Atmospheric particles were clearly scattered on the leaf surface, and the particles were smaller than the stomata. Considering the Pb and Cd contents in the leaves and roots, stomata width-to-length ratio, leaf area size, enrichment factor, and translocation factor, Yidianhongxiancai, Qingxiancai, Baiyuanyexiancai, Nanjingjiangengbai and Sijixiaobaicai were recommended for planting in kitchen gardens and suburban areas as they have low accumulation characteristics. Identifying the influencing factors in the accumulation of heavy metals in vegetables through foliar uptake is important to help plant physiologists/environmentalists/policy makers to select suitable varieties for planting in air-polluted areas and thus reduce their threat to human health.Leafy vegetables cultivated in kitchen gardens and suburban areas often accumulate excessive amounts of heavy metals and pose a threat to human health. For this reason, plenty of studies have focused on low accumulation variety screening. However, identifying specific leafy vegetable varieties according to the foliar uptake of air pollution remains to be explored . Therefore, in this study, the lead (Pb) and cadmium (Cd) contents, leaf morphology, and particle matter contents were analyzed in a micro-area experiment using 20 common vegetables. The results show that the Pb content in leaves ranged from 0.70 to 3.86 mg kg Among all crop varieties, heavy-metal overaccumulation is particularly significant in vegetables and rice. The term “lead vegetables” is often used in China to describe heavily lead-contaminated vegetables, as is the term “cadmium rice” . Lead (P62-2017) ,14. Huan62-2017) . Therefo2.5 [The existing studies mainly focus on heavy-metal uptake from soil by roots and explore the mechanisms involved in root uptake, translocation, and detoxification . On this2.5 . He et a2.5 . Overall2.5 . In addiIpomoea aquatica Forssk), amaranth (Amaranthus tricolor), cabbage (Brassica pekinensis)), and the particulate matter content and leaf morphology were analyzed in a micro-area experiment with the aim of: (1) identifying the degree of Cd and Pb accumulation in atmospheric-plant systems and their risks to the population; (2) exploring the differences in foliar uptake and influencing factors among these varieties; and (3) recommending suitable varieties based on foliar uptake characteristics to maintain food safety.This led is to hypothesize that the accumulation of heavy metals in vegetables may be due to both root uptake from soil and foliar uptake from the atmosphere, especially for leafy vegetables. Therefore, in this study, the Pb and Cd contents in 20 common vegetables , amaranth , and cabbage —were selected and the treatment was repeated three times. Each vegetable was randomly planted in a 2 m × 2 m field. Vegetable seeds were sown on 2 June 2019 and harvested on 2 September 2019. The basic physical and chemical properties of the soil that we analyzed were organic matter (11.6 ± 0.78 g kg−1), pH (8.08 ± 0.06), total nitrogen (0.82 ± 0.20 g kg−1), phosphorus (484 ± 0.18 mg kg−1), potassium (17.4 ± 0.11 g kg−1), total Pb (24.31 ± 0.19 mg kg−1), and total Cd (0.14 ± 0.04 mg kg−1).The study was conducted in the experimental base of Hunan Agricultural University Changsha City, southern China . This area is in a suburban area and beside a city road. The airborne Pb and Cd originated from natural atmospheric deposition, which was mainly emitted from traffic. The leading wind direction in the area is primarily southeasterly in summer and northeasterly in winter. The annual average temperature is 16–18 °C, with an annual average humidity of 70%–80%. The average annual precipitation ranges from 1400 to 1700 mm. Three species of 20 common edible vegetables —water sp−1 with 50% cutoff sizes of <10.0 µm (PM10) and total suspended particles (TSP). The particulate matter samples were digested using an HNO3-HCl (3:1) mixture at 105 ± 5 °C for 2 h, cooled to room temperature, and diluted with deionized water to 30 mL. The aqueous samples were first acidified with sub-boiling quartz distilled 6 M HCl, and then digested using 2 mL HNO3 (1:1) and 1 mL HCl (1:1) at 85 °C until the sample evaporated to 20 mL. Sample replicates and reagent blanks were included in each batch of analysis to ensure the quality of the analysis.The particulate matter samples (PM10 and TSP) were collected by two automatic precipitation and dust fall samplers each day from June 2019 to August 2019. The airflow rate was 28 L min3-H2O2-HF (6:3:3), and plant materials were digested with HNO3-H2O2 (2:1). The digested solutions were diluted to 25 mL with 1% HNO3 solution. All of the samples were stored in the dark at 4 °C and were determined within one month. To ensure the reliability and quality of the data, standard reference soil (GBW07387) and bush twigs and leaves (GBW07602) from the Center of National Reference Materials of China were measured together with soil and plant samples.Vegetable samples were collected at the maturity stage using a crossover method and the samples were rinsed thoroughly with deionized water to remove any attached soil/substrate particles before being separated into shoots and roots. At the same time, surface soil (0–20 cm) samples were collected from each plot. The soil samples were dried at room temperature and homogenized using a mortar and pestle. One portion of the sample was passed through 2 mm sieves to measure the pH content, and another was passed through 0.145 mm sieves to determine the Pb and Cd contents. Subsamples of shoots and roots were firstly dried in oven at 105 °C for 30 min and then stored in oven at 80 °C until reaching a constant weight. They were then crushed with prototype powder. Soil samples were digested with a mixture of HNO−1 and 0.01 µg mL−1 for Pb and Cd, respectively.The Pb and Cd concentrations in solutions were determined using an inductively coupled plasma mass spectrometer . The recovery of spiked standard for each element ranged between 80% and 120% and the detection limits were 0.01 µg mL2) were trimmed from the area between the margin and mid-rib. The small strips were first stored in 2.5% glutaraldehyde solution overnight for fixation (prefixation), and then in osmium tetroxide for post-fixation for 1 h. After being washed twice with buffer solution for 15 min, these samples were passed through a series of acetone solutions  for dehydration. Then, they were dried in a critical point drier (CPD) with CO2 as the carrier gas. The SEM was carried out using a 30 keV, JEOL JSM-6490 LV scanning microscope  with standard automated features such as auto focus/stigmator, auto gun, and auto-contrast with multiple live image display. The samples were coated with carbon using a high vacuum system to wet specimens, the upper surfaces of the leaf segments were studied, and micrographs were taken at various magnifications.Vegetable leaves were observed using a scanning electron microscope (SEM), as described previously . Small sBu et al. (2014) used width-to-length ratio to represent the degree of stomatal opening and closing . The stoAccording to the principle of the mass density formula, a piece of uniform quality paper  was selected, the rectangle was divided, the length and width was recorded, its area (length × width) was calculated, and then it was weighed. Thereafter, the outline of the leaf was drawn on the paper, it was cut out, and then it was weighed. The leaf area was calculated by dividing the weight of the leaf contour paper by the weight of the entire paper and multiplying this by the area of the entire paper . When me−1 day−1)) to the reference dose ). If the EDD is lower than the RfD, there are considered to be no health risk. Reference doses were based on 0.004 and 1 × 10−3 mg kg−1 day−1 for Pb and Cd, respectively [−1 for adults, 66.9 g day−1 for children), conversion factor (0.085 mg kg−1), exposure frequency (365 days year−1), exposure duration (70 years), life expectancy , and average body weight , respectively [The health risk from heavy metals are mainly derived from three pathways: the ingestion of the contaminated crops, inhalation, and dermal contact with the polluted particulate matter. The health risk can be estimated using the ratio of the estimated daily dose EDD=Cectively .−1 for soil, ug m−3 for air). Fe was selected as a reference element for geochemical normalization due to its uniform natural concentration and natural abundance in the Earth’s crust [The enrichment factor (EF) was used to differentiate between natural and anthropogenic sources. It was calculated using the following equation :(2)EF= using a least significant difference (LSD) test at a significance level of  package .−3 day−1 and 0.2050 (0.0568–0.4815) µg m−3 day−1, respectively (−3 day−1 and 0.0017 (0.001–0.0046) µg m−3 day−1, respectively. As compared with the first level of the ambient air quality standard , the concentrations of TSP-Pb and PM10-Pb were both lower than the standard, and first exhibited an increasing trend and then a decreasing trend from July to August. The concentrations of TSP-Cd and PM10-Cd tended to be more stable and evenly distributed throughout the growth period and did not exceed the secondary ambient air quality standard . In addition, the concentrations of Pb and Cd in PM10 were significantly higher than those in TSP, indicating that Pb and Cd were mainly concentrated in fine particles.It can be seen that the concentrations varied greatly at different times. During the test days, the average concentration of Pb in TSP and PM10 was 0.1602 (0.0224–0.4936) µg mectively . The con−1, of which C5 had the lowest concentration (0.22 mg kg−1), while C3 had the highest (57.84 mg kg−1). Overall, the concentration exhibited the following trend: cabbage (14.07 mg kg−1) > water spinach (2.34 mg kg−1) > amaranth (1.09 mg kg−1), and the contents of Pb in roots exhibited the biggest variation among cabbages. The contents in C3 and C2 were significantly higher than C1, and they were significantly higher in these three than in the others cabbage species. Furthermore, there was no significant difference between or among amaranth and water spinach. The contents in leaves ranged from 0.70 to 3.86 mg kg−1, of which C7 had the lowest concentration (0.70 mg kg−1), while B4 had the highest (3.86 mg kg−1). Overall, the concentration exhibited the following trend: amaranth (1.77 mg kg−1) > cabbage (1.38 mg kg−1) > water spinach (1.20 mg kg−1), which was not consistent with Pb in roots. Moreover, the contents of Pb in leaves did not exhibit significant variation among varieties. The accumulation of Pb was biggest in B4, while it was lowest in C12, C8, and C7.The concentrations of Pb in dry-weight roots and leaves are shown in −1 fresh weight. The monitored vegetables contained about 90% water. Therefore, in these 20 varieties, only B4 samples (3.86 mg kg−1) exceeded the MPC and posed potential health risks. Moreover, the C3, C2, and C1 cabbage species were close to the MPC.According to limits of contaminants in food , the maximum permissible concentration (MPC) for Pb in leafy vegetables is 0.3 