Lactuca sativa L.) under sole-source LED lighting of differing spectra. Lighting spectra were provided by differing combinations of LEDs of three different peak wavelengths,  with ratios of B450/R663: 1.25 ± 0.1, B450/R663: 1.25 ± 0.1, and B450/R663 1:1 at two light intensities of photosynthetically active radiation (PAR) (270 μmol m−2 s−1 and 60 μmol m−2 s−1). A further experiment was conducted, in which Blue and Red LEDs were supplemented with Green  with ratios of B435/R663: 1.25 ± 0.1, B450/R663/G520: 1/0.73/0.26, and B450/R663: 1.25 ± 0.1. LED light intensities under the different spectra were adjusted to deliver the same level of PAR (270 ± 20 μmol m−2 s−1). Results from the first experiment showed that increased fraction of blue 435 nm in combination with red light at 663 nm at high irradiance enhanced the physiology of lettuce  and increased the yield while having no significant effect on antioxidant activity. At the lower irradiance, the B435/R663 significantly increased antioxidant activity compared to other spectra. Results from the second experiment showed no significant effect of the spectra of LEDs on the physiology and yield of lettuce, but antioxidant activity was very significantly induced by B450/R663 at the ratio of 1.25 ± 0.1. However, the amount was still less than that obtained by B435/R663 1.25 ± 0.1 from the first experiment. This study indicates that LED light with a spectrum of B435/R663 at a ratio of 1.25 ± 0.1 significantly improves lettuce yield and antioxidant activity.This study focused on the physiology, growth and antioxidant activity response of hydroponically grown lettuce ( Lactuca sativa L.) belongs to the family Composite, and is an important dietary leafy salad vegetable that is primarily consumed fresh or in salad mixes [Lettuce  are controlled, minimizing the interactions with the external climate. There is a significant growing interest in this form of farming because it can be built anywhere, high resource use efficiency  can be achieved with minimum emission of pollutants to the outside environment, the growing environment is not affected by the outside climate and soil fertility, production can be year-round and productivity is over 100 times that of field production, produce quality such as concentrations of phytonutrients can be enhanced through manipulation of the growing environment, especially light quality; produce is pesticide-free and need not be washed before eating; produce has a longer shelf life because the bacterial load is generally less than 300 CFU g−1, which is 1/100 to 1/1000 that of field-grown produce and energy for transportation can be reduced by building PF near urban areas. Light is a key factor and a very important element for the Plant Factories since it has direct impact of growth, yield, and quality of plants.Light-emitting diodes (LEDs) have higher luminous efficiency, long life, and higher efficacy, leading to reduced associated heating  compared−2 s−1 irradiance for 16 h for highest photosynthesis rate and stomatal conductance is R/B = 1 compared to ratios of R/B of 4, 8, 12 with a significant decrease when the ratio of R to B increased from 1 to 12 [It has been reported that lettuce grown under combined Red and Blue LEDs exhibit the highest chlorophyll content, photosynthesis rate , pigment 1 to 12 .Lactuca sativa L.). Lee and Kim [The synergistic effectiveness of the combined Red to Blue ratio can be more clear on lettuce growth in term of leaf area and dry weight when a small quantity of green G light (24%) is added, since green light is better able to penetrate the plant canopy than Red or Blue light . This ma and Kim  also conLactuca sativa L.).However, there is less agreement regarding optimal fraction of either R or B combination effect on lettuce. The current study therefore aimed to investigate the different fraction of R and B LEDs and different RB ratios on the physiology, growth and antioxidant activities in lettuce , then sown and germinated in the greenhouse at Skardon Gardens. When seedlings had their first pair of true leaves, they were transferred to the plant factory facility at the University of Plymouth. The university’s plant factory facility is a converted insulated greenhouse where external light has been excluded. The multi-tier hydroponic growing system consists of gulleys for NFT (nutrient film technique) and is installed with interchangeable LED light units. The plant factory is divided into several multi-shelf hydroponic units, each consisting of three tiers. The distance between tiers is 50 cm, and 16 plants were planted in each tier at a spacing of 20 cm within a gully and 20 cm between gullies. The temperature and humidity were monitored, using Gemini data loggers (Tinytag Plus (part No GP-1590)) and an instantaneous thermometer  at 23 ± 2 °C. The light/dark period was set to 16/8 h.Two experiments were established:−2 s−1) measured using a UPRtek MK350N premium Standalone handheld spectral light meter, Taiwan. Light treatments were as follows:Three lighting treatments were designed and applied at two intensities (high: 270 and low 60 μmol mBlue 435 nm rich treatment: Blue/Red (B435/R663): Blue rich spectrum with 435 nm wavelength used as a source of blue (B/R: 1.25 ± 0.1) .Blue 450 nm rich treatment: Blue/Red B450/R663: Blue rich spectrum with 450 nm wavelength used as a source of blue (B/R: 1.25 ± 0.1) .Red rich treatment: Blue/Red treatment (B/R-rich): Red 663 nm rich light spectrum with 450 nm wavelength used as a source of blue (B/R: 0.72) Blue to Red ratio, (1:1). .−2 s−1 were designed as follows:(1)Blue 450 nm rich treatment: Blue/Red treatment (B-rich/R). Blue rich spectrum with 450 nm wavelength used as a source of blue (B/R: 1.25 ± 0.1) (Blue (450 nm) to Red (663) peak ratio, 1.6:1).(2)Blue, red, green treatment: Blue/Red/Green treatment (B/R/G). Blue rich spectrum with 450 nm wavelength used as a source of blue with (B/R/G: 1.25/1/0.35) (Blue (435 nm) to Red (663) peak ratio, 1.6:1).(3)Red rich treatment: Red (663 nm) rich light spectrum with 450 nm wavelength used as a source of blue (B/R: 0.72) (Blue Red peak ratio 1:1.2).Three lighting treatments at 170 ± 10 μmol m−2 s−1, stomatal conductance mmol m−2 s−1, and transpiration rate mmol m−2 s−1) of planted lettuce to the lighting treatments was measured at two stages of development: at the initial vegetative, stage 4 weeks from the transplanting of plantlets; and the second  harvest stage, conducted 7 weeks after transplanting plantlets to the plant factory setting. The three unfolded top leaves were chosen from five plants from each treatments. Physiological measurements included light-saturated instantaneous maximum photosynthetic rate Amax (μg cm−2 s−1) was measured using an LCi-SD Highly Portable Ambient Photosynthesis System .Physiological response  harvest stage, conducted 7 weeks after transplanting plantlets to the plant factory setting. Morphological measurements of five randomly chosen plants from each treatment were taken. These included leaf number (cmshoot fresh weight (FW); and root fresh weight (RFW) (g), using a sensitive Fisher Scientific SG-402 laboratory balance.3+ TPTZ complex (colourless complex) to Fe2+-tripyridyltriazine (blue coloured complex) formed by the action of electrons donating antioxidants at low pH. This reaction was monitored by measuring the change in absorbance at 595 nm. The Ferric reducing antioxidant power (FRAP) reagent was prepared by mixing 10 part of 300 mM acetate buffer, 1 part of 10 mL TPTZ in 40 mM HCl and 1 part 20 mM FeCl3·6H2O. For the extraction, 0.1 g of freeze dried leaves were weighed and ground using a mortar and homogenized with 4 mL of HEPES buffer and sand purified by acid. From this solution, 0.80 mL was placed in an Eppendorf tube and centrifuged at 13,000 rpm in a microfuge (Micro star 12) for 2 min. The extract was then stored on ice prior to use. The calibration curve was prepared by plotting the absorbance at 595 nm versus different concentrations  of FeSO4. The concentrations of FeSO4 were in turn plotted against a concentration of standard antioxidant Trolox. The blank was prepared by mixing the 0.80 FRAP with 0.10 µL HEPES buffer and the spectrophotometer  set to zero against the blank. From the each of the stored extract samples, 0.10 µΜ of each stored extract were transferred to the cuvettes and 0.80 mL of FRAP reagent added. The FRAP values were obtained by comparing the absorbance at 595 change in the test mixture with those obtained from increasing concentrations of Fe3+ and expressed as mg of Trolox equivalent per gram of sample.The plants  from the second cut were stored in a deep freezer (at −20 °C) and freeze-dried for antioxidant analysis. The total antioxidant activity was analysed using the Ferric Reducing Ability of Plasma (FRAP) assay . The metAll data were subjected to analysis of variance (ANOVA) using Minitab software (version 17), and comparisons of means were made using the least significant difference (LSD) test at a 5% level of probability.p = 0) higher than at second harvest. At both harvest stages, the photosynthesis rate showed a remarkable (p = 0.003) difference between LED treatments. At the first harvest, the B435/R significantly increased assimilation rate by 26% compared to the B450/R, while there were no significant differences between B435/R and B/R-rich. At second harvest, there were significant differences between all LED treatments. The B435/R significantly increased the assimilation rate by about 100 and 32% in comparison to B450/R and B/R-rich, respectively. As with the assimilation rate, stomatal