 values were calculated with non-linear regression fit to a sigmoidal dose–response curve  using GraphPad Prism (v. 4.0.3).BV-2 cells (2 × 102−) which is one of the primary and stable metabolites of NO. Griess reagent  was freshly prepared according to the manufacturer's instructions, that is, 1 g was dissolved in ultrapure water and mixed by inversion for about 4 min. For measuring nitric oxide, after an appropriate incubation time, 100 μl of culture medium was transferred to a transparent 96-well plate and mixed with the same volume of Griess reagent. The plate was incubated for 15 min at room temperature and the absorbance was read at 540 nm using the EnSpire microplate reader . Data were analysed by direct comparison based on the average absorbance of LPS treatment, compound treatment or co-treatment with LPS, performed by normalizing treated cells  to LPS-treated cells set as 100%.The Griess assay is a colorimetric assay to measure the levels of nitrite  according to the following scheme: 1) DMSO ; 2) LPS (1 μg/ml); 3) tested compounds (6.25 μM) 4) 1 h pretreatment with tested compounds followed by LPS 1 μg/ml. The experiment was carried out in triplicate and at least three independent experiments were carried out. Total RNA was extracted from BV-2 cells with TRIzol® reagent , based on the guanidine thiocyanate method described by Chomczynski and Sacchi with some modification . Following LPS stimulation, at each time point, 100 μl of culture medium from each sample was transferred to another plate and mixed with an equal volume of Griess reagent. Simultaneously, on the plate with cells, cell viability was assessed by adding 20 μl of MTS reagent. The cells cultured on a 24-well plate were used for assessing the expression levels of proinflammatory cytokines  by RT-qPCR.To establish an experimental model of neuroinflammation in vitro4 cells were seeded into each well and the following day were inoculated with 1 µg/ml LPS and stimulated for a further 24 h. After that, cells were fixed with 4% PFA for 15 min at room temperature and subsequently washed with PBS. Samples were prepared as for classical IHC staining described above, heated  solution, permeabilized with PBS contained 0.25% Triton-X-100, blocked in 5% normal donkey serum  in PBS and incubated overnight in 4 °C with proper primary antibodies diluted in 5% NDS or SignalBoost™ Immunoreaction Enhancer Kit : anti-IBA1 , anti-B7-2/CD86 , anti-CD206 . After three washes in PBS, triple immunofluorescence was revealed by incubation for 2 h in the appropriate fluorochrome-conjugated secondary antibody: donkey anti-mouse Alexa Fluor488 , donkey anti-rabbit Alexa Fluor594  donkey anti-goat Alexa Fluor647  diluted 1∶500 in 5% NDS. Sections were then washed and coverslipped with an Aquatex mounting medium . Leica TCS SP8 WLL confocal microscope was used to take the pictures. Images were processed in Leica LAS X microscope software.BV-2 cells were cultured using the Nunc™ Lab-Tek™ II 8-well Chamber Slide™ System (Thermo Fisher Scientific), 4 × 10All statistical analyses were carried out using GraphPad Prism 7 with significance determined by One-way ANOVA followed by post-hoc comparisons tests as detailed in the figure legends.IIIa–IIIc were tested for affinity for human H3R (hH3R) in a radioligand binding assay with Nα-methylhistamine as a radioligand in CHO K1 cells stably expressing hH3R. Results are presented as Ki values with SEM . The affinity of this compound was comparable to a derivative with one methoxy group in the phenyl ring, compound IIIa (Ki = 14 nM). It appears that the introduction of the third methoxy group into the 5 position in the phenyl ring (IIIc) slightly decreased the affinity of hH3R (Ki = 24 nM). However, it is still a very active compound. The affinity for hH3R (stably expressed in HEK293 cells) of compound E243 was recently described [hH3R (Ki = 228.2 nM).Compounds EM Table . In geneescribed . This co1 receptor (hH1R) and human H4 receptor (hH4R). Results are collected in Table hH1R and hH4R. A selectivity for hH3R over these receptors was good (> 100) for chalcones IIIb and IIIc (IIIb: Ki (H4R)/Ki (H3R) > 1300, Ki (H1R)/Ki (H3R) > 112; IIIc: Ki (H4R)/Ki (H3R) > 117, Ki (H1R)/Ki (H3R) > 112). However, compound E243 proofed to be non-selective, especially towards H1R: Ki (H4R)/Ki (H3R) > 19 and Ki (H1R)/Ki (H3R) > 5.Selected compounds were further evaluated for their binding affinity at related histamine receptor subtypes: human histamine HIL-1β, IL-6, and TNF-α was dose-dependent in response to LPS range concentration. Therefore, the results demonstrated that the optimal concentration of LPS and the duration of exposure for the next experiment were 1 μg/ml and 24 h, respectively.Initially, our objective was to establish a functional in vitro model of neuroinflammation in murine microglial BV-2 cells. For this, we first examined the effect of increasing concentration of LPS (0.001 – 1 μg/ml) on the viability of BV-2 cells at three time points: 24 h, 45 h, and 69 h. The goal of this experiment was to select an appropriate dose of LPS that would induce an increase in nitric oxide (NO)—a commonly assessed neuroinflammatory endpoint—without causing a cytotoxic effect on cells. Our experimental results showed that LPS in all concentration ranges tested significantly induced NO level after 24 h of treatment without causing any toxic effect on BV-2 cells  in both control and LPS-stimulated BV-2 cells Fig. . Moreove3R ligands in subsequent studies, an MTS assay was performed to investigate cell viability. BV-2 cells were cultured for 24 h in the presence of an increasing concentration of compounds E243 and IIIa–IIIc (twofold dilutions covered the range from 0.78 × 10–6 to 50 × 10–6 M). A dose–response effect is presented on Fig. 50 values are summarized in Table To avoid the toxic effect of H3R ligands on LPS-stimulated BV-2 cells, the level of NO in cell culture medium was measured by the Griess reagent assay. As shown in Fig. E243 and IIIa–IIIc. Compounds E243, IIIa, and IIIb reduced the level of NO by an average of 30% compared to LPS treated cells, while IIIc reduced this level by 61%. Because compound IIIc reduced NO concentration twice more than the other tested compounds, it was selected for further study.To evaluate the anti-inflammatory effects of HIIIc on the expression of pro-inflammatory cytokines, BV-2 cells were treated with LPS in the presence or absence of the compound tested for 24 h. Figure IIIc treatment, indicating that this compound has anti-inflammatory activity. We found that IL-6 mRNA levels after cotreatment with LPS and IIIc were 10 times lower compared to those induced by LPS alone and that was the most significant decline. mRNA levels of IL-1β and TNF-α were on average 2 times and 3 times lower, respectively, than those induced by LPS alone.To investigate the effect of IIIc affected their production using ELISA. The results of this test are presented in Fig. IIIc in the presence of LPS prevents the induction of pro-inflammatory cytokines. The release of IL-1β, IL-6 and TNF-α were significantly lower compared to LPS-stimulated BV-2 cells. These results showed that protein expression results correlate with mRNA expression results, further proving the anti-inflammatory nature of the compound tested.IL-1β, IL-6 and TNF-α are critical pro-inflammatory cytokines in response to LPS. Thus, we decided to check whether 3R antagonists on expression and secretion of pro-inflammatory cytokines in murine microglia after LPS stimulation. LPS is a widely used stimuli to model neuroinflammation associated with neurodegeneration [Our initial studies aimed to examine the effect of Hneration . From thneration . Thus, cneration , 44. For3R antagonists in a functional in vitro model of neuroinflammation. For this purpose, we first optimized the concentration of LPS that is adequate to significantly induced the signs of neuroinflammation. It was shown that 1 μg/ml of LPS stimulated BV-2 cells to remarkably produce the level of NO and expression of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. These results are in line with those obtained by Hosseini et al. who strictly focused on optimization the conditions of inflammation in BV-2 cell [The main challenge for this study was to evaluate the anti-inflammatory efficacy of HV-2 cell . The aboV-2 cell , 47. HerV-2 cell ) such asE243 (a substituted flavone), and compounds IIIa-IIIc  could reduce NO production in LPS-stimulated BV-2 cells. Results indicated that all tested compounds reduced the level of NO, but only one of the chalcones (IIIc) showed the most potent inhibitory activity and lowered this level by as much as 61% compared to control cells (not treated with LPS). The most interesting for us at this stage of investigation was to establish whether this data correspond to the lower level of pro-inflammatory cytokines. The analysis of genes expression was evaluated by real-time qPCR. We found that the IL-6 mRNA level was ten times lower expressed when BV-2 cells were co-treated with IIIc instead of LPS alone. Satisfactory results were also obtained for mRNA levels of IL-1β and TNF-α. It is worth noting that, we also have shown that the results of mRNA expression correlate with that of protein expression evaluated by ELISA. These findings further strongly support our hypothesis that H3R antagonist based on the chalcone scaffold could be valuable in inflammation research.Next we checked whether compound . Yang et al. demonstrated that Safflower yellow, which is a mixture of water soluble chalcone, significantly inhibited the production of IL-1β, IL-6, TNF-α in LPS-stimulated BV2 cells [., it was shown that 2′-hydroxy-3,4,5,3′,4′-pentamethoxychalcone was shown to represent a basic structure with the most potent anti-inflammatory effect, evidenced by complete inhibition of NO and the reduction of various pro-inflammatory cytokines in activated BV-2 cells [hH3R with Ki values < 25 nM. However, the most potent compound IIIb with a dimethoxy substituent on the phenyl ring (Ki = 9.6 nM) and its derivative with one methoxy group on the phenyl ring, compound IIIa (Ki = 14 nM) have not shown inhibitory activity on pro-inflammatory cytokine production in LPS-stimulated BV-2 cells (data not shown). It appears that the introduction of the third methoxy group into the 5 position of the phenyl ring (IIIc) greatly improved anti-inflammatory properties even though it slightly decreased the affinity for hH3R (Ki = 24 nM). However, it is still a very active compound.The anti-neuroinflammatory potential of chalcones has also been documented in other in vitro and in vivo studies . Yang etV2 cells . Moreove-2 cells . This fi-2 cells . Further-2 cells . This isE243. It is somewhat surprising as our previous research described E243 as a multi-target compound against AD [3R antagonism could be beneficial for slowing AD progression [E243 is very interesting as displayed submicromolar activity toward all aforementioned targets . Additionally, we examined the neuroprotective efficacy and showed that pre-treatment of neuroblastoma SH-SY5Y cells for 24 h with E243 (at 1 µM concentration) significantly prevented ROS production (assessed by DCFH-DA assay) almost by 2 times compared to the cells treated with the following neurotoxins H2O2 (300 μM) and okadaic acid (50 nM) . Compound E243 has been proofed as neuroprotective agent to be effective in neuroblastoma cells. The fact that, E243 did not act directly on microglia, might be due to the fact, that its activity is based on different mechanism not related to anti-inflammatory activity. Moreover, this can be partly explained by Qiao et al. who noticed that for anti-inflammatory activity of flavonoids the presence of free hydroxyl group(s) in the phenyl ring  is impo


