Hypericum androsaemum L. extracts by liquid chromatography-high resolution mass spectrometry (LC-HRMS), and investigate the antioxidant and cytotoxic activities of the plant. Cytotoxic activity was evaluated by MTT assay, and apoptosis induction abilities on human prostate adenocarcinoma (PC-3), and hepatocellular carcinoma (Hep G2) cell lines. Accordingly, major secondary metabolites were found as hederagenin (762 ± 70.10 μg/g) in the leaves dichloromethane (LD), herniarin (167 ± 1.50 μg/g) in fruit dichloromethane (FD), (-)-epicatechin (6538 ± 235.36 μg/g) in the leaves methanol (LM), (-)-epigallocatechin gallate (758 ± 20.46 μg/g) in the fruit methanol (FM), and caffeic acid (370 ± 8.88 μg/g) in the fruit water (FW), and (3313 ± 79.51 μg/g) in the leaves water (LW) extracts. LM exerted strong antioxidant activity in DPPH free (IC50 10.94 ± 0.08 μg/mL), and ABTS cation radicals scavenging (IC50 9.09 ± 0.05 μg/mL) activities. FM exhibited cytotoxic activity with IC50 values of 73.23 ± 3.06 µg/mL and 31.64 ± 2.75 µg/mL on PC-3 and Hep G2 cell lines, respectively. Being the richest extract in terms of quillaic acid (630 ± 18.9 μg/g), which is a well-known cytotoxic triterpenoid with proven apoptosis induction ability on different cells, FM extract showed apoptosis induction activity with 64.75% on PC-3 cells at 50 μg/mL concentration. The study provides promising results about the potential of Hypericum androsaemum on cancer prevention.The study aims to determine the secondary metabolites of  Hypericum (Hypericaceae) is a large genus represented by nearly 500 species throughout the world. On the basis of the current knowledge, there are recently 109 taxa evaluated under 20 sections in Turkey, and 48 taxa of them are endemic [1]. The most popular member of the genus is undoubtedly H. perforatum, (common St. John’s wort), which has been used since ancient times mainly for its antidepressant and wound healing effects. Being one of the best-selling herbal medicines in the world, H. perforatum can be found in many stores in different forms of products, such as capsules, ointments, and drops. Incidentally, in the late nineties, preparations made with H. perforatum had made up as much as 25% of all prescriptions prescribed by doctors to treat depression in Germany [2].The genus Hypericum species specifically, and as a consequence, the number of studies conducted to investigate their medical properties has increased exponentially. According to these studies, it can be said that the modern history of Hypericum species is beyond doubt a success story, and the reasons behind the success are rationale [3]. Being another member of the genus Hypericum, Hypericum androsaemum L. (common Tutsan) is an evergreen or semievergreen shrub that is widely known in the Mediterranean basin. It has been reported that the plant has been predominantly used for its sedative, antidepressant, anxiolytic, hepatoprotective, wound healing, antihypertensive, anti haemorrhoidal, and diuretic properties traditionally [4–7]. Differently from H. perforatum, not only the aerial parts but the fleshy and berry-like capsules that ripen from red to black produced by H. androsaemum have also been used for various medical purposes. Since the biological activities of Hypericum species are mainly attributed to their rich mixture of phytochemicals that includes naphthodianthrone derivatives , acylphloroglucinols , flavonoids , tannins, xanthones, and volatile oils [8], it has become crucial to determine the chemical composition of H. androsaemum as well. As a corollary, there are several studies concerning the evaluation of the chemical composition and biological activities of H. androsaemum, including both in vitro and in vivo studies. It must be noted that H. androsaemum is a hypericin-free plant, which can be considered as a major advantage for especially skin-related activities since hypericin is a molecule that can cause phototoxicity, and can also induce activation of hepatic enzymes [9]. Interestingly, H. androsaemum is three times more expensive than H. perforatum in Portugal, due to the high demand and low supply compared to the famous H. perforatum [10].In the past few years, there has been growing interest surrounding H. androsaemum is a quite rich plant in terms of phenolic compounds, including caffeoylquinic acids, chlorogenic acid, and quercetin derivatives . With a better understanding of the chemical composition of the plant, it has been easier to more closely align the research designed to investigate the pharmacological properties. In this context, it has been reported by the studies published to date that H. androsaemum showed antioxidant [11], antidepressant , wound healing and UV protector [9], cytotoxic and immunomodulatory [6], acetylcholinesterase inhibitory [14], antiinflammatory, and anti-Candida [15] effects. Thus far, it has been reported by different studies that H. androsaemum collected from Trabzon, Turkey by LC-HRMS analysis, and evaluation of its antioxidant and anticancer effects. Additionally, and most notably, as the search for safer and more effective agents against cancer, which is indisputably one of the most fatal diseases continues, this study provides a contribution to anticancer research with evidence for anticancer activity of the plant by showing cytotoxicity on prostate adenocarcinoma (PC-3), and hepatocellular carcinoma (Hep G2) cell lines, and significant apoptosis induction on PC-3 cell lines. As it has already been mentioned that both leaves and mature fruits were used traditionally for medical purposes, six extracts; dichloromethane, methanol, water extracts of leaves , and mature fruits  were prepared for the comparison of the activities. Lastly, this study aims to contribute new insights for understanding the therapeutic properties of H. androsaemum.Bearing all this in mind, this current study focuses on the secondary metabolite composition of For LC-HRMS: Caffeic acid (98%), emodin (90%), eupatilin (98%), astragalin (> 97%), (-)-epicatechin (≥ 90%), quercetin (≥ 95%), naringin (≥ 90%), rosmarinic acid (> 96%), naringenin (≥ 95%), chrysin (≥ 96%), fumaric acid (≥ 99%), and rutin (≥ 99%) were purchased from Sigma-Aldrich Germany. (+)-Catechin , (-)-epigallocatechin gallate (EGCG) (> 97%), epigallocatechin (> 97%), acacetin (> 97%), apigenin (> 97%), hederagenin (> 97%), quillaic acid (> 97), hyperoside (> 97%), 3-O-methylquercetin (> 97%), (+)-trans-taxifolin (> 97%), scutellarein (> 97%) were purchased from TRC Canada. Nepetin-7-glucoside (> 97%), dihydrokaempferol (> 97%), and rhamnocitrin (> 97%), were puchased from Phytolab. Luteolin-7-rutinoside , verbascoside , herniarin , hesperidin , (-)-nepetin , apigenin 7-glucoside  were obtained from different companies.For antioxidant activity: ABTS  diammonium salt, purity: 97.5%) and BHT  were purchased from Merck (Germany); DPPH , pyrocatechol (≥ 99%), CuCl2.2H2O (copper (II) chloride dihydrate, ≥ 99%), neocuproine , EDTA , and a-tocopherol (≥ 95.5%) were obtained from Sigma–Aldrich (Germany); Folin Ciocalteu phenol reagent was purchased from Applichem (Germany). All solvents were analytical grade.For cytotoxic activity and apoptotic determination: MTT -2,5-diphenyltetrazolium bromide) was purchased from Biomatik . Dimethylsulfoxide (DMSO) was obtained from Merck . Sodium dodecyl sulfate (SDS) and all the cell culture materials were purchased from Wisent Bioproducts . Cell culture: Prostate adenocarcinoma (PC-3) and hepatocellular carcinoma (Hep G2) cell lines were obtained from American Type Culture Collection . The cells were grown in DMEM-F12 medium, following the manufacturer’s instructions. The subculture was performed when the cells reached 70-80% confluence every 2-3 days.Hypericumandrosaemum L. were collected from Trabzon, Arsin, Çiçekli Village in August 2018. A voucher specimen was deposited in the Herbarium of Faculty of Pharmacy of İstanbul University (ISTE 116811).Leaves and mature fruits of The air-dried plant material (leaves and mature fruits) was ﬁnely powdered, then separately weighed (10 g), and then macerated in methanol (100 mL) for 24 h at room temperature. The macerate was ﬁltered and this procedure was repeated three times. The ﬁltrates were gathered and then the methanol was evaporated under reduced pressure at below 45 °C. The same procedure was applied to obtain the dichloromethane extracts from the leaves and mature fruits. The water extracts of the leaves and mature fruits were prepared by using hot water infusion. The crude methanol extracts and infusions were lyophilized and stored at –20 °C.Thermo Orbitrap Q-Exactive instrument in ESI (Electrospray ionization) Source and Troyasil HS C18 column  were used for LC-HRMS (Liquid Chromatography High-Resolution Mass Spectrometry) analysis. The gradient method  was employed with 0.3 mL/min flow rate at 25 °C. The mobile phase A was formic acid:water  and B is formic acid:methanol . The injection volume was 2 μL. ESI source was utilized to obtain good ionization of small and relatively polar compounds. The scanning ion range was between m/z 85–1500 in the high-resolution mode of the instrument. The compound identification was performed by comparison of retention time of standard compounds (in the range of purity 95%–99%) and HRMS data of Bezmiâlem Vakıf University, Drug Application and Research Center Library (ILMER).The extract was dissolved in the mobile phase , and then internal standard  was added to the final concentration of 3 ppm and volume was filled with mobile phase mixture up to 5 mL. Then, the solution was filtered through a 0.45 m filter and 2 mL was injected into the instrument [16–18].UV spectrophotometer (Shimadzu) was used to determine total phenolic and flavonoid contents. The method which was used to determine the total phenolic contents of the extract was described by Karapandzova et al. [19]. The phenolic compounds concentration was calculated using the following equation obtained from the standard pyrocatechol graphic, and the results were given as micrograms of pyrocatechol equivalents (PEs).2  = 0.9987)Absorbance = 0.0276 pyrocatechol (μg) + 0.0416 (R2  = 0.9992) obtained from standard quercetin graphic.Total flavonoid content determination of the extracts was evaluated according to Karapandzova et al. [19]. The flavonoid compounds concentration was computed using the equation (Absorbance = 0.0277 quercetin (μg) – 0.0434; RA microplate reader  was used for the antioxidant activity assays.50 (The fifty percent inhibitory concentrations) values to express the antioxidant potential of the extracts.The method reported by Karapandzova et al. was applied to determine the DPPH free radical scavenging potential of the extracts [19]. The results were given as IC•+ in the extracts was detailedly defined by Li et al. [20]. IC50 values were calculated to indicate the scavenging capability of ABTS•+ in the extracts.The assay used to determine the scavenging capability of ABTS50 values of the ferrozine – Fe+2 complex formation in the extracts.The metal chelating potential of the extracts was tested using the method of Decker and Welch [21]. Chelating activity was calculated as ICThe method described in detail by Apak et al. was carried out to identify the cupric reducing antioxidant capacity of the extracts [22]. The absorbance values of the extracts read at 450 nm. The results were given as A0.5 values with standard deviations and A0.5 values were calculated by absorbance-concentration graph. 