conductance in plant leaves grown under all LED treatments significantly (p = 0.00) decreased with plants’ maturity (p = 0.00) difference in leaves stomatal conductance between LED treatments at both harvest stages. At the first harvest, the greatest stomatal conductance was under B/R-rich, which increased stomatal conductance by 150 and 400% compared to B435/R and B450/R, respectively. At the second harvest, the greatest stomatal conductance was under B435/R, which was greater by 95 and 290% compared to B450/R and B/R-rich. In contrast to the assimilation rate and stomatal conductance, the transpiration rate increased with plant maturity, (p = 0.066) differences between harvest stages were observed, and there was a significant (p = 0.004) effect of LED treatments on transpiration rate at both harvest stages. At the first harvest stage, the highest transpiration rate was under B/R-rich, which increased by about 33 and 100% compared to B435/R and B450/R, respectively. At the second harvest stage, the highest value was at B/R-rich, followed by B435/R and then B450/R.Assimilation rate in lettuce cultivated under all LED treatments was reduced when the plants reached maturity, , which imaturity  and was aturity, . Signifip = 0.003) increased when plants were grown under combination of B435/R, compared to plants grown under B450/R and B/R-rich. At second harvest, the B435/R increased the plants’ fresh weight by 36 and 14% compared to B450/R and B/R-rich, respectively, and the B/R-rich increased plant fresh weight by 13% as compared to B450/R. Furthermore, the highest root fresh weight was produced by plants cultivated under B435/R, compared to other treatments (p = 0083) increased the root fresh weight by about 50 and 46% as compared to B450/R and B/R-rich, respectively, and, similarly to shoot fresh weight, the B/R-rich increased the root fresh weight by about 25% compared to B450/R. With regard to the number of leaves, no significant effect between all LED treatments were observed. (As shown in eatments . At the bserved. .p = 0.003) under B 435/R compared to B450/R, with no significant differences between B345/R and B/R-rich, which both increased assimilation rate by 25% compared to B450/R at first harvest. However, at the second harvest, the B435/R increased assimilation rate by 135 and 53% compared to B450/R and B/R-rich, respectively, whereas, at the first harvest, the highest (p = 0.072) leaf stomatal conductance was at B450/R, with an increment by 67% compared to other LED treatments. At the second harvest, the highest value of stomatal conductance was obtained by B435/R and B/R-rich, with an increment of 100% compared to B450/R. Although the transpiration rate significantly differed (p = 0.003) between LED treatments, the greatest value at the first harvest was under B435/R, with an increment by 60 and 33% as compared to B450/R and B/R-rich, respectively. Furthermore at the second harvest, the greatest value for the transpiration rate was at B345/R, with an increment of about 114% compared with B450/R, and no significant differences between B435/R and B/R–rich were record.All physiological parameters  for plants cultivated by all LED treatments with lower light intensity progressively decreased with plant maturity , as indip ≥ 0.001) and root fresh weight significantly (p = 0) greater at second harvest than at first harvest. Plants cultivated at both B435/R and B450/R produced significant (p = 0.078) shoot fresh weight , as in h weight  which inh weight . Which wh weight .p = 0.062) differences between all B-rich/R, B-rich/R/G and B/R-rich lights on the plants’ assimilation rates. Stomatal conductance significantly (p = 0.002) has a clear difference between all LEDs treatments, at first harvest B/R-rich increased the stomatal conductance by (20 and 67%) as compared to B-rich/R and B-rich/R/G, respectively. Similarly, the transpiration rate showed significant (p = 0.00) differences between LED treatments. The highest values were obtained under B/R-rich, which increased transpiration rate by 94 and 280%, compared to B-rich/R and B-rich/R/G, respectively, at first harvest.As in the first experiment, all the physiological parameters; assimilation rate, stomatal conductance, and transpiration rate for plants cultivated by all LEDs progressively decreased when plants reached maturity . At bothp = 0.598) differences between LEDs treatments on plant fresh weight (shoot + root fresh weight) Plants grown under all lighting treatment showed an increase in terms of shoots and root fresh weight when plants reached maturity. However, there were no significant (p = 0.296) differences between all LED treatments with high intensity (270 ± 20 µmol m−2 s−1). Lettuce shoot antioxidant activity (in µmol FeSO4 L−1) was recorded, as shown in p = 0) difference in lettuce antioxidant activity (in µmol FeSO4 L−1) when the intensity of lights became lower 60 µmol m−2 s−1 (4 L−1) was at B345/R, followed by B450/R, with increasing rate by about 53 and 420%, compared with B450/R and B/R-rich, respectively. In addition, in the second experiment, as shown in p = 0.004) differences between LED treatment in lettuce shoot antioxidant activity levels (in µmol FeSO4 L−1), the highest level was at B-rich/R, with an increment of 125 and 260%, as compared with B-rich/R/G and B/R-rich, respectively.At second harvest, there were no significant  confirmed that, firstly: using blue light with a wavelength peak of 435 nm enhanced assimilation rate by 26 and 100% at first and second harvest, respectively, and this was also observed at the lower intensity (60 µmol m−2 s−1). The B435/R enhanced assimilation rate by 25 and 135% for the first and second harvest, respectively, more than blue light with a peak of 450 nm. This finding supports our previous study on sweet basil (Ocimum basilicum) [The results from the first experiment at the higher intensity (270 ± 20 µmol msilicum) ,24. The 2 uptake for assimilation [The third confirmation n was that for lettuce shoot fresh weight, using blue light with wavelength peaks of 435 nm and 450 nm at low light intensity had same effect as that for B435/R and B450/R treatments. The same results for lettuce have been recently reported by : they fomilation . Similarmilation  due to tmilation . As a remilation . Blue  blue light with wavelength peak of 435 nm to red light with a wavelength peak 663 nm and at a low intensity of LED light can alter the assimilation translocation between lettuce plant organs. A similar tendency was found in lettuce when the ratio of blue to red increased by 20–50% [In the current research, the root fresh weight at the low light intensity of 60 µmol my 20–50% .At both intensities, there was no significant difference in the number of lettuce leaves among plants cultured under all LEDs treatments. In contrast, the combination of R/B LEDs significantly increased leaf numbers in lettuce ,35,36,37−1 s−1. This was the result of an increase in the assimilation rate of 24.2% compared to R/B LEDs, because green light is able to penetrate the plant canopy and supply energy, especially in plants with overlapping leaves, such as lettuce. Furthermore, [In the second experiment, hermore,  suggestehermore, ,43 consihermore, .4 L−1) under B435/R (1.25 ± 0.1) compared to B450/R (1.25 ± 0.1) B/R-rich (1:1) at the first experiment with low light intensity of 60 µmol m−2 s−1 but not at the high intensity of (270 ± 20 µmol m−2 s−1). Moreover, at B/R-rich (1.25 ± 0.1) compared to B/R/G (1/0.73/0.26) and B435/R (1:1.2) at second experiment (−2 s−1 130 ± 7 µmol m−2 s−1) 12 h photoperiod. The blue light is effective in accumulating secondary metabolism and in promoting the phenolic concentration and antioxidant capacity resulting from an activation of the PAL gateway enzyme in the biosynthesis of phenolic, enhanced by monochromatic blue LEDs [−2 s−1 increased antioxidant activity in lettuce leaves. However, using combined blue and red light led to less change in secondary metabolism and it has been suggested that the metabolic process is more sensitive to change in monochromatic light [In the present study, the levels of antioxidant activity levels in lettuce shoots . However, when a small amount of green light with a wavelength peak at 520 nm is added to the combination of B450/R663 nm and the ratio of B450 nm to Red663 nm is the same or R663/B450 nm = 1.2, all of the LEDs enhanced the assimilation rate by the same amount and produced the same amount of lettuce fresh weight. From these results, it can be concluded that B435/R at high intensity is the best LED for the production of economic yields of hydroponically grown lettuce in the plant factory for production of the highest yields. It was also found that B435/R at the low intensity of 60 µmol m−2 s−1 is the best LED for producing the highest level of antioxidant activity.The results of this study showed that the growth, photosynthesis, and antioxidant activity of lettuce performed better with a combination of blue light with a peak wavelength of 435 nm and red with a peak wavelength of 663 nm with a ratio of (1.25 ± 0.1), than with a combination of blue light with a peak wavelength of 450 nm and red with a peak wavelength of 663 nm with a ratio of (1.25 ± 0.1) at high intensity of (270 ± 20 µmol mGrow LED light system is a growing area for both research and commercial applications. This will increase the capacity of testing more specific wavelengths in both red and blue regions of lights. This is currently is still one of the limitations for a deeper understanding of the plant response to light spectrum.


