Coronavirus disease 2019 (Covid‐19) might present neurological symptoms. We aimed to evaluate the frequency of them at the moment of emergency department (ED) visit and their impact in the prognosis.Retrospective cohort study including all consecutive hospitalized cases between March 8th and April 11th, 2020. Covid‐19 diagnosis was confirmed by polymerase chain reaction test and/or serology. We compared, in patients with and without neurological symptoms on admission, demographic, clinical presentation, and frequency and type of abnormal laboratory values. We analyzed the variables that were associated with in‐hospital all‐cause mortality by Cox‐regression log‐rank test.p = .019). Only four (0.6%) cases had no typical Covid‐19 general symptoms, and only six (1.9%) had a normal laboratory results, for a sensitivity of 98.7% : 96.6%–99.6%) and 98.1% (95% CI: 95.7%–99.2%), respectively. In the multivariate Cox‐regression of mortality predictors, anosmia  and altered mental status  were significant.We included 576 hospitalized patients, 250 (43.3%) female, aged 67.2 years. At the moment of ED visit, 320 (55.6%) described neurological symptoms, including anosmia , myalgia , headache , and altered mental status . Neurological symptoms started the first symptomatic day in 198 (54.2%) cases. Patients with neurological symptoms presented later to the ED (7.9 versus. 6.6 days, Neurological symptoms were the most frequent extrapulmonary symptoms. They were present in half of the Covid‐19 patients at the time of the ED visit. Anosmia on admission was an independent predictor of lower in‐hospital mortality and altered mental status on admission predicted in‐hospital mortality. Half of the Covid‐19 patients described neurological symptoms at the moment of ED presentation. The presence of neurological symptoms at the moment of ED presentation was an independent predictor of mortality. Patients with altered mental status died more and earlier, and patients with anosmia had a lower risk of mortality. The study was done according to the strengthening to the reporting in observational studies in epidemiology (STROBE) guidelines  had confirmed Covid‐19 disease; 2) were hospitalized; and 3) were aged 18 or older. They were excluded if the hospitalization was not done from the emergency department or if there was not any available information about the hospitalization period. The sampling was nonprobabilistic, and all the consecutive confirmed cases were recruited. By the time when the study was done, due to the collapse of the healthcare system, patients with a poor medical condition or those residents of nursing homes were instructed to receive the treatment at home, whenever indicated.2.2Covid‐19 diagnosis was based on real‐time reverse‐transcriptase‐polymerase‐chain‐reaction (RT‐PCR) assay (LightMIx Modular SARS‐CoV (COVID19) E‐gene and LightMIx Modular SARS‐CoV (COVID19) RdRP, Roche Diagnostics S.L.) from oropharyngeal‐nasopharyngeal swab, sputum, or lower respiratory tract sample; or by the presence of anti‐SARS‐CoV‐2 IgM + IgA antibodies  in serological test in patients with clinical symptoms, according to the World Health Organization (WHO) protocols .2.3We collected data from electronic medical records from primary care, emergency department, and hospitalization. According to the local protocol, patients were instructed to contact a specific Covid‐19 line that followed‐up them daily or every other day before and after the hospitalization until decease or curation. In the emergency department, every patient fulfilled a symptom checklist that included anosmia, headache, myalgia, or syncope among other symptoms. During the hospitalization, patients were treated according to the national Covid‐19 management protocol standard of care (SOC) , diabetes , and smoking habit (current or in the preceding 6 months). We assessed the presence of chronic comorbid conditions, including cardiovascular diseases , chronic pulmonary diseases (chronic obstructive pulmonary disease (COPD), asthma, occupational lung diseases, interstitial lung diseases, and pulmonary hypertension), cancer , immunocompromised state , and chronic neurological disorders . The baseline performance of patients was determined by the modified Rankin scale, and we used three points as threshold of disability , increased lactate dehydrogenase (LDH) level (>250 U / L), decreased glomerular filtration rate (GFR) (<90 ml/min/1.73m2), abnormal liver enzymes, including aspartate aminotransferase (AST) (RV < 32 U / L), alanine aminotransferase (ALT) (RV < 33 U / L), and gamma‐glutamyl transferase (GGT) (RV < 40 U / L); increased creatine‐kinase (CPK) (>170 U / L), increased international normalized ratio (INR) (>1.3), D‐dimer (RV < 500 ng / dL), increased C‐reactive protein (CRP) (> 5 mg / L), and increased procalcitonin (PCT) (> 5 ng / mL).We assessed the frequency of abnormal laboratory examinations on admission, including leukocytes : 4–10 cell count x 109 / L), the presence of lymphopenia (<0.9 cell count x 109 / L), the presence of anemia (< 12 g / dL), platelets  or median and interquartile range (IQR). We analyzed the association between qualitative variables with the Chi‐square test, using the Bonferroni method in the multiple comparisons correction, presenting directly the adjusted p‐value. In the contrast of qualitative and quantitative variables, we used the Student t test or the Mann–Whitney U test if the distribution was not normal according to the Kolmogorov–Smirnov test. The statistical signification threshold was 0.05, after adjusting for multiple comparisons. We did a post‐hoc subanalysis in which we compared the frequency of symptoms in patients with nonspecific neurological symptoms  on presentation, compared with the rest of the sample, as well.We present qualitative variables as frequency and percentage. Quantitative variables are presented as mean and standard deviation . Finally, we analyzed the association with a higher mortality with Cox‐regression log‐rank test and logistic regression. We did a univariate regression, and all the variables that had an alpha error equal or lower than 20% were included in the multivariate regression analysis. We describe the hazard ratio (HR), odds ratio (OR), and 95% CI. To minimize the overfitting of the model, we did a sensitivity analysis, and the model was repeated including those variables with an alpha error equal or lower than 10%. Sample size was not calculated in advance, but we estimated the power of our study to be of 95%. The calculation is available in supplementary materials . We anal3SD: 14.7) years, and 250 (43.4%) patients were female. In 320 (55.6%) patients, one or more neurological symptoms were described on admission. Patients with neurological symptoms were younger, with better baseline performance, and with fewer prior cardiac disorders. Table During the study period, 580 patients were screened and 576 were included in the study. Three patients were excluded because they were not admitted from the emergency department and one patient because there was no available information. The mean age of the sample was 67.2 SD: 4.7 yearsp = .758). Diagnosis was confirmed by PCR in 546 (94.8%) of cases and serology in 175 (30.4%). Chest imaging was abnormal in 549 (95.3%) patients.The presumptive source of the contagion was attributed to a close relative in 96 (16.6%) cases, occurred in a retirement home with other positive cases in 64 (11.1%), was related with a journey to a geographical area with infected people in 39 cases (6.7%), was related with healthcare work in 23 cases (3.9%), was related with nonhealthcare work in 21 (3.6%), was nosocomial in 5 (0.8%), and the source was unknown in 328 (56.9%). In patients with neurological symptoms at onset, frequency of unknown source of contagion was 161 (50.3%) compared with 133 (52.0%) in those without neurological symptoms  had neurological symptoms at any point at presentation and admission. Of the 374 patients, 320 (85.6%) cases presented with neurological symptoms in the ED. The most frequent neurological symptoms at the moment of ED presentation were anosmia, in 146 (25.3%) patients, myalgia in 139 (24.1%), headache in 137 (23.8%), and altered mental status in 98 (17.0%). All the neurological symptoms were described more frequently on admission than during hospitalization except for stroke, seizures, and ataxia. Table 3.2SD: 5.5) days, compared with 6.6 (SD: 6.9) in those without (p = .019). Figure Neurological symptoms started the same day as the rest of the Covid‐19 symptoms in 198  patients. By the fourth day of symptoms, neurological symptoms were present in 294 (80.5%) of patients. Figure 3.3p = .001), and light‐headedness , but not asthenia , fever , rash , or cough .Patients with neurological symptoms on admission had more frequently arthralgia, asthenia, fever, light‐headedness, rash, and cough than those without it. Table The presence of general Covid‐19 symptoms in patients presenting with neurological symptoms had a 98.7% (96.6%–99.6%) sensitivity. There were four (0.6%) patients that presented with neurological symptoms and did not have any Covid‐19 symptom on admission (Supplementary Table 3.4p = .018) and glomerular filtration rate (201 (62.8%) versus. 185 (72.3%), p = .021), which were both more frequently abnormal in patients without neurological symptoms on admission. Supplementary Table The frequency of laboratory abnormalities was similar in patients with and without neurological symptoms on admission except for lactate dehydrogenase (187 58.4%) versus. 175 (68.4%), 8.4% versThe presence of laboratory abnormalities in patients presenting with neurological symptoms had a 98.1% (95.7%–99.2%). There were six (1.9%) patients with fully normal laboratory parameters on admission. The combination of lymphopenia, and increased LDH, INR, D‐dimer, CRP, and procalcitonin had a 96.9% (94.1%–98.4%) sensitivity. All the patients with normal laboratory parameters had Covid‐19 symptoms, being the sensitivity of combination of clinical symptoms and laboratory parameters of 100% (99.1%–100%). In patients with neurological symptoms, the laboratory parameters with the higher sensitivity were C‐reactive protein (88.7%), D‐dimer (65.3%), and GFR (62.8%). Supplementary Table 3.5p = .015). Age, mRS, time since symptoms´ onset, hypertension, diabetes, smoking habit, prior history of cardiac disorders, chronic neurological disorders, and immunosuppression were associated with higher odds of mortality. The presence of headache, anosmia, myalgia, asthenia, diarrhea, chest pain, fever, odynophagia, and cough on admission were associated with a lower odds of death, and the presence of altered mental status or dyspnea on admission were associated with higher mortality in the univariate analysis. Supplementary Table The course of the disease corresponded to mild disease in 32 (5.6%) patients, pneumonia in 142 24.7%), severe pneumonia in 269 46.8%), and ADRS in 124 (21.6%). All‐cause in‐hospital mortality rate was 127/576 (22.0%), being lower in patients without neurological symptoms on admission , mRS , chronic neurological disorders , altered mental status , and dyspnea ; while time since symptoms onset , the presence of anosmia , and asthenia  were statistically significant. The restrictive analysis did not alter the results. Supplementary Tables 4In the present study, neurological symptoms were among the most frequent clinical manifestations of Covid‐19, occurring in half of the patients. Only fever (80%), cough (70%), dyspnea (50%), and asthenia (40%) were more frequent than neurological symptoms such as anosmia (25%), myalgia (24%), and headache (23%). It is not widely recognized that Covid‐19 might present with neurological symptoms, as some official guidelines .Authors declare no conflict of interest. The present study was performed without any direct or indirect financial support. East Valladolid Ethics Review Board approved the study (PI‐20–1751). All the datasheets are available for other authors upon reasonable request, by emailing the corresponding author (David García‐Azorín designed and conceptualized the study, played major role in the acquisition of data, analyzed the data, and drafted the manuscript for intellectual content. Javier Trigo, Enrique Martínez‐Pías, Isabel Hernández‐Pérez, Gonzalo Valle‐Peñacoba, Blanca Talavera, Paula Simón‐Campo, Mercedes de Lera, Alba Chavarría‐Miranda, Cristina López‐Sanz, María Gutiérrez‐Sánchez, Elena Martínez‐Velasco, María Pedraza, Álvaro Sierra, and Beatriz Gómez‐Vicente played major role in the acquisition of data and revised the manuscript of intellectual content. Ángel Guerrero and Juan Francisco Arenillas designed and conceptualized the study, analyzed the data, and revised the manuscript for intellectual content.https://publons.com/publon/10.1002/brb3.2058.The peer review history for this article is available at Supplementary MaterialClick here for additional data file.