50), the absorbance values of each sample were compared with the absorbance values of the solvent controls. The IC50 value was computed as the percentage of solvent controls according to the following formula:The extracts were dissolved in DMSO or water depending on solubility in the concentration range of 12.5–100 µg/mL. The negative  solvent (1% DMSO or water) and positive controls (0.1% SDS) were used in all tests. To calculate the 50% inhibition concentration (ICsample × 100) / corrected mean ODsolvent control]Inhibition (%) = 100 + [ was quantified at 590 nm against reference wavelength 670 nm using a microplate reader . All assays were independently repeated three times.The cell viability was determined through the mitochondrial pathway in the MTT assay described by Morgan [23]. The PC-3 cells were seeded in 96-well plates at 1 × 105 cells/well in a 2 mL culture medium into a 6-well plate and incubated overnight. Then, the cells were treated with the highest concentrations of extracts for 24 h and 1% DMSO was used as a negative control. After trypsinization, the cells were collected and washed with cell staining buffer . After washing, the cells were resuspended with the density of 1 × 105 cells/100 μL in annexin V binding buffer and 5 μL FITCH annexin V/10 μL PI were added, mixed, and incubated for 15 min at room temperature. After incubation, 400 μL annexin V binding buffer was added and fluorescence intensity was measured on an ACEA Novo Cyte flow cytometer . The results were analyzed with NovoExpress software and data were expressed as the percent of the total cell amount.The detection of apoptosis was performed with fluorescein isothiocyanate (FITC) annexin V Apoptosis Detection Kit with propidium iodide (PI)  by ACEA Novo Cyte flow cytometer . According to the manufacturer’s instructions, annexin V fluorescent dye binds to phosphatidylserine that is normally found in the inner part of the plasma membrane and externalizes apoptosis. PI directly binds to DNA. The cells are grouped (i) annexin V-negative and PI-negative (live cells), (ii) annexin V-positive and PI-negative (early apoptotic), and (iii) annexin V-positive and PI-positive (late apoptotic or necrotic) . Briefly, the cells were seeded at 2 × 10All tests were repeated in triplicate. A t-test with Microsoft Excel was used to evaluate the results and the results were expressed as mean ± standard deviation. Differences were considered significant at p < 0.05.H. androsaemum collected from Arsin, Trabzon were investigated by LC-HRMS, and the results were given in Table 1. In this study, chemical compositions of dichloromethane, methanol, water extracts of leaves , and mature fruits  of H. androsaemum. In this technique, we have recorded the high-resolution mass spectrometry of each compound together with MS/MS ions simultaneously using Thermo Orbitrap Q Exactive instrument. Quantification of the compounds was carried out by the data of HRMS while for the identification of each metabolite HRMS, MS/MS and RT data of the standards were used. One of the most critical issues of the applied method is the dissolution of plant extracts in the appropriate solvent to get clear and repeatable separation of chromatographic peaks and ionization stability. Regarding previous reports of literature  and the findings clearly showed that one of the best mobile phase solution was determined to be a gradient of acidified methanol and water in the ESI source for those polar compounds.To avoid the similar MS/MS pattern of the triple quadrupole mass spectrometry , it was decided to use high-resolution mass spectrometry and MS/MS screening for the secondary metabolites of the extracts of Validation of the applied method was performed using analytical standards of corresponding compounds (see subsection 2.1) using the target ions (Table 2) and curcumin was used as an internal standard.Calibration curves were obtained from standard calibration solutions. The linearity of the method was assayed by analyzing the calculation of a six-point linear plot in the range of 0.1 mg/L to 10 mg/L with six replicates. The linear regression equation and the squared correlation coefficients were determined and reported in Table 2. The limit of detection (LODs) of the method for each compound was determined according to the following equation: LOD or LOQ (The limit of quantification) = κSDa/b, where 3 for LOQ and κ = 3 for LOD, SDa represents the standard deviation of the intercept, and b represents the slope (Table 2) .Repeatability, and intermediate precision of the developed LC-HRMS used and amount of secondary metabolites were measured in six different extracts for preliminary screening. Then, according to the detected levels of compounds in the extracts, we spiked the extracts to reach the final concentrations as 0.1 mg/L and 0.5 mg/L, and 1 mg/L in 5 mL volumetric flasks. Unspiked plant extracts were also analyzed to determine the target compound concentrations in the blank sample. The recovery of each component at each fortification level was calculated according to the following formula .(1)Recovery %=Measured conncentration - endogeneous concentrationspiked concentrationx100The recoveries of measured secondary metabolites were ranged from 94.5 to 116.4% (Table 2). Mean relative standard deviations (RSD) were found to be 1%–10 % for all and those data were added to the uncertainty budget of all reported compounds.  The repeatability of the applied method was assessed at three concentration levels of the recovery studies. For the intermediate (reproducibility) precision, a set of spiked samples having three concentration levels were analyzed twice a week for a period of 3 weeks and the repeatability of the method evaluated.The same uncertainty evaluation method, the bottom-up approach was applied to obtain the measurement of the uncertainty value for the developed method. The sources for uncertainty were determined as were weighing the sample, calibration graph, and repeatability. Detailed evaluation procedure corresponding equations are given in the previous papers  and to avoid repetition, we summarized the calculation of combined standard measurement uncertainty of target compounds in plant extracts in equation 2. The expanded measurement uncertainty was obtained by multiplying the combined standard measurement uncertainty value with 2 (coverage factor) at a 95% confidence level. The uncertainty value of measurement results is given in Table 2.(2)uc(A)CA=(u(Wss)Wss)2+(u(C0)C0)2+u(Rm)2+u(r)2where uc(A): Combined standard measurement uncertainty of the analyte, CA: Concentration of the target analyte,u(WSS): Combined standard measurement uncertainty of the sample intake,WSS: Weight of the starting sample, u(c0): Combined standard measurement uncertainty of the calibration curve, c0: Determined concentration of the sample by using the calibration curve, u(Rm): Combined standard measurement uncertainty of recovery, u(r): Standard measurement uncertainty of repeatability.Two metabolites, naringenin, and herniarin were identified in all analyzed extracts.LD extract had the highest total flavonoid content among the six studied extracts. As regards the LD extract, 15 constituents were identified. The most abundant secondary metabolite in the LD was hederagenin (762 ± 70.10 μg/g extract) which is a triterpenoid. Incidentally, hederagenin was only found in this one among the 6 studied extracts. Chrysin is another constituent that was determined only in LD with 118 ± 1.41 μg/g extract. Other compounds that were detected in LD were herniarin (150 ± 1.35 μg/g extract), naringenin (82 ± 3.44 μg/g extract), kaempferol (120 ± 1.08 μg/g extract), (-)-epicatechin (314 ± 11.30 μg/g extract), (+)-catechin (103 ± 1.85 μg/g extract), (+)-trans taxifolin (129 ± 3.87 μg/g extract), quercetin (137 ± 1.78 μg/g extract), penduletin (250 ± 4.00 μg/g extract), eupatilin (228 ± 3.19 μg/g extract), (-)-epigallocatechin gallate (193 ± 5.21 μg/g extract), myricitrin (198 ± 6.13 μg/g extract), hyperoside (229 ± 6.87 μg/g extract), and rutin (139 ± 6.25 μg/g extract). In the FD extract, 8 compounds were present. FD is the richest extract among the analyzed extracts in terms of a coumarin derivative, herniarin (167 ± 1.50 μg/g extract). Fumaric acid (67 ± 2.14 μg/g extract), naringenin (77 ± 3.23 μg/g extract), kaempferol (100 ± 0.90 μg/g extract), quercetin (118 ± 1.53 μg/g extract), astragalin (92 ± 3.86 μg/g extract), orientin (81 ± 3.07 μg/g extract), and quercitrin (81 ± 3.88 μg/g extract) are the other phytochemicals that were found in FD extract. H. androsaemum. It must be also noted that, among the six extracts that were investigated, LM had the highest total phenolic content. (-)-Epigallocatechin (157 ± 4.86 μg/g extract), and 3-O-methylquercetin (103 ± 3.09 μg/g extract) are the phytochemicals that were found only in LM. Additionally, LM was found to be richer than the other analyzed extracts in terms of luteolin (59 ± 1.12 μg/g extract), kaempferol (896 ± 8.06 μg/g extract), scutellarein (50 ± 0.70 μg/g extract), (-)-epicatechin (6538 ± 235.36 μg/g extract), (+)-catechin (1122 ± 20.19 μg/g extract), quercetin (804 ± 10.45 μg/g extract), rosmarinic acid (65 ± 2.86 μg/g extract), astragalin (130 ± 5.46 μg/g extract), orientin (124 ± 4.71 μg/g extract), quercitrin (124 ± 5.95 μg/g extract), (-)-epigallocatechin gallate (3035 ± 81.94 μg/g extract), myricitrin (3657 ± 113.36 μg/g extract), hyperoside (3713 ± 111.39 μg/g extract), and rutin (232 ± 10.44 μg/g extract). Besides, fumaric acid (16 ± 0.51 μg/g extract), herniarin (144 ± 1.29 μg/g extract), caffeic acid (134 ± 3.21 μg/g extract), naringenin (86 ± 3.61 μg/g extract), (+)-trans taxifolin (171 ± 5.13 μg/g extract), penduletin (286 ± 4.57 μg/g extract), and eupatilin (266 ± 3.72 μg/g extract) were the other metabolites that were present in LM. A total of 23 different constituents were identified in the LM extract of In FM, 9 compounds were detected. In terms of fumaric acid (107 ± 3.42 μg/g extract), penduletin (336 ± 5.37 μg/g extract), eupatilin (317 ± 4.43 μg/g extract), and quillaic acid (630 ± 18.9 μg/g extract), FM extract was found to be the richest. Also, herniarin (142 ± 1.27 μg/g extract), naringenin (83 ± 3.48 μg/g extract), kaempferol (156 ± 1.40 μg/g extract), and (-)-epigallocatechin gallate (758 ± 20.46 μg/g extract) were identified