Medicago sativa Linn or alfalfa is a tonic plant rich in proteins, vitamins, and minerals that is used to treat many diseases due to its pharmacological properties such as anti-inflammatory and antioxidant activities. So, the aim of this study was to evaluate the efficacy of alfalfa methanolic extract (AME) on the prevention of liver damage caused by nicotine. The total phenols, flavonoids levels, and the free radical scavenging activity of its extract (IC50) were measured. In this study, 30 Wistar rats were randomly divided into 5 groups as control (untreated), N (nicotine only), T1, T2, and T3 . AME  and nicotine  were then administered for 21 days. Weight gain, the liver-to-body weight ratio, liver functional enzymes, and the lipid profile were measured. Moreover, we evaluated oxidative stress, proinflammatory parameters, and histopathological changes in the liver. Total phenols, flavonoids, and IC50 were determined as 51.68 ± 0.62 mg GAE/g, 18.55 ± 1.01 mg QE/g, and 350.91 ± 16.46 μg/ml, respectively. Nicotine changed the measured parameters to abnormal. AME increased weight gain, the liver-to-body weight ratio, and enzymatic antioxidant levels and decreased malondialdehyde, liver functional enzymes, and proinflammatory cytokine levels. The lipid profile and histopathological changes have also been improved by AME in a dose-dependent manner. The results showed that AME in a dose-dependent manner by improving the inflammation and oxidative damage could improve the liver damage caused by nicotine. AccordiIC50 was determined as the concentration of the extract that inhibited 50% of DPPH radicals. Accordingly, it was calculated from the graph of the inhibition percentage against extract concentration. Notably, the assay was performed in triplicate.The toxicity tests were carried out according to the Organization for Economic Cooperation and Development (OECD) test guideline, i.e., OECD Guideline 423 used for the acute oral toxicity test. Before the start of the experiment, the body weight of animals was recorded individually for calculating the proper treatment dosage. To determine the LD50 of AME, 12 Wistar rats 200-250 g) were randomly divided into 4 groups (3 rats per group), and 5, 50, 300, and 2000 mg extract/kg body weight, P.O., were separately administered for each group, respectively. Signs of toxicity and possible death of animals were monitored for 24 hours to calculate the median lethal dose (LD50) of alfalfa. All animals were observed at least once during the first 30 min in the first 24 hours with great consideration given for the first 4 hours following vehicle or alfalfa methanol extract administration, and then they were monitored once a day for 14 days. This observation was done to check the onset of clinical or toxicological symptoms according to the OECD guideline. All observations included changes in the skin and fur, eyes, and mucous membranes, and behavioral patterns were systematically recorded and maintained with an individual record. In addition, consideration was given for observations of convulsions, tremors, diarrhea, salivation, lethargy, sleep, coma, and mortality. At the end of the study, all the animals in all the dose groups were sacrificed and the internal organ-body was compared with values from the control group [0 g were In this study, a 2% solution of nicotine hydrogen tartrate salt was prepared by sterile distilled water, which was then used for intraperitoneal injection (I.P.) at the dose of 0.5 mg/kg .ad libitum access to safe water and rodent feed. Then, the study began by passing 7 days from the animal adaptation to environmental conditions. In the present study, the environmental conditions, use, and euthanasia of animals were considered in terms of the international ethical guidelines for laboratory animals [For this study, 30 male Wistar rats  were purchased from the Pasteur Institute of Iran. Throughout the study, the animals were kept under the controlled environmental conditions in standard cages . When performing the experiment, all animals were given  animals  . Only received distilled water (P.O.);Nicotine Group (N). Only received nicotine ;Treatment Group 1 (T1). Nicotine  + AME ;Treatment Group 2 (T2). Nicotine  + AME ;Treatment Group 3 (T3). Nicotine  + AME  [y, P.O.) , 26.The animals were randomly divided into 5 groups (6 rats per group) as follows:The study period was 21 days for all animals. Once at the beginning and once at the end of the study, the body weight of the animals was measured. At the end of the experimental period, the animals were anesthetized by intraperitoneal injection of ketamine-xylazine  and blood samples were collected from their right ventricle. The obtained blood samples were centrifuged for 15 min at 1000 g. Afterward, the attained serums were aliquoted and then stored at -20°C until processing. After euthanasia, each animal's liver was separated and then weighed. Subsequently, a piece of the left lobe of liver tissue (0.5 g) was immediately mixed with phosphate-buffered saline  in a medium containing ice. Thereafter, it was homogenized by a homogenizer . Subsequently, tissue homogenates were centrifuged for 1 h at 5000 g at 4°C. Finally, the supernatant was stored at -20°C until the measurements of lipid peroxidation, enzymatic antioxidants, and proinflammatory cytokine levels. At the end of the study period, the ratio of liver to body weight was calculated .Serum activity levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (GGT) and serum concentrations of total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) were determined using commercial kits  and the Hitachi 911 autoanalyzer . LDL − Cμl of 10% liver homogenate. This mixture was then placed in a bain-marie for 15 minutes at 95°C. After cooling, the mixture was centrifuged , and the obtained supernatant absorbance was determined at 535 nm. Finally, the MDA level was calculated by a 1.56 × 105 M−1 cm−1 molar extinction coefficient. Notably, all the concentrations were expressed as nmol/g tissue.Malondialdehyde (MDA) levels of tissue homogenates were measured in terms of the Buege and Aust method . Briefly2−· to H2O2 under enzymatic conditions for 120 seconds at 420 nm. Next, CAT activity was measured based on its ability to decompose H2O2 within 1 minute at 405 nm. Also, GPx converted glutathione (GSH) to oxidized glutathione (GSSG). Hence, through the reaction of residual GSH, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was used to determine GPx at 412 nm indirectly. All data were expressed as U/g tissue.In liver homogenates, the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured using colorimetric kits  in terms of the manufacturer's instructions. The basis of the determination of SOD activity was a conversion of Oβ , interleukin-6 , and tumor necrosis factor-α  were quantified using enzyme-linked immunosorbent assay (ELISA) kits  in terms of the manufacturer's protocols at 450 nm . The obtained results were then expressed in pg/ml.In liver homogenates, the levels of interleukin-1μm thick sections of paraffin blocks were cut using a microtome and then stained with hematoxylin and eosin (H&E). Afterward, a semiquantitative scoring system was used to evaluate the histopathological criteria [Hydropic degeneration of hepatocytesKupffer cell proliferationCentral vein congestion and dilation of sinusoidsThe samples obtained from the liver right lobe were fixed for 48 h in 10% phosphate-buffered formalin. After the fixation, the samples were embedded in paraffin. Thereafter, 5 criteria –34. AccoP value < 0.05 was considered the statistical significance level.The study data were reported as mean ± SEM. One-way analysis of variance (one-way ANOVA) followed by Tukey's post hoc test was performed to analyze the parametric data. Moreover, liver histopathological scores were assessed by the Kruskal–Wallis test. In this regard, if the Kruskal–Wallis test was significant, the Mann–Whitney test was then used to perform pairwise comparisons. All graphs and statistical analyses were performed by GraphPad Prism (Version 7.03) software . μg/ml, the inhibitory percentage of the extract increased from 18.94 ± 0.97 up to 77.87 ± 2.07, and for vitamin C, it increased from 36.86 ± 1.82 up to 98.69 ± 0.40.As shown in During the 14 days of the acute toxicity test, the animal's death and body weight changes were not observed. LD50 was higher than the