Ziziphus jujuba Mill. extract (FZM) against the formation of bacterial biofilms (BBFs) in Staphylococcus aureus.To evaluate the in vitro antibacterial effect of flavonoids-rich S. aureus biofilms in vitro. Morphological observation showed a decrease in both biofilm adhesion and thickness. Results of confocal laser scanning microscopy used to detect the thickness of the BBFs showed that FZM treatment reduced the thickness of the BBFs. Furthermore, after the Image-Pro Plus v.6.0 analysis of the fluorescence intensity, FZM treatment reduced the thickness of the BBFs as well as the proportion of green fluorescence. Scanning electron microscopy showed that FZM can disrupt the channels available for substance exchange in the biofilm, thus exposing the bacterial cells and damaging its three-dimensional structures.FZM can effectively inhibit the formation of FZM can inhibit biofilm formation, improve the bacterial pH environment, and eliminate the hydrophobic effect of reactive oxygen species and flavonoids. Ziziphus jujuba Mill.) fruit, commonly called as the sour or round date, has been used for medical purposes for more than 2000 years. It is an important raw material in food and pharmaceutical industries. Approximately 90% of the world’s Z. jujuba is produced in China, where it is widely distributed from Urumqi, Xinjiang. Owing to the extremely strong sedative and tranquilizing effect of this fruit, traditional Chinese medicine often uses the dried seeds for medicinal purposes. The jujube fruit is often wasted, but there are a few studies reporting its medicinal value. The present study analyzed the antibacterial effects of Z. jujuba flavonoid on bacterial biofilms [Jujube . The quality inspection report provided by Xinjiang Maidisen Pharmaceutical Company was identified as the dry and mature fruit of Ziziphus jujuba Mill. var.spinosa (Bunge) Hu ex. H.F. Chow by Miregiuli, the quality inspector of Madison), ethanol anhydrous , brain heart infusion broth , crystal violet , ampicillin (Solarbio No.A8180), normal saline , sodium deoxycholate , 2.5% glutaraldehyde solution , phosphate buffered solution , FITC-Concanavalin A (Sigma No.C7642), propidium iodide , blood agar base medium , cover glass (18 × 18 mm CITO GLAS No.10211818C), slide glass (25 × 75 mm CITO GLAS No.188105), Petri dish , 96-well plates (NEST No.20160120801A), CO2 incubator , biosafety cabinet , microplate reader , confocal laser scanning microscope , electron microscope , scanning electron microscope , S. aureus .Z. jujube fruit was used. Quality control was conducted by Quality Inspector Mireguli from Xinjiang Madison Pharmaceutical Co., Ltd. Jujubes were picked, cleaned, cored, sliced, dried, and ground into 100-mesh powder. The powder was mixed with ethanol  and extracted by reflux for 1.5 h at 70 °C. The supernatant was collected by centrifugation, further concentrated, and dried as jujube extract for further use.In this study, dried ripe S. aureus was streaked with an inoculation needle and incubated for 24 h at 37 °C. Subsequently, an isolated colony was diluted in BHI medium and cultivated at 37 °C for 24 h. After vortexing, the bacterial concentration was adjusted to 1 × 109 CFU/mL using McFarland standards.An isolated colony of standard 2 for 24 h [Biofilms were created by adding 50 μL of the bacterial suspension and 150 μL of the BHI medium containing 1% sucrose to each well of a 96-well plate. They were grown at 37 °C in 5% CO2 for 24 h. The supernatants were removed, and the plates were washed three times with PBS. Plates were dried at room temperature for 30 min, after which 200 μL of 0.1% crystal violet (CV) stain was added to each well and left for 15 min, and wells were rinsed with distilled water until transparent. Next, 2% sodium deoxycholate was added to each well to detach the biofilms, and the OD600nm values were measured.FZM was added to the test group at concentrations of 1.41, 2.81, 5.63, 11.3, 22.5, and 45 μg/mL. Ethanol was used as the negative control, and0.25 mg/mL ampicillin was used as the positive control . There wPlates were prepared as described above. After 24 h of growth, the 96-well plates were stained with CV and fixed with 2.5% glutaraldehyde. The morphological changes were observed under an optical microscope.2, and then, the media was discarded. FZM and control treatments were identical to the ones used above. After incubation for 24 h, the coverslips were rinsed with PBS and fixed with 2.5% glutaraldehyde for 30 min. Subsequently, they were rinsed with PBS, stained with FITC-ConA for 30 min, stained with PI for 15 min, and sealed [Sterilized coverslips treated with concentrated sulfuric acid and 75% ethanol were placed in 15 × 60-mm Petri dishes. To each dish, 0.2 mL of the bacterial suspension and 4.8 mL of BHI medium were added. The dishes were incubated for 24 h at 37 °C in 5% CO2, and the media were then discarded. FZM and control treatments were identical to the ones used above. After rinsing with PBS, the dishes were fixed with 2% glutaric acid, dehydrated with anhydrous ethanol, dried with CO2, and sputter-coated with gold. They were scanned using scanning electron microscopy (SEM) to determine the presence and morphology of BBFs.To 15 × 60-mm Petri dishes, 0.2 mL bacterial suspension and 4.8 mL media were added, bacteria were cultured for 24 h at 37 °C in 5% COt-test was used for evaluating the difference between each group and the control group. The Dunnett T3 test was used for the variance invariance, where α = 0.05. Data were represented as mean ± standard deviation.SPSS 20.0 software was used for statistical analysis. If the data were normal, multiple groups were tested by one-way analysis of variance. If the variance was equal, Dunnett’s The flavonoid concentration in the jujube extract was 1.44 mg/g at 510 nm.p < 0.05). . After S. aureus BFFs were treated with FZM, the number of viable bacteria significantly decreased. At 11.3 μg/mL of FZM, the intensity of fluorescence significantly decreased. The intensity of bacterial fluorescence was 11.3 μg/mL of FZM (10.1 ± 0.03 a.u.). After 5.625 μg.mL− 1 of FZM, the level of fluorescence was stable. The results were significantly different from those of the control group (p < 0.05)  [In the control group, n + red) . The bacS. aureus revealed grape-like clusters in biofilms [Staphylococcus aureus is tightly wrapped on the surface of Petri dish by bacterial biofilm. At 2.81 μg/mL of FZM, large accumulations of S. aureus cells and extracellular mucilage were clearly observed, indicating that there was still biofilm adhesion despite the inhibitory effect of FZM. At 5.63 μg/mL of FZM, visible microvilli were observed around the BBFs, and BBFs transmittance was stronger than that in the negative control group, indicating that FZM had some effects on BBFs at this concentration. At 11.3 μg/mL of FZM, most of the cells shrank and contained broken three-dimensional (3D) structures. Few cells were intact, but these were not coated with mucous and were clear, indicating that this concentration had a strong scavenging effect and that the bacterial structure began to disassemble. The density of S. aureus was significantly lower in the presence of ampicillin (Fig. Different from biofilms formed by other bacteria, S.aureus formed biofilms were more dense, and biofilms , 11. Bacbiofilms . The StaS. aureus is an important nosocomial pathogen in hospitals, and it commonly forms biofilms [biofilms . The curbiofilms . This subiofilms –17. Studbiofilms –21. Therbiofilms .S. aureus biofilm infections [Staphylococcus aureus [Natural active ingredients extracted from plants have unique advantages for the treatment of fections , 23–31. fections , 32. In s aureus . The mecS. aureus biofilms. By increasing the concentration of FZM, the rate of biofilm formation reduced steadily. Biofilm thickness is one of the criteria used to assess the ability of compounds to remove biofilms [In the present study, the results of the microplate assays indicate that FZM effectively inhibits the in vitro formation of biofilms . In our S. aureus biofilms [S. aureus biofilm were gradually destroyed after FZM treatment. Exopolysaccharides were cleared, and the bacteria were gradually exposed. This further confirmed that FZM can significantly improve BBF detachment. It may remove bacterial exopolysaccharides and disrupt the channels available for substance exchange in the biofilm.Current research shows that the removal of adhesion proteins, the increase in bacterial pH, and the destruction of the bacterial quorum sensing system are the key factors for removing biofilms , 27, 35.biofilms , 36–44. FZM can inhibit biofilm formation, improve the bacterial pH environment, and eliminate the hydrophobic effect of reactive oxygen species and flavonoids. Further studies are required to investigate the mechanism of action of FZM on the bacterial quorum sensing system.