The ethyl acetate fraction showed the strongest toxicity against A498 (IC50 = 0.162 µg mL−1) and 786-O (IC50 = 0.271 µg mL−1) cells. With similar toxicity against 786-O, P1 (IC50 = 3.8 µg mL−1) and P3 (IC50 = 3.1 µg mL−1) exhibited greater effect when combined (IC50 = 2.5 µg mL−1). Results support the potential of propolis and its constituents as promising coadjuvants in renal cell carcinoma treatment.Renal cell carcinoma is the most lethal cancer of the urological system due to late diagnosis and treatment resistance. Propolis, a beehive product, is a valuable natural source of compounds with bioactivities and may be a beneficial addition to current anticancer treatments. A Portuguese propolis sample, its fractions ( According to the GLOBOCAN 2020 database, despite not being one of the most prevalent cancers, renal cell carcinoma (RCC) is the most aggressive and lethal cancer of the urological system. By 2040, its incidence and mortality are expected to increase by approximately 54 and 68%, respectively . MetastaThe demand for natural products has been increasing due to their importance in human health and their potential as new drug leads . PropoliThe cytotoxic effect of propolis has been extensively studied against diverse types of cancer cell lines, including breast ,11, prosn-hexane fraction showed no effect on the viability of any of the cell lines at the tested concentrations. On the other hand, the G18.EE-water fraction exhibited the strongest effect on cell viability, with the lowest IC50 values against all cell lines (50 > 30 µg mL−1) and Caki-2 cells. The latter was the least sensitive cancer cell line. Considering all the results  but with higher selectivity indexes.The cytotoxic activity of G18.EE and its fractions was evaluated on three different RCC cell lines-786-O, Caki-2 and A498 . The G18ll lines . However results , the G1850 values obtained ranged from 4.4 to 41 µg mL−1, CaCo2 cells being the least susceptible to both extracts (IC50 > 50 µg mL−1) . From this subfraction (FG), eight FG-derived subfractions were obtained (FG1–FG8). FG8 was selected for additional fractionation using silica column with the same solvent system , resulting in five FG8-derived subfractions (FG8-1–FG8-5). Based on the TLC profiles, three FG-derived subfractions  displayed a single spot each on TLC plate, suggesting the presence of a single compound and thus being selected for the following experiments. Solvents used in the columns for extraction and the respective yields were: FG1 , FG5  and FG8-1   was subjected to VLC, eluting successively with  F1–F22;  were obt F1–F22; . The sub 4.3 mg] .P1, P2, and P3 were analyzed by UPLC-DAD-ESI/MSn following a previously described method [−1 and 2 µL were used for injection. The Ultimate 3000 apparatus was equipped with an Ultimate 3000 Diode Array Detector  and coupled to an ion trap mass spectrometer LTQ XL  equipped with an ESI source. Analysis was run on a Hypersil Gold  C18 column  at 30 °C. The mobile phase was composed of (A) water/formic acid  and (B) acetonitrile/formic acid . The solvent gradient started with 5–40% B (0–15 min), followed by 40–100% B (15–19 min), and returning to the initial conditions. The flow rate was 200 μL min–1 and UV–Vis spectral data for all peaks were accumulated in the range 200–500 nm. The nitrogen (>99% purity) gas pressure used for mass spectrometry analysis was 520 kPa (75 psi). The mass spectrometer was operated in negative-ion mode with ESI needle voltage set at 5.00 kV and an ESI capillary temperature of 275 °C. The full scan covered the mass range from m/z 100 to 2000. CID–MS/MS and MSn experiments were simultaneously acquired for precursor ions using helium as the collision gas with collision energy of 25–35 arbitrary units. Control and data acquisition were carried out with the Thermo Xcalibur Qual Browser data system . Compounds in the subfractions (P1–P3) were identified based on a literature comparison of the ESI-MS/MS data [Subfractions d method . The driP1 were recorded in a Bruker Advance II 400 spectrometer, at 400 MHz (1H) and 100 MHz (13C), at 20 °C (see δ) were recorded in parts per million (ppm) relative to the residual solvent signal (DMSO-d6).NMR spectra of 0 °C see . The cheP1). Yellow amorphous solid. 1H NMR : δ = 3.77 , 3.85 , 6.61 , 6.86 , 7.10 , 8.03 , 10.72 , 13.02 . 13C NMR : δ = 55.5, 59.9, 94.3, 103.0, 104.1, 114.6 (2C), 122.8, 128.3 (2C), 131.4, 152.4, 152.7, 157.4, 162.3, 163.3, 182.1 [5,7-dihydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one . The Caki-2 cell line was cultured in McCOY’s high glucose, L-glutamine, bacto-peptone, and phenol red without sodium pyruvate and HEPES . The A498 was cultured in MEM with 20 mM HEPES without NaHCO3 and L-glutamine . In addition, 1% of an antibiotic preparation  and 10% heating activated fetal bovine serum  were used as supplements for the culture media. Cultures were grown at 37 °C and 5% CO2 in a humidified incubator.Four human renal cell lines-HK2  and 786-O, Caki-2 and A498 (RCC cells) were obtained from the American Type Culture Collection (ATCC). The HK2 and 786-O cell lines were cultured in RPMI 2.0 g/ L NaHCO®) and then were detached from the flask with trypsin  at 37 °C. Medium was then added to the flask for trypsin inactivation. Cells were collected into a 15 mL Falcon tube and centrifuged for 5 min at 900 rpm, at 4 °C. Fresh medium was used to resuspend the cells, and 10 µL of the cell suspension were collected for subsequent addition of 20 µL of trypan blue  for cell counting (Neubauer chamber).To prepare cells for the cytotoxicity assay, flasks with sub-confluent cells were washed with phosphate-buffer 1× , each of the four fractions , each of the three subfractions  and two combinations of the subfractions with equal parts of each subfraction . Controls were performed with 0.25% of DMSO (vehicle) for IC50 determination (concentration that inhibits cell viability in 50%). Following this incubation period, the adherent cells were fixed by adding 50 µL of cold trichloroacetic acid 10%  and incubating for 60 min, at 4 °C. The plates were then washed with deionized water, dried overnight, and 50 µL of 0.1% SRB solution (Sigma–Aldrich) in 1% acetic acid (PanReac) were added to each plate well. After 30 min incubation at room temperature, unbound SRB was removed by washing with 1% acetic acid and air-dried overnight. Bound SRB was solubilized with 100 µL of 10 mM Tris base  and the absorbance was measured at 490 nm in a microplate reader . IC50 values were determined by transforming the absorbance values into a sigmoidal dose-response curve and extracting the concentration of the extracts/fractions needed to inhibit the viability of the kidney cells by 50%, using the GraphPad Prism Software Version 6.0. The selectivity index (SI) was calculated based on the IC50 values obtained for the samples against the cell lines, using the following formula: Propolis cytotoxicity was assessed by the sulforhodamine B (SRB) assay as previously described , which eCytotoxicity is higher for the cancer cell line than for the non-neoplastic HK2 cell line when SI values > 1.50 determination using the GraphPad Prism 5 software for logarithmic transformation after applying a sigmoidal dose-response non-linear regression. For multiple comparisons one-way ANOVA supplemented with post-hoc Tukey test was performed. Different letters mean statistical differences (p < 0.05) between mean values.Experiments were performed in triplicate and repeated at least three times for IC50 values, and importantly, very high selectivity indexes, when compared to the data available in the literature. This suggests that Portuguese propolis from Gerês, as well as its chemical constituents, could be used as alternatives or adjuvants to conventional anticancer treatments. They could be also used to help tackle drug-resistance and toxic effects by reducing the dose of the therapeutic drugs commonly used in RCC treatment.Cancer is one of the leading causes of death worldwide and is expected to cause more than 16 million deaths in 2040 [