oral administration of 2000 mg extract/kg.P < 0.05) compared to that of the C group (262.92 ± 5.99 g). The highest increase in body weight gain was observed in the C group (36.06 ± 3.98 g), and the lowest one was for the N group (12.41 ± 2.41 g). Among the AME-treated groups, the T1 group had the lowest (14.57 ± 2.69 g) and the T3 group had the highest (24.74 ± 2.67 g) increase in weight gain. A significant decrease was also observed in body weight gain among the N, T1, and T2 groups (19.04 ± 5.78 g) compared to the control group (C) (P < 0.05). Notably, there was no significant difference between the T3 and C groups (P > 0.05)  has significantly decreased ( > 0.05) .P < 0.05)  .P < 0.05). Moreover, there was a significant increase in terms of the liver-to-body weight ratio in the T3 group (0.030 ± 0.002 g) when the extract was administered at a dose of 500 mg/kg/day compared to the N and T1 groups (P < 0.05). In addition, no significant difference was observed in the liver-to-body weight ratio between the T3 and C groups (P > 0.05).As shown in Moreover, nicotine intake (the N group) significantly increased the serum activities of ALT, AST, ALP, GGT, and LDH compared to the control group (C group). Administration of this extract has also decreased the serum activities of liver enzymes. Among the AME-treated groups, the most significant decrease in the serum activities of enzymes was observed in the animals receiving the extract at a dose of 500 mg/kg/day (the T3 group) compared to the N group .P < 0.05). Moreover, serum concentrations of TG, HDL-C, and VLDL-C have significantly decreased (P < 0.05). Lipid profiles  of the T3 group have also shown a significant difference compared to those of the N and T1 groups (P < 0.05). In addition, TG, HDL-C, and VLDL-C levels had no significant difference in the group receiving the extract at a dose of 250 mg/kg/day (the T2 group) compared to the other groups (P > 0.05).As shown in P < 0.05). It is noteworthy that MDA, SOD, CAT, and GPx levels of liver tissue in AME-treated groups were dose-dependent, and accordingly, the most significant efficacy of the extract was in the T3 group compared to the N group.As shown in β has significantly increased in the N (725.84 ± 25.70 pg/ml) and T1 (674.91 ± 17.78 pg/ml) groups compared to the C group (311.31 ± 15.50 pg/ml) (P < 0.05). In addition, liver tissue IL-1β concentration showed a significant decrease in the T3 group (454.78 ± 75.58 pg/ml), in which the extract with the dose of 500 mg/kg/day was administered, compared to the T1 and N groups (P < 0.05). Also, the level of IL-1β has significantly decreased in the T2 group (512.37 ± 64.19 pg/ml) compared to the nicotine-receiving group  (The level of IL-1 < 0.05) .P < 0.05). Furthermore, IL-6 concentration in the T3 (184.93 ± 13.99 pg/ml) and T2 (194.82 ± 5.97 pg/ml) groups has significantly decreased compared to that in the animals of the T1 and N groups, which showed a significant increase compared to the C group (P < 0.05).As shown in α concentration in the liver tissue of the T2 (342.72 ± 40.23 pg/ml) group receiving the extract at a dose of 250 mg/kg/day showed a significant decrease compared to that of the T1 (495.14 ± 10.60 pg/ml) and N (558.79 ± 31.82 pg/ml) groups (P < 0.05). Correspondingly, this decrease was also more pronounced when the extract was administered at the dose of 500 mg/kg/day in the T3 group (294.98 ± 40.77 pg/ml) (P < 0.05). The level of liver TNF-α in animals of the N and T1 groups has significantly increased compared to that of the C group (224.97 ± 12.07 pg/ml) (P < 0.05) (The TNF- < 0.05) .P < 0.05). Accordingly, when the extract was administered at doses of 250 and 500 mg/kg/day, the scores of this criterion have significantly decreased in the T2 (1.33 ± 0.21) and T3 (1.17 ± 0.31) groups compared to the N group (P < 0.05), respectively. This decrease was also repeated in the T2 and T3 groups compared to the T1 group (P < 0.05). The criterion scores of hydropic degenerations of hepatocytes have also increased in the T2 and T3 groups compared to the C group; however, it was not significant (P > 0.05)  and T1 (2.67 ± 0.21) groups compared to the C group (0.0 ± 0.0) ( > 0.05) .P > 0.05).As shown in P < 0.05). These scores have significantly decreased in the T3 (0.50 ± 0.22), T2 (0.83 ± 0.17), and T1 (1.00 ± 0.26) groups compared to the N group (P < 0.05)  compared to the C group (0.33 ± 0.21) ( < 0.05) .The histopathological changes of liver tissue of the animals in the studied groups are microscopically shown in Figures Plants are rich sources of biologically active compounds with wide molecular diversities. These sources are known for the development of new drugs –37. In tμg/ml, while the studied concentrations were between 0 and 1000 μg/ml in the DPPH test. Therefore, the IC50 lower than the median indicates the high antioxidant power of alfalfa in inhibiting free radicals. In this regard, the high inhibitory activity of the extract probably resulted from polyphenolic compounds [In recent years, the wide range of the effects of free radicals has interested the attention of scientists, and as a result, it has been proven that free radicals play a remarkable role in the pathogenesis of many diseases such as cancer . Antioxiompounds .In experimental toxicology studies, body weight and the liver-to-body weight ratio are vital and readily available indices when facing oxidative stress . In the The liver plays a key role in the metabolism of drugs, foods, and many compounds, so changing its function endangers a person's health . Liver dSeveral studies have shown a link between heart diseases and lipid profile changes , 59. In MDA is used as an important indicator in the diagnosis of the induced stress within the target tissue . In our In harmony with these results, Taysi et al. reported that nicotine performance at a dose of 0.5 mg/kg intraperitoneally for 21 days caused a significant increase in malondialdehyde, nitric oxide, and glutathione reductase (GR), as well as a decrease in GPx in liver tissue . In addiApparently, alfalfa has various vitamins (such as vitamins C and E), polyphenolic compounds (such as flavonoids and phenols), some types of minerals , some types of phytosterols, and other antioxidant compounds , 70. Theβ, IL-6, and TNF-α) [β, IL-6, and TNF-α). Also, it seems that the induction of nicotine-induced oxidative stress and the production of free radicals have consequently resulted in cell destruction and the invocation of immune cells to the liver, as the target tissue of nicotine [β, IL-6, and TNF-α) [One of the most important indices of tissue damage is inflammation . In thisd TNF-α) , 73. Accd TNF-α) , 75. In nicotine , 77. Thed TNF-α) . In our d TNF-α) .One of the characteristics of working with laboratory animals is access to target tissues in histopathological studies . In prevβ, IL-6, and TNF-α), and it resulted in histopathological changes in the liver tissue. This study showed that AME with phenolic compounds has a high antioxidant power, which can control the mentioned effects of nicotine. This control was revealed through the laboratory findings of the present study. Trapping nicotine-induced free radicals, boosting the body's immune and antioxidant systems, and affecting metabolic pathways are some of the functional reasons for alfalfa. In any case, it should be noted that studies in the experimental phase will be an introduction to the process of supplementary studies.In conclusion, the presence of polyphenolic compounds, as well as the IC50 value below the median concentration, showed the high antioxidant power of AME. Therefore, this plant can be considered an important antiradical in complementary medicine more than before. In this study, nicotine has resulted in weight loss; decreased liver-to-body weight ratios; increased serum activities of liver enzymes ; altered lipid profiles; increased liver MDA levels; decreased levels of SOD, CAT, and GPx antioxidants in liver tissue; and increased hepatic levels of proinflammatory cytokines , heart disease, and metabolic problems, alfalfa can be used as a supplement to suppress oxidative stress that is growing in our lives due to widespread application in all aspects. Moreover, recommending the use of antioxidant compounds, for example, alfalfa, in smokers to prevent the effects of nicotine oxidative stress can be another clinical application of the study.