Annona squamosa (Annona b. and Annona h.) seed, peel, and pulp. We also meausred phenolic, flavonoid, sulfated polysaccharide, tannins, and triterpenoids. Polyphenol identification was determined using RP-HPLC. Results of the antioxidant activity revealed that the highest activity was observed for Annona h. seed extract using DPPH and ABTS assays with IC50 6.07 ± 0.50 and 9.58 ± 0.53 µg/ml, respectively. The antimicrobial activity against various pathogenic strains revealed that the peel extracts of both Annona b. and Annona h. exhibited the best antimicrobial activity. We also assessed the IC50 values for anticancer activity in all six Annona b. and Annona h samples against four cancer cell lines colon (Caco-2), prostate (PC3), liver (HepG-2), and breast (MCF-7) using MTT assay. Annona b. and Annona h seed extracts had the lowest IC50 values for four cancer cell lines with 7.31 ± 0.03 and 15.99 ± 1.25 for PC-3 and MCF-7, respectively. Both seed extracts, Annona b. and Annona h., showed significantly down-regulated mRNA expression of Bcl-2 and up-regulated p53 in all treated cell lines. Apoptosis was evaluated using nuclear staining, flow cytometric analysis, and immunohistochemistry of the proliferation marker (Ki-67). Additional studies are required to characterize the bioactive compounds responsible for the observed activities of Annona seed and determine its mechanism as an anticancer drug.In plants, Fruits and their wastes are the main sources of bioactive compounds. Currently, Annona fruits have attracted the attention of people interested in health-promoting foods due to their phytochemical content that their activities were not studied before. This study aimed to explore the potential antioxidant, antimicrobial, and in vitro anticancer activity of two cultivars  Annonaceae) is a pan-tropical family that contains approximately 130 genera and 2300 species of trees and shrubs1. In the wild, plants from this family produce a large range of exotic fruits and have distinctive varieties, such as graviola fruit (Annona muricata), araticum-do-cerrado or marolo (Annona crassiflora) and conde fruit (Annona squamosa)2. Annona squamosa is belong to the family Annonaceae. The fruit of this plant is called Keshta in Egypt, although it has been called a sugar apple by the English-speaking community. In America and the West Indies areas, this plant (A. squamosa) can be cultivated. This plant is being commercially grown worldwide because of recent exposure to its medicinal properties4. Many ethnic groups use squamosa for the treatment of many chronic illnesses5. Many Annonaceae members are used as anti-cancer6, anti-inflammatory7, anti-HIV8, and anti-bacterial and anti-fungal9 therapies in conventional medicine.The sugar apple family (10. It was expected that the number of dies from cancer will increase to 14.6 million by 203511. Oxidative stress and reactive oxygen species (ROS) are considered as a strong risk factor that trigger inflammatory response and cancer12. Homeostasis of ROS in the human body kept balanced by non-enzymatic and antioxidant manipulators. Imbalance between antioxidants and oxidant modifiers causing damage of cell biomolecules such as lipids, proteins, and DNA13. ROS attack the nucleotide of cell DNA causing mutations and consequently cancer, tumor suppressor genes mutations such as p53 mutation is an example14. Apoptosis is a program cell death, furtiveness of apoptosis resulting in uncontrolled cell division and cancer15. Decision of cell division and apoptosis determined by the balance of proto-oncogene Bcl-2 and tumor suppressor p53 proteins which are involved in many biochemical pathways related to cancer progression16. Nowadays, intensive research efforts are presented to find a new cancer drug can target save apoptosis in cancer cells17. The diversity of cancer types and leading causes increase the demands for new treatment. In United States 1,806,590 new cancer cases and 606,520 cancer deaths only in 202018. At the same time, a new type of natural compound is being discovered every day. In 2006, approximately 50,000 of the newly natural molecules were discovered, this number were grow up to around 326,000 in 2014. Among them, a round 195,000 were biologically active19. However, about 170,000 compounds were toxic. Conventional medicinal herbs and other plants regarded as a huge source of pharmacologically active compounds20. Due to their alkaloid content, Annona plants have been used previously as antibacterial treatment for many related diseases like bacterial caused ulcers, dysentery, and boils21. There have been no previous studies on the biological activities of the different parts  of Egyptian Annona spp. Therefore, the current study explores the phytochemical analysis of Annona cultivated under Egyptian climate conditions as well as elucidation of the antioxidant, antimicrobial, and anticancer activities of its different parts  in vitro.Cancer is the second mortality reason after cardiovascular diseaseAnnona spp. were presented in Table Annona b. and Annona h.) varied from 81.66 ± 1.35 to 83.40 ± 0.61%, respectively. The highest protein content was recorded in Annona b. peels with a concentration of 3.30 ± 0.16%. The fat content ranged from 0.96 ± 0.23 to 29.21 ± 1.12% for Annona b. pulp and Annona b. seeds, respectively. The carbohydrate content ranged from 93.77 ± 1.78 to 94.36 ± 1.26%; the crude fiber content ranged from 66.64 ± 3.92 to 94.36 ± 1.26% for Annona b. pulp and Annona b. pulp, respectively. Finally, the ash content varied from 2.12 ± 0.19 to 3.14 ± 0.28%. Diversity in the gross chemical composition could be attributed to several factors, such as cultivar, ripening stage of fruit, climatic conditions, fertilization and irrigation system, and harvesting time22. It has been reported that the protein content of Annona muricata (L.) peels and seeds were 1.56 ± 0.01 and 2.73 ± 0.10%, respectively24, which is less than current findings in peels and much more in seeds in both species. At the same time, other studies showed that the protein content of Annona crassiflora pulp was 1.38 ± 0.01%25, which was less than the results of the present investigation (2.133 ± 0.33%). Annona b. and Annona h. contained all essential minerals and, Annona b. samples had the highest mineral concentrations, as shown in Table 1. On the other hand, Annona b. pulp showed the highest concentration of all measured minerals except potassium, which found to be high in Annona b. peel (115.78 mg/kg). Annona b. pulp showed high concentration of calcium, copper, iron, magnesium, manganese, sodium, phosphorus, and zinc with values of 84.33, 0.38, 20.73, 56.78, 0.33, 45.24, 55.20, and 0.31%, respectively (Table Annona b. (84.33 mg/kg) and the seed of Annona h. (75.11 mg/kg), which represents approximately 8.4% and 7.5% of the recommended daily intake (1000 mg)26. The pulp of Annona b. showed a considerable concentration of iron (20.72 mg/kg) that represents approximately 133% of the daily requirement (15 mg). Similarly, both Annona species exhibited high concentrations of potassium. The findings of the current investigation emphasized the adequate concentrations of essential minerals in both Annona species such as calcium that is important for healthy bone and teeth. Iron plays a crucial role by preventing anemia and potassium deficiency. Essential minerals are also responsible for the maintenance of an optimal pH balance and blood pressure regulation in the human body.The proximate compositions of different parts of Annona h. . The flavonoid content ranged from 36.05 ± 3.29 to 112.71 ± 4.93 µg QE equivalent/g of dry sample. The seed extract of Annona h. exhibited the highest flavonoid content , followed by the peel extract of Annona b. . The sulfated polysaccharide content ranged from 149.50 ± 3.76 to 186.37 ± 9.02 µg/g, triterpenoid content ranged from 6.20 ± 0.56 to 10.73 ± 0.57 µg/g, and tannin content of different parts of Annona sp. ranged from 0.22 ± 0.07 to 1.92 ± 0.17 µg/g of dry extract. Currently, Annona fruits have attracted the attention of people interested in health-promoting foods due to their content of bioactive compounds. Such bioactive compounds have been reported to have health benefits due to their ability to scavenge harmful free radicals28. Utilization of bioactive compounds such as natural phenolics with antioxidant activity plays a crucial role in the food industry for the formulation of health-promoting foods. The findings of the present work are comparable with those described in the literature28. It is worth mentioning that the matrices studied in the current work were approximately 1.5-fold richer than those described in the previous studies in terms of total phenolic content. Notably, this is the first study exploring the concentrations of tannins, triterpenoids, and sulfated polysaccharides in Annona h. and Annona b., as these aspects have never been reported previously.Phytochemical content in pulps, seeds, and peels of Annona are showed in Fig. Annona b. and Annona h. are shown in Fig. Annona b. seed extract were gallic acid, p-hydroxybenzoic acid, syringic acid, ferulic acid, ellagic acid, benzoic acid, o-coumaric acid, and salicylic acid. These compounds are by far the most abundant compounds, not only in the seed of Annona b. but also in the seed of Annona h. except for ferulic acid, ellagic acid, and benzoic acid. Cinnamic acid was detected in low quantity in Annona b. seed extract and was not found in Annona b. seed extract. Moreover, cinnamic acid derivatives  were detected only in Annona b. seed extract at concentrations of 1.96, 5.08, and 49.02 mg/100 g, respectively. Janicke et al.29 reported that dietary fiber is a rich source of hydroxycinnamic acids, ferulic acid, and p-coumaric acid, all of which may contribute to its protective effect against colon cancer. Besides Pei et al.30 confirmed the antioxidant, anti-inflammatory, anti-mutagenic, anti-ulcer, and anticancer activities of p-coumaric acid. Also, many studies have shown that o-coumaric acid has antioxidant, and antitumor biological activity32.Phenolic compounds of the seed extracts of both 50 values of free radical scavenging activity of the extracts toward DPPH and ABTS. Concerning DPPH, the IC50 values of ascorbic acid and the seed extracts of Annona b. and Annona h. were 6.39 ± 0.04, 7.88 ± 0.28, and 6.07 ± 0.50 µg/ml, respectively, and the peel extract of Annona b. was 61.78 ± 3.16 µg/ml. This finding indicates that the seed extracts exhibited the highest antioxidant capacity; this may be attributed to the greater accumulation of polyphenols and flavonoids in the seed than in other parts of the plant. Regarding ABTS, IC50 values were arranged in ascending order as follows; Annona b. seed, Annona h. peel, and Annona b. pulp, followed by Annona h. pulp, then Annona b. peel. The seed parts of both species exhibited the highest scavenging activity against both DPPH and ABTS free radicals  with high antimicrobial activity could be used to develop some functional food products to increase their shelf-life and boost the overall health benefits.The results of the antimicrobial assay of the peel, seed, and pulp extracts of the two Annona spp. against six pathogenic microorganisms are shown in Table 100 values. The results recorded that the extracts of seed, peel, and pulp of Annona are safe in the range of 654.26 ± 31.50, 551.35 ± 9.70, and 552.49 ± 3.33 µg/ml, respectively for Annona b., and 501.25 ± 0.43, 608.89 ± 6.36, and 493.22 ± 0.93 µg/ml, respectively for Annona h. The safety of an anticancer drug is one of the most important concerns of the physician in the treatment of cancer patients. The anticancer activity expressed as IC50 (concentration of the studied extracts that caused 50% cell death) is usually used to represent the strength and sensitivity of the drug on cancer cell lines. Table Annona b. had the highest anticancer effect against the four studied cancer cell lines . However, there was no significant difference recorded between the IC50 values of the seed extract from Annona b. and Annona h. for four cancer cell lines. There was no detectable anticancer efficiency of pulp and peel extracts of Annona h. on HepG-2 and PC-3 cells as well as peel extract of Annona h. against MCF-7 cells -flow cytometric analysis . The most affected cell lines were PC-3 (51.27 ± 0.1%) and HepG-2 (50.14 ± 0.32%), followed by MCF-7 (49.30 ± 0.07%) and Caco-2 (48.81 ± 0.16%) after Annona b. seed extract treatment. On the other hand, the most affected cell lines were MCF-7 (38.99 ± 0.37%) and Caco-2 (33.05 ± 0.3%) followed by HepG-2 (32.67 ± 0.33%) then PC-3 (29.43 ± 0.035%) after Annona h. seed extract treatment.The safety of different