in the extract. H. androsaemum, 8 metabolites were determined. An anthraquinone derivative emodin was found only in this extract with 44 ± 1.05 μg/g extract. Fumaric acid (44 ± 1.40 μg/g extract), herniarin (117 ± 1.05 μg/g extract), caffeic acid (370 ± 8.88 μg/g extract), naringenin (81 ± 3.40 μg/g extract), (+)-trans taxifolin (76 ± 2.28 μg/g extract), (-)-epigallocatechin gallate (141 ± 3.80 μg/g extract), and quillaic acid (298 ± 8.94 μg/g extract) are the other constituents of the FW extract according to the results. In the FW extract of LW is also found to be quite a rich extract with 18 metabolites in it. The major compound of the extract was caffeic acid with 3313 ± 79.51 μg/g extract. Also, in terms of (+)-trans taxifolin (303 ± 9.09 μg/g extract), LW was more abundant than the other extracts. Herniarin (123 ± 1.10 μg/g extract), naringenin (88 ± 3.69 μg/g extract), luteolin (57 ± 1.08 μg/g extract), kaempferol (175 ± 1.57 μg/g extract), scutellarein (48 ± 0.67 μg/g extract), dihydrokaempferol (3 ± 0.11 μg/g extract), (-)-epicatechin (882 ± 31.75 μg/g extract), (+)-catechin (172 ± 3.09 μg/g extract), quercetin (183 ± 2.37 μg/g extract), penduletin (170 ± 2.72 μg/g extract), eupatilin (146 ± 2.04 μg/g extract), rosmarinic acid (62 ± 2.72 μg/g extract), (-)-epigallocatechin gallate (376 ± 10.15 μg/g extract), myricitrin (158 ± 4.89 μg/g extract), hyperoside (188 ± 5.64 μg/g extract), and rutin (66 ± 2.97 μg/g extract) were found to be present in LW extract.H. androsaemum growing in Turkey. In a previous study, ethanol extract of aerial parts of the plant was obtained and the chemical composition of the extract was analyzed using an LC/ESI/MS/MS method. According to the results of the study, rutin, hyperoside, quercitrin, and quercetin were identified in the extracts [31]. Another study was also conducted in 2017 to determine the secondary metabolites of H. androsaemum that were collected from different altitudes of Turkey by HPLC. The results showed that chlorogenic acid, neochlorogenic acid, caffeic acid, hyperoside, isoquercitrin, quercitrin, quercetin, (+)-catechin, and (-)-epicatechin were identified in the whole plant material of H. androsaemum sampled at different altitudes of a valley in Turkey [32].A few number of studies have been carried on the investigations on chemical constituents of H. androsaemum is one of the most popular members of the Hypericaceae family, and not only distributed in Turkey but also widely and wildly grows in many parts of Europe. On that account, there have been studies focused on the phytochemical composition of the plant that was harvested from different countries as well. One of those studies that was conducted in Portugal showed that caffeoylquinic acid and quercetin derivatives were found to be the major compounds in the water infusion of the plant [11]. In another study, the HPLC/DAD analysis of methanol extracts of H. androsaemum harvested from Portugal revealed a phenolic fingerprint composed of different compounds including mainly caffeoylquinic acid, quercetin derivatives, and kaempferol [33]. The phytochemical profiles of ethanol extracts of different Hypericum species from Sicily-Italy were screened by HPLC-DAD-MS in 2018, and the results showed that the H. androsaemum ethanol extract contained caffeoylquinic acid, catechin, and a few quercetin derivatives [34]. According to another study, quinic acid, chlorogenic acid, and quercetin derivatives were identified in methanol extracts of aerial parts of the plant from Portugal [35]. According to the study about the chemical characterization of the ethanol extract from red berry-like fruits of H. androsaemum, the extract was found to be rich in terms of shikimic acid and chlorogenic acid. Moreover, neochlorogenic acid, gallic acid, catechin, epicatechin, rutin, and hyperoside were found to be present in the extract. This study was influential, regarding this was the first report about the marker bioactive compounds of H. androsaemum red berries, and more importantly, the significant phytochemicals that exhibit a wide range of biological activities such as chlorogenic acid was found to be much more abundant compared to the leaves extracts [7]. The chromatographic profile of the H. androsaemum ethanol:water extract was also reported by a previous study. Accordingly, protocatechuic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, epicatechin, taxifolin, quercetin-3-O-glucuronide, and quercetin-3-O-glucoside were identified in the extract. The extracts also exerted antioxidant, cytotoxic, antiinflammatory, and anti-Candida activities, which were attributed to the major phenolic constituents, namely 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid [15]. Along with these studies, there have been few other studies to investigate the constituents of the extracts prepared with the berries of the plants. Shikimic acid, gallic acid, chlorogenic acid, catechin, epicatechin, rutin, and hyperoside were determined predominantly .As mentioned in the introduction part before, Within this context, the results of this study are in agreement with the literature, moreover, it can be seen that many of the phytochemical compounds were determined for the first time within this current analysis. H. androsaemum were given as micrograms of pyrocatechol (PE) and quercetin (QE) equivalents, respectively. The total phenolic content of the extracts was higher than the total flavonoid content except for LD. LM had the highest total phenolic content with 108.51 ± 1.28 μg pyrocatechol equivalent/mg extract, and LD had the highest total flavonoid content with 86.74 ± 2.29 μg quercetin equivalent/mg extract. The total phenolic and flavonoid contents values are given in Table 3. Generally, the total phenolic and flavonoid contents of leaf extracts are found to be higher than the fruit extracts. In the study of Keskin et al., total phenolic contents of flower, fruits, and seed methanol extracts of H. retusum were investigated. The total phenolic content of fruit methanol extract of H. retusum was found to be lower than the other extract [36]. In the study of Sekeroglu et al., ethanol extract of the leaves of H. perforatum had the highest total phenolic content among the flower, stem, and leaf extracts [37]. Another study indicated that methanol extract of H. lydium had higher total phenolic content than the water extract [38]. In the study of Saddiqe et al., the total flavonoid content of the dichloromethane extract of H. androsaemum was found to be higher than its total phenolic content [39]. According to the literature survey, total phenolic and flavonoid content results in this study are parallel with the other studies about total phenolic and flavonoid contents of different Hypericum species.The concentrations of total phenolic and flavonoid of water, methanol, and dichloromethane extracts prepared from leaves, and fruits of 50 (concentration of 50% inhibition) and CUPRAC results given as A0.5 values (Table 3). DPPH free radical scavenging, ABTS cation radical scavenging, and metal chelating activities results given as IC50 10.94 mg/mL. LW also showed strong DPPH free radical scavenging activity, even stronger than BHT with IC50 29.83 mg/mL. Keskin et al. reported DPPH free radical scavenging activities of flower, fruit and, seed extracts of H. retusum. Fruit extract showed weaker activity than the other extracts [36]. In the results of the current study, all fruit extracts of H. androsaemum showed weaker activity than all leaves extracts. Boga et al. reported methanol extract of H. capitatum var. capitatum had the highest DPPH free radical scavenging activity among the petroleum ether, acetone, methanol, and water extracts with IC50 16.82 mg/mL [40]. DPPH free radical activities of ethanol and water extracts of flower, stem, and leaf of H. perforatum were studied, and all ethanol extracts showed better activities than water extracts. The leaf ethanol extract also showed the best activity with 84.0% inhibition value at 1 mg/mL concentration [37]. The results obtained from the current study showed that there was parallelism with literature results.In DPPH free radical scavenging activity, LM showed the best activity among all extracts and also from the standards a-TOC and BHT, with IC50 10.88 ± 0.44 and 9.09 ± 05 mg/mL values, respectively, and methanol extract showed better activity than a-TOC which was used as standard. Methanol extract of H. humifusum with 180.87 mg TEs/g for ABTS exerted strong activity [41]. In the study of Eruygur et al., in ABTS cation radical scavenging assay, the methanol extract showed better activity (IC50 16.63 μg/mL) than the water extract (IC50 20.48 μg/mL) [38]. In ABTS cation radical scavenging assay, the methanol and water extracts of the whole H. capitatum var. capitatum showed very well activities with IC50 9.24 and 9.76 μg/mL values, respectively. These values are almost identical to the result of the previous study conducted by Boga et al. (2016) [40]. In the study of Kang et al., ABTS radical scavenging activity of petroleum ether, ethyl acetate, and methanol extracts of the whole plants of H. ascyron were studied and methanol extract showed the best activity among all extracts with IC50 24.55 μg/mL value [42]. There is only some literature about ABTS cation radical scavenging activity on Hypericum species and results are very similar to the current study results. LW and LM showed strong antioxidant activity in ABTS cation radical scavenging assays with IC50 77.61 ± 0.79 mg/mL value. LM showed moderate metal chelating activity (IC50 96.56 ± 1.29 mg/mL). In the study of Maltas et al., methanol extracts of H. salsugineum, H. aviculariifolium, and H. perforatum showed moderate metal chelating activities [43].FW demonstrated the best metal chelating activity among all extracts with ICH. lanuginosum, H. capitatum var. capitatum showed better activity than the other extract unlike our results . Furthermore, the study of Ersoy et al. showed that the methanol extracts of three different Hypericum species especially H. calycinum have good cupric ion reducing antioxidant capacity [45].LM demonstrated significant cupric reducing antioxidant capacity (CUPRAC) and showed better activity than a-TOC that was used as standard at all tested concentrations. LW was also found to have good cupric reducing antioxidant capacity. In different studies, water extracts of H. androsaemum was measured on two cancer cell lines, namely prostate adenocarcinoma (PC-3) and hepatocellular carcinoma (Hep G2) cell lines. 