This study aimed to design an effective nanoparticle-based carrier for the oral delivery of fisetin (FST) with improved biopharmaceutical properties. FST-loaded nanoparticles were prepared with polyvinyl alcohol (PVA) and poly(lactic-co-glycolic acid) (PLGA) by the interfacial deposition method. A central composite design of two independent variables, the concentration of PVA and the amount of PLGA, was applied for the optimization of the preparative parameter. The responses, including average particle size, polydispersity index, encapsulation efficiency, and zeta potential, were assessed. The optimized formulation possessed a mean particle size of 187.9 nm, the polydispersity index of 0.121, encapsulation efficiency of 79.3%, and zeta potential of −29.2 mV. The morphological observation demonstrated a globular shape for particles. Differential scanning calorimetry and powder X-ray diffraction studies confirmed that the encapsulated FST was presented as the amorphous state. The dissolution test indicated a 3.06-fold increase for the accumulating concentrations, and the everted gut sac test showed a 4.9-fold gain for permeability at the duodenum region. In conclusion, the optimized FST-loaded nanoparticle formulation in this work can be developed as an efficient oral delivery system of FST to improve its biopharmaceutic properties. Drosophila and R6/2 mouse model, to demonstrate that FST has the potential to treat Huntington’s disease through the inhibition of the ERK pathway. A recent study has examined various flavonoid compounds using oxidative and genotoxic stress tests in the senescent murine model, and the results indicated that FST supplementation is significantly beneficial to aged wild-type mice by regaining their tissue homeostasis, reducing the age-related pathology, and extending lifespan, which proves FST has senolytic effect [Fisetin   is a natc effect . The heac effect .Despite FST’s profound health-promoting effects, the efficient oral delivery of FST is still problematic because the low aqueous solubility (less than 1.0 mg/mL) limits its dissolution and oral bioavailability ,13. In aFor the development of ideal nanoparticle formulation, various preparative parameters, such as the amount of polymer and surfactant, should be taken into consideration, because these parameters affect the resulting particle size, size distribution, encapsulation efficiency, and zeta potential, which are critical factors for the dissolution rate, permeability, and stability. The design of experiment (DOE) approach is a method to optimize the preparative variables for the development of FST-loaded PLGA nanoparticles (FST–NP). DOE may provide an alternative to assess the impacts of many factors on responses more efficiently than one-factor-at-a-time (OFAT) experiments . The advAn HPLC–DAD method was used for quantification of FST in the formulation and in vitro release study, and the analytical chromatogram is shown in The development of nanoformulations with desired properties relies on comprehensive process parameters. We conducted CCD to discuss the effect of PVA concentration and the amount of PLGA. In this experimental design, the total number of experimental groups is 13. p-value of X2 (PLGA amount) and X22 are below 0.05, indicating they would have an influence on particle size and concluded to Equation (1). In Particle size plays an important role in this study and has a crucial influence on the characterization of FST–NP. In the study, the ANOVA statistical analysis and model fitting were conducted to realize the relationship between factors and responses of FST–NP. In this model, the p-values of X1 (PVA concentration), X2 (PLGA amount), X1X2, X12, and X22 are all less than 0.05, suggesting that these variables have significant differences in the EE response (Equation (2)). In EE would display the preparation efficiency of this research. For EE, the p-value of X22 (square of PLGA amount) is below 0.05, indicating FST–NP is significant affected by the amount of PLGA (Equation (3)). In Zeta potential provides information about the stability for FST–NP dispersion. The higher the absolute value of this response is, the better stability of FST–NP performs because of electrostatic repulsion. For zeta potential, the p-values of variables X2 (PLGA amount), X1X2, and X22 are less than 0.05, which means that these variables have significant effects on PDI (Equation 4). In PDI is a measure of homogeneity of particle size and is generally expressed as smaller than 0.3 for a homogenous population of nanocarriers . After AThe results show that the optimal formulation contains a PVA concentration of 0.5% and a PLGA amount of 75.3 mg. According to this formulation, PLGA nanoparticles were prepared, and the particle size, encapsulation efficiency, zeta potential, and PDI were displayed in After optimization, it seems that a particle size of 187.9 nm, with a PDI of 0.121, is comparable ,31. As lThe particle size measured by DLS is 187.9 ± 6.1 nm A. The paFor characterization of FST–NP, the differential scanning calorimetry was executed to confirm its thermal performance. The endothermic peaks of the FST appeared at 132 °C and 343 °C for water loss and the melting point of FST, respectively [w/w) ratio group have obvious diffraction peaks owing to different dilute ratios. For FST–NP, there are no peaks observed in the PXRD pattern, speculating that FST may be evenly dispersed in PLGA and become amorphous after preparation.Powder X-ray diffraction was used to affirm the crystallinity of FST–NP. In −1 (OH stretching), 3356 cm−1 (OH stretching), 1615 cm−1 (C=O stretching), 1447 cm−1 (aromatic C=C stretching), 1280 cm−1 (–OH bending), 1120 cm−1 (C–O stretching) and 854 cm−1 (out of plane C–H bending) [−1 (C–H stretching), 2950 cm−1 (C–H stretching), and 1760 cm−1 (C=O stretching) [−1, 2950 cm−1, and 1760 cm−1 did not change and was similar to that of blank PLGA nanoparticle because of the relatively low content of FST. Compared with FST, the characteristic peaks showed displacement from 3356 cm−1 to 3370 cm−1, and from 1615 cm−1 to 1622 cm−1. It showed that FST is present in the matrix of PLGA, and there may be some interactions among them [FT–IR was used to confirm that FST was encapsulated in PLGA nanoparticles. In bending) ,34,35. Tetching) . In the In the pH 1.2, pH 6.8, and pH 7.4 release medium, the release of FST–NP are all higher than the FST standard, and the release reaches a stable level after the first hour . In thisIn In this study, the stability test of FST–NP was conducted at 5 ± 3 °C and 25 ± 2 °C, 60 ± 5% RH and observed the particle size, PDI, and FST content of FST–NP after 0, 3, 7, 14, 21, 30 and 60 days. In a previous study, FST was demonstrated to have a concerning level of stability in alkaline conditions, and the half-life of FST in pH 7.5 condition at 37 °C is only 3.4 h . This prThe utilization of flavonoids as therapeutic or health-promoting agents is generally hindered by their inherent properties, such as inferior water solubility, low intestinal permeability, and insufficient stability, which limits the oral absorption of flavonoids. The enhancing of saturation solubility and/or membrane permeability is an effective strategy necessary to improve the oral bioavailability of flavonoids according to the theory of the biopharmaceutical classification system . There aBased on the results above, FST–NP enhances dissolution performance and intestinal permeation via nanoscale size, higher surface area, and better biocompatibility to prolong FST–NP retention time on the intestine. In addition, FST–NP is stable within 60 days to prevent degradation. In this way, FST–NP may enhance oral bioavailability because of characterization improvement and be a potential agent to make therapy efficient.