Trapa quadrispinosa Roxb.) husk and to further explore the predominant compounds and their mechanisms on α-glycosidase inhibition by virtual screening and molecular dynamics. A total of 29 substances were identified and quantified in this study. Tannins were the main constituents of water caltrop husk extract. All of the free phenolic (FP), esterified phenolic (EP) and insoluble-bound phenolic (BP) fractions exhibited good antioxidant activities, and the BP had the highest radical scavenging ability with IC50 values of 0.82 ± 0.12 μg/mL (ABTS) and 1.15 ± 0.02 μg/mL (DPPH), respectively (p < 0.05). However, compared with the EP and BP, the FP showed the strongest inhibition towards the α-glycosidase and the IC50 value of FP was 1.43 ± 0.12 μg/mL. The 1,2,6-trigalloylglucose and α-glycosidase complex had better root mean square deviations (RMSD) stability via molecular dynamics simulation study. Results obtained from this study may provide a good potential natural resource for the improvement of oxidative stress injury and blood glucose control in diabetes mellitus, which could expand the use of water caltrop husk and improve its economic value.The aim of this study was to investigate the phenolic profiles, antioxidant activities and α-glycosidase inhibitory activities of three different phenolic fractions from water caltrop ( Diabetes is a serious chronic disease characterized by hyperglycemia, which is the third leading killer worldwide after cardiovascular disease and cancer . ProlongCynara Scolymus L.) have strong antioxidant activity −m/z = 183.0293) was identified as 4-O-methylgallic acid, and the characteristic fragment at m/z of 140.0140 and 111.0076 [12 ([M-H]−m/z = 635.0903) was identified as trigalloylglucose and its characteristic fragment ions were 169.0134 and 313.0568 [18 ([M-H]−m/z = 787.1014) was identified as tetragalloylglucose, and the characteristic fragments at m/z of 465.0674, 617.0793 and 635.0897 [23 ([M-H]− m/z = 939.1132) was identified as pentagalloylglucose, and the characteristic fragment ion ([M-H]− m/z = 617.0793) of compound 23 resulted from loss of galloyl moiety 322, indicating the presence of condensed gallic acid as a group on glucose [1 and 8  were higher peaks in EP and BP. Compound 1 ([M-H]−m/z = 169.0136) was identified as gallic acid by references, and the characteristic fragment ion ([M-H]−m/z = 125.0234) of compound 1 was due to the loss of CO2 (44Da) [8 ([M-H]−m/z = 321.0259) was identified as digallic acid by references, and the characteristic fragment ion of 169.0134 −m/z = 197.0453), both of which contained fragment ions with an m/z of 123.0074 in their MS2 fragment ions, a characteristic ion for syringic acid in the negative ion mode [24 was identified as quercitrin ([M-H]−m/z = of 447.0941), and the characteristic fragment ion ([M-H]−m/z = 300.0277) was due to the loss of the rhamnose moiety [Phytochemical compounds of three phenolic fractions from water caltrop husk were comparatively identified by using UHPLC-ESI-HRMS/MS in the negative mode. The ion current chromatograms are illustrated in 111.0076 ,28. Comp313.0568 ,29. Comp635.0897 ,17,29,30 glucose ,29,30. H2 (44Da) ,30. Comp169.0134 ,30. Meanion mode ,28. Comp1, 5, 12, 16, 18 and 23 were the most abundant components in FP, accounting for 33.79%, 17.03%, 14.43%, 9.10%, 6.35% and 5.48% of the total identified phenolic compounds in the extract, respectively. Compounds 1 and 8 were the most abundant components in EP, accounting for 78.87% and 14.29% of the total identified phenolic compounds in the extract, respectively. Compounds 1, 5 and 8 in BP were the most abundant components, which accounted for 74.90%, 15.48% and 5.40% of the total identified phenolic compounds in the extract, respectively. Similar to the results of previous studies, the main phenolic components in different fractions of the same plant material were different [5, 12, 18 and 23, while compound 1 was the main compound in both EP and BP. The contents of compound 1 in EP and BP were 8.91 times and 11.58 times that in FP, respectively. This may be due to the fact that many insoluble gallotannins bound to plant cell walls or other biomacromolecules were hydrolyzed by strong alkali to produce compound 1  during the extraction of EP and BP [The quantitative results of FP, EP and BP are summarized in ifferent ,17. In wP and BP .Rhus chinensis Mill.) fruits, raspberry pomace and walnut kernel, in which FP contained the highest phenolic content [p > 0.05). The flavonoid content of BP was significantly higher than that of FP and EP, and the flavonoid content was 99.90 ± 0.35 mgRE/g. Many plants are rich in phenolic compounds, which could be measured by Folin–Ciocalteu reagent method. Compared with those previous reports [The total phenolics content was expressed by mg gallic acid equivalents (GAE)/g dry weight of water caltrop husk extract. The content of total flavonoids was expressed by mg rutin equivalents (RE)/g dry weight of water caltrop husk extract. The highest phenolic content in FP was 406.06 ± 3.94 mg GAE/g, while the phenolic contents in EP and BP were 0.68 and 0.88 times that in FP, respectively. The results were consistent with the quantitative results. There was abundant phenolic in water caltrop husk, and it mainly exists in the form of FP. These findings are similar to those reported in previous studies for Chinese sumac . The IC50 values of FP, EP and BP were 1.86 ± 0.01 μg/mL, 2.14 ± 0.07 μg/mL and 1.15 ± 0.02 μg/mL, respectively. The correlation coefficients of TPC, TFC and the ABTS radical scavenging ability were analyzed with Pearson’s correlation analysis and the results are shown in p < 0.01), which was consistent with previous studies [The ABTS radical scavenging ability is one of the commonly used methods to evaluate the antioxidant activity of certain substances ,35. Figu studies ,37.50 values of the FP, EP and BP were 1.04 ± 0.02 μg/mL, 1.34 ± 0.04 μg/mL and 0.82 ± 0.12 μg/mL, respectively (p < 0.05). The general trend of DPPH free radical scavenging activities of the phenolic extracts in the three different forms from water caltrop husk were similar to that of ABTS. The correlation coefficients of TPC, TFC and the DPPH free radical scavenging ability by Pearson’s correlation analysis are shown in p < 0.01), which was consistent with the findings reported in previous studies [DPPH is a stable free radical, and the evaluation of its scavenging ability is an important indicator to test the antioxidant capacity of natural compounds ,35. As s studies ,37.As mentioned above, a large number of studies have shown that elevated levels of oxidative stress can cause cell and tissue damage, thereby resulting in chronic inflammation and metabolic diseases ,2,3,4,5.50 = 1.43 ± 0.12 μg/mL. Both EP and BP showed weaker inhibitory activity on α-glycosidase than FP, with IC50 values of 71.01 ± 1.31 μg/mL and 23.01 ± 0.90 μg/mL, respectively. When the sample concentration reached 100 μg/mL, the α-glycosidase inhibition rate of EP was 54.88%, and the α-glycosidase inhibition rate of BP was 75.24%. The inhibitory effects towards α-glycosidase showed a trend of FP > BP > EP. As shown in Rhus chinensis Mill.) fruits [50 value of acarbose (a positive control) was 0.27 ± 0.02 μg/mL (p < 0.05).Hyperglycemia is an important feature of diabetes, which seriously affects human health. α-Glycosidase is a key enzyme for digesting carbohydrate in the intestinal tract. Inhibition of α-glycosidase activity can reduce carbohydrate digestion and absorption, thus delaying postprandial blood glucose rise . Figure ) fruits ,36,38. IAs mentioned above, α-glycosidase can directly participate in the metabolism of starch and glycogen to hydrolyze glycosidic bonds and release glucose, which is an indispensable enzyme in human glucose metabolism ,12. Its As shown in This study used 100-ns molecular dynamics simulations to further investigate the stability of the trigalloylglucose and α-glycosidase complex or tetragalloylglucose and α-glycosidase complex. The α-glycosidase was used as a control protein. Ultimately, the differences between those compound-enzyme complexes with different conformations were investigated.The RMSD was used to determine the average deviation between the conformation and initial conformation of the complex in the system at the specific time, and it was generally used to evaluate whether the complex had reached a stable state in this system . Figure The RMSF was used to analyze the fluctuation of the receptors’ residues by their reference position during the simulation period . As show50 values of 0.82 ± 0.12 μg/mL (ABTS) and 1.15 ± 0.02 μg/mL (DPPH), followed by the FP whose IC50 values were 1.86 ± 0.01 μg/mL (ABTS) and 1.04 ± 0.02 μg/mL (DPPH). Additionally, the IC50 values for the antioxidant activity of EP were 2.14 ± 0.07 μg/mL (ABTS) and 1.34 ± 0.04 μg/mL (DPPH). Pearson correlation analysis showed that TPC and TFC had a significant positive correlation with the two in vitro antioxidant activities. When compared with the EP and BP, the FP showed the strongest inhibition towards the α-glycosidase and the IC50 value of FP was 1.43 ± 0.12 μg/mL, and the IC50 values of EP and BP inhibiting α-glycosidase were 71.01 ± 1.31 μg/mL and 23.01 ± 0.90 μg/mL, respectively. By molecular docking and molecular dynamics analysis, all three isomers of trigalloylglucose or tetragalloylglucose can combine with α-glycosidase through hydrogen bonds and hydrophobic interaction forces to form stable complexes. Among them, the complex formed by 1,2,6-trigalloylglucose and α-glycosidase was the most stable. Although molecular dynamics is currently becoming a powerful and flexible investigative tool in the most diverse fields of structural biology and materials science, further studies should be conducted in the future to evaluate the effects of water caltrop husk extracts even in vivo. In sum, this study may provide a potential natural resource for the improvement of oxidative stress injury and blood glucose control in diabetes mellitus, and expand the use of water caltrop husk to improve its economic value.In this study, a total of 29 substances were identified and quantified. Tannins were the main constituents of water caltrop husk extract. They mainly existed in the form of trigalloylglucose, tetragalloylglucose and pentagalloylglucose. All of the free phenolic (FP), esterified phenolic (EP) and insoluble-bound phenolic (BP) fractions exhibited good antioxidant activities, and the BP had the highest radical scavenging ability with IC