0.1% SDS was used as the positive control. The results are given in Table 4, as the percentage of dead cells, and also the calculated half-maximal inhibitory concentration value (IC50) and comparing them by statistical analysis.In the current study, cytotoxicity of FM, FD, FW, LM, LD, and LW extracts of 50 value. According to the results, FM was the most effective cytotoxic among the six studied extracts on both cell lines with IC50 values of 73.23 µg/mL on PC-3 cell lines, and 31.64 µg/mL on Hep G2 cell lines. FD exhibited weak cytotoxic activity on Hep G2 cell lines (IC50 81.18 µg/mL). It must be noted that due to the solubility problems it has not been possible to test the cytotoxicity of these extracts with higher concentrations. The cytotoxic potential of the extracts may increase with prolonged exposure.As known, a higher cytotoxic effect is indicated by a lower ICH. androsaemum are becoming more intriguing with low or no toxicity and improved efficacy in the treatment of the disease.A remarkable fact that, out of 121 prescription drugs that were being used for cancer treatment, 90 of them are known as plant-based drugs [46]. Plant extracts that are rich in phenolic compounds, especially in flavonoids, have been proven to be strong candidates in the treatment of cancer, and they are considered as new horizons for cancer therapeutics. The conclusion reached by a variety of scientists is, there is a positive correlation between flavonoid intake and reduced incidence of cancer. For instance, they were found to be effective in the treatment of prostate cancer, which is one of the major medical burdens, being the most common cancer in males in Europe and North America, via modulating the apoptotic pathway . On that account, flavonoid-polyphenol anticancer agents including In the past years, a relatively large number of studies were devoted to different aspects of different biological activities, especially the cytotoxicity of triterpenoids. Therefore, there are numerous studies about the cytotoxicity of triterpenoids with promising results. Notwithstanding the fact that most studies were carried out on cell lines from one to five, which can be considered as a narrow range of cell lines, some studies investigated their cytotoxic activity on a wide panel of them, even up to 59 cell lines and they provided evidence for their cytotoxicity [49]. As mentioned above, the obtained results of this study suggest that the FM extract exhibited higher cytotoxic activity than the other extracts , and this significant difference in activity seems to be due to the presence of a certain compound, which is quillaic acid, a triterpenoid. Therefore, the results indicate that the anticancer activity of the extract could be related to quillaic acid.Hypericum species is substantially increased because there are studies that give information about investigations on cytotoxic activities of several Hypericum species. For instance, it is known that H. pamphylicum showed no cytotoxic activity against human cervical cancer (HeLa and NRK-52E) cell lines [50]. In another study, the cytotoxic effect against A549 and PC3 human lung cancer cell lines demonstrated by H. adenotrichum and H. olympicum was shown using the Comet assay. It was also reported that H. adenotrichum exhibited stronger cytotoxic activity than H. olympicum in a dose-dependent manner [51]. H. japonicum was also found to have cytotoxic effects, the methanol extract of the plant caused cytotoxicity on human acute monocytic leukemia (THP-1) cell lines [52]. There is another study about the cytotoxicity of H. olympicum and it has also given evidence about its cytotoxic activity on MCF-7 and MDA-MB-231 breast cancer cell lines [53]. Moreover, H. triquetrifolium methanol extract was shown to have a cytotoxic effect on different cell types including large cell lung carcinoma (COR-L23), hepatocellular carcinoma (Hep G2), renal adenocarcinoma (ACHN), amelanotic melanoma C32 cell lines [54]. H. scabrum is also among Hypericum species with cytotoxic activity on HT-29 and Hep G2 cell lines [55]. H. androsaemum has also been evaluated for its cytotoxic properties and reportedly, the red and black berry infusions demonstrated cytotoxic effect on colon carcinoma cells, such as HCT15 and CO115 cell lines [6]. In another study, H. androsaemum ethanol extract was found to be the most effective cytotoxic among 16 different Hypericum species on human glioblastoma A1235 cell lines [56]. Furthermore, a strong cytotoxic effect on NIH/3T3 murine fibroblasts was revealed by a study conducted in 2018 [34]. Over and above that, it was found to exhibit a cytotoxic effect on HeLa, Hep G2, MCF-7 (breast adenocarcinoma), and NCI-H460  cell lines [15]. To sum up, it would be accurate to say that the results of this study are in accordance with previous studies.On the other hand, it can be said that the understanding of anticancer properties with Apoptosis is a term that was first used in 1972 by scientists to describe a mode of cell death regulated by endogenously driven mechanisms, and a characteristic morphology [57]. One of the most important advances in basic cancer research was the discovery that apoptosis has a profound effect on the malignant phenotype, and it has been reported that most cytotoxic agents induce apoptosis [58].H. androsaemum has been one of the candidates. Liver cancer is another most frequently occurring cancer in the world, being the second most common cause of cancer mortality [59]. It has been shown by the previous studies that H. androsaemum extracts induce apoptosis in human colorectal cells [60]. Human lung carcinoma A549, melanoma SK-Mel-2, and mouse skin melanoma B16F1 cells [61]. Prostate cancer is the fifth most common cause of cancer death, and the most commonly diagnosed cancer in men globally, which is interestingly more common in the developed countries [48]. In the search of new natural products as agents against this disease, being traditionally used against prostate problems and quite rich in terms of flavonoids, In this current study, the apoptosis assay was performed at different concentrations in the range of 12.5–200 µg/mL for the extracts. Treatment of 12.5, 25, and 50 µg/mL doses of FM on PC-3 cell lines, total apoptotic cells were detected as 3.29%, 6.69%, and 64.75%, respectively. Following the application of 12.5 and 25 μg/mL doses of FM on Hep G2 cells, 8.48% and 27.64% total apoptotic cells were observed, respectively. When 100 and 200 μg/mL of LM were applied on PC-3 cells, the apoptotic cell death was detected as 8.23% and 10.54%, respectively. 10.56% and 17.39% total apoptotic cells were determined after the application of 100 and 200 μg/mL of LM on Hep G2 cells, respectively. On the other hand, the dichloromethane extracts did not induce apoptosis on both cell lines. Considering the results of the Annexin V assay , FM extract showed remarkable selective apoptotic induction on PC-3 cells at 50 μg/mL concentration with nearly 65%.Evaluating the chemical compounds of the FM extract compared to other studied extracts, it may be observed that the FM was the richest in terms of quillaic acid (630 ± 18.9 μg/g extract). As mentioned before, quillaic acid is a triterpenoid, and there is evidence about its apoptosis induction potential. In particular, in a study that was carried out to investigate its anticancer potential, it has been reported that quillaic acid was effective on the induction of apoptosis in human colon cancer cells [62], human Jurkat T lymphocytes [63], and gastric cancer AGS cells [64]. Furthermore, in 2005, it was reported that quillaic acid glycosides were shown to induce many apoptotic manifestations in cancer cells and therefore cancer-related activities are among the most popular properties of triterpenoids in the last decade [65]. Another major compound of the FM extract is (-)-epigallocatechin gallate, which is also proven to induce apoptosis [66]. However, EGCG could also act as an antiapoptotic agent in higher concentrations [67]. This can explain the lesser apoptotic activity of the LM extract, which stood out as the richest in terms of EGCG (3035 ± 81.94 μg/g extract). Hypericum species on different cell lines. For example, H. ascyron demonstrated apoptotic induction on HeLa cells [68]. H. japonicum ethyl acetate extract induced apoptosis on mouse H22 liver cancer cell lines in vivo [69]. H. adenotrichum and H. olympicum methanol extracts also induced apoptosis on human PC3 lung cancer cells [56]. Additionally, H. scabrum methanol extract and petroleum ether fraction were shown to reveal apoptosis induction capacity [55]. More importantly, H. androsaemum was also found effective in apoptosis induction in previous studies. It has been reported that aqueous extract of the leaves induced apoptosis on HCT115 and CO115 human colon carcinoma cell lines through MAPkinase and PI3K/Akt pathway .There are studies conducted by different scientists from all over the world investigating the apoptotic induction activity of various H. androsaemum to the best knowledge, and its apoptotic induction ability on Hep G2 and PC-3 cell lines, therefore it can be said that this could provide a new horizon for cancer therapeutics.Based on the results of this present study, which is the first report about the determination of quillaic acid in H. androsaemum is one of the most popular members of Hypericum species. A plethora of studies, most of which focus on the other pharmacological properties of them, but there are also a few that hint at their anticancer activity, heralded an unprecedented and widespread interest in these plants. In this study, aiming to contribute by providing new knowledge on the evaluation of antioxidant and anticancer properties of this ethnobotanically valuable plant with its phytochemicals substantiated by LC-HRMS, six extracts prepared from H. androsaemum from Turkey were investigated. Accordingly, LM was found as the richest extract among them with 23 different constituents in it. On top of that, it had the highest total phenolic content. Expectedly, LM was found to be the strongest antioxidant mainly due to its rich phenolic content. Regarding the anticancer properties, FM was found to be significantly the most effective on apoptosis induction on PC-3 cell lines, also the strongest cytotoxic on PC-3 and Hep G2 cell lines among the six studied extracts. The anticancer effect of this extract may be due to quillaic acid, which is a triterpenoid, and was shown to have significant apoptosis induction ability and cytotoxic effect on different cell lines by different studies. In drawing things to close, being traditionally used against several problems, the evidence provided with this study point to the fact that H. androsaemum could be a very promising plant in the prevention of cancer.Nurdan Yazıcı Bektas: investigation, methodology, reviewing, software. Ezgi Ersoy: writing-original draft, conceptualization, methodology. Mehmet Boga: investigation, conceptualization, methodology, supervision, writing-reviewing and editing. Tugce Boran: investigation. Ercan Cinar: investigation. Gul Ozhan: supervision. Ahmet Ceyhan Goren: supervision, validation. Esra Eroglu Ozkan: conceptualization, methodology, visualization, writing-reviewing, supervision and editing.