® RG 502H) was purchased from Evonik Industries AG . Poly   and sodium phosphate monobasic were purchased from Sigma-Aldrich Co. . Acetone was purchased from Echo Chemical Co. . HPLC grade acetonitrile was obtained from Fisher Scientific .Fisetin (purity 96%) was purchased from Alfa Aesar . Luteolin (purity 98%) was from Tokyo Chemical Industries . PLGA  with a ratio of 35:65 (v/v), and the flow rate was set to 1.2 mL/min. The injection volume was 20 μL and detected at 358 nm. The run time of FST analysis was 10 min. Analytical method validation for FST analysis was performed according to our previous work [In vitro FST content was determined by high-performance liquid chromatography (HPLC), coupled with a diode array detector (DAD) modified from the previous study . The HPLous work .v/v) methanol as stock solution, and diluted to the concentration of 50.0, 25.0, 12.5, 5.0, 1.25, and 0.5 μg/mL with 50% (v/v) methanol. After dilution, A 25 μL luteolin standard (100.0 μg/mL) was added as internal standard for the preparation of calibration curves.For the standard preparation, FST (5.0 mg) standard was dissolved in 50% (2PO4 (pH 2.5) with a ratio of 23:77 (v/v). For detection, Ag/AgCl was chosen to be the reference electrode, applied potential was set to +1.0 V, the filter was 0.1 Hz, and the range was 20 nA. The determination of FST in the permeability study was determined by HPLC coupled with an LC–4C amperometric detector . The HPLC system  consisted of a D-5160 pump, an L-5260 autosampler, and a BAS LC–4C electrochemical detector . For separation, the same column, flow rate, and injection volume were used as mentioned above. The mobile phase consisted of acetonitrile and 0.05 M NaHg for 30 min, the supernatant was removed and FST–NP was collected. The FST–NP was prepared by the nanoprecipitation method . PLGA an1) and PLGA amount (X2), and responses included particle size (Y1), size distribution expressed by the polydispersity index (Y2), encapsulation efficiency (Y3), and zeta potential (Y4), as shown in 1), maximized encapsulation efficiency (Y2), zeta potential (Y3) < −20 mV and minimized polydispersity index (Y4). The Design-Expert 6.0.3 software  was applied to conduct RSM based on CCD experiments to optimize the FST–NP formulations. The chosen factors were the concentration of PVA  with the following equation: The EE of FST–NP was evaluated based on previous work . Brieflyw/v) phosphotungstic acid aqueous solution, then removed excess phosphotungstic acid, dried, and measurements were determined by TEM.Appearance, shape, and particle size of the FST–NP were observed by transmission electron microscope (TEM)  . FST–NP Differential scanning calorimetry (DSC)  was executed to observe melting point and glass transition temperature to evaluate the thermal behavior of FST–NP. In this study, DSC was applied to evaluate the FST, blank PLGA nanoparticles, physical mixture, and FST–NP. The 10 mg sample was weighed in the aluminum pan and heat from 30 °C to 500 °C with a heating rate of 10 °C/min.Powder X-ray diffraction (PXRD)  could be executed to evaluate the change of the crystal form of the FST after preparation. In this study, the PXRD was applied to evaluate the FST, blank PLGA nanoparticles, physical mixture, and FST–NP. The sample was ground before analysis, and the diffraction angle was between 5° and 50° at the scan rate of 0.1°/sec.−1.In this study, Fourier transform–infrared spectroscopy (FT–IR)  was executed to observe the functional groups and bonding of the FST, blank PLGA nanoparticles, and FST–NP. Sample powder mixed with potassium bromide was compressed into thin tablets for determination. The range of wavenumber was from 4000 to 400 cmg for 10 min and determined by HPLC–DAD  to assess the release of the FST–NP in pH 1.2, pH 6.8, and pH 7.4 medium. The sample equal to 4.0 mg of FST would be added to a 100 mL dissolution medium. The temperature was maintained at 37 ± 0.5 °C with a stirring speed of 100 rpm. A total of 500 μL of dissolution medium was collected and refilled 500 μL fresh medium at 1, 4, 8, 12, 24, 48, 72, 96, and 120 h. The withdrawn samples were centrifuged at 13,000× HPLC–DAD . The resThe everted gut sac model ,54 was eThe samples were dispensed into sample vials, sealed with rubber stoppers and aluminum caps, and kept at 25 ± 2 °C, 60 ± 5% relative humidity (RH), and 5 ± 3 °C, respectively. The particle size, distribution, and FST content were measured at 0, 3, 7, 14, 21, 30, and 60 days.Other stability tests for FST were conducted in different pH conditions. FST standard was put in pH 1.2, 6.8, and 7.4 medium with the final concentration of 5 μg/mL for 72 h, and the temperature was maintained at 37 ± 0.5 °C. The sample was withdrawn at 1, 4, 12, 24, 48, and 72 h, and determined by HPLC–DAD.t-test; p < 0.05 was considered a significant statistical difference.The experiment results are expressed as mean ± standard deviation. Differences between formulations used SPSS v20  and were compared by w/v) PVA, and 75.3 mg of PLGA, resulting in particles with an average size of 187.9 nm, PDI of 0.121, negative surface charge −29.2 mV, and EE of 79.3%. Morphological examination by TEM illustrates globular-shaped nanoparticles. According to the DSC and XRD analysis, FST is enclosed in the PLGA particles as an amorphous state. The FST–NP demonstrates a 3.06-fold increase in the dissolution rate and a 4.9-fold increase in the permeability at the duodenum region. The above results indicate that the FST–NP has great potential to be developed as an oral carrier for efficiently delivering FST with improved biopharmaceutical properties. This study proposed an efficient nanoparticle-based system to improve the biopharmaceutical properties of FST for oral delivery. A straightforward interfacial deposition method was employed to prepare the FST–NP, and the preparative parameters were optimized by the RSM based on CCD to achieve appropriate characteristics, including particle size, PDI, EE, and zeta potential. The optimized FST–NP formulation consists of 5 mg FST, 0.5% (


To date, little is known about the effects of combined organic substrate addition and N fertilization on the diazotroph community composition and structure in purple soils. We investigated the effects of N fertilization and straw mulching on diazotroph communities by quantifying and sequencing the nifH gene in wheat rhizosphere. The abundance and richness of diazotrophs were greater the higher the fertilization level in the mulched treatments, whereas in the nonmulched treatments (NSMs), richness was lowest with the highest N fertilization level. The abundance and α-diversity of diazotrophs correlated with most of the soil properties but not with pH. At the genus level, the relative abundances of Azospirillum, Bacillus, and Geobacter were higher in the NSMs and those of Pseudacidovorax, Skermanella, Azospira, Paraburkholderia, Azotobacter, Desulfovibrio, Klebsiella, and Pelomonas in the mulched treatments. The differences in community composition between the mulched and the NSMs were associated with differences in soil temperature and soil organic carbon and available potassium contents and C:N ratio. Overall, straw mulching and N fertilization were associated with changes in diazotroph community composition and higher abundance of nifH gene in alkaline purple soils.Diazotrophs that carry out the biological fixation of atmospheric dinitrogen (N N transilable N . Diazotrilable N . Diazotrilable N . The nifmicrobes .Agricultural management practices, e.g., mulching and fertilization, affect the physiochemical properties of soil . DiazotrnifH-containing microbes in agricultural soils  evaluate the effects of N fertilization and straw mulching on the abundance, diversity, and community structure of diazotrophs; (2) identify the diazotroph genera affected by N fertilization and straw mulching; and (3) determine the relationships between dominant diazotroph genera and soil properties. We hypothesized that fertilization and straw incorporation would change soil properties, in particular the C and N levels, that would further affect the diazotroph communities; higher C to N ratios were expected to be associated with higher abundance and α-diversity of diazotrophs.For this study, we established a field experiment to monitor the effects of inorganic N fertilizer and straw mulching on the community composition of diazotrophs in purple soil. We applied quantitative PCR (qPCR) and high-throughput sequencing targeting the –1, total nitrogen (TN) content 0.83 g kg–1, total phosphorus (TP) content 0.86 g kg–1, and total potassium content 13.96 g kg–1.The straw mulch no-tillage experiment was established in 2015 at Renshou