mg kg−1 in the roots of the 20 varieties, of which C1 had the lowest concentration (0.10 mg kg−1), while C3 had the highest (13.58 mg kg−1). Overall, the concentration exhibited the following trend: cabbage (0.67 mg kg−1) > amaranth (0.42 mg kg−1) > water spinach (0.26 mg kg−1), and the contents of Cd in roots exhibited the biggest variation among cabbages. C1 was significantly higher than C3 and C2, while these three were significantly higher than the other cabbage species; moreover, C8 was significantly higher than C5 and C4. In addition, there was no significant difference between or among water spinach. B5 was significantly higher than B4, B2, and B1 in amaranth. The contents ranged from 0.21 to 0.99 mg kg−1 in leaves of the 20 varieties, of which A2 had the lowest concentration (0.21 mg kg−1), while C2 had the highest (0.99 mg kg−1). Overall, the concentration exhibited the following trend: cabbage (0.67 mg kg−1) > amaranth (0.42 mg kg−1) > water spinach (0.26 mg kg−1). This was consistent with that for Cd in roots.The concentration of Cd in dry-weight roots and leaves are shown in −1 fresh weight. The monitored vegetables contained about 90% water. Therefore, the contents of Cd in all 20 varieties did not exceed the standard.According to limits of contaminants in food , the maximum permissible concentration (MPC) of Cd in leafy vegetables is 0.2 mg kg−5, 2.16 × 10−5, and 1.68 × 10−5 mg kg−1 day−1 for adults and 2.78 × 10−5, 4.11 × 10−5, and 3.20 × 10−5 mg kg−1 day−1 for children, respectively, with the following trend: amaranth> cabbage> water spinach. However, for Cd, the average daily intakes of the three major types of vegetables were 3.17 × 10−6, 5.10 × 10−6, and 8.14 × 10−6 mg kg−1 day−1 for adults and 6.04 × 10−6, 9.71 × 10−6, and 1.55 × 10−5 mg kg−1 day−1 for children, respectively, with the following trend: cabbage> amaranth> water spinach. In general, the reference doses for Pb and Cd are 0.004 and 1 × 10−3 mg kg−1 day−1, respectively [The estimated daily dose (EDD) describes the contents of a certain element in the body’s daily vegetable intake, expressed as the amount of accumulated contaminants in the human body during daily vegetable intake. It can be seen from ectively ,28. All The enrichment factor (EF) was calculated as the heavy-metal concentrations in shoots as compared to the concentrations in soil/atmosphere. The EFs of Pb and Cd in shoots as compared to atmospheric deposition and soil are shown in Allium ascalonicum L. (leafy vegetables) was 1.258, exhibiting higher a value than Raphanus sativus L. and Daucus carota L. (non-leafy vegetables) [Enrichment factor (EF), which refers to the content of elements in plants as compared to that in the environment, can be used to evaluate the ability of heavy-metal accumulation . It is wetables) . TherefoTranslocation factor (TF), which refers to the content of elements in leaves compared to in roots, can be used to evaluate the ability of heavy-metal translocation. A TF >1.0 indicates preferential partitioning of metals from roots to shoots . TherefoThe scanning electron micrographs of the 20 varieties leafy vegetables are shown in −1) > cabbage (1.38 mg kg−1) > water spinach (1.20 mg kg−1) for Pb, and cabbage (0.67 mg kg−1) > amaranth (0.42 mg kg−1) > water spinach (0.26 mg kg−1) for Cd  used the width-to-length ratio to represent the degree of stomatal opening and closing . This st) for Cd , which i14 used tracea L. . TherefoIn order to explore the influencing factors of heavy-metal accumulation in leafy vegetables, stomata width-to-length ratio, leaf area size, EF, and TF were measured. Combined with various indicators, a clustering heat map was generated to illustrate the phenomenon . It can pakchoi was due to the high transpiration rate, which helped to translocate Cd from roots to leaves [In addition to foliar uptake, heavy-metal uptake from roots was another major pathway of heavy-metal accumulation in leaves. It can be seen that C2 and C3 in o leaves . TherefoIn order to explore the influencing factors of Pb and Cd accumulation in leafy vegetables and screen suitable varieties based on foliar uptake, 20 varieties of leafy vegetables were grown in a micro-area experiment. This study creatively investigated the contribution of foliar uptake to heavy-metal accumulation in leafy vegetables and explored the effect of leaf area and stomata size on heavy-metal accumulation in leafy vegetables. It can be seen that foliar uptake was an important source of Pb and Cd accumulation in leafy vegetables. The study indicated that water spinach accumulated enormous amounts of Pb through foliar uptake, and the amount mainly depended on the size of stomata. B1, B2, B5, C4, and C12 demonstrated a weaker ability to absorb heavy metals through foliar uptake and are recommended to be planted in areas prone to air pollution. Furthermore, as a result of the strong root uptake, C1, C2, and C3 were deemed unsuitable for planting in areas with contaminated soil. The results provide technical support for the safe production of leafy vegetables. However, the various factors that affect foliar uptake are complicated and include leaf inclination, branch density, plant canopy structure, leaf area, and stomata size, etc. Therefore, further studies, including microscopic or quantitative methods, are required to explore the mechanism of foliar uptake and quantify its contribution to heavy-metal accumulation in leaves.


An adequate hydration status is critical to ensure efficiency during mental and physical activities. Our goal was to assess the hydration status of a Spanish group of aeronautical military men and to determine the association of hydration status with body composition and anxiety.A total of 188 men were evaluated through a validated hydration questionnaire, anthropometric and biochemical parameters, and an anxiety questionnaire. Based on these methods, the criteria of hydration were established.P = 0.026), weight [(84.7 ± 10.5) vs. (80.5 ± 10.2) kg], body mass index [(26.3 ± 3.1) vs. (25.2 ± 2.8)]kg/m2, body fat [(22.3 ± 5.6) vs. (18.3 ± 6.5)] %, urine specific gravity, and urine color. Using a logistic binary regression model, hydration status was related significantly with the percentage of body fat (P = 0.004), but no relation was found with age, comorbidities, or medications.Of the total sample, 81% met the hydration criteria , and 19% did not meet the hydration criteria . Subjects not meeting the hydration criteria had lower urine pH, negative water balance, and lower water intake. The latter also had higher anxiety status  and negatively with body fat (kg) , percentage of body fat , and waist/hip ratio . Based on a linear regression model, total water intake/weight was related significantly with percentage of body fat (P = 0.001) and percentage of body water content (P = 0.035). No relation was found, however, with waist/hip ratio, age, comorbidities, or medications.Furthermore, total water intake/weight was positively correlated with percentage of body water (These findings all suggest a relationship between hydration status and body composition but also set the bases for future studies that relate hydration status and anxiety status. These results can be used to improve the hydration status and body composition of military personnel. Optimal functioning of the human body requires an adequate hydration level  given byTo date, no study has assessed the hydration status (HS) of military personnel in Spain. For these reasons, the purpose of the present study was to evaluate, for the first time, the hydration status of an aeronautical military personnel group in Spain, through a complete and validated questionnaire of hydration  as well Centro de Instrucción de Medicina Aeroespacial (CIMA), Madrid. From March 2018 to June 2018 , subjects from all over Spain were informed of the study and invited to participate. The inclusion criteria were good mental and physical health . Exclusion criteria were (a) diseases or any special health conditions that limited their flight license, and/or (b) diseases related to hydration status, including renal impairment, urinary tract infection, water balance disease, and diabetes. This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Clinical Research Ethics Committee of the CEU San Pablo University (Madrid). The corresponding ethics code is 119/16/08. All the aeronautical personnel who participated in the study did so on a voluntary basis, and their written informed consents were obtained prior to the study.The present study was a cross-sectional observational study of a sample of Spanish aeronautical military personnel who attend annual medical check-ups at the official The military aeronautical personnel attended the CIMA for a 2- to 3-day medical check-up period. Recruitment started the first day of medical check-ups for each group and included a volunteer recruitment briefing with infographics of the procedure of the study. From March 2018 to June 2018, volunteers that expressed interest in participating in the study received the study protocol in writing as well as verbal responses to any questions. Of the total number of subjects, 6% did not consent to participate in the study or could not participate because they did not meet the inclusion criteria.During the first day, the participants completed the self-reported questionnaires under the supervision of trained dietitian personnel.The “hydration status questionnaire” (HSQ), validated by the Nutrition and Food Science research group of CEU San Pablo University  includedA specifically designed questionnaire evaluating physical activity through a brief register of working day and non-working day activities was used. In this questionnaire, a total of 24 h were divided into sleeping, eating, work activity, sitting or lying , walking or other modes of transport, other activities , and sports (specifying which). The total daily energy expenditure (TDEE) was calculated according to their respective metabolic equivalent (MET) intensity levels  , in whic:00–16:00Armstrong et al.  indicate2 (m2), and compared to WHO reference values . Differences between normally distributed variables were derived using Student’s Correlation analysis was performed using Spearman’s coefficient or Pearson’s coefficient according to variable normality. All the statistical analyses were performed using SPSS 24.0 Software .A linear regression model was constructed to explore the associations between water intake adjusted by body weight as a dependent variable, and the body composition variables age, co-morbidity, and medications as independent predictors. Furthermore, a logistic binary regression model was used to explore associations between hydration status and body composition variables (body fat percentage), age, co-morbidity, and medication as independent predictors.2 was considered in the lower range of overweight according to reference values. However, we observed a percentage of body fat (19.1 ± 6.5) % established as normal in adult men. Furthermore, the waist/hip ratio (0.87 ± 0.05) in the total sample showed no risk factors