It was not known whether this observed reduction of inflammation after VNS in intestinal inflammation was mediated by direct innervation of the gut or if the signals are relayed through the spleen. To investigate this, we studied the VNS effect on the small bowel lesions of splenectomized rats and splenic nerve stimulation (SNS) in intact rats. We observed that VNS reduced small bowel inflammation also in splenectomized rats but SNS alone failed to significantly reduce small bowel lesion area. Interestingly, VNS significantly reduced small bowel lesion area for 48 h when indomethacin administration was delayed. Thus, 1 min of electrical activation of the vagus nerve reduced indomethacin-induced intestinal lesion area by a spleen-independent mechanism. The surprisingly long-lasting and spleen-independent effect of VNS on the intestinal response to indomethacin challenge has important implications on our understanding of neural control of intestinal inflammation and its potential translation to improved therapies for IBD.Crohn’s disease is a chronic, idiopathic condition characterized by intestinal inflammation and debilitating gastrointestinal symptomatology. Previous studies of inflammatory bowel disease (IBD), primarily in colitis, have shown reduced inflammation after electrical or pharmacological activation of the vagus nerve, but the scope and kinetics of this effect are incompletely understood. To investigate this, we studied the effect of electrical vagus nerve stimulation (VNS) in a rat model of indomethacin-induced small intestinal inflammation. 1 min of VNS significantly reduced small bowel total inflammatory lesion area [(mean ± SEM) sham: 124 ± 14 mm Crohn’s disease and ulcerative colitis are two debilitating inflammatory bowel diseases (IBD) characterized by abdominal pain, weight loss, and frequent bowel movements. The hallmarks of Crohn’s disease are the involvement of the small bowel and the transmural inflammation in contrast to ulcerative colitis, which is limited to the colon and presents as a more superficial intestinal inflammation with bloody stools. IBD often affects patients relatively early in life, commonly with onset between 15 and 30 years of age . Many ofinflammatory reflex is an example of a neural circuit that involves signaling in the vagus nerve that regulates both organ-specific and systemic immune activity (+)-expressing T cells, and alpha7 nicotinic acetylcholine receptors (α7 nAChR) on innate immune cells are essential for inhibition of systemic release of TNF-α in inflammation .ad libitum to Harlan Teklad Rodent Chow  and fresh municipal tap water. This study was approved by FIMR’s Institutional Animal Care and Use Committee.Male Sprague Dawley rats (6–8 weeks old) were obtained from Taconic Farms, Inc.,  and the study was conducted at The Feinstein Institutes for Medical Research . Animal care including room, cage and equipment sanitation conformed to the guidelines cited in the Guide for the Care and Use of Laboratory Animals, and the applicable standard operating procedures of FIMR. Animals were housed in a laboratory environment with temperatures ranging between 19.5 and 24.5°C and relative humidity between 30 and 70%. Automatic timers provided 12 h of light and 12 h of dark. Animals were allowed access The surgery and method used for VNS has been previously described . The ratStimulator -driven charge-balanced biphasic pulses were generated using a bipolar current source and were capacitively isolated with > 1 uF ceramic capacitors on both electrode outputs. Rats were stimulated with a pulse waveform amplitude of 1 mA, 200 μs pulse width, and 50 μs inter-pulse-interval, previously demonstrated to activate vagus nerve fibers , for 60 The surgery and method used for splenic nerve stimulation (SNS) has been previously described . The ratThe rats were anesthetized and secured in supine position. A bipolar cuff electrode was secured around the left carotid sheath as described above. The rats were gently repositioned to a right lateral decubitus position and the spleen exposed by a 3 cm incision on the left abdomen above the spleen. The major splenic blood vessels were isolated, ligated with 4–0 silk suture, cut distally to the suture, and the spleen removed. The incision was subsequently sutured in two layers with 4–0 braided silk, and electrical or sham VNS was performed as described above.2 asphyxiation 24 h post induction of enteropathy. Blood was collected via cardiac puncture and clotted at room temperature for 1 h. The intestine from the proximal jejunum to the cecum was removed, cleaned, and formalin-fixed. The distal jejunum was assessed for histopathology.We used an enteropathy model known to produce lesions in the small intestine . 0.5 h aFor analysis of inflammatory mediators in the intestine and serum, Evans blue was withheld from a set of animals. In these animals, a standardized 2.54 cm section of the region between the distal jejunum and proximal half of ilium was cleaned, snap frozen in liquid nitrogen, and homogenized in a tissue protein extraction reagent  containing a protease/phosphatase inhibitor cocktail . Lesion area in these rats was not quantitated.Fixed intestines were flat-mounted, photographed, and the total lesion area was quantitated by a blinded scorer using digital morphometry . Photomicrographs were acquired of fixed jejunal samples that were embedded in paraffin and stained with hematoxylin and eosin. Chlorination and peroxidation activity were determined using EnzChek Myeloperoxidase Activity Assay Kit  according to the manufacturer’s instructions. Intestinal IL-23 normalized to protein and serum HMGB1 concentrations were measured by western blot. Serum TNF was measured by ELISA . Concentrations of select other mediators of intestinal inflammation were measured by a quantitative multiplexed electrochemiluminescence assay .t-test or ANOVA with Bonferroni’s post hoc analysis. Hedges’ g was used to calculate effect size for Student’s t-test to measure magnitude of differences between groups1. Biomarker data was assumed to be normally distributed and Grubbs’ outlier test (α = 0.05) was used. Reduction from mean level of sham biomarkers in serum and tissue were analyzed with Student’s t-test. Data are shown as percentage of sham (mean ± SEM) unless otherwise specified and number of animals (n) are reported per group. p < 0.05 was considered significant. Statistical calculations were performed using the Prism 8 software .Differences in lesion areas between groups were analyzed by Student’s 2, VNS: 62 ± 14 mm2, p < 0.002; effect size = 1.0)  . Microsce = 1.0) .p = 0.02; effect size = 1.3] and chlorination activity  were significantly reduced in VNS-treated compared with sham animals.Myeloperoxidase (MPO) peroxidation activity, an established marker of neutrophil infiltration , was sigp = 0.003; effect size = 2.2; p = 0.0006; effect size = 2.3; p = 0.04; effect size = 0.9; p = 0.03; IL-1β sham: 100 ± 27, VNS: 31 ± 11, p = 0.03; IL-4 sham: 100 ± 34, VNS: 23 ± 11, p = 0.04; p = 0.4; KC sham: 100 ± 36, VNS: 51 ± 15 p = 0.1; TNF sham: 100 ± 28, VNS: 55 ± 16, p = 0.1; Serum TNF levels were significantly lower in VNS-treated rats compared with sham [(% of sham ± SEM) sham: 100 ± 16, VNS: 30 ± 8, 2, VNS: 15 ± 11 mm2, p = 0.02; effect size = 1.1; p = 0.01; effect size 1.2; 2 ± SEM) sham: 93 ± 13, SNS: 93 ± 22, p = 0.5; effect size = 0.01; p > 0.9; effect size 1.3; In certain contexts, the effects of VNS on cytokines and inflammation require the splenic nerve and an intact spleen . To dete2 ± SEM) sham: 124 ± 14, 24 h post-VNS: 62 ± 14, p = 0.02; vs. 48 h post-VNS: 49 ± 10, p = 0.03; 2 ± SEM) sham: 124 ± 14 vs. 72 h post-VNS: 130 ± 27, p > 0.9; vs. 96 h post-VNS: 108 ± 28, p > 0.9; In endotoxemia, a brief episode of stimulation, lasting seconds to minutes, has a sustained effect on the release of pro-inflammatory cytokines . To inveHere, we found that electrical stimulation of the vagus nerve reduced indomethacin-induced acute small bowel inflammation by a spleen-independent mechanism. Reduction of small bowel lesioning remained for 48 h after a single episode of VNS.The findings here that electrical VNS reduced small bowel inflammation align with previous studies that show that signals in the vagus nerve regulate levels of pro-inflammatory cytokines and inflammation in a wide range of organs and scenarios . ReductiThe mechanism for vagus nerve regulation of intestinal inflammation is not fully understood. The vagus nerve directly innervates the gastrointestinal tract and is a key conduit for the bi-directional communication between the enteric nervous system and the central nervous system . The maiAvailable data indicate that signals in the vagus nerve regulate gut inflammation by different routes, spleen-independent and spleen-dependent. One spleen-independent conduit involves the vagus nerve, efferent sympathetic nerves and myenteric efferent fibers that regulate macrophages in the intestinal wall . Evidenc+ T cells, capable of acetylcholine biosynthesis, are found in the innervated Peyer’s patches of the small intestine of rodents , Takeda, Ferring, Orion Pharma, Otsuka, Tillotts, Novartis, Pfizer, and Janssen, received research funding from AbbVie and MSD, and has been a former shareholder of Emune AB. These funders were not involved in the study design, data analyses, data interpretation, and the writing of the report. PO has received honoraria for lectures from Ferring and Janssen and is a shareholder of Emune AB. AC and PO were supported by MedTechLabs, Stockholm. These funders were not involved in the study design, data analyses, data interpretation, and the writing of the report.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A gas chromatography–mass spectrometry method for the determination of four polycyclic aromatic hydrocarbon markers in Chinese medicine raw material, extracts, and health food products was established; In spiked-recovery experiments, the average recovery was about 78.6–107.6% with a precision of 2.3–10.5%. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 μg/kg and 0.7 μg/kg, respectively. When the developed method was utilized to determine PAH4 contents in 12 locally available health food products, 3 samples contained over 10.0 μg/kg BaP, and 5 samples contained over 50.0 μg/kg PAH4. The European Union (EU) limits for BaP and PAH4 are 10 and 50.0 μg/kg, respectively; therefore, more attention must be drawn to the exposure risk of BaP and PAH4 in CMRMs, their extracts, and health food products. According to the risk assessment based on the Margin of Exposure (MOE) method, it is recognized that the products mentioned in this study pose a low risk.The four polycyclic aromatic hydrocarbon markers (PAH4) of benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP) are indicators showing polycyclic aromatic hydrocarbon (PAH) contamination levels in Chinese medicine raw materials (CMRMs), extracts and health food products; Samples of herbal medicine, herbal extracts, and food supplements were extracted with  Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of organic compounds that contain two or more fused aromatic rings composed of carbon and hydrogen atoms . PAHs arRhizoma coptidis products [The Chinese medicine raw materials (CMRMs) are the raw material of Chinese medicinal plants and herbs; the materials can be used as medicine after cooking or prepared to product by factory. The utilization of CMRMs, usually plants and herbs or their extracts, in everyday food is common in China, especially those CMRMs mentioned in the article “One Root of Medicine and Food” . To dateproducts  while otproducts ,15. Consn-propyl ethylenediamine (PSA), SDB (styrene-divinylbenzene copolymer), silica, aminopropyl, C18 SPE columns, and others [18 and PSA for dry fermented sausage [The SPE columns used for purification include Florisil, EUPAH SPE (styrene-divinylbenzene polymer), d others ,20,21. Ud others , or othed others , C18 and sausage , animal- sausage , salmon, sausage . Accordi12-PAH4 were measured. In the measurement range of 0.005 µg/mL to 0.01 µg/mL, the concentration ratio of PAH4 to 0.05 µg/mL internal standard D12- PAH4 was set to the abscissa x, and the peak area ratio of PAH4 to internal standard D12-PAH4 (response ratio) was set to the ordinate y to obtain a linear