We sought to identify a usable biomarker from blood samples to characterize early-stage Alzheimer’s disease (AD) patients, in order to facilitate rapid diagnosis, early therapeutic intervention, and monitoring of clinical trials. We compared metabolites from blood plasma in early-stage Alzheimer’s disease patients with blood plasma from healthy controls using two different analytical platforms: Amino Acid Analyzer and Tandem Mass-Spectrometer. Early-stage Alzheimer’s patient blood samples were obtained during an FDA-approved Phase IIa clinical trial . Participants included 25 early-stage Alzheimer’s patients and 25 healthy controls in the United States. We measured concentrations of 2-aminoethyl dihydrogen phosphate and taurine in blood plasma samples. We found that plasma concentrations of a phospholipid metabolite, 2-aminoethyl dihydrogen phosphate, normalized by taurine concentrations, distinguish blood samples of patients with early-stage AD. This possible new Alzheimer’s biomarker may supplement clinical diagnosis for early detection of the disease. Onset of Alzheimer’s disease (AD) symptoms is correlated with accumulations of misfolded proteins and protein fragments, particularly amyloidβ42 (Aβ42) plaques and a dense tauopathy of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau deposits in specific brain regions . There iVarious biomarkers associated with later stages of AD have been suggested  includinWe have sponsored an FDA-approved Phase IIa clinical trial of L-serine at the Department of Neurology, Geisel School of Medicine, Dartmouth  for early-stage Alzheimer’s disease patients. At the time they receive their initial diagnosis, based on the Clinical Dementia Rating (CDR) score, patients are offered entry into the clinical trial. We hypothesized that a unique metabolic biomarker of early Alzheimer’s disease could be identified by examining the physiological amino acids and nitrogen containing compounds within these early disease state blood samples. Using an automated Amino Acid Analyzer along with confirmation from tandem mass-spectroscopy, we examined metabolites displaying clear differences between AD and control blood plasma samples. We found that the concentration of 2-aminoethyl dihydrogen phosphate normalized by taurine concentrations in blood plasma samples reliably identifies early-stage AD patients.We compared initial blood plasma samples collected from the antecubital area of the arm from early-stage Alzheimer’s disease patients  with blood plasma from healthy controls , with analysis blinded. Plasma came from initial blood draws from our Phase IIa human clinical trial for early-stage Alzheimer’s patients, and control samples . The control subjects had a mean age of 39 (range 20–62 years) while the Alzheimer’s patients had a mean age of 71 years (range 57–82). The study was approved by the IRB . Informed consent was obtained from all participants. All methods were performed in accordance with the relevant guidelines and regulations.Blood plasma was collected in K2-EDTA tubes and centrifuged immediately at 2000 x g for 15 minutes at 4°C. Time between blood collection and freezing was less than 1 hour and the sample was stored at -80°C and shipped on dry ice. The plasma sample was thawed at 4°C and combined with an equal volume of cold 10% (w/v) trichloroacetic acid . The sample was left to precipitate at room temperature for two hours followed by centrifugation at 14,000 x g for 5 min. The supernatant was removed by pipette and filtered using a centrifuge filter  at 14,000 x g for 5 min.2>/ = 99%). In addition, retention time checks were conducted for 2-aminoethyl dihydrogen phosphate  and taurine (Sigma-Aldrich T0625-10G). A lower end curve  demonstrated that 2-aminoethyl dihydrogen phosphate was linear to 1.4 μmol/L (R2 = 99.9%). The limits of detection (LOD) and lower limits of quantification (LOQ) were calculated using the EPA method [The underivatized sample (20 μL) was injected into a Hitachi Amino Acid Analyzer L8900 equipped with a Hitachi Reaction column  at 135°C, a high-speed physiological fluid analysis analytical column Li-form resin #2622SC 6 mm ID x 40 L 060928C (PN855-4515), and AmmoniaFilter column . Hitachi pre-made buffers  were used as follows: (B1) PF-1/AN0-5031, (B2) PF-2/AN0-5032, (B3) PF-3/AN0-5033, (B4) PF-4/AN0-5034, (B6) PF-5/AN0-5035, (R1) ninhydrin solution , and (R2) ninhydrin-buffer of lithium acetate dihydrate . Wash solutions included (B5) 5% ethanol , (R3) 10% methanol , and (C1) 10% methanol. Separation was achieved with a flow rate for pump 1 of 0.54 mL/min and 0.47 mL/min for pump 2, reactor column set to 135°C, and a 146 min gradient elution: 0.00 min = 100% B1, column oven temp 35°C, 50% R1, 50% R2; 16.0 min = 100% B1; 16.1 min = 81% B1, 19% B2, column oven 58°C; 41.0 min = column oven 32°C; 57.0 min = column oven 70°C; 69.5 min = column oven 65°C; 70.0 min = 15% B1, 75% B2, 10% B3; 88.0 min = column oven 60°C; 94.0 min = 20% B2, 80% B4; 94.1 min = 25% B2, 75% B4; 109.0 min = 25% B2, 75% B4, column oven 70°C; 109.1 min = 100% B4; 123.0 min = 100% B4; 123.1 min = 100% B6; 127.0 min = 50% R1, 50% R2; 127.1 min = 100% R3; 129.0 min = 100% B6, 129.1 min = 100% B1; 131.0 min = column oven 35°C; 132.0 min = 100% R3; 132.1 min = 50% R1, 50% R2; 146.0 min = 100% B1. Amino acid standards  were mixed to equal concentrations and complete standard curves run at concentrations of 5, 10, 25, 50 100, 250, 500, 1000 μmol/L. All amino acids curves were linear within this range (Rm/z collision energy (CE) 17.1, 312 to 142 m/z CE 17.1, 312 to 312 m/z CE 2, RF lens of 90). This experiment was repeated a second time to ensure reproducibility. The limit of detection (LOD) for this method was 0.004 μg/ml and the lower limit of quantification (LOQ) was 0.01 μg/ml. A standard curve was prepared with concentrations 20 pg/ml, 40 pg/ml, 0.2 ng/ml, 0.4 ng/ml, 2 ng/ml, 4 ng/ml, 10 ng/ml, 20 ng/ml, 0.1 μg/ml, 0.2 μg/ml, and linearity had an R2 value of 99%.Confirmation of 2-aminoethyl dihydrogen phosphate in the samples was conducted through tandem mass spectrometry. Plasma samples from AD patients and controls, with analysis blinded as to treatment, were deproteinated as above and derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate  following manufacturers recommendations. AD blood samples were combined as were control samples and analyzed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer with multiple injections per sample. The mass spectrometer was equipped with a Thermo Vanquish pump, autosampler, H-ESI probe, and heated column compartment set to 65°C. An OPTON-10005 Genius 3022 dual N2 generator  supplied purified nitrogen to the mass spectrometer. Separation was achieved using a Kinetex C-18 column ; and gradient elution  ≥ 99% adjusted to pH 5.0 with glacial acetic acid (Fisher Scientific A385-500), B = 100% methanol ≥ 99.9% : initial conditions 99.8% A, 1.0 min 99.8% A, 1.5 min 40% A, 2.0 min 25%A, 2.5 min 25%A, 2.6 min 10% A, 5.5 min 10%A, 6 min 98%A, 8 min 98%A all set with curve 5. The mass spectrometer was run in positive mode (4400 V) with the following settings: spray voltage = static; sheath gas = 5.6 Arb; aux gas = 23.5 Arb; sweep gas = 1.2 Arb; ion transfer tube = 150°C; vaporization temperature = 450°C; Q1 resolution = 0.7 FWHM; Q3 resolution = 0.7 FWHM; and CID gas = 2 mTorr. Scans for derivatized 2-aminoethyl dihydrogen phosphate were conducted with the following transitions (312 to 171.2 −1(x), where x is the original ratio. Two hypotheses were tested using a two-tailed t-test for samples with unequal variances:The concentration of 2-aminoethyl dihydrogen phosphate, which elutes at 2.1 minutes on the Amino Acid Analyzer chromatogram, was normalized for each sample by the concentration of taurine, an aminosulfonic acid which elutes immediately before at 1.6 minutes . A ratio0: The normalized means of 2-aminoethyl dihydrogen phosphate are equal for early-stage Alzheimer’s disease patients and controls.H1: The normalized means of 2-aminoethyl dihydrogen phosphate are different for early-stage Alzheimer’s disease patients and controls.H−1(x) because the data were not normally distributed. The mean of the sin−1(x) transformed ratio of 2-aminoethyl dihydrogen phosphate to taurine for controls was 42.3 (sd = 69.9), while the mean of the sin−1(x) transformed ratios for early-stage Alzheimer’s patients was 185.5 (sd = 57.0) with a t statistic of -7.9, df = 48. H0 was therefore rejected, with p<0.000000001. We note that the absence of an analyte detection does not mean that it is not present, only that while using the stated methods that it is below the limit of detection. Since a value below the LOD approximates a zero compared to the high concentrations reported in other samples (See The mean ratio of 2-aminoethyl dihydrogen phosphate to taurine for Alzheimer’s patients was 0.37 (sd = 0.13) compared to a mean of 0.07 for controls (sd = 0.12) . The datples See . Therefoples See  and 2.Confirmation of the presence of 2-aminoethyl dihydrogen phosphate in the blood plasma of early-stage AD patients was conducted by tandem mass spectrometry . 2-aminoO-phosphorylethanolamine, colamine phosphoric acid, ethanolamine O-phosphate 2, O-phosphoethanolamine, O-phosphocolamine, and colamine phosphoric acid. This molecule is important in the structure and function of cellular membranes. It is a precursor in the biosynthesis of phosphatidylethanolamine and phosphatidylcholine. It is essential for the formation of mammalian glycosylphosphatidylinositol-anchored proteins (GPI) which bind other proteins to the plasma membrane [2-aminoethyl dihydrogen phosphate is the IUPAC name for a molecule previously referred to in the literature by other names including membrane . GPI maymembrane . The promembrane .In cell culture, 2-aminoethyl dihydrogen phosphate has been shown to inhibit mitochondrial respiration and induce apoptosis by disrupting the mitochondrial membrane potential . Physiolhttps://neurology.testcatalog.org/show/AAQP). Our study suggests that the concentration of 2-aminoethyl dihydrogen phosphate normalized by taurine concentrations in blood plasma samples could potentially be added to the CDR scale as a diagnostic tool for early-stage AD.Normal concentrations of 2-aminoethyl dihydrogen phosphate for healthy adults range from “not detected” to up to 69 μmoles/L –28. The Elevated 2-aminoethyl dihydrogen phosphate levels, primarily in urine, also occur in patients diagnosed with a rare inherited disease known as hypophosphatasia . The disde novo sphingolipid biosynthesis pathway where serine palmitoyltransferase catalyzes the reaction of L-serine with palmitoyl-Coenzyme A to form sphingoid bases. Breakdown of both sphinganine and sphingosine leads to the production of 2-aminoethyl dihydrogen phosphate [In vitro, an increase in sphinganine kinase decreases free sphinganine which results in the disruption of axonal growth in cultured hippocampal neurons [If the sphingolipid biosynthesis pathway is disrupted in early-stage AD, an increase in the downstream 2-aminoethyl dihydrogen phosphate could result. A need for additional L-serine to drive the pathway forward could also occur, based on the hosphate . In vitr neurons . Sphingo neurons .There are limitations to our study. Since there is evidence of Alzheimer’s latency prior to diagnosis  measurabThe concentration of 2-aminoethyl dihydrogen phosphate normalized by the concentration of taurine in blood plasma reliably distinguished blood samples of early-stage AD patients from controls in a blinded analysis. If verified with larger sample sizes, the quantification of 2-aminoethyl dihydrogen phosphate could potentially assist in the diagnosis of early-stage Alzheimer’s disease when used in conjunction with the patient’s CDR score and other potential AD biomarkers.