for cardiovascular disease. Percentage of body water (60.4 ± 7.5)% in the total sample was also in the range of normal values 55–65% , with a TDEE of 3158.7 (333.9) kcal/d. Of the total sample, 18% presented co-morbidities, and 16% took medications unrelated to hydration status. The body weight of the participants was (81.6 ± 10.3) kg, while body height was (178.7 ± 6.2) cm. A total of 50.0% of the sample showed normal weight values, 42.6% were overweight, and 7.4% were categorized as obese according to their BMI values. Mean BMI (25.4 ± 2.9) kg/ms 55–65% .Moreover, blood indices  were within the physiological range , as werer = 0.357, P = 0.000) and negatively with body fat , percentage of body fat , waist , hip , and waist/hip ratio .Water from beverages (2709.5 ± 943.6) ml/d, water from foods (795.5 ± 413.7) ml/d, total water intake (3508.9 ± 1112.1) ml/d, water elimination (3402.4 ± 1086.5) ml/d, water balance (88.5 ± 1294.3) ml/d, and total water intake/weight (43.7 ± 14.9) ml/kg were estimated using the HSQ. Total water intake/weight was correlated positively with percentage of body water (P = 0.001) and percentage of body water (P = 0.035). No relation was found with waist/hip ratio, age, comorbidities, or medications.To evaluate the independent relation between total water intake/weight and body composition variables , age, co-morbidities, and medications, a linear regression model was used Table . The tot2, body fat [(22.3 ± 5.6) vs. (18.3 ± 6.5)]%, and waist/hip ratio [(0.9 ± 0.1) vs. (0.87 ± 0.1)], and lower body water [(58.1 ± 7.2) vs. (60.9 ± 7.5)]% than subjects that met the hydration criteria.It was found that 81% of the total population met the hydration criteria, whereas 19% did not. Table P = 0.002), water from food (P = 0.005), total water intake (P = 0.000), water balance (P = 0.034), and total water intake/weight (P = 0.000); than subjects that met the hydration criteria. Also differences were showed in urine markers, between subjects that did not meet the hydration criteria and subjects that meet the hydration criteria: pH ; specific gravity , and urine color . However, differences in blood indices were not observed in either group .Results of WI, WE, and WB are presented in Table P = 0.004), whereas no relation was found with age, comorbidity, or medications.To evaluate the independent relation between HS and body composition variables (percentage of body fat), age, comorbidities, and medications, a logistic binary regression model was used Table . The HS Anxiety results showed a low level of anxiety in the total population: score 3  for STAI-State, and score 2  for STAI-Trait score. No correlation between STAI (STAI-State and STAI-Trait) and any HS variable was found.P = 0.026), where subjects that did not meet hydration criteria had higher scores [4  vs. 3 ] than the group that met the hydration criteria. However, no differences were shown in the anxiety test STAI-Trait between subjects that did not meet hydration criteria and subjects that met the hydration criteria .In the HS category, significant differences were noted in the anxiety test STAI-State  , the NatOn the other hand, mean daily WE was 3402.4 ml/d for the whole sample. WE data were in accordance with the current literature, which is 1500–3100) ml/d for adults , 15. In 00–3100 mLaboratory data for urine  are considered good markers to evaluate HS , 52. In Blood parameters (hematocrit and hemoglobin) were analyzed due to their potential as HS markers ; howeverIn our population, 81% of the participants met the established hydration criteria, and 19% did not meet the hydration criteria. Lower results were observed in Belgian military personnel, 13.6% of the sample were classified as dehydrated (using USG of first urine of the morning) . A US miRegarding anthropometric parameters, BMI suggested overweight values in the total sample. Conversely, percent body fat was considered in the range of healthy values. Several other studies found similar results and an inability to distinguish the different contributions of body weight and fat and non-fat tissue, which explains why the BMI might overestimate adiposity in muscular and lean bodies in active people , 57. BodIn our study, subjects that did not meet the hydration criteria had significantly higher scores in STAI-State than subjects that met the hydration criteria, indicating a possible relationship between these two variables that should be carefully further evaluated. However, in other studies, the relationship between hypohydration status and degraded mood was consistent , 59. In The strengths of this study include the novelty of researching a vulnerable and high-risk group, such as aeronautical military personnel, as well as the careful protocol and methodology used.Even though the present study’s protocol was well controlled, some limitations should be acknowledged. Regarding the HS, the selection of hydration markers may have limitations. The most important limitation refers to the impossibility of directly assessing water consumption, since it was estimated through a validated questionnaire. Other limitations include the measurement of USG through reagent strips instead of refractometry, and the non-availability of 24 h urine samples.Our results indicate that there is a clear association between hydration status and body composition, but also set the bases for future studies that relate hydration status and anxiety status in the military personnel evaluated. These results can be used to highlight the importance of an adequate hydration status and body composition for military personnel, to maintain physical and mental performance for their professional career and development.


Zanthoxylum nitidum (Roxb.) DC. (ZN), with strong effects of anti-inflammation and antioxidant activities is treated as a core herb in traditional Chinese medicine (TCM) preparation for treating stomachache, toothache, and rheumatoid arthritis. However, the active ingredients of ZN are not fully clarified due to its chemical complexity. In the present study, a double spectrum–effect analysis strategy was developed and applied to explore the bioactive components in herbs, and ZN was used as an example. Here, the chemical components in ZN were rapidly and comprehensively profiled based on the mass defect filtering-based structure classification (MDFSC) and diagnostic fragment-ion-based extension approaches. Furthermore, the fingerprints of 20 batches of ZN samples were analyzed by high-performance liquid chromatography, and the anti-inflammatory and antioxidant activities of the 20 batches of ZN samples were studied. Finally, the partial least squares regression (PLSR), gray relational analysis models, and Spearman’s rank correlation coefficient (SRCC) were applied to discover the bioactive compounds in ZN. As a result, a total of 48 compounds were identified or tentatively characterized in ZN, including 35 alkaloids, seven coumarins, three phenolic acids, two flavonoids, and one lignan. The results achieved by three prediction models indicated that peaks 4, 12, and 17 were the potential anti-inflammatory compounds in ZN, whereas peaks 3, 5, 7, 12, and 13 were involved in the antioxidant activity. Among them, peaks 4, 5, 7, and 12 were identified as nitidine, chelerythrine, hesperidin, and oxynitidine by comparison with the standards and other references. The data in the current study achieved by double spectrum–effect analysis strategy had great importance to improve the quality standardization of ZN, and the method might be an efficiency tool for the discovery of active components in a complex system, such as TCMs. Generally, traditional Chinese medicines (TCMs) achieve their therapeutic effects by initial interactions via multicomponents and multitargets. To understand how it works, it is necessary for researchers to study the relationship between TCMs’ compounds and their efficacy from a holistic perspective . A pharmZanthoxylum nitidum (Roxb.) DC. belongs to the genus Zanthoxylum of family Rutaceae -grade acetonitrile and methanol were bought from MEDIA . Formic acid was HPLC grade and purchased from Aladdin . Chelerythrine, diosmin, hesperidin, nitidine chloride, sanguinarine, magnoflorine, and dihydrochelerythrine, all with purity of ≥98%, were obtained from Chengdu Push Bio-Technology . Deionized water was purified using a Milli-Q water purification system . Dulbecco’s modified Eagle’s medium (DMEM) was produced by Gibco , and DPPH was produced by Tokyo Chemical Industry .After being ground into powder and air-dried (filtered with a 50-mesh sieve), each ZN sample (5 g) was dissolved with 100 ml of 80% ethanol and extracted three times at 3, 2, and 1 h on a water bath at 80°C. Then a Rotavapor OSB-2200 from EYELA Co.  was used to remove the solvent. The extracts were powdered by an FD5-series vacuum freeze dryer  and then stored for use.The comprehensive characterization of ZN samples was conducted on an Agilent 6545 UHPLC Q-TOF MS  with the monitoring of Agilent LC-QTOF/MS Mass Hunter Workstation Acquisition Software Version B.05.01 . Tested samples were separated on an Agilent Zorbax clipse Plus C18 column . The mobile phase consisted of acetonitrile . The mobile phases were 0.1% formic acid (A) and acetonitrile (B) with the following gradient elutions: 5–15% B linear 0–3 min, 15–20% B linear 3–6 min, 20–22% B linear 6–7 min, 22% B isocratic 7–9 min, 22–26% B linear 9–12 min, 26–28% B linear 12–13 min, 28% B isocratic 13–15 min, 28–34% B linear 15–16 min, 34–38% B linear 16–18 min, 38–40% B linear 18–20 min, 40–55% B linear 20–24 min, 55–65% B linear 24–25 min, 65–85% B linear 25–28 min, and 85–95% B linear 28–30 min. The injection volume and the detection wavelength were set as 0.6 μl and at 254 nm, respectively. The flow rate was 0.6 ml/min, and the column temperature was maintained at 35°C.2) pressure, 30 psig; drying gas flow rate, 8.0 L/min; drying gas (N2) temperature, 320°C; shealth gas flow rate, 12.0 L/min; shealth gas (N2) temperature, 350°C; OCT RF V, 750 V; skimmer, 65 V; fragmentor, 135 V. The scan ranges for product ions were m/z 100–3,000, and the collision energy was set at 20, 40, and 60 V. Before the analysis, mass spectrometer of the TOF was calibrated at m/z 121.0508 and 922.0098 in positive ion mode to ensure the mass accuracy. Data acquisition and analysis were obtained by Agilent LC-MS MassHunter Workstation Software (version B.08.00).Both MS and MS/MS were performed in positive ion mode, and the ion source is dual AJS ESI. The MS parameters were set as follows: capillary voltage, 3,500 V; nebulizer gas . Chromatographic separation was conducted on a column of symmetry columns . The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile (B). The gradient conditions are shown in vitro screening method for evaluating the radical scavenging activity  was measured using nitric oxide assay kit  according to the manufacturer’s instructions. The OD value was measured at 540 nm with a microplate reader . The calculation formula of inhibition rate of NO production is as follows:m is the NO concentration of the model