Equation. The monitoring ion and linear Equation of each compound are shown in GC–MS parameters and PAH analysis conditions utilized were those found in GB 5009.265 . The masn-hexane and acetone systems; however, some samples were difficult to process and did not disperse finely in the extraction solvent, leading to incomplete extraction. To address this clumping issue, anhydrous ethanol and water were firstly used to wet the CMRMs. After samples are fully wetted and dispersed, organic solvents can effectively extract the PAHs from the sample. In this study, anhydrous ethanol and hexane were employed for sample wetting and sample extraction, respectively.The extraction of PAHs from traditional CMRMs, using direct organic solvent extraction, required cyclohexane, ethyl acetate, and mixed Due to the various plant parts required in traditional Chinese medicines , the extracts of CMRMs , especia18 SPE column were used for PAHs purification from grains and low-water food samples. For high fat or oil samples, a Florisil SPE column was selected. In this study, a 2 g/12 mL Florisil column was used because it can effectively adsorb chlorophyll, lutein, and some of the interfering oils. The EUPAH SPE column can specifically adsorb PAHs and remove the interference of alkane and acidic compounds, thus enabling quantitative analysis. For the pigment-rich samples, it was necessary to decrease the sample load to avoid overloading the SPE column.In “National Food Safety Standard Determination of Polycyclic Aromatic Hydrocarbons in Foods”, a PSA column and a CPAHs are ubiquitous environmental pollutants, and external PAH contamination must be strictly controlled during the experimental process. Reagents, vessels, concentrators, and consumable laboratory supplies are the main sources of external contamination. To reduce the PAH4 blank background value, pollutants were removed from vessels using high-temperature baking at 300 °C or hexane washing. Reducing reagent consumption and the step-count can also effectively control the blank value. In this study, the blanks contained less PAH4 than the detection limit of the method, which effectively ensured the reliability of the results. The blank test mass chromatogram of the developed method is shown in Dendrobium candidum, Dendrobium candidum extract, and Dendrobium candidum-containing health food product (n = 6 for each group) for a recovery test. The accuracy and precision of the method are shown in Dendrobium candidum, Dendrobium candidum extract, and Dendrobium candidum-containing health food product, respectively. These results meet the criteria specified in EU regulation recovery between 50% and 120%. Therefore, when a 1.00 g sample is selected, the LOQ and LOD of the four PAHs compounds are 2.0 μg/kg and 0.7 μg/kg, respectively.Three concentrations of PAH4  were spiked into three sample groupings: unprocessed Rhodiola rosea extract and spirulina. The results showed that the spirulina contained more BaP and PH4 than the Rhodiola rosea extract , an estimate of the lowest dose (95% confidence level) that causes no more than a 10% cancer incidence in rodents, was used to obtain the MOE. Taking primary hepatocellular carcinoma as the toxicity effect endpoint to assess the risk of PAHs intake in the population, the BMDL10 of BaP and PAH4 were 70 µg/kg bw/d and 340 µg/kg bw/d, respectively.The MOE method is used to assess the risk characteristics for substances with genetic toxicity, which is published by the U.S. Environmental Protection Agency (U.S. EPA) and preferred by both the World Health Organization (WHO) and the European Food Safety Authority (EFSA) for the risk evaluation of carcinogens. Generally, it was assumed that a MOE of 10,000 or higher would be of low concern for public health and might be considered of low priority for risk management actions . The benHealth food products are usually recommended for consumption at 3 g/day. According to 12-PAH4  mixed internal standard solution (XA20950902CY PAH-Mix9) were purchased from Dr. Ehrenstorfer GmbH Corp .The 10.0 μg/mL PAH4  mixed standard solution (LA20950183CY PAH-Mix183) and the 100 μg/mL D18 SPE columns were purchased from Kang Yuan Technology Co., LTD. . Acetone, isooctane, ethyl acetate, and n-hexane of chromatography grade were purchased from Merck, USA. Analytical grade anhydrous ethanol, anhydrous ether, petroleum ether, methylene chloride, concentrated aqueous ammonia, potassium hydroxide, and anhydrous sodium sulfate were purchased from Sinopharm . Ultrapure water was obtained using a Milli-Q water polishing system. To reduce the blank background value, vessels, glass tubes, and containers were baked at 300 °C or washed with hexane. Total of 12 representative CMRM extracts and health food samples, including 2 Dendrobium candidum extract (contain polysaccharides), 1 Astragalus mongholicus extract (contain flavonoids), 1 Codonopsis pilosula extract (contain glycosides), 1 fructus alpiniae oxyphyllae extract , 1 Gastrodia elata extract (contain glycosides), 1 Ganoderma lucidum extract (contain terpenoids), 2 Dendrobium candidum-containing health foods, 2 Ganoderma lucidum-containing health foods, and 1 Codonopsis pilosula-containing health food as well as Rhodiola rosea extract, Lotus leaf extract, and spirulina were purchased from a local market in Hangzhou, China.Florisil (2 g/12 mL), EUPAH SPE , n-propyl ethylenediamine (PSA), and CAn Agilent 6890–5973 gas chromatography–mass spectrometer (GC–MS), equipped with a DB-PAHEU capillary column (20 m × 0.18 mm inner diameter × 0.14 μm film thickness) part number 121–9627, was used for sample analysis. The injection port temperature was 280 °C.The column temperature was initially set to 80 °C for 2 min, then increased to 250 °C in 10 °C/min intervals, maintained at 250 °C for 2 min, and finally set to 320 °C in 8 °C/min intervals and maintained at 320 °C for 5 min. High purity helium (99.999%) was used as the carrier gas. Splitless sample injection (1 μL) was employed, and the sample was ionized by an electron ionization source at 70 eV and 230 °C, with a 250 V electron multiplier and a 280 °C transmission line temperature. The monitoring ions are listed in Additional equipment was obtained from their respective manufacturers as follows: multisample vortex oscillator ; traditional Chinese medicine ultrafine pulverizer ; high-speed centrifuge ; solid-phase extraction manifold , nitrogen blowing instrument ; analytical balance with a sensitivity of 0.01 g ; and ultrasonic cleaner .12-PAH4 mixed internal standard stock solution was prepared by diluting 100.0 μg/mL D12-PAH4 mixed internal standard solution (0.02 mL) with acetone (10.0 mL). The D12-PAH4 stock solution was stored in the dark at 4 °C. Five working solutions containing 1.0, 2.0, 5.0, 10.0, or 20 ng PAH4 and 10 ng D12-PAH4 were prepared for the GC–MS analysis as internal standards by aliquoting 5 μL, 10 μL, 25 μL, 50 μL, or 100 μL of the 0.2 μg/mL PAH4 standard solution to five volumetric flasks already containing 50 μL of 0.2 μg/mL D12-PAH4 mixed standard solution; the final working solutions containing 0.0025, 0.005, 0.01, 0.025, 0.050 μg/mL PAH4 and 0.025 μg/mL D12-PAH4, respectively. All the stock solutions were stored in the dark at 4 °C.The 0.2 μg/mL PAH4 mixed standard stock solution was prepared by diluting 10.0 μg/mL PAH4 mixed standard solution (0.2 mL) with acetone (9.8 mL). The 0.2 μg/mL DSolid CMRM samples were crushed using a superfine pulverizer and stored in 50 mL plastic centrifuge tubes until required. Chinese medicine extracts were directly subject to the extraction procedure without additional processing.12-PAH4 mixed internal standard solution as well as 10 mL anhydrous ethanol were added. The mixture was subject to ultrasonic treatment for 5 min, and then 10 mL of water was added. The resulting mixture was vortexed for 1 min and ultrasonicated for another 5 min. Finally, 10 mL of n-hexane was slowly added, and the mixture was vortexed for an additional 5 min. After centrifugation at 9000× g for 3 min, the organic n-hexane phase was collected for further purification.The PAH extraction procedure was as follows: 2.00 g of sample was placed in a 50 mL centrifuge tube, and 50 μL of 0.2 μg/mL DBefore purification, the Lotus leaf extract without purification, purified by the Florisil column and by the Florisil and EUPAH SPE columns, were analyzed for comparison of the purification method.n-hexane (5 mL) was passed through to activate the SPE column. The n-hexane sample extract about 10 mL collected previously was directly loaded on the activated column, and the eluent was collected in a 15 mL centrifuge tube for PAHs collection. Then, in order to fully collect PAHs, another 5 mL of methylene chloride n-hexane solution  was eluted through the column, and the eluent was collected in the same 15 mL tube. The collected combination liquid (about 15 mL) was concentrated under a stream of N2 at 50 °C and reconstituted in 2 mL n-hexane. The EUPAH SPE column was first activated with 3 mL methylene chloride and 3 mL n-hexane. Thereafter, the reconstituted 2 mL n-hexane sample was loaded to the column and eluted at 1.0 mL/min. n-Hexane (4 mL) was passed through to remove the impurities, then 5 mL of methylene chloride: ethyl acetate solution  was eluted through the column. The eluent was collected in a 10 mL glass tube and concentrated under a stream of N2 at 40 °C. The obtained residue was reconstituted in 200 μL acetone: isooctane  and transferred to a sample vial for GC–MS analysis. Blank test controls were conducted together with the samples for analyses.The SPE purification procedure  was as fDendrobium candidum, Dendrobium candidum extract, and Dendrobium candidum-containing health food products (n = 6 for each group) for a recovery test. The limit of detection (LOD) at a signal-to-noise ratio (S/N) of 3 and the limit of quantification (LOQ) at an S/N of 10 were determined from the S/N ratio check function on low PAH-containing samples in MSD ChemStation software (Agilent Technologies).Concentrations of PAH4  were spiked into unprocessed The experimental data are represented as mean ± standard deviation (SD) of three independent replicates. The calculation of SD and linear equations were conducted with the SPSS 20.0 software package .The MOE value is the ratio between the reference dose calculated by the dose–response relation curve and the human dietary exposure estimated by the mathematical model. In order to estimate MOE, the chronic daily intake (CDI) of PAHs using the BaP equivalent concentration should be calculated according to the Environmental Protection Agency (EPA) . The rat10 is Benchmark Dose Level 10, which means the lowest dose that causes adverse reactions in 10% of the population, the BMDL10 value of BaP and PAH4 were of 70 µg/kg bw/d, and 340 µg/kg bw/d, respectively [The MOE calculation formula is as follows:ectively .n-hexane and sequentially cleaned up by SPE on Florisil and then a EUPAH SPE column.An isotope internal standard dilution combined with a GC–MS detection method was developed to determine the content of PAH4 in CMRM-containing extracts and health food products. CMRM samples were extracted with In spiked samples containing 2.0 μg/kg, 10.0 μg/kg, or 20.0 μg/kg PAH4, the average recovery was 78.6–107.6% with a precision of 2.3–10.5%, which can meet the detection requirement limits for BaP (10.0 μg/kg) and PAH4 (50.0 μg/kg) in herbal medicine, and food supplements containing botanicals according to EU regulation 2015/1933. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 μg/kg and 0.7 μg/kg, respectively. This method for PAH4 analysis was applied to 12 commercially available CMRM-containing food products. Using the tolerance limits established by EU regulation 2015/1933, three samples exceeded the tolerance limit for BaP content, and five samples exceeded the tolerance limit for total PAH4 content. Finally, the method of exposure limits was utilized to assess the PAHs exposure risk in the tested samples; it is recognized that the products mentioned in this study are of low risk. To encourage the development of better processing methods in CMRM-containing health product production that can remove PAHs and decrease PAH contamination, regulations and limits for safe levels of PAH in CMRM-containing products should be enacted.


Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.Nitrous oxide (N NirK is a homotrimer with a predicted molecular mass of about 35 kDa per monomer that contains type 1 and type 2 Cu centres. The catalytic subunit of cNor, NorB, contains heme b and a binuclear active centre (heme b3 and FeB). NorC is a membrane-anchored protein (16 kDa) that contains heme c. Finally, the catalytic subunit of Nos, NosZ (120–160 kDa), is a homodimer Cu-containing enzyme with two distinct Cu centres (CuA and CuZ). nditions . DenitriC operon , a Cu-coirK gene , a cytocD operon , and a CLX genes . Nap is B. diazoefficiens requires both oxygen limitation and the presence of nitrate or a derived nitrogen oxide (NOx), this control being mediated by the FixLJ-FixK2-NnrR regulatory cascade 2 cofactor and a [4Fe-4S] centre  centre ,19. Ther] centre . Under Ce medium ,38. ContnirK gene expression, protein levels decreased under Cu-L conditions compared with Cu-S. However, this effect was more pronounced in the periplasm than in the cytosolic fraction. In contrast, in Cu-H grown cells, NirK was expressed more in the periplasm than in the cytoplasm. The sequence of B. diazoefficiens NirK shows the sequence 3TRRAALI9 in the N-terminal region ). Next, 50 µL of that solution was added to each reaction tube. After incubation for 10–20 min at 30 °C, the reaction was stopped by vigorous shaking until the blue colour disappeared from the samples. Controls were prepared by vigorously shaking immediately after the addition of dithionite. MV+-NR and MV+-NIR activities were expressed as nmol NO2− produced or consumed·(mg protein·min)−1. Two biological replicates for each Cu condition were used. Three sets of 250-mL flasks were inoculated at an initial OD. (2003) . The rea600 of 0.05 in BVMN and incubated at 30 °C and 170 rpm under microoxic conditions for 3 days. Cells were then harvested and washed in a similar way as for the MV+-NR and MV+-NIR activity assays and resuspended in 1.5 mL of the same buffer. NO consumption rates were determined using a 2-mm ISONOP NO electrode APOLLO 4000® , following Cabrera et al. (2016) [−1 of Aspergillus niger glucose oxidase and 250 units mL−1 of bovine liver catalase. Once a steady base line was obtained, 50 µL of a saturated NO solution of 1.91 mM (at 20 °C) [−1. Two biological replicates for each Cu condition were used. A set of 500-mL flasks was inoculated at an initial OD. (2016) . The reat 20 °C)  was adde600 of 0.05 in BVMN and incubated at 30 °C and 170 rpm under microoxic conditions for 3 days. Cells were then harvested, washed in a similar way as for the MV+-NR and MV+-NIR activity assays, and resuspended in 1.5 mL of the same buffer. N2O consumption rates were measured as essentially described by Jiménez-Leiva et al. (2019) [v/v) O2 and 98% (v/v) N2 and then, 500 µL of 2% (v/v) N2O from a gas mixture with 98% (v/v) N2 were injected in each tube. Next, all the tubes were incubated at 30 °C with agitation for 30 min to ensure equilibrium between gas and liquid phases. After this incubation, a volume of cell suspension corresponding to 0.3–0.4 mg protein was injected manually into each reaction tube. When reaction tubes were set up, 1-mL aliquots were taken from the headspace of each tube at the start of the reaction and four hours after incubation at 30 °C. N2O measurements and concentration calculations were performed as described in 2OR activity was expressed as nmol N2O consumed·(mg protein·h)−1. Two biological replicates for each Cu condition were used. Three sets of 250-mL flasks were inoculated at an initial OD. (2019) . The ass600 of 0.05 in BVMN and incubated under microoxic conditions at 30 °C and 170 rpm for 3 days. Cells were then collected at 8000× g for 8 min at 4 °C. After sedimentation, cells were washed once with a solution of 150 mM KCl in Tris-HCl 50 mM . Washed cells were finally resuspended in 1.5 mL of fractionation buffer, consisting of wash buffer containing 1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). Cell fractionation, membrane isolation, sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and heme-staining were performed as essentially described by Delgado et al. (2003) [g for 10 min at 4 °C to remove cell debris. These extracts were immediately centrifuged at 140,000× g for 45 min at 4 °C using a tabletop ultracentrifuge Optima Max . Cell membranes were resuspended in fractionation buffer and a volume equivalent to 30 µg of protein was mixed with freshly prepared SDS loading dye 6x , adding distilled water up to a final volume of 12 µL. Samples prepared were consecutively loaded without boiling onto 4%-stacking and 14%-resolving-SDS-PAGE polyacrylamide gels, resolved and transferred to a nitrocellulose membrane using Trans-Blot Turbo System (Bio-Rad). Heme-dependent peroxidase activity was detected using the AmershamTM ECL SelectTM Western Blotting Detection Reagent Kit  and chemiluminescence signals were revealed with a ChemiDoc XRS+ System. Images were analysed operating with the Quantity One and Image LabTM softwares (Bio-Rad). Two biological replicates for each Cu condition were analysed. A set of 500-mL flasks was inoculated at an initial OD. (2003) . Cells wB. diazoefficiens 110spc4 at an initial OD600 of 0.05 in BVMN and incubated under microoxic conditions at 30 °C and 170 rpm for 3 days. Additionally, napA, nirK and nosZ mutant strains were used as the negative control in the experiments, being inoculated at an initial OD600 of 0.2, 0.2 and 0.05, respectively, in order to collect a sufficient cell mass of each strain. Cells were then harvested at 3740× g for 15 min at 4 °C. Periplasmic proteins were isolated following a protocol modified from Felgate et al. (2012) [−1 lysozyme was added and the mix was incubated at 30 °C for 2 h. Subsequently, cells were centrifuged at 12,000× g for 15 min to separate periplasmic proteins from spheroplasts. The periplasmic fraction was then consecutively concentrated up to 4 mL and 250 µL using Amicon® Ultra tubes of 3 kDa and 10 kDa molecular weight cut-off (MWCO), respectively. After centrifugation of the tubes at 7500× g for 20 min, periplasmic proteins were stored at −20 °C until use. The subsequent spheroplasts fractionation was performed as described in g for 5 min, and were then loaded onto 4%-stacking and 14%-resolving-SDS-PAGE polyacrylamide gels, resolved and transferred to a nitrocellulose membrane using Trans-Blot Turbo System (Bio-Rad). NapA immunoblot analyses were performed according to Delgado et al., 2003 [P. pantotrophus NapA at a dilution rate of 1:750 [B. diazoefficiens  at a dilution rate of 1:1000, while the secondary antibody utilised was a horseradish peroxidase (HRP)-conjugated anti-rabbit one (Sigma-Aldrich) at a dilution rate of 1:3500. NosZ immunoblotting was carried out as previously described [P. denitrificans at a dilution rate of 1:1000 together with a HRP-donkey anti-sheep secondary antibody (Sigma-Aldrich) at a dilution rate of 1:3500. Detection of peroxidase activity and image processing were performed as described in A set of 500-mL flasks was inoculated with . (2012) . Cells wl., 2003  using anescribed ,27,34, upost-hoc Tukey HSD test at p ≤ 0.05 with SPSS software was performed. The total number of replicates is given in each figure. Data were checked for normal distribution according to Kolmogorov–Smirnov and Shapiro–Wilk tests. We then performed inferential statistics to test null hypotheses applying a parametric ANOVA for unpaired treatments. Next, a B. diazoefficiens. Taken together, our results suggest that Cu not only affects enzymatic activity of Nap, NirK, cNor or Nos enzymes, but also may act as an essential factor in the regulation of the denitrification gene expression, as well as mediate the transport and maturation of the Cu-dependent NirK and NosZ enzymes, respectively. Therefore, Cu could be involved in the denitrification regulatory network and not only acts as a mere enzymatic cofactor of the Cu-dependent enzymes, but also as an important regulatory signal of this process. The main goal of the present work was to investigate the influence of Cu on denitrification in the soybean endosymbiont


The therapeutic approach is crucial to prostate cancer prognosis. We describe treatments and outcomes for a Spanish cohort of patients with prostate cancer during the first 12 months after diagnosis and identify the factors that influenced the treatment they received.This multicenter prospective cohort study included patients with prostate cancer followed up for 12 months after diagnosis. Treatment was stratified by factors such as hospital, age group (<70 and ≥70 years), and D’Amico cancer risk classification. The outcomes were Eastern Cooperative Oncology Group (ECOG) performance status, adverse events (AEs), and mortality. The patient characteristics associated with the different treatment modalities were analyzed using multivariate logistic regression.P < .001); 230 (48.9%) patients had at least 1 AE, and 12 (2.6%) patients died.We included 470 men from 7 Spanish tertiary hospitals (mean (standard deviation) age 67.8 (7.6) years), 373 (79.4%) of which received treatment  as follows: surgery ; radiotherapy (RT) ; and hormone therapy (HT) . The remaining patients (n = 97) were allocated to no treatment, that is, watchful waiting (14.0%) or active surveillance (5.7%). HT was the most frequently administered treatment during follow-up and RT plus HT was the most common therapeutic combination. Surgery was more frequent in patients aged <70, with lower histologic tumor grades, Gleason scores <7, and lower prostate-specific antigen levels; while RT was more frequent in patients aged ≥70 with histologic tumor grade 4, and higher ECOG scores. HT was more frequent in patients aged ≥70, with histologic tumor grades 3 to 4, Gleason score ≥8, ECOG ≥1, and higher prostate-specific antigen levels. The number of fully active patients (ECOG score 0) decreased significantly during follow-up, from 75.3% at diagnosis to 65.1% at 12 months (Surgery or RT were the main curative options. A fifth of the patients received no treatment. Palliative HT was more frequently administered to older patients with higher tumor grades and higher Gleason scores. Close to half of the patients experienced an AE related to their treatment. Prognosis depends not only on patient characteristics, but also on the ability of healthcare systems to timely detect and treat patients with prostate cancer.Prostate cancer is the most common cancer in men in both Western Europe  and in Spain, with an estimated incidence of 171.4 and 147.9 patients per 100,000 men per year, respectively.,3 it is important to conduct studies that focus on how patients are treated in the real world and the association between their characteristics and the specific treatment modalities. Some initiatives have been recently published in this regard, such as an international prospective cohort study that reported the characteristics of cancer patients and toxicity related to radiotherapy (RT). Another international initiative has been developed in Asia in a cohort of patients with advanced prostate cancer.Once diagnosed, patients are generally treated according to their performance status, clinical cancer stage, tumor characteristics, and—ideally—individual values and preferences. In addition to making recommendations to guide clinical management of prostate cancer patients, The current article reports the results of the first 12-month follow-up after diagnosis of this cohort of patients; we describe the treatments and outcomes, and identify the factors that influenced the therapy received during this follow-up period.We also published the baseline clinical characteristics, diagnoses, and factors affecting patient care intervals for a prospective cohort study of the incidence of prostate cancer in Spain.2We conducted a multicenter observational cohort study on patients with prostate cancer attending 7 tertiary hospitals in Spain: Hospital Universitario 12 de Octubre ; Hospital Universitario Ramón y Cajal  in Madrid; Hospital Universitario Donostia  in Donostia-San Sebastián; Hospital General Universitario de Valencia  in Valencia; Hospital Universitario Virgen de las Nieves  in Granada; Fundació Puigvert-Hospital de la Santa Creu i Sant Pau  (co-ordinating center); and Hospital del Mar  in Barcelona. The protocol was approved by all centers’ Research Ethics Committees.Patients with prostate cancer were consecutively enrolled from October 2010 to September 2011. Inclusion criteria were patients with histologically proven and newly diagnosed prostate cancer at any stage of the disease, who were being treated at any of the 7 participating hospitals, and who had provided their