parts of Annona on normal human lung fibroblast cells (WI-38 cell line) represented by the EC42. The National Cancer Institute has collected about 35,000 plant samples from 20 countries and has screened around 114,000 extracts for anticancer activity43. Aidy et al.44 reported the anticancer potential activity of Annona muricata against breast, prostate, pancreatic, liver, lung, and colon cancers. Also, the anticancer properties of graviola (Annona muricata) were reported by Islam et al.45. Another report mentioned the anticancer potential of Annona muricata L. leaf extract46. Annona b. and Annona h. seed extracts and its polyphenols may be potentiating their anticancer activity. Polyphenols from diet have previously been documented to interfere many of biochemical pathways involved in cancer progression47. Furthermore, polyphenols may act as immune system booster, as well as help in living cells protection from ROS damage.Natural products are still important sources to discover new anticancer drugs48. Thus, this in vitro study aimed to evaluate the anticancer potential of two Egyptian Annona species fruits and their byproducts as a first step for use in medical applications.Many clinical trials of polyphenols treatments confirming the protective mechanism of them due to variations in dose, timing, and other conflicting factors16. Both studied seed extracts of Annona b. and Annona h. significantly down-regulated the expression of Bcl-2 and up-regulated p53 mRNA expression in PC-3, HepG-2, Caco-2, and MCF-7 treated cell lines. Annona b. seed extract suppressed Bcl-2 expression (0.23–0.48) in the treated human cancer cell lines by a higher extent than Annona h (0.47–0.82) as shown in Fig. Annona b. enhanced the expression of p53 by more than fourfold, which was higher than in Annona h-treated cancer cells (2.15 to 3.10-fold)  in comparison with brown color-stained untreated control cells . The potent inhibitory effect of seed extract of Annona b. on Ki-67 may attribute to their stronger apoptotic activity  (Annona b.) and the cultivar Abdel Razik (a hybrid of two species: Annona cherimola × A. squamosa) (Annona h.) were purchased from Mansheya Market, Alexandria, Egypt. The fruits were identified and authenticated by Prof. Atef M. Ibrahim at the Pomology Department, Faculty of Agriculture, Alexandria University. After identification, the plant material was processed for extraction. The collection of Annona Fruits comply with relevant institutional, national, and international guidelines.Fruits of g for 25 min. then filtered, lyophilized, and stored in a freezer at − 20 °C until further use.Peel, seeds, and pulp samples were oven-dried at 45 °C. The dried samples were ground separately into powder and stored at 4 °C until further use. The powdered samples were extracted with 1:20 w/v of distilled water and then heated at 70 °C for 30 min with stirring. The extracts were centrifuged at 10,000 × 52. The crude fat and fiber contents were estimated in accordance with Folch et al.53, and Prosky et al.54, respectively. The mineral content, including Na, K, Ca, Mg, Cu, Mn, Zn, and Fe, was measured by microwave plasma atomic emission spectrometry (MP-AES) , according to AOAC55. Phosphorus content was determined spectrophotometrically56. The mineral content was expressed as mg/kg of dry weight (dw). All measurements were carried out in triplicate.Crude protein content determination, in dried powder form, was conducted using the Kjeldahl procedure according to Nelson and Sommers57. The total flavonoid content of the extracts was quantified by a colorimetric method described by Sakanaka et al.58. Sulfated polysaccharides were measured by the toluidine blue assay as described by Albano and Mourao59. The triterpenoid content was assessed colorimetrically via reaction of the triterpenoids with vanillin using ursolic acid as a standard60. The tannin content was determined colorimetrically following the method described by Price et al.61. All measurements were carried out in triplicate.The total phenolic content was determined using Folin-Ciocalteu reagent, as described by Dewanto et al.Annona h. and Annona b. seed were carried out by following the procedure described by Tomaino et al.62 using an RP-HPLC system  at 284 nm wavelengths with a C18 column  maintained at 35 °C. The values for the main phenolic compounds were expressed in mg/100 g.Quantitative analyses of the phenolic content of the water extracts of the Annona h. and Annona b. were used to evaluate their antioxidant effects. Evaluation of the antiradical effect of each extract was done by estimating the IC50 values (50% inhibitory concentration). The reducing power of the extracts was determined according to the method described by Yen and Duh63, absorbance was measured at 700 nm. The reducing power was expressed as ascorbic acid equivalents (ASE)/mg. ASE is the reducing power of 1 mg of sample, which is equivalent to the reducing power of 1 nmol of ascorbic acid. The DPPH assay was carried out according to the method described by Shimada et al.64, and the absorbance of the solution at 517 nm was noted. For the ABTS radical scavenging assay, the method described by Re et al.65 was adopted, and the absorbance was measured at 734 nm after 7 min. All experiments were carried out in triplicate.Determination of reducing power and the antiradical potentials  of different Annona parts of Ann. s. and Ann. h. against various pathogenic strains , Alexandria, Egypt); Candida albicans ATCC MYA-2876, Klebsiella pneumoniae ATCC12296, Salmonella senftenberg ATCC 8400, and Staphylococcus aureus NCTC 10,788 were obtained from Microbial Resource Center (MIRCEN), Ain Shams Univ. (ASU), Hadak Shobura, Cairo, Egypt. Antibacterial activity was evaluated by measuring the diameters (mm) of the inhibition zones using a caliper. The tests were performed in triplicate67.The agar well diffusion method was used to measure the antimicrobial activity of the peel, seeds, and pulp extracts of 3 cells per well in a 96 well microtitre plate. The cells were treated with and without the serial extract dilutions after a 24 h incubation for cell attachment. The cytotoxicity assay was performed after 48 h, according to Mosmann68 using MTT reagent . The effective safe concentration (EC100) value (at 100% cell viability) of each tested extract was estimated by Graphpad Instat software (version 6.01).Extract cytotoxicity was investigated using human lung fibroblast WI-38 cells (American Type Culture Collection (ATCC), USA) grown in DMEM  containing 5% fetal bovine serum  by seeding as 3 × 102 incubator at 37 °C. The sensitivity of tumor cells to different Annona part samples was assessed using MTT assay. The anticancer activity of each tested sample was expressed as an IC50 value that causes 50% death for cancer cells and estimated by Graphpad Instat software. The morphological changes of all used cell lines were investigated by phase contrast inverted microscope  after Annona seed extract treatment.Four human cancer cell lines  were used for this investigation. A colon cancer line (Caco-2) and a prostate cancer line (PC3) were maintained as adherent cell cultures in DMEM medium , while the HepG-2 (liver cancer line) and MCF-7 (breast cancer line) were cultivated in RPMI 1640 medium. These two media are supplemented with 10% fetal bovine serum. After cell seeding and attachment, cells were treated with and without the serial dilutions of extracts for 48 h in 5% CO2 incubator for 24 h. Then the cancer cell lines  were treated with the most active part of Annona h. and Annona b. (seed extracts) for 48 h. After washing the cells with phosphate buffer saline (PBS), the cells were stained with dual fluorescent nuclear dye of EB/AO . The untreated and treated cancer cells were investigated using a fluorescence phase-contrast microscope .Cancer cells were seeded in six well plates and incubated for cell attachment in 5% COFor flow cytometric detection, the untreated and treated human cancer cell lines  were harvested by trypsinization then washed and resuspended in PBS. The mechanism of cancer cell death was analyzed by staining cells with fluorescein isothiocyanate (FITC)-annexin V and PI  for 15 min in the dark. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min and resuspended in PBS. Cell death rates were detected by flow cytometry  using a FITC signal detector (FL1) for annexin-stained apoptotic cells and the phycoerythrin emission signal detector (FL2) for PI-stained necrotic cells .Annona b. and Annona h. seed extracts separately. Total RNAs of all treated and untreated samples were extracted using a Gene JET RNA Purification Kit . The purity and the quantity of extracted RNA samples were determined using a UV-spectrophotometer, and cDNA was synthesized from mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR), using a cDNA Synthesis Kit . Real time PCR was performed by amplification of cDNA for each sample using the following primers (Forward/Reverse) were 5′-TAACAGTTCCTGCATGGGCGGC-3′/5′-AGGACAGGCACAAACACGCACC-3′ and 5′- TCCGATCAGGAAGGCTAGAGTT-3′/5′-TCGGTCTCCTAAAAGCAGGC-3′ for p53 and Bcl-2, respectively. In addition, the following primers (5′-GTGGGCCGCTCTAGGCACCAA-3′/5′- CTCTTTGATGTCACGCACGATTTC-3′) were used for detecting the β- actin housing gene. The 2−ΔΔCT equation was used to estimate changes in gene expression for p53 and Bcl-2 before and after treatment of cancer cells with Annona extracts.Quantitative gene expression of p53 and Bcl-2 were determined in cancer cells after treatment with 2O2 for 10 min, then washed in PBS buffer twice for 3 min and placed in 10 mM citrate buffer (pH 6), followed by heating at 60 °C for 10–20 min. After cooling and washing in PBS, the slides were separately soaked overnight in primary antibody (anti-Ki-67). Slides were washed in PBS, covered with biotinylated goat anti-polyvalent secondary antibody for 10 min, and then streptavidin peroxidase was added. After 10 min, the substrate of the secondary antibody  was added followed by washing in PBS and placed in a hematoxylin bath for 1–4 min. This was followed with a PBS wash (1 min) and then water (3 min). The percentage of immunostained cells was evaluated by imaging analysis software cellSens (CellSens Dimension 1.12) using a phase-contrast microscope 69.After trypsinization, the untreated and treated HepG-2 cells were centrifuged and washed with PBS buffer and then 10% formalin in PBS was added to cell pellets. The fixed cell specimens were dehydrated in ascending grades of alcohol and immersed in xylene for one hour (three times), followed by impregnation in melted paraffin to form solid paraffin blocks. Next, a rotator microtome was used to cut each block into 3–5 μm thick sections transferred onto positively charged slides. Slides were dried at 60–70 °C for 1–2 h then dewaxed by immersion in xylene three times for 5 min and rehydrated in descending grades of ethanol. After that, slides were incubated in 3% Hp)- values < 0.05 considered statistically significant.Data were expressed as mean ± standard error of the mean by the multiple comparison one-way analysis of variance (ANOVA) and Tukey’s test using the SPSS16 software program with probability , may be responsible for their apoptosis-dependent anticancer behavior against (Caco-2), prostate (PC3), liver (HepG-2), and breast (MCF-7) cancer cell lines. This is the first report on the possible molecular mechanism of action Annona b. seed extract has on cancer cell proliferation. Additional investigations will be required to characterize the bioactive compounds that may be responsible for the observed activities of the Annona seed to determine the potential mechanisms that make it an anticancer drug. Furthermore, Annona seed extract could be used in other food application and the manufacturing of nutraceuticals.Nutritional composition analysis of two Annona species peels, seeds, and pulp containing relatively high contents from protein, fat, fibers, carbohydrates, and good mineral quantity was done. The high potassium and calcium levels mean the species parts may be used for food supplementation, to increase the quality of food. Based on the current data the significantly high levels of phenolic compounds in