group, Cs is the NO concentration of the samples, and Cn is the NO concentration of the normal control group. Finally, the original cells in 96-well plates were used to determine the cell viability by an MTT assay (Raw264.7 cells (100 μl) were seeded at 2.5 × 10TT assay .Twenty batches of ZN samples were chemically profiled and matched automatically by using the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012.” Then the common peaks of 20 fingerprints were calculated by the multipoint method, and the control chromatogram was generated automatically by the average method.The HCA of different batches of ZN samples was carried out with the SPSS statistics software . The between-group linkage method and the squared Euclidean distance were used to establish the clusters.TPT approximates to the common peak areas and UQT to the activity values, and E and F contains the residuals of the regression model.PLSR is the statistical method, which is related to principal component regression, but is not a hyperplane that looks for the maximum variance between the response variable and the independent variable . It is hThe Grey relational analysis is often used to measure the degree of correlation among factors according to the degree of similarity or dissimilarity between the development trends of factors, which is also called “grey correlation degree” . The antρ) is a widely used method to measure the degree of correlation between two variables. A positive correlation coefficient value of ρ (from 0 to 1) implies that the two variables are positively correlated. On the contrary (from −1 to 0), there is a negative correlation. Besides, a correlation coefficient value of 0 implies that the two variables are not related . The formula for the calculation of the relational degree is as follows:In TCMs, the same chemical structure compounds can be classified as a category with a unique mother skeleton. These compounds with similar structure group share identical diagnostic ions and mass defect value, which could make the structural identification more efficient . In thisWith a well-preset mass defect window, the MDFSC approach can quickly find out the class of compound based on their similar mother skeleton over certain mass ranges, and can also obtain new compounds never found before . Conside/z, which correspond to jatrorrhizine.On the basis of existing literatures  and authCombined with the MDFSC approach and DFIBE, the structure-diagnostic ion-oriented strategy was employed to rapidly speculate the chemical constituents, and in total, 48 compounds were inferred from ZN. Among them, alkaloids accounted for 35 of the 48, which indicate the significance of alkaloids. All the detailed information is listed in m/z 332.09, 318.08, and 290.08 were presented, and consecutive losses of CH2 (14 Da), CO (28 Da), and O (16 Da) could be easily found. Nitidine ([M]+ at m/z 348.1246) is the typical compound, eluted at 8.71 min, and its molecular formula was determined as C21H18NO4+. In the positive MS/MS spectra, its MS/MS spectra had fragment peaks of 332.0926 [M-O]+ and 318.0763 [M-O-CH2]+, which correspond to the core substructures of benzophenanthrolines. Its tentative fragmentation pathway is shown in For benzophenanthrolines, their basic carbon framework consists of a benzene and a phenanthridine. Benzophenanthrolines are the most studied category of compounds in ZN, such as NC and chelerythrine . In the +, [M+H−30]+, and [M+H−16]+ were available in the MS/MS spectra of quinoline, and certain product ions at m/z 200.03, 184.04 were the basis of DFIBE judgment. According to the patterns above, skimmianine ([M+H]+ at m/z 260.0925) was identified rapidly. It was eluted at 9.63 min, and its molecular formula was determined as C14H13NO4. It had fragment peaks of 200.0661[M+H-CH2+H2-O+H2-CH2O]+ and 184.0390 [M+H-CH2+H2-O+H2-CH2O-CH2+H2]+, which were equal to the core substructures. Then the skimmianine was identified with the support of integrating data bases , C (12 Da), CO (28 Da), and O (16 Da) could be easily found (+ at m/z 338.1400), which was eluted at 5.47 min, and its main molecular formula was determined as C20H20NO4+. Its fragment peaks contain 322.1091 [M+H-CH4]+, 294.1130 [M+H-CH4-CO]+, and 280.0972 [M+H-CH4-CO-CH2]+. After integrating data bases, jatrorrhizine was confirmed. During the whole characterization of isoquinoline, we found that the overall structure and fragmentation pathway of magnoflorine is very interesting. Tentative fragmentation pathways of jatrorrhizine and magnoflorine are shown in For isoquinoline, the basic carbon framework consists of a phenanthridine connecting to pyridine. Its MS/MS spectra provided several certain product ions at ly found . The typWith the assistance of MDFSC and DFIBE approaches, several alkaloid glycosides and alkaloids of larger relative molecular weights were found in ZN. It is attractive that the mother nucleuses of these compounds are similar to the known alkaloids we discovered. Take compound 40, for example; its mother nucleus is nitidine, which is described in the previous section, and its tentative fragmentation pathway is shown in + at m/z 314.1760). It is a benzylisoquinoline alkaloid, and is eluted at 3.14 min in high polarity. Its fragment peaks contain 269.1174 [M+H-C2H8N]+, 237.0909 [M+H-C3H12NO]+, 107.0489 [M+H-C12H18NO2]+, etc. After integrating the databases and DFIBE approach, magnocurarine B was identified. Its tentative fragmentation pathway is shown in Several alkaloids do not belong to the above four categories or cannot be found in medium to low polarity, such as magnocurarine B . HPLC fingerprints were provided in Based on the SPSS 19.0 software, we took the relative area of each common peak as the index for HCA . The samin vitro. Moreover, as is shown in The inhibition of NO production is positively correlated with the anti-inflammatory activity . Thus, t50 value is the indicator to evaluate the antioxidant property of ZN samples, and it is negatively correlated with the antioxidant activity , S12 (47.21 ± 0.33 μg/ml), and S20 (55.22 ± 0.64 μg/ml) were lower than 60 μg/ml . Then the PLSR loading scatters were performed  from common peak areas and the Y matrix of DPPH and the NO inhibition activities were used in this model , 12 (oxynitidine), and 17 might be the potential anti-inflammatory compounds in ZN, and peaks 3, 5 (chelerythrine), 7 (hesperidin), 12, and 13 might contribute to the antioxidant activity of ZN. Our study not only revealed the plant metabolites of anti-inflammatory and antioxidant activities of ZN, but it could also be an applicable data support for the discovery of active components of other TCMs. Our future studies will perform more comprehensive and reliable methods to verify the anti-inflammatory and antioxidant activities of ZN in vitro and in vivo.In order to solve the challenge of discovering bioactive compounds in ZN, a comprehensive filtering approach and a spectrum–effect relationship were performed in our work. A total of 48 compounds were identified from ZN by the structure-diagnostic ion-oriented network, and 35 of them were alkaloids. During the identification of chemical components in ZN by integrating the MDFSC and DFIBE approaches, some new alkaloid analogs were found, such as compounds


Cordia dichotoma Forst. (Boraginaceae) has potent pharmacological impact. Meanwhile, its effect on fertility is unclear.Cordia fresh fruits hydroethanolic extract on fertility.This study investigates the effect of n = 30). The first group was negative control, and the second, third, and fourth groups received 125, 250, and 500 mg extract/kg bodyweight for 56 days. After 56 days, Cordia force-feeding stopped, and all groups were kept under laboratory conditions for another month to study the recovering effect.120 Wistar albino male rats were divided into four groups , compared to the negative control. Also, significant depletion on follicle-stimulating hormone (2.66 ± 0.21 mIU/L), luteinizing hormone (1.07 ± 0.06 mIU/L), and testosterone (2.69 ± 0.13 nmol/L) level was recorded, compared to the negative control. Cordia negative effect showed on histopathological studies of testes, prostate, and seminal vesicles. Fortunately, these adverse effects of Cordia recovered remarkably after stopping administration for one month.After day 56, extract at 500 mg/kg significantly reduced sperm total count, motility%, and alive%, to 47.60 ± 2.27 × 10Cordia antifertility effect may be due to its hypocholesterolemic effect, where cholesterol, the steroid cycle precursor, was significantly reduced. This study can be incorporated in clinical research after being repeated on another small experimental animal, their offspring, and one large experimental animal, then going to a clinical study that we plan to do in the future. Fertility is a multi-factorial character; infertility is the diminished or absent capacity to produce viable offspring. Functional causes of infertility are secondary to the essential nutritional, hereditary, stress, and work factors Abraham . Male inCordia dichotoma Forst. (Boraginaceae) grows in Egypt and has been known as Mokhate trees since the Pharaoh's era . All other chemicals were of analytical grade. Kits for spectrophotometric analysis were obtained from Bio Diagnostic . ELISA kit for insulin was purchased from Epitope Diagnostic Inc Company, San Diego, USA. ELISA kits for follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) were purchased from AB diagnostic system Gmbh, Berlin, Germany. ELISA kit for testosterone (TS) hormone was purchased from XEMA Co. Ltd, Moscow, Russia.C. dichotoma fruits were collected from Cordia trees in Sharkia Governorate, Egypt, during August 2018. The plant was identified by the Department of Plant Taxonomy at the Ministry of culture and Orman Botanical Garden, Giza, Egypt. A voucher specimen (no. 1-18-5) was deposited in the herbarium of Mazhar Shehab Botanical Garden, Giza, Egypt. Washed fruits (1 kg) were crushed using a blender (Toshiba) and were exhaustively extracted with 70% ethyl alcohol (3 L) by shacked soaking several times (4 times) during 1 month at room temperature. The filtrate was collected every 1 week, evaporated by a rotary evaporator at 40 °C, and then lyophilised. The remaining residue (200 g) was kept under −20 °C.3 solution (10 mL) was added, and volume was completed to 25 mL with distilled water. Gallic acid (standard solution)  was prepared with the same procedure. The reaction was incubated (90 min at room temperature) and read at 550 nm with an Ultraviolet (UV)/Visible spectrophotometer against blank.The total phenol content was determined according to Singleton et al.  and was 2 (0.30 mL) were taken in a 10 mL volumetric flask. 