informed consent.; prostate-specific antigen (PSA) value at diagnosis; total Gleason score; clinical cancer stage (I–IV); tumor stage (T(Tumor size) N (lymph Nodes ) M (Metastasis) Classification of Malignant Tumors Staging System); intervention, that is surgery, RT, hormone therapy (HT), or watchful waiting/active surveillance ; Eastern Cooperative Oncology Group (ECOG) performance status score; adverse events (AEs) based in the Common Terminology Criteria for Adverse Events, and mortality during follow-up.We collected the following data: age; body mass index; World Health Organization (WHO) histologic tumor grade (1–4) groups: low risk ; intermediate risk ; and high risk . We stratified the analysis by age (<70 years and ≥70 years), initial treatment, D’Amico risk, hospital, and ECOG score.Using TNM tumor stages, PSA values, and Gleason scores, we classified patients into the following D’Amico risk and the fact that clinical treatment guideline recommendations depend on whether the patient's life expectancy is more or less than 10 years at the time of diagnosis.We established the cut-off age at 70 years based on a national male life expectancy of 80 years,For the descriptive analyses, we used relative frequencies for categorical variables, and either mean and standard deviation or median and interquartile range for continuous variables, depending on the skewness of the data distribution.P value was greater than .05, excluding first the variable with higher P value. The potential predictors included in the maximal model were selected based on clinical plausibility, and included age, body mass index, WHO histological tumor grade, TNM tumor stage, total Gleason score, and PSA value at diagnosis. We report the odds ratio and the corresponding 95% confidence interval. We used the non-parametric Friedman test for repeated measures to estimate ECOG within-patient change across follow-up assessments . This analysis was performed on the overall cohort and stratified by received treatment modality .The proportion of missing values for each variable is reported. To study baseline patient characteristics associated with specific treatment modalities, we used 3 independent multivariate logistic regression models. Each model had a binary outcome measure representing whether a patient received as first-line treatment the modalities: surgery (model 1), RT (model 2), or hormonal therapy (model 3). We performed a backward elimination strategy to fit the most final parsimonious model containing the clinical and demographic factors significantly associated to the odds of receiving each one of the treatment alternatives. At each iteration, we excluded a variable from the model when its Data were analyzed using SPSS statistical software version 20.0  and Stata v12 software .3In total, we recruited 470 patients: 451 (96.0%) of them completed the 12-month follow-up, 12 (2.6%) died, and 7 dropped out of the study  and 3/4 . Most patients (62.5%) had T1b-T2b tumors. Over half of the patients (55.1%) scored ≤6 on the Gleason scale, 26.8% scored 7, and 15.7% scored ≥8  were allocated to watchful waiting and 373 (79.4%) were treated as follows: surgery n = 163; 34.7%); RT ; and HT   received a combination of treatments, most frequently RT plus HT n = 72; 15.3%) ; and among those receiving HT, 18.2%, 20.2%, and 61.8% had low, intermediate, and high risk, respectively (P < .001). In this age group there was no difference in the odds of patients receiving RT (P = .098) or being assigned to watchful waiting (0.748) (Table P = .044); among those receiving RT, 36.4%, 28.8%, and 34.8% had low, intermediate, and high risk, respectively (P = .030); and among those receiving HT, 17.3%, 18.5%, and 64.2% had low, intermediate, and high risk, respectively (P < .001). In this age group, there was no difference in the odds of patients being assigned to watchful waiting (0.756) , F (63.6%), and G (50.7%); RT for hospitals A (22.9%) and B (38.5%); and HT for hospitals D (42.3%) and E (50.0%). HT was the most prescribed treatment in the 12-month follow-up, although its use ranged from 5.5% in hospital B to 50% in hospital E.The multivariate logistic regression analysis Table  revealedA total of 230 AEs were reported, with 48.9% of patients experiencing at least one Table . By inte4 HT was also administered in one third of patients as palliative treatment. During the 12-month follow-up, HT was the most frequent treatment and RT plus HT was the most frequent treatment combination.This prospective cohort study describes healthcare practices at 7 Spanish hospitals for newly diagnosed prostate cancer patients, focusing on primary therapy and patient-relevant clinical outcomes observed within the first 12 months since diagnosis. The majority of the 470 included patients were under 70 years old and 81% had localized prostate cancer. Primarily, surgery or RT were the initial treatment modalities for 1 in 3 patients, as per guideline recommendations. in a 6-month follow-up study. Our watchful waiting percentage decreased to 14% at 12-month follow-up, as the remaining 5.7% were moved to active surveillance. In contrast, Hoffman et al described that more patients were in active surveillance at 18-month follow-up, while fewer than 2% remained in watchful waiting.One fifth of patients was assigned to watchful waiting after diagnosis, a similar rate to that reported by Hoffman et alIn our study a high proportion of patients with T1b-T2b tumor stage underwent surgery and received either RT or HT. A lower-than-expected proportion of patients with T2c-T4 tumor stage received RT or HT. surgery was more frequent in younger patients (<70 years) with low or intermediate cancer risk (D’Amico classification), whereas HT was more frequent in high-risk cases and older patients (≥70 years). However, RT administration was only influenced significantly by the risk classification in older patients and those with higher histological grade.In agreement with guideline recommendations, However, we found that certain factors influenced decision-making regarding different therapeutic options: patients who underwent surgery were younger and had better prognostic factors ; patients treated with RT had higher histological tumor grades and poorer ECOG scores; and lastly, patients who received palliative HT were older and had poorer prognostic factors .According to a recently published clinical trial, there is no single best treatment option for localized prostate cancer, as overall survival is similar for patients undergoing radical prostatectomy, RT, or active surveillance.,5 and our study only focused on incident prostate cancer patients. One international multicenter cohort study focused on patients receiving RT. Twenty-seven percent of included patients underwent a prostatectomy before RT, whereas in our study the proportion was only 1.5%. The proportion of patients receiving RT and HT also differed between studies—69% and 15%, respectively. Another international multicenter cohort study included advanced prostate cancer, whereas our study mainly included localized prostate cancer.Other cohort studies have been published recently, but their results are not completely comparable to our study. These studies included prevalent and incident patients,While the Spanish National Health System is a public system providing universal coverage and free-of-charge treatments to patients, we found important differences in therapeutic choices between the 7 participating hospitals, with some preferring surgery, whereas others preferred RT or HT. This variability was probably related to patient characteristics and differing hospital criteria regarding treatments.,15 such as urinary incontinence and impotence for surgery, impotence and cystitis for RT, and hot flushes for HT.At baseline, 3 quarters of patients had a good performance status that worsened over the follow-up period. Only a small proportion of patients (2.6%) died during the first year, mainly due to cancer-related reasons. The most common AEs match those reported in previous studies,Regarding limitations, our study may be affected by potential information bias, given that our data were prospectively obtained from hospital records and participants. However, we consider this limitation of little actual relevance.A main strength of our study is that our patient sample is probably representative of the annual incident cases of patients diagnosed with prostate cancer in Spain, since they were recruited in 7 hospitals located in 5 different regions. In addition, the prospective nature of the study guarantees greater data consistency and accuracy, and so overcomes the typical shortcoming of retrospective data collection affecting similar studies carried out elsewhere. The relatively small number of patients lost to follow-up (4.2%) reinforces the validity of our results. A longer follow-up will undoubtedly be useful in further assessing the impact of diagnosis and therapy on prostate cancer patients.5Surgery and RT were the most common curative options used on initial diagnosis of prostate cancer. Watchful waiting was applied to 1 in 5 patients after diagnosis. Palliative HT was the most prescribed follow-up treatment. Surgery was more frequently indicated in younger patients with better prognostic factors. HT was more frequent in older patients, with more advanced tumor stages and higher Gleason scores. Around half of the patients experienced an AE related to the treatment. Performance status decreased steadily in the first year after diagnosis. The treatments administered by the participating hospitals varied widely.Ailish Maher and Andrea Cervera Alepuz revised the English in a version of this manuscript. Maria José Martinez Zapata is funded by a Miguel Servet research contract (CPII20/00023).EMPARO Study Group: Coordinating investigator: Xavier Bonfill Cosp .Project manager: María José Martínez Zapata .Clinical research assistants: Alborada Martínez ; Enrique Morales Olivera ; Esther Canovas, Laura Muñoz, Gemma Mas, René Acosta, Ekaterina Popova ; Irma Ospina ; María José Velázquez ; Tamara Ruiz Merlo ; Gael Combarros Herman, Judit Tirado Muñoz .Statistical analysis: Robin W.M. Vernooij ; Javier Zamora and Claudia Coscia Requena .Co-investigators: Barcelona, SpainAlbert Frances ; Carola Orrego Villagran, Rosa Suñol (Instituto Universitario Avedis Donabedian); Dimelza Osorio, Gemma Sancho Pardo, Ignasi Bolívar, José Pablo Maroto, María Jesús Quintana, Cristina Martin ; Ferran Algaba, Palou Redorta, Salvador Esquena (Fundació Puigvert); Jordi Bachs ; María José Martínez Zapata ; Montserrat Ferrer Fores, Stefanie Schmidt, Olatz Garin, Virginia Becerra Bachito, Yolanda Pardo .Bilbao, SpainAmaia Martínez Galarza, José Ignacio Pijoán Zubizarreta .Granada, SpainArmando Suárez Pacheco, Cesar García López, José Manuel Cozar Olmo ; Carmen Martínez, Daysy Chang Chan, María José Sánchez Pérez .Madrid, SpainAna Isabel Díaz Moratinos, Angel Montero Luis, Asunción Hervás, Carmen Vallejo Ocaña, Costantino Varona, Javier Burgos, Javier Zamora, Jose Alfredo Polo Rubio, Luis López-Fando Lavalle, Miguel Angel Jimenez Cidre, Muriel García, Alfonso, Nieves Plana Farras, Rosa Morera Lopez, Sonsoles Sancho Garcia, Victor Abraira, Victoria Gomez Dos Santos ; Agustín Gómez de la Cámara, Javier de la Cruz, Juan Passas Martínez, Humberto García Muñoz, María Ángeles Cabeza Rodríguez .San Sebastián, SpainIrune Ruiz Díaz, José Ignacio Emparanza, Juan Pablo Sanz Jaka .Valencia, SpainAgustín Llopis González, María Morales ; Carlos Camps, Cristina Caballero Díaz, Emilio Marqués Vidal, Francisco Sánchez Ballester, Joaquín Ulises Juan Escudero, Jorge Pastor Peidro, José López Torrecilla, María Macarena Ramos Campos, Miguel Martorell Cebollada .Conceptualization: Xavier Bonfill.Data curation: Maria Jose Martinez-Zapata.Formal analysis: Robin WM Vernooij, Víctor Abraira, Javier Zamora.Funding acquisition: Xavier Bonfill, Javier Zamora.Investigation: Xavier Bonfill, Maria Jose Martinez-Zapata, María José Sánchez, María Morales-Suárez-Varela, José Ignacio Emparanza, Montse Ferrer, José Ignacio Pijoan, Joan Palou.Methodology: Xavier Bonfill, Maria Jose Martinez-Zapata, Eva Madrid, Víctor Abraira, Javier Zamora.Supervision: Xavier Bonfill, Maria Jose Martinez-Zapata.Validation: Maria Jose Martinez-Zapata, Robin WM Vernooij.Writing – original draft: Xavier Bonfill, Maria Jose Martinez-Zapata, Robin WM Vernooij, María Morales-Suárez-Varela, Eva Madrid, Javier Zamora.Writing – review & editing: Xavier Bonfill, Maria Jose Martinez-Zapata, Robin WM Vernooij, María José Sánchez, María Morales-Suárez-Varela, José Ignacio Emparanza, Montse Ferrer, José Ignacio Pijoan, Joan Palou, Eva Madrid, Víctor Abraira, Javier Zamora.