experimental base  of the Agricultural College of Sichuan Agricultural University, China. The station is located in an area with a humid subtropical monsoon climate, an annual average temperature of 17.4°C and a mean annual precipitation of 1,009 mm. The soil is a typical alkaline purple soil. The basic physicochemical properties of the 0–20-cm soil layer in 2015 were as follows: pH 7.82 (soil:water = 1:2.5), organic carbon content 9.78 g kg2 (6 m × 5 m) replicate plots per treatment. The straw mulching rates were 0 (NSM: no straw mulching) and 8,000 (SM: straw mulching) kg ha–1. N fertilizer levels were 0 (N0), 120 (N1), and 180 (N2) kg ha–1. P and K fertilizers were applied to all the plots as 75 kg P2O5 ha–1 and 75 kg K2O ha–1, respectively.The split plot design experiment had straw mulching as the main factor and nitrogen level as the subplot factor with four 30 mSoil samples were collected on March 20, 2019, at the wheat anthesis stage. Fifteen wheat plants per plot were collected gently using a spade, and the soil attached to the roots was pooled into one composite rhizosphere soil sample per plot. The samples were transported on ice in a constant-temperature box to the laboratory and stored at −80°C.4+-N and NO3–-N were extracted from fresh soil with 2 mol L–1 KCl and determined using colorimetry  , and available potassium (AK) contents were analyzed as described previously . NH4+-N , China) . Soil teDNA was extracted from 0.5 g of soil using the Fast DNA Spin Kit for Soil  according to the manufacturer’s instructions. The purity and concentration of the extracted DNA were detected by using 2% agarose gel electrophoresis and a Nanodrop 2000 spectrophotometer . The extracted DNA was stored at −20°C.nifH gene copy number was determined using real-time qPCR on an ABI7500 Fast Real-Time PCR System (Applied Biosystems Inc. United States). The nifH fragments were amplified in triplicate using primers PolF (5′-TGCGAYCCSAARGCBGACTC-3′) and PolR (5′-ATSGCCATCATYTCRCCGGA-3′) (2O). Amplification was initiated by denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and reading the plate at 80°C (The CGGA-3′) . Amplifi at 80°C .nifH gene fragment from mixed soil DNA samples. The nifH fragment was amplified as described above using pmD®18-T Vector  according to the manufacturer’s instructions. Based on a translated nucleotide query (nifH from Ruminococcus sp. (sequence ID MBE6845760.1). Plasmid DNA was extracted using a Plasmid Miniprep Kit , and the plasmid concentration was measured with a spectrophotometer   . As the H copies . Since NH copies , the planifH amplicons of approximately 360 bp were amplified using the primers PolF with a unique sample identifying barcode and PolR. Amplification was done in triplicate 25 μl reactions including 1 μl of each 10 μM primer, 2 μl (20 ng) of template DNA or 2 μl sterilized distilled water in the negative control, and 12.5 μl of MasterMix containing Taq DNA polymerase, PCR buffer, Mg2+ and dNTPs , and 8.5 μl double distilled water (ddH2O). The amplification program included initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 60°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10 min  test. The nifH abundance data were log10 transformed and checked for normality prior to the test. The relationships between soil properties, nifH abundance, and α-diversity were estimated using Pearson correlation analysis. β-Diversity was visualized using weighted UniFrac distance-based principal coordinate analysis (PCoA) in R package “ape” . Differences in soil properties, ge “ape” . Hierarcge “ape” . Corrploge “ape” . The relge “ape” . Linear ge “ape” .nifH gene copy numbers ranged from 3.2 × 107 to 4.7 × 108 per g soil, with the lowest and the highest numbers in the NSMN2 and SMN2 treatment, respectively  (P < 0.05) (P < 0.05) (The ectively . In the  < 0.05) . Compare < 0.05) . Further < 0.05) .nifH communities were distinct in all the six mulching–fertilizer level combinations (P < 0.05) (P < 0.05) (The  < 0.05) . The fir < 0.05) . The mul < 0.05) . Similar < 0.05) , showingProteobacteria accounted for 88.5–92.9% of the total relative abundance in all the treatments  (The relative abundance of the phylum eatments . The releatments . In the eatments . At the e > 3.5) .4+-N, NO3–-N, AP, and AK contents and C:N ratio and negatively with soil temperature (P < 0.05), and the number of observed species correlated positively with SOC, AN, NO3–-N, AP, and AK contents and C:N ratio and negatively with soil temperature (P < 0.05)   . The abu < 0.05) . The rel < 0.05) . Differe < 0.05) . The gencontents .Azospirillum and Azotobacter) or in symbiosis   . The abuizobium) . Fertiliizobium) . We assezotrophs . The N fzotrophs . We foun4+-N, NO3–-N, AP, and AK contents and C:N ratio, and the number of observed species was associated with differences in soil temperature; SOC, AN, NO3–-N, AP, and AK contents; and C:N ratio. In agreement, the results of RDA suggested that the differences in diazotroph communities were associated with differences in soil temperature; NH4+-N, AN, NO3–-N, AP, and TN contents; and C:N ratio. Especially, the differences between the mulched and NSMs possibly resulted from the higher SOC and AK contents in the mulched treatments.The availability of soil nutrients, which is highly responsive to straw and fertilizer input, is crucial for the establishment of soil diazotroph community structure . In our Azospirillum within this class were high. Azospirillum is the best characterized genus of plant growth-promoting rhizobacteria and increased the yield and dry weight of wheat  GenBank database with the accession number All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Catunaregam spinosa (Thunb.) Tirveng. (Syn. Randia dumetorum (Retz.) Lam.), belonging to the Rubiaceae family, is distributed in south Asian countries. It is used as a traditional medicine to treat gastrointestinal and hepatic problems and as an anti-inflammatory and antimicrobial agent. The main aim of this review is to collect and analyze the available scientific information on traditional uses, phytochemistry, and pharmacological activities of C. spinosa. The scientific information related to C. spinosa was collected from various resources and databases such as SciFinder, Scopus, PubMed, and other databases. C. spinosa was found to be an important crude drug of the traditional medicinal systems such as Ayurveda. It was found to be used by the people of India as an alternative medicine, while the fruit of this plant was found to be used in dietary regimens as well. Active phytochemicals such as catunarosides, randianin, and several other saponins and triterpenoids possess various pharmacological activities such as anti-inflammatory, hepatoprotective, antibacterial, and immunomodulatory activities. Many studies have been performed to isolate the active compounds; however, there is a need for more activity-guided isolation studies. Various in vitro studies showed promising results but there are not many studies related to mechanism of actions using animal models. Hence, future studies on C. spinosa should focus on correlating the traditional uses with active phytoconstituents and modern pharmacological activities. Humans have been dependent on plants since the ancient times for various reasons such as food, shelter, and medicine . MedicinCatunaregam belonging to family Rubiaceae -2β-hydroxy-olean-12-en-28-oic acid, 3-O-[β-D-glucopyranosyl-(1⟶2)-β-D-glucopyranosyl]-olean-12-en-28-oic acid, and 3-O-[β-L-rhamnopyranosyl-(1⟶3)-β-D-glucuronopyranosyl-(1⟶2)-β-D-glucopyranosyl-(1⟶2)-β-D-glucopyranosyl]-12-en-28-oic acid, and two triterpenoids, oleanolic acid and 3β,23-dihydroxy-olean-12-ene-28-oic acid, were isolated from fruits [C. spinosa, the compounds named catunaregin and epicatunaregin were isolated [C. spinosa [2Various flavonoids, alkaloids, tannins, lignans, terpenoids, and volatile oils have been reported from this plant. The triterpene, saponins , and irim fruits . From thisolated . Many trisolated  from theisolated . 