Malignant peritoneal mesothelioma is a rare tumor entity. Although cytoreductive surgery and hyperthermic intraperitoneal chemotherapy have increased overall survival, its prognosis remains poor. Established chemotherapeutics include mitomycin C (MMC) and cisplatin (CP), both characterized by severe side effects. GP-2250 is a novel antineoplastic agent, currently under clinical investigation. This in vitro study aims to investigate effects of GP-2250 including combinations with CP and MMC on malignant mesothelioma. JL-1 and MSTO-211H mesothelioma cell lines were treated with increasing doses of GP-2250, CP, MMC and combination therapies of GP-2250 + CP/MMC. Microscopic effects were documented, and a flow-cytometric apoptosis/necrosis assay was performed. Synergistic and antagonistic effects were analyzed by computing the combination index by Chou-Talalay. GP-2250 showed an antiadhesive effect on JL-1 and MSTO-211H spheroids. It had a dose-dependent cytotoxic effect on both monolayer and spheroid cultured cells, inducing apoptosis and necrosis. Combination treatments of GP-2250 with MMC and CP led to significant reductions of the effective doses of CP/MMC. Synergistic and additive effects were observed. GP-2250 showed promising antineoplastic effects on malignant mesothelioma cells in vitro especially in combination with CP/MMC. This forms the basis for further in vivo and clinical investigations in order to broaden treatment options. Malignant mesothelioma (MM) is a rare, aggressive tumor entity of serous membranes, the mesothelium, featuring predominantly pleura and peritoneum  ,2. MesotPeritoneal MM is characterized by a highly aggressive growth pattern, a poor prognosis and its resistance towards standard tumor therapy . TypicalUntreated peritoneal MM leads within an average of 6 to 16 months to death after initial diagnosis. Due to its rarity, establishing a standard therapy regimen has been challenging. Currently, there are almost exclusively retrospective case reports of single institutions or multi-institutional cohort studies without a randomized controlled study design . CytoredCRS and HIPEC utilizing established chemotherapeutics have a high rate of lesser and severe complications including pulmonal, renal and cardiac complications; hematologic toxicities and sepsis with multiorgan dysfunction, tied to the drugs’ toxicity . In lighA recent development in antitumorigenic substances is the oxathiazinane GP-2250 . This study is the first to investigate the possible application of the novel substance GP-2250 in MM therapy.GP-2250s cytotoxic effects on the MM cells lines JL-1 and MSTO-211H in a monolayer and spheroid model were analyzed using flow cytometry measuring the percentage portions of viable, apoptotic and necrotic. Spheroids are very dense, three-dimensional (3-D) tumor cell formations, mimicking the characteristics of solid tumors far closer than monolayer cultures and bridging the gap between in vitro monolayer cultures and in vivo studies ,21,22,23Both JL-1 and MSTO-211H cells formed stable spheroids (sph.), which passed through the spheroid-formations assay unaffectedly . When inGP-2250 showed both with monolayer MM cells and with MM sph. a dose-dependent decrease in viable cells and an increase in apoptotic and necrotic cells .50) was computed at 603.7 µmol/L [321.21–1134.98 µmol/L 95% confidence interval] for JL-1 mnl. and at 1193.04 µmol/L [598.23–2379.26 µmol/L 95% confidence interval] for MSTO-211H mnl. cells (JL-1 monolayer (mnl.) cells showed a first significant reduction of viable cells after a 48 h treatment with 250 µmol/L GP-2250, MSTO-211H mnl. cells at 500 µmol/L GP-2250 . Maximuml. cells .MM cells in spheroids showed higher resistance to GP-2250 compared to their respective monolayer cultures .50 was computed for JL-1 sph. at 949.64 µmol/L [684.72–1317.05 µmol/L 95% confidence interval] and for MSTO-211H-sph. at 2587.99 µmol/L [1547.46–328.18 µmol/L 95% confidence interval]. In JL-1 sph., apoptotic cells outweighed necrotic cells, except for treatment with 1500 µmol/L GP-2250. In MSTO-211H sph., necrotic cells slightly outweighed apoptotic cells. The respective ED50’s of the GP-2250 monotherapies are shown in The first significant reduction of viable cells in JL-1 sph. was achieved by treatment with 250 µmol/L GP-2250 and with 1000 µmol/L GP-2250 in MSTO-211H sph. At a maximum dose of 1500 µmol/L GP-2250 (JL-1 sph.) and 2500 µmol/L (MSTO-211H sph.), viable cells were reduced from approximately 70% to 21.03% ± 5.78% and 43.76 ± 6.01%, respectively. An EDCytotoxic effects of a combination treatment of GP-2250 and MMC on both cell lines in a spheroid model were analyzed. Concentrations with low to moderate effects in monotherapy according to previous works  were cho57. Decrease under combination treatment was statistically significant compared to the respective monotherapies with 1000 µmol/L GP-2250 and 2.5 µmol/L MMC. This applied for the effect under treatment with 1000 µmol/L GP-2250 + 0.5 µmol/L MMC, as well, which equated to an ED46. Hence, a reduction of ED50 of MMC was achieved from 5–10 µmol/L MMC in monotherapy to 0.5–2.5 µmol/L MMC in a combination therapy. Both mentioned combinations additionally showed synergistic effects. Conversely, treatments with 500 µmol/L GP-2250 displayed an antagonistic (+0.5 µmol/L MMC) and additive (+2.5 µmol/L MMC) effect.All tested combinations of GP-2250 plus MMC showed a significant decrease of viable MSTO-211H cells compared to the untreated control b. Apopto36). This was the only treatment displaying a significant difference in viable cells compared to its respective monotherapies. Synergistic effects were not observed, and instead, the treatments showed antagonistic effects.For JL-1 cells, 250 and 500 µmol/L GP-2250 were combined with either 1 or 5 µmol/L MMC e). TreatConcerning the moiety of necrotic and apoptotic cells of JL-1 sph., none of the combinations proved to show a significant increase of the shares of necrotic or apoptotic cells in comparison to each monotherapy.p < 0.05, each). No such difference was observed concerning the moiety of necrotic cells.In MSTO-211H sph., only the combination of 1000 µmol/L GP-2250 with 0.5 µmol/L MMC led to a significant rise of apoptotic cells to 34.3% ± 6.1% in comparison to each of its monotherapies . Therefore, the ≥ED50 of CP was lowered from 50 µmol/L in a monotherapy to 30 µmol/L in combination with 500 µmol/L GP-2250.With JL-1 sph., 250 and 500 µmol/L GP-2250 were combined with 5 and 20 µmol/L CP c,d. As op < 0.01, each), whereas no such differences were observed for the other concentration. The share of necrotic cells increased significantly for the combinations of 250 µmol/L and 500 µmol/L GP-2250 with 30 µmol/L CP (21.9 ± 0.9% and 25.0 ± 2.3%) as well as 500 µmol/L GP-2250 and 40 µmol/L CP (29.0 ± 1.6%) in comparison to each monotherapy .Concerning the moiety of apoptotic cells in JL-1 sph., only a combination of 500 µmol/L GP-2250 and 30 or 40 µmol/L, respectively, led to a significant rise of apoptotic cells (44.3 ± 3.5 and 55.9 ± 3.2%) compared to each monotherapy . Concerning the moiety of necrotic cells, none of the combinations proved to significantly increase the shares in comparison to each of its monotherapies.In MSTO-211H sph., only the combination of 1000µmol GP-2250 and 5 µmol/L CP proved to significantly increase the share of apoptotic cells to 30.0 ± 2.3% versus 19.1 ± 7.2% for 1000 µmol/L GP-2250 and 19.9 ± 3.1%  and 500 µmol/L GP-2250 (MSTO-211H mnl.) led to a significant reduction of viable cells. Amounts of 1000 µmol/L GP-2250 (JL-1 mnl.) and 2000 µmol/L GP-2250 (MSTO-211H mnl.) reduced viable cells to less than 20%. This is consistent with observations of Buchholz, Majchrzak-Stiller et al. who measured the portion of vital pancreatic carcinoma cells treated with 1500 µmol/L GP-2250 at 7.6–28.5% depending on the cell line. MSTO-211H mnl. treated with the same dose had a vital percentage portion of 52.86% (±4.09%). JL-1 mnl. were not treated with 1500 µmol/L, as 1000 µmol/L GP-2250 already reduced the viable cells to 17.07% (±10.60%). In conclusion, the intensity of GP-2250s cytotoxic effect is conditional to the tumor entity and to the cell line.After promising results using monolayer cells, further experiments were conducted using MM spheroid cultures. This 3-D model bridges the gap between monolayer in vitro studies and in vivo models as it simulates tumor microenvironment, nutrition supply, gene expression, extracellular matrix and response to treatment ,21,22,27MM spheroids were treated with increasing doses of GP-2250 over 48 h. The data showed an anti-adhesive microscopic effect as well as a cytotoxic effect on MM spheroids.50) and by 70% (ED70) at 1000 µmol/L and 1500 µmol/L GP-2250, respectively. In contrast, MSTO-211H sph. did not reach an ED50 even when treated with 2500 µmol/L GP-2250. A clear change in the spheroids’ outer integrity was observed after treatment with 750 µmol/L (JL-1 sph.) and 1000 µmol/L GP-2250 (MSTO-211H sph.)  and 1000 µmol/L GP-2250 (MSTO-211H sph.) . JL-1 spIn the present study, the ratio of apoptotic mnl. cells outweighed the necrotic cells under treatment with GP-2250 in both, JL-1 and MSTO-211H. Similarly, JL-1 spheroids showed initially an increasing portion of apoptotic cells up to concentrations of 1500 µmol/L, where necrotic cells outweighed the apoptotic portion. At this dose, vital MSTO-211H cells were at 45.19 ± 2.02%, again with the necrotic ratio slightly higher than the apoptotic ratio. All in all, JL-1 sph. were more sensitive towards GP-2250 than MSTO-11H spheroids. To date, there are no other published studies investigating the effect of GP-2250 on tumor spheroids.Buchholz, Majchrzak-Stiller et al. 2017 