Evidence about ω-3 polyunsaturated fatty acids (ω-3 PUFAs) and oral cancer risk were limited. We aimed to evaluate the association of erythrocyte ω-3 PUFAs with the risk of oral cancer in a population from China.Erythrocyte ω-3 PUFAs of 236 oral cancer patients and 300 controls were determined by gas chromatography. Restricted cubic spline and logistic regression were used to analyze the association between erythrocyte ω-3 PUFAs and oral cancer risk. The crude and adjusted OR with 95% CI was calculated. Stratification analysis was performed to explore the potential interaction between ω-3 PUFAs and other traditional risk factors such as smoking and drinking.OR of EPA, DHA, and ω-3 index were 0.52 (95% CI: 0.35–0.76), 0.19 (95% CI: 0.08–0.44), 0.20 (95% CI: 0.09–0.44), respectively. Stratification analysis showed that the adverse correlation between EPA and oral cancer was only significant in the non-smoking group, while the adverse correlation of ɑ-linolenic acid (ALA), EPA, and DHA were only significant in the non-drinking group. General multiplicative interactions were observed between ω-3 PUFAs and smoking or drinking.Eicosapentaenoic acids (EPA), docosahexaenoic acids (DHA) and ω-3 index were negatively but non-linearly related to risk of oral cancer as observed by restricted cubic spline. The adjusted Adverse but non-linear associations were observed between erythrocyte EPA, DHA, ω-3 index, and oral cancer risk. Additionally, there were multiplicative interactions between ω-3 PUFAs and other behavior factors such as smoking and drinking. The protective effect of ω-3 PUFAs maybe more significant in the non-smoking or non-drinking population. It has been a major public health problem in less developed countries. The recognized etiologic factors of oral cancer include age, sex, smoking, drinking3, HPV4, consumption of betel nut5, etc. However, in our previous studies, we observed that a large proportion (about 50%) of oral cancer patients were not exposed to the above behavior risk factors, indicating that there are other unrevealed important factors. In the past decades, increasing interest has arisen regarding to the relation between lipid metabolism, especially fatty acids, and carcinogenesis.Oral cancer is one of the most common malignant tumors of the head and neck cancer (HNC), with an increasing incidence globally. There are 354,864 new cancer cases and 177,384 deaths per year worldwide with higher rates in developing countries6. Reduced ω-3 PUFAs in erythrocyte membrane have been reported to be associated with higher coronary heart disease risk9 and could also increase risk of neuropsychiatric10. In addition, increasing evidence suggest that dietary or blood ω-3 PUFAs are associated with a variety of cancers, such as HNC, esophageal cancer11, colorectal cancer13, and breast cancer15. Moreover, those observed associations are largely independent of other known risk factors.Erythrocyte membrane fatty acids are objective and stable biomarkers of fatty acids exposure. Compared with serum or plasma fatty acid, erythrocyte membrane fatty acid can reflect the medium to long-term fatty acid exposure status of human body. Erythrocyte membrane fatty acid composition is determined by a combination of fatty acid diet intake and metabolism. All major classes of fatty acids can be found in erythrocyte, which include saturated, monounsaturated, trans unsaturated, and polyunsaturated (both ω-3 and ω-6 families). Among all the major classes of fatty acids, ω-3 PUFAs have got the most research interest. ω-3 PUFAs include ɑ-linolenic acid , eicosapentaenoicacids , docosapentaenoic acids , docosahexaenoic acids , and ω-3 indexNevertheless, the role of dietary and nutritional factor in the etiology of oral cancer has been largely neglected, and there has been very limited evidence about the association between ω-3 PUFAs and oral cancer. To clarify the potential association may help to elucidate other underlying etiology of oral cancer which cannot be explained by revealed risk factors. Therefore, we conducted a case control study: (1) to assess the association between erythrocyte ω-3 PUFAs and risk of oral cancer, (2) to evaluate if there are interactions between erythrocyte ω-3 PUFAs and other reported risk factors, such as smoking and drinking in oral cancer.A hospital-based case-control study was conducted with patients who were recruited from the First Affiliated Hospital of Fujian Medical University from September 2010 to January 2019 in Fujian province, China. The inclusion criteria was as follows: (1) histologically confirmed primary oral cancer; (2) Chinese population with residence in Fujian Province at least for 10 years; and (3) age between 20 and 80 years old; Patients with recurrent or metastasized cancer, with previous chemotherapy or radiotherapy treatment were excluded. Control participants were recruited from the health examination center of the same hospital during the same period. Current or past experience of cancer was excluded. Finally, 236 patients and 300 control participants were admitted to the study.All participants provided a signed informed consent. The study protocol was approved by the Institutional Review Board of Fujian Medical University  and conducted in accordance with the ethical standards described in the Declaration of Helsinki. Information of demographic characteristics were collected using a structured questionnaire for both case and control group by face to face interview.Approximately 5 mL fasting blood sample was collected for each participants in an EDTA tube. All the blood samples of oral cancer patient were obtained the second day after patients were admitted to the hospital, to insure that the exposure detected was prior to any drug treatment or examination. After collection, the blood samples were centrifuged at 1500 rpm for 10 min at 4 °C to separate the erythrocytes.16). Fatty acids methyl esters (FAME) were obtained by incubating with 14% boron trifluoride ether/methanol  solution at 60 °C for 10 h. FAME was extracted into 1 ml hexane, vaporized to dryness and redissolved in hexane for gas chromatography analysis17.Erythrocytes were haemolysed in hypotonic Tris–HCl buffer, pH = 7.4, at 4 °C for 2 h. A membrane pellet was obtained by ultra centrifugation  from which lipids were extracted with chloroform/methanol , and the extract was dried in N2 fitted with flame ionization and DB-23 fame column . The carrier gas was nitrogen and the inlet worked in a split model with a split ratio of 10:1. The injection temperature was 250 °C and detection temperature was 280 °C. The starting temperature of the column was 120 °C. After 5 min, the temperature was programmed from 120 to 175 °C at a rate of 10 °C/min, the temperature was continuously increased 5 °C/min up to 210 °C and finally up to 230 °C.Individual fatty acids was identified by comparison with known standards , and expressed as a percentage of total fatty acids quantified from peak areas. The coefficients of variation for ALA, EPA, DPA, and DHA were 1.03%, 0.77%, 1.24%, 1.32%, respectively.18.Four individual fatty acids, ALA, EPA, DPA, DHA were included in the study and expressed as percentages of total fatty acids. Total ω-3 PUFAs refers to ALA + EPA + DPA + DHA and ω-3 index refers to EPA + DHAχ2 test or Fisher’s exact test. For the comparison of continuous variables between the two groups, t-test was performed for normally distributed data, while Wilcoxon rank sum test was adopted for data with skewed distribution. Restricted cubic spline was used to reveal the potential non-linear relationship between erythrocyte ω-3 PUFAs and risk of oral cancer. Then erythrocyte ω-3 PUFAs were included in unconditional logistic regression model as a continuous variable or as a dichotomous variable categorized according to the median of control group. The crude and adjusted OR with 95% CI was calculated to evaluate the effect of erythrocyte ω-3 PUFAs on oral cancer risk. All of the data were analyzed using Stata software version 13.1. All P-values were two-sided and P < 0.05 was considered as statistically significant.Distribution of demographic characteristic between case and control were examined using A total of 236 patients and 300 control participants were included in the study. Details of demographic characteristics of all participants are listed in Table ω-3 PUFAs were presented as median and inter quartile range in Table OR with 95% CI was calculated using logistic regression. Erythrocyte ω-3 PUFAs were included in unconditional logistic regression model as continuous variable, or as dichotomous variable categorized according to the median of the control group. As shown in Table OR of which were 0.51 (95% CI: 0.29–0.91), 0.54 (95% CI: 0.37–0.79), 0.23 (95% CI: 0.09–0.57), 0.19 (95% CI: 0.08–0.45), 0.12 (95% CI: 0.04–0.32), 0.21 (95% CI: 0.09–0.47), respectively. In the multivariate model which included erythrocyte ω-3 PUFAs as dichotomous variable, consistent adverse association was observed for EPA, DHA, total ω-3 PUFAs, and ω-3 index, adjusted OR of which were 0.62 (95% CI: 0.41–0.92), 0.61 (95% CI: 0.41–0.90), 0.66 (95% CI: 0.45–0.99), and 0.64 (95% CI: 0.43–0.94), respectively.Next, the crude and adjusted OR of which were 0.63 (95% CI: 0.39–1.00). Multiplicative interaction was observed between smoking and ALA, EPA, and DHA (Pinteraction < 0.05) (Table Pinteraction < 0.05), and the significant protective effect of ALA, EPA, and DHA were only observed in the non-drinking group, OR of which were 0.63 (95% CI: 0.41–0.99), 0.57 (95% CI: 0.36–0.88), 0.64 (95% CI: 0.41–0.98), respectively  Table . Similar19. Kim et al. performed a prospective systematic review and meta-analysis, results of which showed that the combined RR of the highest and lowest levels of ω-3 PUFAs was 0.79 (95% CI: 0.64–0.98) in colorectal cancer13. Meanwhile, another meta-analysis noted a significant adverse association between high serum DPA and total prostate cancer risk 20. However, there are also evidence supporting that plasma ω-3 PUFAs are not associated with cancer risk22 or even positively associated with cancer risk23. Bassett et al. performed a case-cohort analysis with 1717 men and 464 prostate cancer cases to investigate the association between fatty acids in plasma and prostate cancer risk, and no association was found. And not coincidentally, Chaje’s et al. found that plasma phospholipid fatty acid EPA, DPA, DHA had no association with gastric cancer risk24. Brasky et al. conducted a case-cohort study which included 2273 men and explored the association between plasma long-chain ω-3 PUFAs and the risk of prostate cancer. They noted that long-chain ω-3 PUFAs (DPA and DHA) were positively correlated with the risk of prostate cancer23. In our study, we observed that erythrocyte ALA, EPA, DPA, DHA, total ω-3 PUFAs, and ω-3 index was significantly higher in control participants than oral cancer patients. Non-linear association was observed between ALA, EPA, DHA, total ω-3 PUFAs, ω-3 index, and oral cancer. The risk of oral cancer decreased with the increase of ALA, DPA, DHA, total ω-3 PUFAs, and ω-3 index. To the best of our knowledge, reports of the association between serum or erythrocyte membrane ω-3 PUFAs and oral cancer are rare. Our findings are consistent with two HNC studies which involved dietary fatty acid intake. One study from Italy and Switzerland illustrated that higher intake of ω-PUFAs was associated with the decrease of oral cancer risk, the multivariate odds ratios (OR) for the highest quintile of ω-3 PUFAs compared to the lowest one was 0.525. The other study from the US also reported higher intake of dietary ω-PUFAs was associated with decreased HNC risk11.The potential role of ω-3 PUFAs in carcinogenesis has long been controversial. A number of studies have reported adverse association between blood ω-3 PUFAs and cancer risk. Matejcic et al. conducted a cohort study to explore the association between plasma fatty acids and the risk of pancreatic cancer, they found that plasma ω-3 polyunsaturated fatty acid α-linolenic acid and docosapentaenoic acid was negatively correlated with pancreatic cancer26. Another in vivo experiment noted that ω-3 PUFAs play an antioxidant role in immortalized mouse Schwann cells through nuclear factor-related factor 2 pathway27. Furthermore, studies exploring the relationship between plasma PUFAs and biomarkers of oxidative stress of breast cancer found that ω-PUFAs can modulate the critical processes of breast carcinogenesis with oxidation and inflammation28.In addition to epidemiological studies, some in vitro and in vivo experiments have also confirmed the protective effects of ω-PUFAs in tumorigenesis. A study explored the effects of ω-PUFAs on tumorigenic keratinocyte lines—epidermal  and oral (tongue) noted that ω-PUFAs can activate ERK1/2 pathway through EGFR, causing both reduced proliferation and apoptosis29. Possible explanation is that the general dietary intake of nutrients in smokers is lower than that of non-smokers30. We also found that the adverse correlation between EPA and oral cancer was only significant in the non-smoking group, while the adverse correlation of