10% AlCl3 (0.3 mL) was added after 5 min. NaOH (2 mL 1 M) was added after 5 min and diluted to 10 mL with distilled water. Quercetin (standard solution)  was prepared with the same procedure. The reaction was read at 510 nm against blank.Total flavonoid content was measured according to Lin and Tang  and was g of extract was extracted in 50 mL of 10% acetic acid solution in absolute ethanol by shaken for 4 h. Then, concentrated NH4OH was added drop by drop to precipitate the alkaloids. The precipitate (in weighted filter paper) was filtrated off and washed with 1% NH4OH solution. The filter paper with alkaloids was dried at 60 °C for 60 min and reweighted. By weight difference, the weight of alkaloids was determined.The gravimetric method of Onwuka  was used× 4.00 mm, 5 µ) column according to Kim et al.  equipped with Shimadzu UV-Vis detector (SPD-10Avp) at 330 nm, LC-16ADVP binary pump, DCou-14 A degasser, and Phenomenex RP-18  was estimated at 10,000 mg/kg.Acute toxicity of the 425 OECD  for acutad libitum over three months.The experiment was conducted in the animal house at the National Research Centre, Dokki, Giza, Egypt. Permission was obtained from the Ethics Committee of the National Research Centre under registration No. 19/093. Adult male Wistar albino rats (120 rats) ranging from 150 to 170 g in weight were obtained from the Central Animal House at the National Research Centre, Giza, Egypt. Animals were maintained in plastic cages under laboratory conditions . Water and food were available Cordia force-feeding stopped, and the four groups were kept under laboratory conditions for another month to study the recovery effect.The extract three doses of 500, 250, 125 mg/kg body weight were dissolved in normal saline and were administrated by gastric tube. Experimental animals were adapted for 1 week under laboratory conditions. The 120-rats were divided into four groups, each group 30 rats. The first group was the negative control group; the rats were force-fed normal saline for day 56. The second, third, and fourth groups were e force-fed the extract as 125, 250, 500 mg/kg body weight, respectively, for 56 days. After day 56, g body weight) . Testes, prostate, and seminal vesicles were kept in 10% formalin for histopathological examination.After 28-days, day-56, and day-90, the experimental period, ten rats from each group were fasted and maintained with tap water overnight. Rats were anaesthetised by injecting ketamine and xylazine . Glucose concentration was estimated immediately, using Gluco Star Test Strip .eosin–nigrosine stain to evaluate sperm viability, where eosin-stained dead sperm pink and live sperm remained unstained , glutathione peroxidase (GPx), and catalase (CAT) were determined according to Griffith (2O2) were estimated according to Ohkawa et al. , glutathione reductase (GR), glutathione-Griffith , GoldberGriffith , Paglia Griffith , Habig eGriffith , Beers aGriffith , respecta et al. , Chance a et al. , respect were prepared and stained with Haematoxylin and Eosin n = 10. A one-way ANOVA test was performed to compare different groups, followed by Duncan's post hoc test, and all comparisons were significant when p ≤ 0.05 using software COSTAT .Data were presented as mean ± SE (standard error), Cordia fresh fruit and hydroethanolic extract of fresh fruits (70%). The total phenolic content in fresh fruits and Cordia extract was 2.95 ± 0.13 and 3.01 ± 0.18 mg gallic acid/g extract. The total flavonoid content of the fresh fruits and extract was evaluated as 3.87 ± 0.17 and 4.26 ± 0.14 mg quercetin/g extract, respectively. Total alkaloids of Cordia fresh fruit and extract were 6.50 ± 0.25 and 56.95 ± 0.75 mg total alkaloids/g.This study determined the total phenols, flavonoids, and alkaloids of p-qumaric (0.37%), syringic (4.82%), vanillic (6.19%), and salicylic acid (3.28%). In addition to three flavonoids, including rutin (7.92%), quercetin (5.19%), and kaempferol (10.64%) , chlorogenic acid (0.12%), caffeic acid (1.35%), rosemarinic acid (35.78%), (10.64%) .Cordia fruits extract (1–7 g/kg body weight) to albino mice groups did not produce any mortality or dangerous changes during the first 48 h. After the first 48 h, no mortality or detrimental changes were observed during the following 14 days with these concentrations, compared to the negative control. The mortality started with concentration at 8 g/kg/day until 10 g/kg/day. The concentration that killed 50% of animals during the first 48 h was estimated at 10 g/kg/day.Oral administration of a single dose of graded concentrations of p ≤ 0.05). In addition, indirect relation between Cordia concentration and MBWG was noticed, where MBWG decreased when Cordia doses increased  (ncreased .p ≤ 0.05) decreased compared with the control group. After day 56, the bodyweight of both medium and high doses significantly (p ≤ 0.05) decreased than the control group, and the three groups showed a decrease in recovery month compared with the control.After 28 days, the bodyweight of the high dose group significantly . p ≤ 0.05) reduced as a response to Cordia administration compared to the corresponding values of the negative control (p ≤ 0.05).Data presented in Cordia-treated groups significantly declined after administration of the extract for 28-days, and this reduction continued for 56 days, compared to the negative control (p ≤ 0.05). The same observation was recorded at the recovery stage.Creatinine, uric acid, and urea levels of Cordia extract showed a hypoglycaemic effect in a dependent manner that represented through a significant (p ≤ 0.05) reduction in blood glucose levels concurrent with a significant (p ≤ 0.05) rise in serum insulin, compared to the negative control values  in comparison with the negative control level at the same time (85.75 ± 1.39 mg/dL). At the recovery stage, blood glucose of Cordia-groups was significantly increased compared to previous values but was still lower than that of the negative control.On the contrary, serum insulin of eriment. . The lowCordia-treated groups were significantly elevated after administering the extract for 28 and 56 days, compared to the negative control (Cordia dose recorded the highest insulin level (29.04 ± 0.95 mIU/mL) during the experimental period. Cordia-groups' insulin levels were significantly higher than that of the negative control at the recovery stage.Serum insulin levels of  control . After 2Cordia was in a dependent manner. TC, TG, VLDL-C, and LDL-C levels of Cordia-treated groups significantly declined after administering the extract for 28 and 56 days (p ≤ 0.05). The hypolipidemic effect of Cordia after administration for 56 days was higher than its effect after 28-day. After day 56, the highest dose of Cordia produced the lowest values of TC, TG, and VLDL-C  during the experimental period. At the recovery stage, TC, TG, VLDL-C, and LDL-C levels of Cordia-treated groups significantly elevated more than recorded at 28 and 56 days. However, they were still lower than the negative control at the same stage (p ≤ 0.05).The extract exhibited a hypolipidemic impact on the treatment and recovery stages compared to the negative control . The hypCordia-treated groups significantly magnified after administration Cordia extract for 28 and 56 days, compared to each negative control. After day 56, the highest dose of Cordia produced the highest HDL-C level (50.87 ± 1.11 mg/dL) during the experimental period. The HDL-C level did not change significantly at the recovery stage compared to the negative control at the same stage.On the contrary, HDL-C levels of Cordia treatments had significant antioxidant properties, which parallelly increased by Cordia doses increased during treatment and recovery stages compared to the negative control (p ≤ 0.05) . The ameliorative effect of Cordia extract on antioxidant biomarkers prolonged the recovery stage. The highest GSH concentration, GR, GST, and GPx activities were recorded at the recovery stage  ( ≤ 0.05) . Adminisry stage . CAT act ≤ 0.05) .Cordia treatments recorded a significant (p ≤ 0.05) effect on FSH, LH, and TS production of all Cordia-treated groups, compared to the negative control. The general trend was observed, Cordia significantly decreased these hormones during the treatment stage and significantly increased them during the recovery stage.Data in Cordia treatment showed a negative effect on FSH and LH concentration, where gonadotropic cells produced FSH and LH less than negative control after 28-day (p ≤ 0.05), and the highest dose of Cordia caused the highest reduction in FSH , in comparison with the negative control . Ongoing Cordia administration (at medium and high doses) for 56 days showed the same trend and decreased FSH and LH levels significantly. The highest dose of Cordia recorded the lowest FSH and LH concentration , in comparison with the negative control . Thus, although Cordia caused a significant decrease in the FSH concentration after administration for 56 days, compared with the negative control, the hormone concentration after 56 days from the administration was higher than that after 28 days. On the contrary, FSH and LH levels of Cordia-treated groups significantly (p ≤ 0.05) elevated during the recovery stage compared to the negative control levels. The highest dose of Cordia caused the highest elevation in FSH and LH level , in comparison to the negative control .Cordia for 28-days, TS concentration did not significantly change compared to the negative control. After administration of the extract for 56 days, TS of all Cordia treated groups significantly decreased compared to the negative control. In the recovery stage, TS levels of all Cordia-treated groups were significantly elevated. The highest TS level was recorded with the group treated with the highest dose of Cordia (500 mg/kg); 4.05 ± 0.18 nmol/L, compared to TS of negative control (3.12 ± 0.26 nmol/L) at the same time.After administering low and high doses of Cordia treatment had a significant effect on male fertility. Cordia showed a negative impact on the seminal quality of treated rats compared to the negative control. Furthermore, almost Cordia recorded a significant (p ≤ 0.05) reduction of sperm concentration, sperm motility, and alive sperm% concurrent with a substantial elevation of abnormality% in the treatment stage that considered antifertility effect. Fortunately, rats overcame the harmful impact of Cordia on fertility during the recovery stage . Medium and high doses dramatically declined sperm production that reached 64.00 ± 1.33 × 106 and 52.04 ± 3.64 × 106 sperm/mL, respectively, with 8.35% and 25.48% reduction less than the negative control. After 56 days, Cordia at 250 and 500 mg/kg dramatically decreased sperm concentration (61.50 ± 3.62 × 106 and 47.60 ± 2.27 × 106 sperm/mL with 15.08 and 34.27 reduction percentage), compared to the negative control (72.42 ± 5.75 × 106 sperm/mL). Whereas, force-fed low dose significantly (p ≤ 0.05) raised sperm concentration to 79.50 ± 5.67 × 106 sperm/mL in comparison to the negative control (72.42 ± 5.75 × 106 sperm/mL). In the recovery stage, reversible effect was observed, where the highest dose of Cordia recorded the highest sperm concentration (76.20 ± 4.64 × 106 sperm/mL) with a 15.45% increase compared to the negative control (66.00 ± 6.93 × 106 sperm/mL) (Sperm concentration of perm/mL) .Cordia treatment, it significantly reduced (p ≤ 0.05) sperm motility compared to the negative control. Furthermore, administration