initially measured an increase of apoptotic pancreatic carcinoma cells as well, but with an increasing dose of GP-2250, they observed a preponderance of necrotic cells .In monolayer, the moiety of apoptotic cells always surpassed the share of necrotic cells. In spheroids, even in untreated controls, the share of viable cells was lower, and moieties of necrotic and apoptotic cells were higher compared to monolayer cells. Additionally, the share of necrotic cells rose with increasing doses of GP-2250, surpassing the shares of apoptotic cells. This is tied to the intrinsic structure of the 3-D spheroid model where a proliferative, a dormant and a necrotic zone along a gradient of nutrients from the outside to the center can be differentiated .Another explanatory approach is associated with the experimental design. Physiological apoptosis is a highly controlled, adenosine triphosphate (ATP)-dependent process with subsequent phagocytosis of the degradation products. In absence of inflammatory cells and thus phagocytosis in in vitro cell culture, secondary necrosis occurs. The presently utilized flow cytometry markers Annexin-V and Propidium Iodide (PI) cannot differentiate between primary and secondary necrosis as PI intercalates with exposed DNA, found in both necrotic and late apoptotic cells. This might lead to false positive measurement of necrotic cells.Secondly, Buchholz, Majchrzak-Stiller et al. suggest that GP-2250 might initiate programmed necrosis. This process is a caspases-independent programmed cell death initiated by cell stress or activation of death receptors, leading to excessive levels of reactive oxygen species (ROS) ,29. GP-250 were higher in spheroids than in monolayers of both cell lines. While in monolayer all cells are equally exposed to media and chemotherapeutics, this is not the case in 3-D structured spheroids. Thus, higher concentrations of chemotherapeutics are needed in order to expose especially the inner zones to an effective dose. Furthermore, the increased cellular adhesion of tumor cells in spheroids has been proven to reduce sensitivity towards cytotoxic drugs [Concentrations needed to achieve an EDic drugs ,30,31,32The cytotoxic effect of GP-2250 on MM spheroids was compared to the effect of standard therapeutics CP and MMC. In addition, we investigated possible combination therapies of GP-2250 plus CP or MMC.Currently, there is no general standard therapy regimen for MM due to its small incidence and the lack of randomized multicenter studies. Most commonly used is a combination of MMC, CP or doxorubicin . AlthougBoth MMC and CP interact with DNA synthesis ,34, showCP as well as MMC are characterized by severe side effects and poor tolerability ,14,38,3950 of MMC was lowered from 5–10 µmol/L to 0.5–2.5 µmol/L. Amounts of 1000 µmol/L GP-2250 plus 0.5 µmol/L MMC as well as 2.5 µmol/L MMC showed synergistic effects.This is the first study to investigate a possible combination of GP-2250 with CP or MMC. When combining GP-2250 with MMC and CP, their respective doses were significantly lowered while still achieving the same decrease in viable cells. With MSTO-211H sph., ≥ED50 of CP was lowered from 10 µmol/L CP in a monotherapy to 5–7 µmol/L CP in the combination therapy with a synergistic effect. Concerning JL-1 sph., 500 µmol/L GP-2250 + 40 µmol/L CP showed a synergistic effect. An amount of 500 µmol/L GP-2250 + 30 µmol/L CP led to an additive effect and lowered the ED50 from 50 to 30 µmol/L CP. In total, our experiments combining GP-2250 with CP or MMC for treating MM spheroids showed promising results. Concerning synergistic effects, the present study cannot make general statements. Both combinations showed synergistic, additive as well as antagonistic effects . The other analysed cell line was MSTO-211H, derived from pleura effusions of a 62-year-old male diagnosed with malignant biphasic mesothelioma .2 in humidified atmosphere.MSTO-211H cells were maintained in RPMI-1640, JL-1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM). Both mediums were supplemented with 10% Fetal Bovine Serum (FBS), 1% Penicillin/ Streptomycin and 1% L-Glutamine . Cells were maintained as monolayer in 100 mm cell culture dishes at 37 °C with 5% COReagents: GP-2250  was stored as dry powder at room temperature. It was freshly prepared every two weeks by dissolving in double distilled water, set to a physiological pH, sterile filtered and stored protected from light.Mitomycin C (MMC) was purchased from Selleck Chemicals LLC, Munich, Germany, as dry powder and dissolved in dimethyl sulfoxide (DMSO) as described in the manufacturer specifications. The solution was divided in aliquots and stored at −20 °C. A new aliquot was used for each treatment. Cisplatin  was dissolved according to manufactures instructions in 0.9% sodium chloride and set to a physiological pH every two weeks by the St. Josef-Hospital Bochum pharmacy and was stored protected from light at 4 °C.Spheroid cultures and spheroid formation assay: To generate spheroids, cells were seeded in ultra-low attachment surface 6-well plates  with 400.000 cells per well in 2 mL serum-free stem cell medium. The medium was composed of 95% DMEM/F12  supplemented with 2,3% Penicillin/Streptomycin , 2% B27-Supplement , 1% Minimal Essential Medium Non-Essential Amino Acids  and 0,02% basic Fibroblast Growth Factor . Spheroids were cultured for 4 days (JL-1) and 5 days (MSTO-211H) and stirred twice during that period. JL-1 spheroids were cultured for 4 days only due to their higher density. Afterwards, a spheroid formation assay was performed as portrayed in Cytotoxicity analysis using flow cytometry: Each treatment was conducted on 3–5 independent experiments with consecutive passages. After an incubation period of 48 h followed by microscopic documentation, a cell viability assay was performed. Monolayer cells were detached using trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02% in phosphate-buffered saline (PBS) , and spheroids had to be separated using Accumax . Cells were resuspended in 200 µL binding buffer . Cells were then marked with 10 µL Annexin V-FITC  for 15 min at room temperature protected from light. A volume of 10 µL Propidium Iodide  was added, and the percentages of viable (Annexin V-FITC and PI negative), apoptotic  and necrotic cells (Annexin V-FITC and PI positive) were determined using flow cytometry . The resulting dot plots were analyzed using CellQuest Pro software .Statistics: The results of flow cytometry analysis were expressed via means ± standard deviation (SD). Comparison of means of normal distributed results of two groups was computed using the unpaired t-test by GraphPad QuickCalcs . p values < 0.05 were considered statistically significant and marked in graphics as followed: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Analysis of symbiotic effects was conducted by computing the combination index (CI) by Chou-Talalay using CalcuSyn, version 2.11 . A CI < 0.9 was classified as a symbiotic effect (+), CI = 0.9–1.1 as an additive effect (±) and CI > 1.1 as an antagonistic effect (-). The mean effective dose was also computed by CalcuSyn. Analysis was performed as described by Chou [ by Chou ,45.50. Thus, dose reduction and thereby potentially improved tolerability and decrease of side effects at increased effects were achieved. Further studies are needed to elucidate GP-2250’s mechanism of action. Furthermore, the effect of GP-2250 on MM cells should be investigated in vivo, as well as in combination with other malignant peritoneal mesothelioma standard therapeutics such as docetaxel, irinotecan or doxorubicin. This study forms the basis for further in vivo testing and clinical trials in order to broaden treatment options in a highly vulnerable cohort of patients.This study proved anti-adhesive and cytotoxic effects of GP-2250 on MM monolayer cells and MM spheroids, inducing apoptosis and necrosis. Combination with CP and MMC led to synergistic and additive effects as well as to a significant reduction of ED


Kaempferia galanga (L.) is one of the prospective therapeutic plants with an aromatic rhizome, and belongs to the Zingiberaceae family. This herb is commonly used by local practitioners in traditional Asian medicine.Kaempferia galanga rhizome essential oil rich in ethyl p-methoxy cinnamate (EMCKG) was evaluated using GC/MS for chemical composition analysis. EMCKG was analyzed for its possible antimicrobial, neurodegenerative inhibitory, acetylcholinesterase, anti-inflammatory, and antioxidant activities as well as for the genotoxic effects using the standard methodologies. ANOVA and post hoc was performed to test the statistical significance of the study.In the present investigation, the novel 50 = 15.64 ± 0.263 µg/mL; ABTS assay IC50 = 16.93 ± 0.228 µg/mL) antioxidant activity than standard ascorbic acid . Similarly, EMCKG showed comparable activity in albumin denaturation (IC50 = 2.93 ± 0.59 µg/mL) and protease inhibitor assay (IC50 = 17.143 ± 0.506 µg/mL) to that of standard sodium diclofenac . The EMCKG exhibited a dose-dependent antimicrobial activity pattern with the highest inhibitory activity at 500 µg/mL against Staphylococcus aureus and considerable anticholinesterase activities (IC50 = 21.94 ± 0.109 µg/mL) compared to the standard galanthamine (IC50 = 27.18 ± 0.511 µg/mL). EMCKG also showed strong anti-diabetic activity (IC50 = 18.503 ± 0.480 µg/mL) and anti-tyrosinase activity (IC50 = 14.756 ± 0.325 µg/mL) as compared to the standards used (acarbose IC50 = 20.39 ± 0.231 µg/mL and kojic acid IC50 = 17.73 ± 0.192 µg/mL) in the study. Genotoxicity analysis of EMCKG revealed that at 1 µg/mL concentration has no toxic effects in mitosis of Allium cepa roots . The ANOVA confirmed