ALA, EPA, and DHA were only significant in the non-drinking group, which may be attributed to the fact that the anti-inflammatory and protective effect of ω-3 PUFAs was partially counteracted by the damage caused by smoking or drinking31. Future studies are needed to better understand the mechanism underlying the interaction between ω-3 PUFAs, smoking and drinking.Moreover, we observed general interaction between ω-3 PUFAs and traditional risk factors such as smoking and drinking. Erythrocyte EPA, DHA, and ω-3 index were lower in smokers compared with non-smokers. This is consistent with reports by Zehr et al.There are several limitations for this study. Firstly, this is a case-control study, since there is a long latency period which means that exposure data must be available for a period of several decades or more in order to identify true overall risks. The erythrocyte fatty acids data was not collected prospectively. Erythrocyte fatty acids may be affected by changes in dietary patterns or other behaviors due to symptoms from the disease. Case-control studies can be affected by reverse causality and cannot establish temporality. Thus, the associations observed between erythrocyte fatty acids and oral cancer should be interpreted cautiously. Secondly, as for the control of confounding factors, age, sex, ethnic group, marital status, education level, origin, family history of cancer, BMI, occupation, smoking, drinking were adjusted. But we did not collect information of medication affecting fatty acid levels, and this may cause potential confounding. Lastly, the sample size of this study is limited, further study can increase the sample size to confirm the current findings.The risk of oral cancer decreased with the increase of ω-3 PUFAs. There were adverse association between erythrocyte EPA, DHA, total ω-3 PUFAs, ω-3 index, and oral cancer risk. Additionally, multiplicative interactions between ω-3 PUFAs and other behavior factors such as smoking and drinking were observed. The protective effect of ω-3 PUFAs may be more significant in the non-smoking or non-drinking population. Future prospective research are needed to confirm the current findings.Supplemental tables


The determinations of polyphenols, the antioxidant capacity, and the yield indicators from the stalks (petioles) of rhubarb were made at each out of the 10 harvests carried out. The highest yield (59.16 t·ha−1) was recorded under the chemical fertilization. The total polyphenols content and antioxidant capacity varied widely from 533.86 mg GAE·g−1 d.w. and 136.86 mmol Trolox·g−1 d.w., respectively in the unfertilized control at the last harvest, up to 3966.56 mg GAE·g−1 d.w. and 1953.97 mmol Trolox·g−1 d.w. respectively under the organic fertilization at the four harvest. From the results of our investigation, it can be inferred that the chemical fertilization was the most effective in terms of yield, whereas the sustainable nutritional management based on organic fertilizer supply led to higher antioxidant compounds and activity.In recent years, rhubarb is being increasingly cultivated, as it provides early yields when the vegetables supply to market is deficient and shows high levels of both polyphenols content and antioxidant capacity in edible parts. In 2017, we investigated crops of the rhubarb cultivar Victoria to the fifth year of production. Comparisons were performed between three root phase fertilizations—chemical (NPK 16-16-16 Rheum rhabarbarum L.) is a perennial plant [Rhubarb  and the lowest for Victoria cv from autumn harvest (86.57 mg·g−1 d.w.) [Rumex acetosa [Rumex scutatus [Rumex crispus [Rumex japonicus [Rumex hastatus [Rumex ecklonianus [Rumex tingitanus [Rumex sanguineus [Rumex acetosella [Rumex maderensis [Rumex obtusifolius [It should be emphasized that literature data regarding the effect of fertilizers  as well as of the harvesting times on the polyphenol content of the rhubarb stalks are missing. Kalisz et al. analyzed the polyphenol content of two cultivars  in the spring and autumn seasons, recording the highest value of flavan-3-ols for Red Malinowy cultivar from spring harvest (195.98 mg·g−1 d.w.) . Studies acetosa ,21,22, Rscutatus , Rumex c crispus ,25, Rumeaponicus , Rumex hhastatus ,28,29, Rlonianus , Rumex tngitanus , Rumex snguineus , Rumex aetosella , Rumex mderensis , and Rumsifolius , but no The main objectives of this research were to assess: (1) the influence of the chemical, organic, and biological fertilization on the dynamic yield and their quality ; and (2) the optimal harvesting periods, correspondent to the highest plant nutrient content, in order to promote fertilization measures for sustainable agriculture.−1 d.w., followed by chemical fertilization with a total polyphenol content of 1320.62 mg GAE·g−1 d.w. . The organic fertilization (Og) provided 1477.95 mg GAE·gg−1 d.w. . The rol4), with a value of 2450.86 mg GAE·g−1 d.w., followed by fifth harvest (R5) with a total polyphenol content of 1819.34 mg GAE·g−1 d.w. The lowest polyphenol content was recorded at the last harvest (R10), i.e., 670.92 mg GAE·g−1 d.w. Statistically, lower values were recorded at the eighth harvest time (R8) with a total polyphenol content of 1015.14 mg GAE·g−1 d.w. The highest polyphenol content was recorded at the fourth harvesting period , followed by the Og treatment, with the value of 728.05 mmol Trolox·g−1 d.w. (232.47% compared to C). Ch fertilization resulted in an antioxidant activity of 478.48 mmol Trolox·g−1 d.w. (152.52% compared to variant C).Regarding the influence of fertilization on antioxidant activity (AC), the highest values were recorded when fertilizing with Mo, with the value of 877.07 mmol Trolox·g−1 d.w., in the case of R10, up to 1079.76 mmol Trolox·g−1 d.w. in the case of the fourth harvesting season (R4). Higher statistically significant values of AC compared to C were recorded at R5 (834.44 mmol Trolox·g−1 d.w.) and at R2 (589.75 mmol Trolox·g−1 d.w.). Statistically lower values of AC compared to C were recorded at R8 (467.39 mmol Trolox·g−1 d.w.) and R9 (477.34 mmol Trolox·g−1 d.w.).Regarding the influence of the harvesting season on the antioxidant activity, the values ranged in the wide interval from 293.09 mmol Trolox·g4 and R5).From the data presented in Polygonaceae familly species such as English spinach [Data obtained from this study are in agreement with those reported for other  spinach  and sorr spinach .Regarding the influence of fertilization and harvesting time on the total polyphenols content, statistically significant differences were recorded . −1 d.w., in the case of R10, unfertilized, up to 3966.56 mg GAE·g−1 d.w., in the case of R10, Og fertilization. , while at R4, the highest value was recorded (2183.38 mg GAE·g−1 d.w.).Indeed, the total polyphenols broadly varied from 433.86 mg GAE·gization. . In the Rheum harvested in two different years of production (2007 and 2009). The total polyphenols content ranged from 673 mg GAE·g−1 d.w., in the case of Loher Blut of the Rheum officinale species harvested in 2007, up to 4173 mg GAE·g−1 d.w., in the case of the Plum Hutt cv of the Rheum rhabarbarum L. species, harvested in 2009 [The data obtained in this experiment are in agreement with those obtained by Takeoka et al. who analyzed the content of polyphenols in the petioles of 29 different species of the genus  in 2009 . Higher  in 2009 .−1 d.w., in the case of C, from R10, to 1953.97 mmol Trolox·g−1 d.w., in the case of Og fertilization, from R4 , while the highest values were recorded at R5 (1614.49 mmol Trolox·g−1 d.w.). As far as Og is concerned, the antioxidant activity depended on harvesting time and varied widely from 338.78 mmol Trolox·g−1 d.w., in the case of R6, to 1953.97 mmol Trolox·g−1 d.w., in case R4. Then, the lowest values at the last harvest (136.86 mmol Trolox·g−1 d.w.) and the highest ones were obtained at R4 (688.76 mmol Trolox·g−1 d.w.).Regarding the influence of Ch on harvesting times, the antioxidant capacity ranged from 300.55 mmol Trolox·gSolfanelli et al. justify the higher content of polyphenolic compounds by the fact that in warmer periods, with less precipitation, the sugar content increases in plants and thus the activity of AMF is higher .Studies on the influence of different fertilizers on the antioxidant capacity of the petioles of rhubarb are scant. Zhou et al. determined the antioxidant activity of commonly consumed vegetables in Colorado and found significant variation among individual samples of each vegetable tested. They ranked the antioxidant activity of the vegetables as follows: rhubarb > green bean; tomato > potato; kale > spinach > broccoli .Rheum. The antioxidant capacity ranged from 463 µmol Trolox·g−1 d.w., in the case of Rheum officinale, up to 1242 µmol Trolox·g−1 d.w., in the case of Valentine cv, of the species Rheum rhabarbarum L. [Takeoka et al. analyzed the antioxidant capacity of the 29 species of the genus barum L. .Populus sp., high TPC and AC are also accounted for by water stress and higher temperatures [In perennial species, as in eratures .In general, polyphenols are secondary metabolism compounds that are produced by plants for defensive purposes under increased biotic or abiotic stress.The phenolic compounds content and composition were positively influenced by the application of fertilizers and the harvesting season, and in this respect, p-coumaric acid, ferulic acid, isoquercitrin, rutoside, and quercetrol were analyzed .−1, in the case of Ch, to 15.46 µg·g−1, in the case of the Mo fertilization. Control and Og treatment had higher values of p-coumaric acid, of 17.44% and 11.86%, respectively, compared to the Ch treatment.The values of p-coumaric ranged from 9.86 µg·g−1, in the case of Ch, to 31.45 µg·g−1, in the case of the Mo treatment. Control and Og treatments showed increases of 24.87%, respectively 2.58%, compared to Ch.Ferulic acid widely varied from 21.67 µg·gp ≤ 0.05) by biological fertilization. Values ranged from 37.04 µg·g−1, in the case of Ch, to 78.65 µg·g−1, in the case of Mo. Control and Og fertilization resulted in increases of 84.12%, respectively 66.90%, compared to the Ch treatment.Isoquercitrin was significantly influenced  phenol content as follows: the highest p-coumaric acid content was obtained at R7, ferulic acid at R8, isoquercitrin at R6, rutozide at R4, and quercetrol in R9. The lowest content of p-coumaric acid was recorded at first harvest (R1), while the lowest content of ferulic acid was recorded at R5, the lowest content of isoquercitrin and rutoside were recorded at the last harvest (R10), and the lowest content of quercetrol was recorded at R9.The harvesting time significantly influenced . Lower values of rutoside were achieved by R8 and R9, with increases of 43.54% and 59.4% respectively compared to R10.The content of rutoside in the petioles of resale varied in wide limits of 196.02 µg·g−1, in the case of R1, to 21.64 µg·g−1, in the case of R9. Higher values were obtained by R10 and R4, with increases of 90.5% and 88% respectively compared to R1. Quercetrol achieved increases of 1.42% and 4.54% respectively in the R2 and R3 harvest periods.The content of quercetrol in the rhubarb petioles ranged from 11.22 µg·gFertilization with Mo had a significant positive influence on p-coumaric acid, ferulic, isoquercitin, and quercetrol.The positive influence of biological fertilizers on the content of polyphenols has also been demonstrated in species such as tomatoes ,45 or peThe type of fertilization and the harvesting season significantly influenced the content of the five polyphenols  .−1, in the case of the Ch treatment, from the eighth period (R8), to 46.14 µg·g−1, in the case of fertilization with Mo from R7.P-coumaric acid content widely varied from 4.00 µg·g−1, in the case of Ch fertilization from R5, to 51.58 µg·g−1, in the case of Mo from R7.Ferulic acid fluctuated from 12.16 µg·g−1, in the case of the Og fertilization, from R6, to 148.78 µg·g−1, in the case of the control, which was also from R6.In addition, isoquercitrin was also significantly influenced by the fertilizer and harvesting period. It ranged in wide limits from 25.50 µg·g−1 in the case of the control from R7 to 1442.24 µg·g−1 in the case of Og fertilization from the R4 harvest time.The values of rutoside fluctuated from 121.06 µg·g−1 in the case of the control of R7 to 33.20 µg·g−1 in the case of the Ch treatment of R7 and Og from R10.The content of quercetrol ranged from 6.78 µg·gThe content of biologically active compounds depends mainly on the cultivation method as well as cultivar and harvest time . Many stThe fertilization treatments significantly influenced the number of petioles rhubarb per plant, their average weight per plant, the average weight per plant, and the yield dynamics .2, in the case of the control plant, to 11.14, in the case of Ch.The number of petioles per plant ranged from 9.2−1, in the case of the control plant, to 42.92 g·plant−1, in the case of Mo.The average weight of the petioles per plant was not significantly influenced by fertilization. However, this ranged from 39.11 g·plant−1, in the case of the unfermented treatment, to 59.16 t·ha−1, in the case of Ch, although the differences between treatments were not significant.The total yield ranged from 47.27 t·haCynara cardunculus L. [The influence of different types of fertilizers on production indicators has also been demonstrated in vegetable species such as culus L. ,53.