of Cordia for either 28 or 56 days significantly decreased sperm motility compared to the negative control. The weakest sperms occurred in the 500 mg/kg group during the treatment stage. On the contrary, sperm of 500 mg/kg group during the recovery stage became the fastest, compared to the motility of negative control; 65.00 ± 1.29% and 52.00 ± 2.49%, respectively.Concerning the antifertility effect of Cordia for 28 days significantly (p ≤ 0.05) raised alive sperm percentage, except for high dose, compared to the negative control percentage. Low and medium doses of Cordia significantly protected sperms and remained alive more than in the negative control; 84.76 ± 1.49, 78.33 ± 1.81, and 72.00 ± 2.37%, respectively. Continuing administration of Cordia for 56 days significantly decreased alive sperm than that of the negative control, except low dose that caused a non-significant decrease. Cordia-treated animals showed a reversible effect in the recovery stage that represented a significant elevation in alive sperm%, compared to the negative control.Fortunately, administration of Cordia significantly increased sperms abnormality percentage compared to the negative control in the treatment and recovery stage. The abnormality percentage increased parallel to Cordia doses increased. A high dose of Cordia only significantly (p ≤ 0.05) decreased sperm abnormality percentage lesser than negative control in the recovery stage; 17.33 ± 0.15 and 19.60 ± 0.50%, respectively.p ≤ 0.05) affected with Cordia administration . However, after day 56 and day 90, Cordia did not cause a significant change in the relative weight of tests compared to the negative control.Compared to the negative control, the relative weight of reproductive organs was significantly , compared to the negative control . Finally, at the recovery stage, the relative weight of epididymis of Cordia-animals significantly decreased. Meanwhile, the relative weight prostate was increased considerably compared to the negative control animals.After 28 days, epididymis and prostate relative weight did not significantly change compared to the negative control. Meanwhile, after day 56, epididymis and prostate relative weight were raised considerably in groups treated with high doses of Cordia did not significantly change the relative weight of the seminal vesicle after 28-days. However, after 56 days, a high dose significantly increased the relative weight of the seminal vesicle, compared to the negative control; 0.583 ± 0.02 and 0.384 ± 0.02%, respectively (p ≤ 0.05). Thus, at the recovery stage, the relative weight of the seminal vesicle of treated animals was close to the relative weight of the negative control.Cordia for 28-day showed mild atrophy of seminiferous tubules associated with mild interstitial edema  showed mild hyperplasia of the lining epithelium, respectively  showed normal seminiferous tubules associated with mild interstitial edema  that promotes FSH and LH hormones from pituitary glands. Leydig cells in interstitial tissue of tests produce TS under the LH effect. FSH stimulates Sertoli function. FSH and TS synergistically work to produce sperm cells in seminiferous tubules by meiotic division. Produced sperms pass through the epididymis, those secret substances for sperm maturation. Finally, seminal vesicles and prostate glands secret semen components as fructose, citrate, inositol, and prostaglandins. Success spermatogenesis process is dependent on the harmony between the previous steps. Therefore, any pathological alternation in the male reproductive system may interfere with fertility via impairment of TS level or disturbing spermatogenesis and sperm maturation Cooper .de novo synthesis. Therefore, depletion in TC due to genetic modification in the mouse model led to infertility  is vital for the male reproductive system. TC is the precursor of steroid synthesis, the major TS source, crucial for normal spermatogenesis. The steroidogenesis process is responsible for TS production from TC via several steps as de novo synthesis was elevated during the development of pachytene, leptotene, and zygotene stages to increase germ cells' diameter and surface area (Sèdes et al. Indeed, sperm production is strongly associated with TC on the excellent mass production of germ cells during spermatogenesis. Therefore, TC Additionally, lipids strongly affect spermatozoa post-testicular maturation. In the epididymis, the lipid composition of spermatozoa gives it greater membrane fluidity. The epididymis also synthase lipid vesicles to facilitate protein transfer to spermatozoa. Finally, spermatozoa capacitation, the post-testicular maturation, strongly depends on the lipid composition of the spermatic membrane (Jin and Yang Cordia extract reduced TC, the precursor of the steroidogenesis process, by about 20.21 and 29.02% after administration for 28 and 56 days, respectively. Therefore, the high dose recorded the highest antifertility effect. Also, Cordia extract showed a hypoglycaemic effect. Glucose converts to pyruvate and lactate to provide post-mitotic germ cells with energy reduced after administration for 28 days, 56 days, and recovery stages.From the previous explanation, we can observe the importance of the lipid profile of animals for fertility and normal embryonic development resulting insufficient sperm count, good motility percentage, acceptable alive percentage, and low sperm abnormality, which lead to successful fertility. Unfortunately, in this study, Cordia fruits may be attributed to the reduction of FSH and LH that reflected TS production and seminal quality. Besides the hypolipidemic and hypoglycaemic effects of Cordia that reduced cholesterol, the steroid cycle's precursor.Finally, we can conclude that the antifertility effect that appeared by Cordia harmed male fertility during two months of dosing. A significant deficit in sperm count, motility%, alive%, concurrent with a substantial increase in abnormalities%, were recorded. Also, it significantly reduced FSH, LH, and testosterone levels. In addition, a negative alternation in reproductive organs, including tests, prostate, and seminal vesicles. Fortunately, animals can restore their normal fertility within one month from stopping Cordia administration. The antifertility effect of Cordia may be attributed to its hypolipidemic effect. Where Cordia significantly minified the cholesterol concentration of treated rats. Therefore, Cordia is considered safe for animals because it remains in the field for two months annually. If the animals eat Cordia within two months, then they can restore their fertility within one month.


Candidatus Nitrosocosmicus.” However, the mechanism of its ammonia tolerance has not been reported. In this study, the AOA species “Candidatus Nitrosocosmicus agrestis,” obtained from agricultural soil, was determined to be able to tolerate high concentrations of NH3 . In the genome of this strain, which was recovered from metagenomic data, a full set of genes for the pathways of polysaccharide metabolism, urea hydrolysis, arginine synthesis, and polyamine synthesis was identified. Among them, the genes encoding cytoplasmic carbonic anhydrase (CA) and a potential polyamine transporter (drug/metabolite exporter [DME]) were found to be unique to the genus “Ca. Nitrosocosmicus.” When “Ca. Nitrosocosmicus agrestis” was grown with high levels of ammonia, the genes that participate in CO2/HCO3− conversion, glutamate/glutamine syntheses, arginine synthesis, polyamine synthesis, and polyamine excretion were significantly upregulated, and the polyamines, including putrescine and spermidine, had significant levels of production. Based on genome analysis, gene expression quantification, and polyamine determination, we propose that the production and excretion of polyamines is probably one of the reasons for the ammonia tolerance of “Ca. Nitrosocosmicus agrestis,” and even of the genus “Ca. Nitrosocosmicus.”Ammonia tolerance is a universal characteristic among the ammonia-oxidizing bacteria (AOB); in contrast, the known species of ammonia-oxidizing archaea (AOA) have been regarded as ammonia sensitive, until the identification of the genus “IMPORTANCE Ammonia tolerance of AOA is usually much lower than that of the AOB, which makes the AOB rather than AOA a predominant ammonia oxidizer in agricultural soils, contributing to global N2O emission. Recently, some AOA species from the genus “Ca. Nitrosocosmicus” were also found to have high ammonia tolerance. However, the reported mechanism for the ammonia tolerance is very rare and indeterminate for AOB and for AOA species. In this study, an ammonia-tolerant AOA strain of the species “Ca. Nitrosocosmicus agrestis” was identified and its potential mechanisms for ammonia tolerance were explored. This study will be of benefit for determining more of the ecological role of AOA in agricultural soils or other environments. Nitrosopumilales, “Candidatus Nitrosotaleales,” “Candidatus Nitrosocaldales,” and Nitrososphaerales within the phylum Thaumarchaeota, play a major role in the global nitrogen cycle by mediating the conversion of ammonia to nitrite , composed of  nitrite –3. AOA anitrite –, 4. The reported , 6. Alth ammonia –9.2O), contributing to global N2O emission from agricultural soils (–Because the ammonia affinity of AOA is dozens or hundreds of times higher than that of the ammonia-oxidizing bacteria (AOB) –12, memb–al soils –19.2O production  to Noduction –19; they% to Noduuction ––. In part ammonia , 8 , respectively. Download FIG S1, PDF file, 0.4 MB.Heat maps showing pairwise average nucleotide identity (ANI) and average amino acid identity (AAI) values inferred from the available AOA genomes. The ANI and AAI were calculated using JSpeciesWS and compareM , which is shorter than that of “Candidatus Nitrosocosmicus exaquare” G61 (51.7 h), “Candidatus Nitrosocosmicus franklandus” C13 (40 h), and “Candidatus Nitrosocosmicus oleophilus” MY3 (77.4 h)  and “Ca. Nitrosocosmicus oleophilus” MY3 (3.2 × 106 cells ml−1) but lower than that of “Ca. Nitrosocosmicus exaquare” G61 (5.6 × 107 cells ml−1)  9. After 7.4 h 9. ls ml−1) 9.Ca. Nitrosocosmicus agrestis” was observed to be optimal at 37°C and nearly inhibited at 42°C , which is much higher than the levels of many other terrestrial AOA strains, such as “Candidatus Nitrosotenuis aquarius” AQ6f (0.1%) (Candidatus Nitrosotenuis cloacae” SAT1 (0.03%) (Candidatus Nitrosotenuis uzonensis” N4 (0.1%) (Candidatus Nitrosoarchaeum koreensis” MY1 (0.4%)  , “Candid (0.03%) , “Candid4 (0.1%) , and “Ca1 (0.4%) . Ammonia1 (0.4%) .Ca. Nitrosocosmicus agrestis” was also able to use urea as the sole energy and nitrogen source; the ammonia oxidation rate when urea was the sole energy source was only 1/10 of that when ammonia was the energy source, while the relative expression levels of genes relating to urea transport and hydrolysis were conversely 11 to 13 times higher (50s) of nitrification inhibitors, including allylthiourea (ATU), dicyanodiamide (DCD), and 3,4-dimethylpyrazole phosphate (DMPP), for “Ca. Nitrosocosmicus agrestis” were determined to be 445.1, 947.1 and 488.0 μM, respectively (–50 of nitrapyrin (NP) for “Ca. Nitrosocosmicus agrestis” was only 0.599 μM  and ammonia (B) concentration in the ammonia oxidation process and relative gene expression levels in response to urea (C). 