that except for the anticholinesterase activity, there is insignificant difference for essential oil and standards used for all the other bioactivities thus confirming their interchangeable applicability.GC/MS analysis identified ethyl p-methoxy cinnamate as the major component of EMCKG essential oil with an area percentage of 66.39%. The EMCKG exhibited moderate (DPPH assay ICCurrent research provides the basis for the fact that besides being a rich source of ethyl p-methoxycinnamate, EMCKG has the potential for future formulation and development of an inexpensive skin-care agent and for the preparation of anti-diabetic drugs. Kaempferia galanga (L.) is a high-value aromatic medicinal plant with numerous uses from its medicinal properties point of view. It is an aromatic rhizomatous herb belonging to the Zingiberaceae family. The name of the plant was cross-checked with http://www.theplantlist.org on 1st March 2021. The rhizome is tuberous with a camphoraceous odor along with some bitter aromatic taste. It is distributed widely in the tropics and sub-tropics of Asia as well as Africa 6); aluminum chloride (AlCl3); tris-buffer (C4H11NO3); ethyl methanesulfonate (C3H8O3S); ascorbic acid (C6H8O6); sodium carbonate (Na2CO3); ferric chloride (FeCl3); acetocarmine (CH3CO2H); ethylene diamine tetraacetic acid (EDTA) (C10H16N2O8); 2,2-diphenyl 1 picrylhydrazyl (C18H12N5O6); potassium persulfate (K2S2O8); MHA-Mueller Hinton Agar and PDA-Potato Dextrose agar were procured from HiMedia (India). ABTS  (C18H18N4O6S4); trichloroacetic acid (C2HCl3O2); ferrous chloride (FeCl2), ferrozine (C20H12N4Na2O6S2) and diclofenac sodium salt (C14H10Cl2NNaO2) were purchased from Sigma Aldrich (Germany). A fresh egg of a hen used for albumin and all the other unlabeled chemicals were obtained from the market of Jorhat, Assam India.The chemicals used were HPLC grade methanol and ethanol; potassium ferricyanide  is cultivated at the experimental farm of CSIR-NEIST, Jorhat, India  under the supervision of the plant breeder Dr. Mohan Lal, CSIR-NEIST Jorhat. A voucher specimen was placed in the CSIR-NEIST Jorhat departmental herbarium vide herbarium no RRLJKN-17. For the essential oil isolation matured rhizomes of K. galanga (cv. Jor Lab K-2) were harvested during December 2020. Collected rhizomes were washed, to remove the sand particles which were cut into smaller slices. The sliced rhizomes were shade dried for 7 days followed by oven drying at the temperature of 70 °C for 3 days until a constant mass was obtained. The essential oil was isolated from dried rhizomes using the Clevenger apparatus as per Ethyl p-methoxycinnamate rich The GC/MS analysis of EMCKG was done as per 50i.e., 50% inhibition was analyzed using MS Excel.The EMCKG was evaluated for anti-inflammatory activity as per 50i.e., 50% inhibition was analyzed using MS Excel.Protease inhibitor activity was analyzed using a standard method as per Staphylococcus aureus, Micrococcus luteus, Streptococcus mutans, Bacillus subtilis and Bacillus cereus with ATCC-11632, ATCC-10240, ATCC-25175, ATCC-11774 and ATCC-10876, respectively. Salmonella typhimurium and Salmonella enteric with ATCC-13311, and ATCC-35664 were used as gram-negative bacteria. In addition to those four fungal strains namely, Aspergillus niger, Fusarium keratoplasticum, Aspergillus oryzae, Saccharomyces cerevisiae, Aspergillus fumigatus, and Candida albicans with ATCC-16885, ATCC-36031, ATCC-10124, ATCC-9763, ATCC-204305, ATCC-66027 respectively were employed for evaluation of the antifungal activity. Standard Ciprofloxacin  and Fluconazole  each at 10 µg/disc were used as standard drugs.The EMCKG antimicrobial activities were analyzed using both gram-positive and gram-negative bacteria. The gram-positive bacteria used were The EMCKG was analyzed for their antimicrobial activity as per viz., 50, 100, 250 and 500 μg/mL. The broth was taken as blank with a control that is free from the essential oil. Data were recorded at an interval of 1 h continuous up to 8 h. The measurements of the optical density (OD600) were taken using the Eppendorf Bio spectrometer basic. Time was taken along the horizontal axis and the OD of the supernatants was taken against the vertical axis.The bacterial growth curve experiment was performed to analyze the bactericidal capabilities of EMCKG essential oil. The experiment was performed as per the protocol of 50i.e., 50% inhibition was analyzed using MS Excel.The EMCKG was analyzed for acetylcholinesterase inhibitory (AChE) activity using a standard modified methodology as per 50i.e., 50% inhibition was analyzed using MS Excel.The antidiabetic activity of EMCKG was analyzed using standard methodology as per 50i.e., 50% inhibition was analyzed using MS Excel.Tyrosinase inhibitory assay was conducted as per 50i.e., 50% inhibition was analyzed using MS Excel.DPPH free radical scavenging activity of EMCKG was performed as per 50i.e., 50% inhibition was analyzed using MS Excel.The ABTS assay was performed following the protocol as per 50i.e., 50% inhibition was analyzed using MS Excel.The metal chelating activity assay was conducted for the antioxidant activity of EMCKG as per 50i.e., 50% inhibition was analyzed using MS Excel.The reducing power of EMCKG was evaluated using a standard protocol as mentioned by The genotoxicity of EMCKG was analyzed using a standard method as per p ≤ 0.05 the difference was considered statistically significant). The ANOVA analysis was done followed by a post hoc analysis.The standard deviation (SD) was analyzed using MS Excel. Each of the experiments was performed in triplicates to reduce experimental errors  and 3-carene (9.60%). The other compounds present were in minor (<5%) amounts. The minor compounds present were endo-borneol (2.76%), camphene (1.66%), α-pinene (1.68%), eucalyptol (1.24%), benzyl benzoate (0.49%), p-cymene (1.12%), D-limonene (1.06%), pentadecane (1.00%), cis-p-mentha-2,8-dien-1-ol (0.80%), and bornyl acetate (0.72%)  as the primary major compound followed by  (0.72%) . The cin50 value of 2.93 µg/mL as compared to that of standard sodium diclofenac 19.18 µg/mL for protein denaturation assay, while protease inhibitor assay revealed IC50 of 17.14 µg/mL for EMCKG and 23.87 µg/mL for standard sodium diclofenac  followed by ethyl trans-p-methoxycinnamate (14.3%), eucalyptol (10.57%), δ-3-carene (5.12%), and n-pentadecane (4.8%), respectively  (42.9%) and 2 (COX-2) (57.82%) whereas the standard indomethacin similarly inhibited COX-1 (82.8%) and COX-2 (54.6%)  value of 10 mg/kg . A previ (54.6%) . It was isorders .K. galanga was found to have the maximum quantity of ethyl-p-methoxycinnamate. Against acute inflammation, in rats, petroleum ether extract was found effective at 300 mg/kg concentration. While petroleum ether extract at a concentration of 100 mg/kg was found to effectively reverse the inflammation   against S. epidermidis and S. aureus; while for P. acne, the concentrations are 0.6%, 1.2%, and 2.4%  . Anotherla typhi . Kaempfeand 2.4% . Another. flavus . HoweverC. albicans, gram-negative, positive bacteria with the zone of inhibition of 31.0, 8.0–12.0 and 12.0–16.0 mm respectively. It was found to be stronger than that of standard antifungal Clotrimazole which had an inhibition zone of 25.0 mm against C. albicans  and acetylcholinesterase (AChE). This study is significant in Alzheimer’s disease treatment /100 g for leaves and 17 mg AA/100 g for rhizome respectively , 26 (CP rhizome), and 19.5 µg/mL (IVP rhizome). Additionally, the hydrogen peroxide scavenging activity revealed a similar good antioxidant property against H2O2, with IC50 values of 29 , 24.5 (for oil samples of IVP rhizome), and 21.5 µg/mL (for ascorbic acid)  . A previextracts . Similarextracts . The metondition . Hence aK. galanga essential oil is safe as it did not affect much the mitotic index and chromosomal aberration rates in the Allium cepa assay. So far, K. galanga crude rhizome extracts were tested for various toxicological studies such as contact toxicity, insecticidal effects, acute and sub-acute toxicities in rats, etc. (K. galanga essential oil and this present report is the first of its kind. The present investigation showed that up to the concentration of 1 µg/mL the essential oil does not possess any genotoxic effect. Hence, this investigation result could give a safety tag of up to 1 µg/mL concentration of the essential oil. Therefore, in the near future formulation of the drug with potential effects could be designed by limiting the concentration up to the safety mark.Genotoxicity assay of EMCKG made it clear that at 1 µg/mL concentration the use of ts, etc. . So far K. galanga rhizome essential oil was found to be rich in ethyl p-methoxycinnamate. Thus, an ethyl p-methoxycinnamate rich variety of K. galanga would be highly valuable and is required to meet the high industrial demand for raw materials for ethyl p-methoxycinnamate. The essential oil was found to possess a significant number of bioactivities like antioxidant, anti-inflammation, neurodegenerative disorders inhibitor, anti-diabetic, skin whitening as well as antimicrobial potential which further increases its pharmaceutical and industrial value in addition to validating its ethnopharmacological application. The essential oil also emerged as a very suitable source for raw materials in the formulation of anti-diabetic, skin whitening agents. From a toxicological point of view, the essential oil was found to be safe up to the concentration of 1 µg/mL. For the use of essential oil to formulate products or drugs it needs to go through further pre-clinical as well as deep clinical trials.The studied 10.7717/peerj.14606/supp-1Supplemental Information 1Click here for additional data file.