−1, in the case of R4, up to 8044.75 kg·ha−1, in the case of the first harvesting season (R1) .The total quantity of stalks ranged from 1199.75 kg·hason (R1) . The higSalvia officinalis L., where phenophases led to higher production at the beginning of the harvesting period [Mouna et al. conducted a study on g period .−1, in the case of the control at R4, up to 11,997 kg·ha−1, in the case of chemical fertilization (Ch) from the ninth harvest (R9). Highest values were recorded under Ch fertilization at R7 and that with Og from R9, which showed positive increases, compared to the control from the fourth harvest time (R4)  .Corroborating the dynamics of the production with the climatic conditions, we can say that the production at the perennial plants has its peak at the beginning of the vegetation period when the average temperature increases above 5 °C and the soil has sufficient accumulated humidity from the winter season. The vegetative buds bloom in greater numbers, but they grow slower, they are more succulent, the petioles reach the normal size in a longer period, and they accumulate water in greater quantity, when the harvest is bigger. After harvesting and in more arid onditions, the plants grow slower; they recover in a longer period, because they start from dormant buds or those that appear in the respective year of culture.The application of chemical fertilizers, which have high solubility, made the production from the second part of the vegetation period bigger, and between the control and the types of fertilization at the beginning of the year, there were not very big differences, because the first petioles initially grow from the accumulated reserves in rhizomes in the previous year.Rheum rhabarbarum L.) using root cuttings of the Victoria cv. The cambic chernozem soil is characterized by a medium fertility, with 3.1% organic matter, 32% of clay and pH = 6.6. During the experimental period, the average temperature was 18.54 °C, the precipitation was 293.5 mm, and the relative humidity of the air was 66.8%  on rhubarb (as 66.8% .−2). Due to the dry agricultural year, three irrigations were practiced, each with 250 m3·ha−1, when the soil available water decreased below 80%. Flowering stems were removed 4 to 5 days after planting in order to favor the development of the edible part [The planting was practiced in 2012, with plants spaced 0.75 m along the rows, which were 1.00 m apart (density of 1.33 plants·mble part . During ®), and Organic (Orgevit®) treatments were compared to an unfertilized control (C). Chemical NPK 16-16-16 was produced by Ameropa Company® (Romania), Micoseeds MB® was from MsBiotech , and Orgevit® was from MeMon BV (Netherlands). The biological product consists of an arbuscular mycorrhizal fungus (Glomus spp.), PGPR , and a fungus (Trichoderma sp.) in different proportions. Organic fertilizer is chicken manure formulate with pH 7, 4% N, 2.5% P2O5, 2.3% K2O, 1% MgO, 0.02% Fe, 0.01% Mn, 0.01% B, 0.01% Zn, 0.001% Cu, and 0.001% Mo. All fertilizers were supplied to the soil. Thus, the Ch fertilizer was applied in an amount of 425 kg ha−1. The product beneficial microorganisms-based Micoseed MB (Mo) was used in the dose of 60 kg ha−1. Og was applied at the dose of 2400 kg ha−1. Ch and Og fertilizers were supplied after the first petiole harvest (03.04) in five stages ; Micoseeds MB® was applied in two stages, the first before planting (16.03) and the second one day after the first harvest (04.04) according to producer recommendations. For the determination of the doses of the fertilizers Og and Ch, the chemical composition of each product was taken into account, and for the Og, it was considered that 70% of the active substance of the product is assimilated in the first year after application.Three types of fertilization were compared in a split plot design experiment with three replications. Chemical (NPK = 16-16-16), Mycorrhizal-based formulate .In order to prepare the material for the laboratory analyses, 10 stalks from each repetition were selected randomly for a total of 40 pieces. The petioles were cut into 1 cm fragments for drying under normal weather conditions. The drying was carried out on a Sanyo oven, type MOV-112F, at a temperature of 70 °C up to the total loss of water from the dry material in order to determine the quantity of water and the dry substance. The samples were crushed into small fragments of 0.1–1 mm. The last step consisted in the packaging and labeling of variants in order to carry out laboratory analyses to determine the polyphenol content and antioxidant capacity in plants.In this application, the presence and content of different phenolic compounds in 70% ethanolic extracts were studied using an HPLC-MS, method which allows the simultaneous detection of several phenolic compounds with a single column pass .v/v). The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 min; then, it had 42% methanol for the next 3 min. The flow rate was 1 mL·min−1, and the injection volume was 5 μL [The identification and quantification of polyphenolic compounds was carried out using an Agilent Technologies 1100 HPLC Series system  equipped with a G13311A binary gradient pump, G1322A degasser, column thermostat, G1316A UV detector, and G1313A autosampler. The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap SL). For the separation, a reverse-phase analytical column was employed  and the work temperature was set at 48 °C. The detection of the compounds was performed in both UV and MS mode ,58,59. Twas 5 μL ,61.−1). A daily prepared solution of 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) showed a final absorption at 520 nm of 1.8 AU. The mixture was shaken and allowed to stand for 1 h at room temperature; then, the absorption was measured at 520 nm in a Lambda 25 spectrophotometer. The antiradical activity of the sample is inversely correlated with its purple color intensity. Aqueous solutions of Trolox at various concentrations were used for calibration (0.15–1.15 mmol·L−1). The results were expressed as μmol equivalents of Trolox  per g of sample  [The rhubarb stalks’ antioxidant capacity was assessed in terms of radical scavenging activity, following the procedure described by Re et al.  with sliapacity) .p ≤ 0.05 probability level.Data statistical processing was carried out by one-way and two-way ANOVA, and mean separations were performed through Tukey’s test using a SPSS version 21, referring to The present study confirms that fertilizer management method may have a significant impact on the phenolic composition of rhubarb. The fact that there are qualitative variations between polyphenols at the same harvest date, under same environmental conditions, on the same cultivar indicates that they are influenced by the treatment used.However, in addition, harvesting time under climatic conditions has an important effect on the quality and quantity of bioactive compounds.The research shows that the biological fertilizer had a positive effect on the total phenol content, directly influencing the antioxidant capacity from rhubarb, compared to the chemical and organic fertilizers, which are very important in the human diet.Biological fertilization provides satisfactory nutritional conditions for the accumulation of ferulic acid, p-coumaric acid, isoquercitrin, and quercetrol compared with rutoside, which accumulates in the highest quantities under organic fertilization. Polyphenolic compounds accumulate in petiols of rhubarb as follows: rutozide > isoquercitrin > ferulic acid > quercetrol > p-coumaric acid.Chemical fertilization provided the highest yields, regardless of the harvesting period, but the differences with respect to organic and biological treatments were not, which means that there are favorable conditions for using the two fertilizers in sustainable agriculture, without restriction.The treatments carried out and the harvesting period in accordance with the climatic conditions emphasize that there are the premises of promoting organic and biological treatments for a type of non-polluting, sustainable agriculture.


Adjustable crop load primarily involves bud manipulation, and usually switches from vegetative to reproductive buds. While this switch is not fully understood, it is still controlled by the ratio of hormones, which promote or inhibit bud formation. To determine the reasons for biennial bearing, the effect of apple rootstock, scion cultivar, crop load, as well as metabolic changes of endogenous phytohormones , and soluble sugars  were evaluated, and their connections with return bloom and yield of apple tree buds were analyzed. Cultivars “Ligol” and “Auksis” were tested on five rootstocks contrasting in induced vigor: semi-dwarfing M.26; dwarfing M.9, B.396, and P 67; and super-dwarfing P 22. Crop load levels were adjusted before flowering, leaving 75, 113, and 150 fruits per tree. Principal component analysis (PCA) scatter plot of the metabolic response of phytohormones and sugars indicated that the effect of the semi-dwarfing M.26 rootstock was significantly different from that of the dwarfing M.9 and P 67, as well as the super-dwarfing P 22 rootstocks in both varieties. The most intensive crop load (150 fruits per tree) produced a significantly different response compared to less intensive crop loads (113 and 75) in both varieties. In contrast to soluble sugar accumulation, increased crop load resulted in an increased accumulation of phytohormones, except for ABA. Dwarfing rootstocks M.9, B.396, and P 67, as well as super-dwarf P 22 produced an altered accumulation of promoter phytohormones, while the more vigorous semi-dwarfing M.26 rootstock induced a higher content of glucose and inhibitory phytohormones, by increasing content of IAA, ABA, and GAs. The most significant decrease in return bloom resulted from the highest crop load in “Auksis” grafted on M.9 and P 22 rootstocks. Average difference in flower number between crop loads of 75 and 150 fruits per tree in “Ligol” was 68%, while this difference reached ~ 90% for P 22, and ~ 75% for M.9 and M.26 rootstocks. Return bloom was dependent on the previous year’s crop load, cultivar, and rootstock. Apple rootstocks are categorized according to tree vigor, i.e., dwarfing, semi-dwarfing, semi-vigorous, and vigorous. As the production of dwarfing rootstocks has greatly increased the yield efficiency, fruit size, and quality of the commercial apple orchard, focus on rootstock-scion interactions have become increasingly important. Studies performed in Lithuania investigating the effect of rootstock on fruit quality, particularly with respect to bioactive compound accumulation, revealed that a significantly higher phenol content was found in “Ligol” fruits on P 61 and P 22 rootstocks, compared to “Auksis” fruits produced on P 67 rootstock . RootstoThe effect of different rootstocks on apple tree productivity was established in our rootstock trials performed in three countries. M.9 rootstock was the most productive under Lithuanian climate conditions; Pure 1, P 60, and B.9 were most productive in Latvia and Bulboga, while B.146 and M.26 were the most productive in Estonia . SignifiRootstock-cultivar interaction was found to be significant in evaluating the effect of rootstock on apple bearing stability . AlternaMdTFL1-1 which acts as a flowering repressor in apple  levels were found in root and xylem exudates of scion cultivars grafted on dwarfing rootstock, compared to those grafted on semi-vigorous rootstocks  produced by seeds was confirmed in heavily cropped “Fuji” trees  in 2014–2016. Apple cultivars “Ligol” (Poland) and ““Auksis” (Lithuania) on five rootstocks with contrasting induced vigor were tested: semi-dwarf M.26; dwarf M.9, B.396, and P 67; and super-dwarf P 22. The orchard was planted in 2005, at 4 m × 1.5 m intervals. Trees were trained as slender spindles. Pest and disease management was carried out according to the rules of integrated plant protection .