1A, 0.1A, and 0A correspond to 1 mM, 0.1 mM, and 0 mM NHCopyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01003-20.3FIG S3Ca. Nitrosocosmicus agrestis.” The relative ammonia oxidation rate was calculated based on the nitrite concentration on the eighth day. All error bars indicate standard errors of the means for biological triplicates. DCD, dicyanodiamide; ATU, allylthiourea; DMPP, 3,4-dimethylpyrazole phosphate; NP, nitrapyrin. Download FIG S3, TIF file, 0.4 MB.Influences of nitrification inhibitor on the ammonia oxidation activity of “Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ca. Nitrosocosmicus agrestis” decreased with increases in the initial ammonia concentration; about 75%, 35%, and 5% of the activity were maintained when the concentration of un-ionized ammonia (NH3) reached 400, 800, and 1,592 μM, respectively  than the other AOA strains. The inhibitory concentration of NH3 for strains of the genus “Ca. Nitrosocosmicus” was determined to range from 796 to 3,894 μM; except for strains of “Ca. Nitrosocosmicus” and the terrestrial strains Nitrososphaera viennensis EN76 and “Ca. Nitrosotenuis aquarius” AQ6f, the inhibitory concentrations of NH3 for other strains are less than 500 μM , tricarboxylic acid cycle, gluconeogenesis, and nonoxidative pentose phosphate pathways, are presented  and urea hydrolysis , which participate in energy metabolism, are also found in the genome ; bin 2 is the genome of “Ca. Nitrosocosmicus agrestis,” bin 3 is the genome of Ensifer sp., and bin 4 consists of contigs of “Ca. Nitrosocosmicus” but did not pass the quality check of CheckM. Download FIG S4, TIF file, 0.3 MB.Classification and coverage of metagenome contigs (A) and composition of bins from the “Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.01003-20.8TABLE S1Ca. Nitrosocosmicus agrestis” genome and genes for polyamine metabolism and transport in the coexisting heterotroph Ensifer sp. strain SSB1. Download Table S1, XLSX file, 0.04 MB.Functional genes for carbon metabolism, energy metabolism, transporters, carbohydrate-active enzyme, and nitrogen metabolism in the “Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2 to HCO3− , which is responsible for the reversible hydration of COto HCO3− , 36. Thrgrestis” ; they wegrestis” . Until nsotalea” . In contsotalea”  37). Th. Th2 to 10.1128/mSystems.01003-20.5FIG S5FIG S5, TIF file, 1.7 MB.Evolution analysis of β (A) and γ (B)-carbonic anhydrase based on amino acid sequence. The software was IQ-TREE, the method used was maximum likelihood, the model was LG+I+G4, and the ultrafast bootstrap value was 1,000. Download Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3) or is transferred from the extracellular environment by ammonium transporters (Amt) (in the form of NH4+) is integrated into glutamine/glutamate syntheses. A single copy of the gene encoding Amt was identified in the genome of “Ca. Nitrosocosmicus agrestis” . Al. Al3) orgrestis” . When aris steps .10.1128/mSystems.01003-20.6FIG S6N. europaea Rh protein based on the program MUSCLE (B). The residues that constitute the proposed ammonium-binding site are marked with a red asterisk. Download FIG S6, TIF file, 2.8 MB.Evolution analysis of Amt amino acid sequences in the different AOA genomes (A). The software was IQ-TREE, the method used was maximum likelihood, the model was LG+F+R3, and the ultrafast bootstrap value was 1,000. Sequence alignment of the AOA Amt protein to the Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ca. Nitrosocosmicus agrestis” . The method was BLASTp, and the matrix was BLOSUM45. Download FIG S7, TIF file, 1.1 MB.Evolution analysis of DME transporter in the different AOA genomes (A) and evolution analysis of polyamine exporters and AOA DME transporters (B). The functionally identified polyamine exporters in bacteria and yeasts are in blue; the speculated polyamine exporters in “Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3), the expression patterns of genes involved in ammonia oxidation, arginine synthesis, polyamine synthesis, glutamine/glutamate syntheses, carbonic anhydrase, and polyamines transport when “Ca. Nitrosocosmicus agrestis” responded to high ammonia had significant changes. For example, amoA and the Amt gene were significantly downregulated at 796 μM NH3; however, all of the genes participating in arginine synthesis, polyamine synthesis, and polyamine excretion had significant upregulation, with values ranging from 1.28 to 5.63 times  decreased by 17% in contrast to that seen with low levels of ammonia (7.96 μM NH3) (During chemoautotrophic growth of “ectively . The ave μM NH3) ; however μM NH3) . It is sCa. Nitrosocosmicus agrestis” cultures grown with different concentrations of ammonia were determined by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Polyamines including putrescine, cadaverine, and spermidine were identified, with concentrations ranging from 1.37 to 10.25 μg liter−1 (3), cadaverine production was not significantly different but spermidine production was about three times higher; notably, putrescine was detected only with a high level of ammonia (796 μM NH3) and was identified as the predominant polyamine in the culture  proteins, belonging to the Amt/MEP/Rh (ammonium transporter/methylammonium-ammonium permease/rhesus protein) family and acting as bidirectional transporters of NH4+/NH3, have been reported in Nitrosomonas  to form NH4+. However, if it presents Amt-1, NH4+ transport into the cytoplasm via Amt-1 would be the main way for ammonia to enter cells. At this stage, the assimilation of NH4+ and bicarbonate will produce a large amount of H+, which in turn will decrease the cytoplasmic pH and significantly suppress cell growth.The first notable finding in this regard is the presence of only the Amt-2 transporter in genomes ; Fig. S6 ammonia , suggestCa. Nitrosocosmicus”  and arginine syntheses were significantly upregulated under high-ammonia conditions, which suggested that the NH3 that diffuses through the cell membrane was probably used for arginine synthesis and further polyamine synthesis.The third mechanism is the presence of an incomplete ornithine cycle pathway in the genome of “Ca. Nitrosocosmicus agrestis” probably uses DME as an exporter of polyamine. The DME genes of “Ca. Nitrosocosmicus agrestis” that are closely related to the bacterial putative DME were determined to be upregulated 2.5- to 7.5-fold when growing with high levels of ammonia  from cells would not be a mechanism of ammonia tolerance. However, polyamines, including putrescine, cadaverine, and spermidine, were identified in the supernatant of “nditions , suggestd yeasts . It is pCa. Nitrosocosmicus agrestis” grown with either a high or low concentration of ammonia were far from corresponding to the amount of ammonia oxidized. Several explanations for this observation are possible. First, the role of polyamines is probably as an active molecule, like their functions in bacteria and archaea  bacteria belonging to the Rhizobiales  and Ensifer adhaerens Casida A (6 copies)  , 59. In radation  60, 61), 61Ca. NCa. Nitrosocosmicus” and heterotrophs has barely been reported, and so some unknown or complex interactions between them may occur, with the result that many terrestrial AOA species are uncultivated or even unclassified  of all life and widely distributed among the hyperthermophilic, acidophilic, and thermoacidophilic archaea, suggesting their potential role in the response to adverse environmental conditions . The polassified . The polCa. Nitrosocosmicus agrestis” strain SS was obtained from agricultural soil by using a two-step strategy and maintained in a mineral salts medium (MSM) (“um (MSM) . The cul3 (the diffused CO2 is sufficient for SS growth) but was buffered with 10 mM MES [2-(N-morpholino)ethanesulfonic acid], HEPES, or Tris to maintain the pH in the ranges of 6 to 7, 7 to 8, and 8 to 9, respectively. Concentrations of nitrite and ammonium were determined by the standard Griess-Ilosvay method and the indophenol blue method, respectively (Ca. Nitrosocosmicus agrestis” and 1369F/1492R for bacteria), following the method described in our previous study  was inoculated into the fresh MSM without supplying quartz and subjected to the determination of physiological characteristics. To avoid pH deviation due to hydrolysis or dissociation of bicarbonate, the MSM used for pH characterization was not supplied with NaHCOectively . The conectively . The meaus study .in situ hybridization (FISH) was observed on a scanning confocal microscope ; transmission electron microscopy (TEM) was performed on an H-7650 transmission electron microscope  at an accelerating voltage of 80 kV; scanning electron microscopy (SEM) was performed using a Merlin field emission scanning electron microscope .Fluorescence The genomic DNA from 3,000 ml batch cultures was extracted according to a previously described protocol . Purifiede novo assembled using metaSPAdes v3.13.0 , respectively.The raw data were length and quality filtered using Trimmomatic v0.38  and de n v3.13.0  with 127 v3.13.0  was used v3.13.0  with the v3.13.0 . Scaffol v3.13.0 . Gene pr v3.13.0  and Micr v3.13.0 . KEGG pa v3.13.0 . Putativ v3.13.0  and dbCA v3.13.0 , respectpeciesWS  and compA phylogenomic tree was obtained using the automatically generated alignment of 43 concatenated universal marker protein sequences, which were identified by CheckM v1.013 . The bes−ΔΔCT method was used to estimate fold change in gene expression, normalized to the endogenous control 16S rRNA. Primer pairs ; the synthesis of first-strand cDNA was performed using the PrimeScript RT reagent kit with gDNA Eraser . Quantitative PCR were performed on an ABI 7500 Fast real-time PCR system  using TransStart Tip Green qPCR SuperMix . The reaction conditions were as follows: 1 min at 94°C and 40 cycles of 10 s at 94°C and 34 s at 60°C. The 2er pairs  were des10.1128/mSystems.01003-20.9TABLE S2Table S2, XLSX file, 0.01 MB.Primers used for quantitative PCR. Download Copyright © 2021 Liu et al.2021Liu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 5 min, the pellet was collected and used for protein measurement by using a bicinchoninic acid (BCA) protein assay kit .To measure the total protein in AOA cells, about 20 ml of culture was collected and filtered through a 0.22-μm cellulose filter. The filter with cells was cut into pieces and ground in a 2-ml tube containing 0.5 g quartz sand, and 500 μl of lysis buffer (TE buffer containing 1% Triton X-100) was added to the tube and mixed for 5 min using the Vortex Adapter . After centrifugation at 5,000 × g for 15 min. The supernatant was filtered through a 0.22-μm cellulose filter again and used for the quantitative analysis of amino acids or polyamines.An 800-μl portion of the supernatant was collected and filtered through a 0.22-μm cellulose filter. The filtrate was mixed with 200 μl of sulfosalicylic acid (10%) and incubated at 4°C for 1 h, following centrifugation at 14,000 × 3COONH4 in H2O (A) and 0.2% HCOOH in H2O (vol/vol) (B). Amino acids in the supernatant were determined by using the amino acid analyzer A300-advanced .Concentrations of putrescine, cadaverine, spermidine, and spermine in the supernatant were determined using the LC-20A HPLC system  coupled with an API 4000 Quantiva triple-quadrupole tandem mass spectrometer  according to a previous study . HPLC sen = 3) as noted. Student's t test was used to calculate the P value as noted. P values of <0.05 and <0.01 were considered to indicate significant differences and highly significant differences, respectively.Results are expressed as means and standard errors (VUYS00000000 for “Ca. Nitrosocosmicus agrestis” and JAEPRP000000000 for heterotroph Ensifer sp. SSB1.The genomes described in this study have been deposited in NCBI under GenBank accession number


Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. is a medicinal plant used in Traditional Chinese Medicine (TCM) for thousands of years. However, due to the overexploitation, this species is considered to be endangered and is included in the Red List, e.g., in the Republic of Korea. Therefore, a new source of this important plant in Europe is needed. The aim of this study was to develop pharmacognostic and phytochemical parameters of the fruits. The content of polyphenols  and phenolic acids in the different parts of the fruits, as well as tocopherols, fatty acids in the oil, and volatile constituents were studied by the means of chromatographic techniques . To the best of our knowledge, no information is available on the content of eleutherosides and phenolic acids in the pericarp and seeds. The highest sum of eleutheroside B and E was detected in the whole fruits (1.4 mg/g), next in the pericarp (1.23 mg/g) and the seeds (0.85 mg/g). Amongst chlorogenic acid derivatives , 3-CQA was predominant in the whole fruits (1.08 mg/g), next in the pericarp (0.66 mg/g), and the seeds (0.076 mg/g). The oil was rich in linoleic acid (C18:3 (n-3), 18.24%), ursolic acid (35.72 mg/g), and α-tocopherol (8.36 mg/g). The presence of druses and yellow oil droplets in the inner zone of the mesocarp and chromoplasts in the outer zone can be used as anatomical markers. These studies provide a phytochemical proof for accumulation of polyphenols mainly in the pericarp, and these structures may be taken into consideration as their source subjected to extraction to obtain polyphenol-rich extracts. Plant-based metabolites have served as lead compounds for many important drugs, such as morphine, digoxin, quinine, hyoscyamine, salicylic acid, and artemisinin . AccordiEleutherococcus senticosus (Siberian ginseng). The plant is native to Russia, the Far East, China, Korea, and Japan. The fruits of E. senticosus have been used in Russia for many years as a tonic on the central nervous system and as an adaptogen. Modern research is focused on elucidation of the pharmacological activities of wild fruits, including their antioxidant, antimicrobial, and anticancer effects. An acidified 80% methanol extract showed high xanthine oxidase and AChE inhibitory activities. The anticancer activity of the extract was also proven by screening various human cell lines, including LNCaP (prostate cells), MOLT-4F (leukemia cells), A549 (lung cells), ACHN  HCT-15 (colon cells), and SW-620 (colon cells). The latest reports indicate their immunostimulatory and anti-inflammatory activities and an increase in the number of leukocytes. The intractum from the fruits was found to stimulate human leukocyte resistance to the VSV (Vesicular Stomatitis Virus) infection via reduction of viral replication, which might be associated with increased secretion of interleukin 10 (IL-10). Besides medical applications, the fruits are industrially used in the production of phyto-jam (0.8–1.6 kg/100 kg of the product). Considering the data mentioned above, the consumption of wild edible fruits of E. senticosus by local communities in many developing countries is gaining increasing interest . The same steps were made for extraction of the pericarp and seeds.Mature fruits of 4-year-old The anatomical structure of the fruits was examined microscopically using a compound microscope coupled with a camera, evaluated, and photographed (40X). Chloral hydrate was used as a reagent. Computer images were captured using software ProgRes CapturePro 2.8—Jenoptik optical system.The analyses were performed on an EliteLaChrom chromatograph with a PDA detector and EZChrom Elite software . The following chromatographic system was used: an RP18e LiChrospher 100 column   at 25 °C; mixtures of water (solvent A) and acetonitrile (solvent B) both acidified with 0.025% of trifluoroacetic acid were used as the mobile phase. The compounds were separated by gradient elution with the following program: 0.0–8.0 min A 90%, B 10%; 8.1–18.0 min A 90–80%, B 10–20%; 18.1–30.0 min A 80%; B 20%; 30.1–45.0 min A 0%, B 100%; 45.1–60.0 min A 90%, B 10%. The flow rate was 1.0 mL/min. Data were collected between 190 and 400 nm. The identity of compounds was established by comparison of retention times and UV spectra with the corresponding standards. Quantitative analysis was performed at λ = 264 nm for eleutheroside B and λ = 206 nm for eleutheroside E and E1. The chromatographic parameters and calibration data for quantification of the investigated eleutherosides are provided in The analyses were performed on an EliteLaChrom chromatograph with a PDA detector and EZChrom Elite software . The chromatographic system was as follows: a C18 reversed phase core-shell column  , a mixture of water (solvent A) and acetonitrile (solvent B) with 0.025% of trifluoroacetic acid, and the following gradient elution program: 0.0–5.0 min A 95%, B 5%; 5–60 min A from 5 to 20% and B from 95–80%. The flow rate of the mobile phase was 1.0 mL/min and the temperature of thermostat was set at 25 °C. Data were collected between 210 and 400 nm. The identity of compounds was established by comparison of retention times and UV spectra with the corresponding standards. Quantitative analysis was performed at λ = 326 nm for chlorogenic acids and λ = 260 nm for protocatechuic acid. The chromatographic parameters and calibration data for quantification of the investigated phenolic acids are provided in E. senticosus fruits were placed in a 15 mL sealed vial and extracted using a fiber coated with 50/30 µm divinylbenzene–carboxen–polydimethylsiloxane . The 2-undecanone–2 mg/mL in water  was used as an internal standard. Equilibration was performed at 60 °C for 15 min, the fiber exposition time was 15 min, and the thermal desorption was 3 min at 250 °C directly in the gas chromatography (GC) injection port. All analyses were performed in triplicate. The gas chromatography (GC) analysis was performed using Agilent 7890B GC coupled with a 7000GC/TQ system mass spectrometer . Separation was carried out on an HP-5 MS column; 30m × 0.25 mm × 0.25 µm  at a constant helium flow of 1 mL/min. The injector temperature was set at 250 °C and the sample was applied in a split mode (70:1). The temperature program was 50 °C for 1 min, followed by 5 °C/min to 120 °C, 8 °C/min to 200 °C, then to 250 °C in 16 °C/min and held isothermal for 2 min. The MS source, transfer line, and quadrupole temperature were set at 230 °C, 320 °C, and 150 °C, respectively. The mass spectra were collected in a scan mode from m/z 30–400 and the ionization voltage was 70 eV. Data acquisition was performed using Agilent MassHunter Workstation software (version B.08.00). The identification of the compounds was based on a comparison of fragmentation patterns with the NIST17 mass spectra library and they were matched with retention index (RI) obtained by calculation relative to the n-alkane standard . The quantitative analysis (expressed as percentages of each compound) was carried out by peak area normalization measurements without a correction factor.Head space-solid phase microextraction (HS–SPME) was conducted according to Zielińska et al.  with sliAir-dried and pulverized fruits (5 g) were extracted four times with hexane (4 × 30 mL) using an ultra-sonic bath (4 × 15 min.) The extracts were combined and evaporated in a rotary evaporator. The oil (50 mg) was placed in a volumetric flask (5 mL) and dissolved in 2-propanol. The solution was filtered through a 0.25 µm polyamide membrane filter before HPLC analysis. The oil (10 mg) was dissolved in 500 μL of TBME and derivatized by the addition of 250 μL of TMSH. The whole sample was shaken vigorously, and the GC–MS analysis was performed.v/v/v); eluent flow rate 1.0 mL/min; column temperature 10 °C. The injection volume was 10 μL. The analysis was based on chromatograms recorded with the PDA detector. Data were collected between 200 and 400 nm. The quantitative analysis was performed at λ = 200 nm.The analysis was performed on a VWR Hitachi Chromaster 600 chromatograph with a 5430 Diode Array Detector, a 5440 FL Detector, and EZChrom Elite software . A RP18e LiChrospher 100 column   was used for the analyses. The identity of compounds was established by comparison of retention times and PDA spectra with the corresponding standards. Ursolic acid was determined using a previously published methodology . An isocv/v). The eluent flow rate was 1.2 mL/min. The column temperature was set at 30 °C. The injection volume was 5 μL. The quantitative analysis was performed using a fluorescence detector with an excitation wavelength at λ = 296 nm and an emission wavelength at λ = 330 nm.Tocopherols were determined using an isocratic system. The mobile phase consisted of acetonitrile and methanol (5:95 m/z to 500 m/z) was used as a detector connected with the GC. The temperature of the MS source and the MS quadrupole was set at 230 °C and 150 °C, respectively. Identification of the constituents was based on a comparison of their mass spectra with the mass spectra library NIST resources and retention times with standards.The analysis was performed using an Agilent GC–MSD system (GC/MSD 6890N/5975) equipped with a HP-88 Agilent capillary column , MSD ChemStation ver. E.02.02.1431 software , and a split–splitless injector. The oven temperature was programmed from 110 °C to 190 °C with 8 °C/min, held for 2 min at 110 °C and 13 min at 190 °C. The temperature of the injector was 250 °C. The injection volume was 1 μL . Helium was used as a carrier gas at a flow rate of 1.2 mL/min. A quadrupole mass spectrometer with electron ionization (EI) at 70 eV and with a full scan type acquisition mode . The evaluations were analyzed for one-way analysis of variance. Statistical differences between the treatment groups were estimated by Spearman’s (R) and Person’s (r) test. All statistical tests were carried out at a significance level of α = 0.05.The practical aspect of the present results may be the application of the fruits as an ingredient of plant-based products used to treat immune-related diseases, as confirmed by Graczyk et al. . With th