The objective of the current study is to determine the pharmacokinetics (PK) of fb-PMT in experimental animals, such as mice, rats, and monkeys. NP751 was quantified using a propylene diamine-modified tetraiodothyroacetic acid (DAT) as an internal standard. The limit of quantification (LOQ) for fb-PMT was 1.5 ng/μL and the recovery efficiency was 93.9% with the developed method. The peak plasma concentration (Cmax) and the area under the curve (AUC) results at different doses in mice, rats and monkeys suggest that pharmacokinetics of NP751 is dose-dependent within the dose ranges administered. Results indicate that NP751 has comparable PK parameters that provides enough exposure as a molecularly tumor targeted molecule in multiple species and is a promising anticancer therapeutic.We have recently reported on the development of fb-PMT (NP751), a conjugate of the thyroid hormone metabolite tetraiodothyroacetic acid (tetrac) and monodisperse polyethylene glycol 36. It exhibited high affinity for thyrointegrin α Our pre0.23 nM) . We alsoThe objective of this study was to develop a quantitative LC-MS/MS method for fb-PMT and to evaluate the PK parameters of fb-PMT, such as peak plasma concentration (Cmax) and area under the curve (AUC), in mice, rats and monkeys. This required a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for fb-PMT for bioanalytical assays. Although it had a high molecular weight (Mw 2522.9), we were able to develop a sensitive multiple-reaction-monitoring (MRM) method using a pseudo-molecular ion for monodisperse fb-PMT, in contrast to polydisperse P-bi-TAT, which required collision-induced dissociation (CID), of the precursor ion .fb-PMT was synthesized and purified as described earlier at a contract manufacturing organization  under Good Manufacturing Practices (GMP) . Diaminofb-PMT-treatment of mice was carried out in the animal facility of the Veterans Affairs (VA) Medical Center , and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the VA. Mice were housed under controlled conditions . Mice were allowed to acclimatize for at least 1 week prior to the start of treatments. fb-PMT  was administered subcutaneously to 4 mice, and plasma was collected at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20 and 24 h. Plasma samples were stored at −80°C for further analysis.The rat study was conducted by Inotiv . Briefly, fb-PMT  was administered subcutaneously to male and female Sprague Dawley rats (n = 3 each) daily for 14 days. Samples were collected at the following target timepoints on Day 1: pre-dose, 0.5, 1, 3, 6, 9, 12 and 24 h following the completion of dosing. Samples were collected at the following target timepoints on Day 28: pre-dose, 0.5, 1, 3, 6, 9, 12 and 24 h following the completion of dosing. Plasma samples were stored at -80°C for further analysis.The monkey study was conducted by a contract research organization. Briefly, fb-PMT  was administered subcutaneously to male and female cynomolgus monkeys (n = 2 each) daily for 28 days. Samples were collected at the following target timepoints on Day 1: pre-dose, 0.5, 1, 3, 6, 9, 12 and 24 h following the completion of dosing. Samples were collected at the following target timepoints on Day 28: pre-dose, 0.5, 1, 3, 6, 9, 12 and 24 h following the completion of dosing. Plasma samples were stored at -80 °C for further analysis.50 μL of mouse, rat, or monkey plasma with or without fb-PMT and an internal standard (IS) of DAT (150 ng/ml) were vortex-mixed with 1 ml of acetonitrile/water (80/20 v/v) for 30 min. After centrifugation, solvent was evaporated under a nitrogen stream at 50°C. Extracts were reconstituted with 50 μL of acetonitrile/water (80/20 v/v).An API-4000 mass spectrometer  equipped with Shimadzu UPLC system  was used for LC-MS/MS analyses. A Kinetex 2.6 μm Biphenyl 100 LS column  was used for reversed-phase separation. Mobile phases were (A) water containing 0.1% formic acid and 5% acetonitrile and (B) acetonitrile with 0.1% formic acid. The flow rate was 0.4 ml/min, and the gradient was linear from 25% B to 95% B for 2–4 min. The oven temperature was 40°C and the injection volume was 5 μL. Electro-spray ionization (ESI) was used in positive MRM mode. Mass transitions for analytes were Q1/Q3: 841.991/109.26 (fb-PMT), 862.9/845.7 (IS). The operative parameters of the mass spectrometer were as follows: declustering potentials (DP): 56 V; entrance potentials (EP): 10; collision energies (CE): 90 eV; collision cell exit potential (CXP), 6 V; curtain gas (CUR), 10 psi; gas 1  40 psi; gas 2  40 psi; ion spray voltage (IS), 5500 V; temperature (TEM), 500 °C; collision activate dissociation (CAD) gas: 9 psi; dwell time: 150 ms. Nitrogen was used for the curtain, source, and exhaust gases. A standard curve was obtained using standard solutions of fb-PMT with concentrations of 3000, 1000, 300, 100, 30, 10, 3, and 1 ng/ml.Recovery efficiency was calculated with the response of extracts from spiked plasma sample (sample) and blank plasma sample (blank) with spike after extract as follows:Recovery efficiency (%) = Response in sample/Response in blank * 100LOQ = 10 x SD/slopeLOD = 3 x SD/slopeThe limit of quantification (LOQ) and the limit of detection (LOD) was determined using data of six injections of a standard solution (9.8 ng/ml). LOQ and LOD were calculated using the standard deviations (SD) of signals for the analyte and the slope of linear regression curve as follows:p < 0.05 were considered statistically significant.It was performed with GraphPad Prism 7 software. Data are presented as mean ± SD. For comparison between groups, ANOVA was used. *- of fb-PMT was detected in negative mode (data not shown). The MW of fb-PMT calculated with the m/z of the deprotonated pseudo-molecular ion was consistent with the theoretical values. While observing the molecular ion was useful for confirming molecular weight, the intensity of the deprotonated molecular ion [M-H]- was very weak, so it was not applicable to MRM.The ionization of fb-PMT was studied in positive and negative modes. A deprotonated pseudo-molecular ion [M-H]In the positive ion mode, a triply charged ion of fb-PMT at m/z 842.0 was detected, which was much stronger than singly or doubly charged ions . The MRMThe time course of fb-PMT concentrations in mouse plasma is shown in The time courses of fb-PMT concentrations in rat plasma are shown in The time course of fb-PMT concentrations in monkey plasma is shown in ALL technique for PEGylated gemcitabine (Mw 70–76 kDa) and conducted its PK study in rats , mean peak (Cmax) exposures to fb-PMT and mean total (AUC) exposures increased in a dose related manner .Following SC administration, fb-PMT was rapidly distributed into systemic circulation resulting in quantifiable plasma concentrations at 1 h post-dose in all measured mice, and generally remained quantifiable through 12 h. Mean time to peak concentration (Tmax) occurred 1 h post-dose at all four doses tested. Following Tmax, plasma fb-PMT concentrations decreased in a multiphasic manner with mean TFollowing daily SC administration for 28 days, fb-PMT was rapidly distributed into systemic circulation resulting in quantifiable plasma concentrations at 0.5 h post-dose in all measured rats on both Day 1 and Day 28, and generally remained quantifiable through 12 h. Predose fb-PMT concentrations were not observed at all dose levels on Day 1, and at the 5 and 15 mg/kg dose levels on Day 28. There were quantifiable pre-dose fb-PMT concentrations at the 30 mg/kg dose level across both sexes on Day 28. Composite mean time to peak concentration (Tmax) occurred 1 h post-dose on Day 1 and 1–3 h post-dose on Day 28. Elevated Day 28 plasma concentrations at the 12 h relative to the 9 h timepoint were observed for the females administered 5 and 30 mg/kg.1/2) values ranging from 0.87 to 1.7 h. Female composite mean peak (Cmax) and total (AUClast) exposures to fb-PMT were comparable (within 2-fold) to the male exposures on Day 1 and Day 28. Across the dose range (5–30 mg/kg), composite mean peak (Cmax) and total (AUClast) exposures to fb-PMT generally increased in a dose proportional manner on Day 1 and Day 28. Accumulation was not observed based on RA of Cmax and AUClast, which ranged from 0.458 to 0.875 and 0.828 to 1.24, respectively.Following daily SC administration of fb-PMT for 28 days, female and male fb-PMT plasma concentrations declined in a multiphasic manner with individual composite mean half-life  occurred 1–6 h post-dose on Day 1 and occurred 1–3 h post-dose on Day 28. Following Tmax, plasma fb-PMT concentrations decreased in a multiphasic manner with mean Tlast) exposures to fb-PMT were comparable (within 2-fold) to the male exposures on Day 1 and Day 28. Across the dose range (2.5–15 mg/kg), mean peak (Cmax) exposures to fb-PMT increased in a dose proportional manner and mean total (AUClast) exposures to fb-PMT generally increased in a dose proportional manner on Day 1 and Day 28. Accumulation was not observed based on Ratio of accumulation (RA) of Cmax and AUC, which ranged from 0.744 to 1.08 and 0.821 to 0.862, respectively  and total  Medical Center, Albany, NY, United States in accordance with current institutional guidelines for humane animal treatment and approved by the VA IACUC (protocol number 545017).