−1 for “Auksis”, and 27, 41, and 54 t ha−1 for “Ligol”, respectively.Thinning of flower clusters and fruitlets was carried out before flowering at the pink bud stage and corrected after the June drop by adjusting three crop load levels: 75, 113, and 150 fruits per tree. Taking the average fruit weight into account, the expected yield at these crop levels was 21, 32, and 43 t haReturn bloom was evaluated a year following crop load adjustment, at the balloon stage of flowering, by counting all the flower clusters per tree.Bud development in September and November was defined according to 2 and extracted with 5 ml of cold 50% (v/v) acetonitrile. An internal standard solution mixture containing isotope-labeled phytohormones  was added to the samples. Samples were purified using Chromabond HLB SPE cartridges . Briefly, cartridges were conditioned with 2 ml MeOH and 2 ml H2O. Samples were applied to the cartridges, and the pass-through was collected. Sample elution was carried out with 30% acetonitrile, and each eluted sample was collected together with the pass-through. Samples were dried in a vacuum concentrator and dissolved in 50 µl of 30% (v/v) acetonitrile.Approximately 0.5 g of fresh plant tissue (90–120 buds per treatment) was ground with liquid N1, GA4, and GA7, IAA, JA, ABA, and zeatin) was performed using an Acquity BEH C18 column . The mobile phase was A: water [with 0.02% (v/v) acetic acid], and B: acetonitrile with 0.02% (v/v) acetic acid, at a flow rate of 0.1 ml min−1. Gradient was maintained at 5% B for 3 min, raised to 40% B in 12 min, raised to 90% B in 1 min, maintained at 90% B for 1.5 min, and equilibrated at 5% B for 4 min, before starting the next injection. Hormones were detected with the Bruker Ultra HTC ion trap mass spectrometer in negative mode, using multiple reaction monitoring (MRM).Phytohormone analysis was performed in buds collected in mid-September, according to the procedure described by 2O. The extraction process was carried out for 4 h at 20°C with mixing. Samples were centrifuged at 14,000 × g for 15 min. A clean-up step was performed prior to the chromatographic analysis. Briefly, 1 ml of supernatant was mixed with 1 ml of 0.01% (w/v) ammonium acetate in acetonitrile, and incubated for 30 min at +4 °C. The samples were centrifuged at 14,000 × g for 15 min and filtered through a 0.22-µm PTFE syringe filter . The analyses were performed using a Shimadzu HPLC instrument (Japan) equipped with an evaporative light scattering detector (ELSD). The separation of fructose, glucose, and sucrose was performed using a Shodex VG-50 4D HPLC column with deionized water (mobile phase A) and acetonitrile (mobile phase B) gradient. The gradient was maintained at 88% B for 13 min, changed linearly to 70% B in 9 min, maintained at 70% B for 1 min, raised back to 88% B in 2 min, and the column was equilibrated to 88% B for 5 min. The flow rate was set at 0.8 ml min−1.Approximately 0.5 g of fresh plant tissue (90–120 buds per treatment) was ground and diluted with deionized HThe experiment was carried out in a randomized complete block design with three replicates and one single tree in each plot. Data was processed using the XLStat software. Analysis of variance (ANOVA) was carried out along with the post-hoc Tukey’s HSD test for statistical analyses. Differences were considered to be significant at p < 0.05. Multivariate principal component analysis (PCA) was also performed. The agglomerative hierarchical cluster (AHC) analysis was used to generate similarity cluster diagrams based on the rootstock, crop load and metabolites similarity, and variables similarity.Evaluation of the effect of rootstock on apple return bloom showed that the average highest number of flower clusters for both tested cultivars was recorded in trees on M.9 and P 67 rootstocks Table 1.Evaluation of the effect of crop load on apple return bloom showed that both cultivars responded in the same way, i.e., the higher the crop load established in the previous year, the lower the recorded return bloom. Significant differences were established among all crop load levels. However, suppression of return bloom was different for both cultivars when comparing the lowest and highest crop levels. The return bloom of “Ligol” at a crop load of 150 fruits per tree was lower by 68%, compared to that obtained at a crop load of 75 fruits per tree, while return bloom of “Auksis” was lower only by 24%.Evaluation of the effect of rootstock and crop load interactions on cultivar response produced varying results. Return bloom of “Ligol” on all rootstocks significantly depended on crop load. Only crop load levels of 113 and 150 fruits per tree, established on B.396 rootstock, did not significantly differ. Differences between crop load effect on return bloom of “Auksis” were less pronounced. Significant differences were recorded by comparing only the highest and lowest crop load levels, with no differences found between adjacent crop load levels on almost all rootstocks, and no differences detected among all the levels on B.396 rootstock.Generative bud development in September and November was defined for both varieties Table 2.−1 FW in both varieties. Among the inhibitor phytohormones the highest accumulation was observed in IAA and GA7. IAA concentrations ranged from 36 to 270 ng g−1 FW, and from 76 to 310 ng g−1 FW, while GA7 concentrations ranged from 36 to 187 ng g−1 FW, and from 96 to 354 ng g−1 FW in “Ligol” and “Auksis”, respectively , soluble sugars , ratio of promoter to inhibitor phytohormones, and return bloom, in response to rootstocks with different growth vigor, and crop load. The first two factors (F1 vs. F2) of the PCA, as shown in the correlation circle  concentrations in both varieties. Other rootstock and crop load combinations distinguished in medium to high JA, GA7, and IAA concentrations. The return bloom was in the same cluster with IAA and GA7 (C3) in “Auksis” (7 in C3 clusters (7) resulted in higher values of return bloom and vice versa. Other tested phytohormones and sugars were in C1 in both varieties. In contrast to “Auksis”, during generative bud development in September  levels induces growth cessation and bud set. GA4 treatment led to the enhancement of several energy metabolism pathways, including those associated with sugar metabolism  and soluble sugars (glucose and sorbitol)  . Fadón eorbitol) . Li et a1 accumulation  produced a significantly different response compared to less intensive crop loads (113 and 75) in both varieties. In contrast to soluble sugar accumulation, increased crop load resulted in increased levels of phytohormones, but did not affect ABA accumulation.Dwarfing rootstocks M.9, B.396, and P 67, as well as the super-dwarf P 22 resulted in altered accumulation of promoter phytohormones, while the more vigorous semi-dwarfing M.26 rootstock induced a higher content of glucose in “Ligol” buds, of glucose and sorbitol in “Auksis” buds, and of inhibitory phytohormones, by increasing the levels of IAA, ABA, and GAs. Therefore, soluble sugars, especially glucose, and hormonal pathways were interconnected and acted together to control flower induction, while being both rootstock- and crop load-dependent.Apple return bloom was dependent both on rootstock and crop load. Interactions between rootstocks and cultivars were established: M.9 rootstock determined the highest return bloom for “Auksis”, and P 67 rootstock for “Ligol”. The lowest return bloom was recorded on the super dwarfing P 22 rootstock in “Ligol”, and on the dwarfing B.396 in “Auksis”. Comparing the effect of rootstock on return bloom under different crop load levels showed that the most significant decrease in return bloom resulted from the highest crop load in “Auksis” grafted on M.9 and P 22 rootstocks, and in “Ligol” grafted on P 22 rootstock.All datasets presented in this study are included in the article/DK—trial setup, field trials, data analysis, and writing of the manuscript. GS—chromatographic analysis, data analysis, and writing of the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by a grant (No. MIP-036-2014) from the Research Council of Lithuania.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nature Communications 10.1038/s41467-020-18551-0, published online 1 October 2020.Correction to: 4− in Fig. 1b, which should read 85, which should be HOIO2.The manuscript contains two instances in which chemical compounds are incorrectly presented. These are HSOFurthermore, there should not be a hyphen between 30 and nm on page 6, line 2. In addition, there is inconsistent text alignment in the “Instrumentation and measurements” section in which full justified and left aligned text are mixed.This has now been corrected in both the PDF and HTML versions of the Article.


Apis mellifera (AmCA) spermatheca and inhibitory effects of pesticides belonging to various classes, such as carbamates, thiophosphates, and pyrethroids, were investigated herein. The inhibitory effects of methomyl, oxamyl, deltamethrin, cypermethrin, dichlorodiphenyltrichloroethane (DDT) and diazinon on AmCA were analysed. These pesticides showed effective in vitro inhibition of the enzyme, at sub-micromolar levels. The IC50 values for these pesticides ranged between of 0.0023 and 0.0385 μM. The CA inhibition mechanism with these compounds is unknown at the moment, but most of them contain ester functionalities which may be hydrolysed by the enzyme with the formation of intermediates that can either react with amino acid residues or bid to the zinc ion from the active site.Carbonic anhydrase  plays crucial physiological roles in many different organisms, such as in pH regulation, ion transport, and metabolic processes. CA was isolated from the European bee  Some pesticides are persistent organic contaminants in the soil and environment. They also constitute one of the most significant causes of pollution worldwide2/bicarbonate levels2 to HCO3− interconversion, thus, playing vital functions in various biochemical/physiological processes, including physiological pH regulation, gas balancing, calcification, photosynthesis, metabolism, etcCarbonic anhydrases  are found in almost all living organisms and control pH and COPesticides and fungicides interfering with rainwater, irrigation water, or groundwater, plants, can inhibit particular enzymes2.2.1.All chemicals for the affinity system were provided by Sigma-Aldrich . Other reagents were obtained from Merck .2.2.g for 30 min. The precipitate portion was discarded and the supernatant portion was separated and used in subsequent studies.Bee spermatheca samples were washed three times with 50 mM Tris/Sulphate (pH 7.8). Spermatecha samples taken from 200 bees were combined and homogenised with liquid nitrogen, then placed in the same buffer and centrifuged at 4 °C, 15,000 2.3.2SO4 (pH 8.7) and the gel was washed with 25 mM Tris-HCl/22 mM Na2SO4 (pH 8.7). Finally, elution was performed with 1 M NaCl/25 mM Na2HPO4 (pH 6.3). The temperature was kept at 4 °C during all experiments.The enzyme was purified using Sepharose-4B-sulfanilamide affinity gel prepared by our group2.4.2 hydrase assay50) of the enzyme activity were thus obtained graphically using a regression software. The enzyme concentration in the assay system was 8.9 nM.The effects of methomyl, oxamyl, deltamethrin, dichlorodiphenyltrichloroethane (DTT), cypermethrin and diazinon on AmCA activities were assayed colorimetrically using the CO3.Apis mellifera) colonies poses an important problem in many developed countriesThe collapse of honeybee , cypermethrin, and diazinon, respectively  rates found in this study50 for the inhibition of AmCA with pesticides presented in It should be mentioned that a detailed study on pesticide residues in bees was conducted by Sanchez-Bayo and GokaThere has been a great interest in recent studies focussed on the effects of several classes of chemicals on CA enzymesThis also brings us to the possible mechanism of action of these pesticides investigated here. The carbamates, thiophosphates, and pyrethroids investigated here  possess 2N–COCOOH in the case of the second carbamate). The pyrethroids may also be hydrolysed at their ester functionality, with the generation of carboxylic acids and alcohols which were shown to act as CAIs,,,The two carbamates from 4.Apis mellifera spermatheca by affinity chromatography. Pesticides such as methomyl, oxamyl, deltamethrin, and DDT showed inhibition effects, comparable to those of cypermethrin and diazinon. The IC50 values were determined as 0.0023 ± 0.0001, 0.0025 ± 0.0001, 0.0028 ± 0.0001, 0.0034 ± 0.0001, 0.0078 ± 0.0003 and 0.0385 ± 0.0012 μM, respectively (in vivo (or in the hive) validation.AmCA was purified from ectively . Our res