11-Meth spinosa . The bioC. spinosa extracts and compounds have been subjected to various pharmacological activity evaluations. This plant has been studied widely for its pharmacological activities, mainly antibacterial, antioxidant, anti-inflammatory, antimicrobial, anticancer, and antifertility activities. Few studies have reported sedative and analgesic activities. Most of the activities are based on random screening rather than traditional knowledge, which cannot provide the correlation between ethnomedicine and modern pharmacological activities. Some studies carried out on C. spinosa are discussed in the following section.Due to the widespread traditional uses, C. spinosa showed prominent antibacterial activity against various species of Proteus, Staphylococcus, Clostridium, Salmonella, Vibrio, Bacillus, Escherichia, and Pseudomonas [The extract obtained from the fruit bark of udomonas .C. spinosa was tested against human pathogenic bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis; and the minimum inhibitory concentration (MIC) was found to be 0.07, 0.08, 0.88, 3, and 0.73 mg/mL, respectively, and the zone of inhibition ranged from 15 to 20 mm [The water extract of the seed of to 20 mm .C. spinosa showed a potent effect against Gram-negative bacteria, that is, K. pneumoniae, E. coli, and S. typhi, as compared to Gram-positive bacteria, that is, B. subtilis and S. aureus [In another study, the antimicrobial activity was performed by the broth dilution method and disc diffusion method against various Gram-positive and Gram-negative bacteria. The methanolic extract of leaves of . aureus .C. spinosa was subjected to its antimicrobial activity by the disc diffusion method, where it was tested against P. aeruginosa, E. coli, S. pyogenes, S. typhi, S. pyogenes, and B. subtilis. The activity of the extract was tested as compared to the standard Chloramphenicol. One hundred µg/mL of the ethanolic extract showed more potency and highest zone of inhibition of 28.0 mm against the Bacillus and Klebsiella pneumoniae of 24.0 mm and the lowest was that of Streptococcus pyogenes. In chloroform extract, the maximum zone of inhibition of 26 mm was recorded for Bacillus subtilis followed by Pseudomonas aeruginosa of 16 mm and it was lower for the remaining bacteria. In the aqueous extract, the maximum zone of inhibition of 18 mm was recorded for Klebsiella pneumonia followed by Bacillus subtilis of 14.0 mm and it was lower for other bacteria [The fruit extract of bacteria .C. spinosa at the dose of 200 mg/kg was studied in male albino Wistar rats with alcohol (28.5%) induced liver injury. The biochemical parameter and histopathology of the liver were accessed before and after treatment and were found to be significant to retrieve the parameters towards the normal stage. The treatment with standard silymarin and C. spinosa extract showed a decrease in the elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DB), and triglyceride (TG) as compared to rats exposed to alcohol only. The histopathological examination showed severe necrosis in cells treated with alcohol only, while reduced necrosis was found in the rats treated with C. spinosa extracts [The hepatoprotective potential of methanolic extract of the fruit of extracts .4) induced hepatic damaged rats [C. spinosa leaf and bark extracts at 400 mg/kg for 15 days showed a significant decrease in various biochemical parameters such as ALP, AST, ALT, LDH, albumin, and total and direct bilirubin and an increase in total protein levels as compared to the CCl4 only treated group.A similar study was performed in carbon tetrachloride  was 1.7 µg/mL. The EC50 value of the extract for nitric-oxide scavenging assay was 16.8 µg/mL as compared to the standard (curcumin: 11.2 µg/mL). Similarly, the EC50 value of the extract for superoxide anion activity was 8.4 µg/mL compared with the standard (ascorbic acid: 12.5 µg/mL) [Methanolic extracts obtained from the fruit bark of 5 µg/mL) .50 values of 45.12 µg/mL as compared to ascorbic acid (34.91 µg/mL). Similarly, in the ferrous ion chelating activity study, the extract had the IC50 value of 64.70 µg/mL as compared to ascorbic acid (30.96 µg/mL) [In another study to evaluate the DPPH free radical scavenging activity for the methanolic extract of leaves, the extract showed the IC6 µg/mL) .+ scavenging assay were performed to determine the antioxidant potential of leaves of C. spinosa [50 value recorded was 0.154 ± 0.14 and 0.175 ± 0.12 mg/mL, respectively. Another study recorded the IC50 value of stem and leaves of C. spinosa as 8.08 and 5.35 mg/mL, respectively, as compared to the IC50 value of ascorbic acid (<2 mg/mL) [The DPPH and ABTS  spinosa  . The IC2 mg/mL) .in vitro analysis. The in vivo antioxidant properties and their mechanism of action are yet to be studied.All these antioxidant activity evaluation studies were based on the C. spinosa were studied in the carrageenan-induced inflammation test in the hind paw of rats. The inflammation was inhibited by 31.69, 35.11, and 41.62% at 1, 3, and 5 h, respectively, by 200 mg/kg of a dose of ethanolic extract of leaves of C. spinosa, which was comparable to the inhibition of standard drug indomethacin (10 mg/kg) [Anti-inflammatory activities of 0 mg/kg) .C. spinosa fruit extract were studied in the streptozotocin-induced male Wistar diabetic rats. The group of rats treated with standard drug gliclazide and C. spinosa fruit extract showed a decreased level of glucose as compared to the diabetic control group. The value of blood sugar decreased from 88.5 ± 6.86 to 74.16 ± 4.02 in gliclazide (50 mg/kg) treated group in between 60 and 120 min, while C. spinosa fruit extract (400 mg/kg) decreased the blood sugar level from 106.16 ± 7.8 to 87.5 ± 5.00 mg/dL [C. spinosa and their characterization can lead to the development of potential drugs in the future.Antihyperglycemic activities of methanolic extract of 00 mg/dL . The detC. spinosa at concentrations of 10, 50, and 100 μM and found to be significant for preventing cell migration. A maximum decrease of 65.4% in cell migration was recorded for 100 μM. The next invasion assay was done to demonstrate the antiangiogenesis activity. In this assay, catunaregin significantly decreased the invasion of HUVECs induced by VEGF. The highest decrease of the invasion of HUVECs (61.3%) was recorded for the concentration of 100 μM [The wound healing was performed in the HUVECs cell and it was treated with catunaregin obtained from the plant f 100 μM .C. spinosa was determined in the catalepsy in mice induced by clonidine and haloperidol. In this study, the forepaw of mice was placed in the bar and the time required to remove the paw from the bar was recorded. The catalepsy recorded for clonidine was 229.2 ± 14.88 sec., which was significantly decreased to 135.2 ± 4.84 seconds when treated with 720 mg/kg of ethanolic extract of fruits of C. spinosa similar to that of standard chlorpheniramine maleate [The anticataleptic efficiency of the ethanolic extract of fruits of  maleate .C. spinosa was studied in Pheretima posthuma. The extract of C. spinosa (200 mg/mL) was tested against worms for its ability to paralyze and kill the worms. The study showed that the ethanolic extract revels comparable results similar to albendazole (10 mg/mL), a standard drug as a positive control [The anthelmintic activity of the fruit extract of  control .C. spinosa such as treating ulcers, asthma, abortifacient, curing piles, jaundice, and others on which no pharmacological studies have been made. Further, research can be done on the wide range of pharmaceutical activities related to its ethnomedicinal value.There are several other ethnomedicinal practices of C. spinosa at a dose of 2000 mg/kg in a Swiss albino mice model were found to be nonlethal [C. spinosa was reported by Jangwan et al. [C. spinosa in Wistar albino rats, no mortality was observed. However, when administrating the different doses of 200 mg/kg, 2000 mg/kg, and 5000 mg/kg continuously up to 14 days, the mortality observed was 0%, 25%, and 50%, respectively. The result was supported by a change in biochemical parameters and histopathology reports [Acute toxicity study showed that the aqueous and alcoholic extracts of onlethal . Anothern et al. . Upon ad reports .C. spinosa leaf extract and lethal concentration (LC50) was determined. The LC50 values of water and ethanol extract were 0.94 and 0.23 µg/ml, respectively [50 value was found to be 1.32 µg/mL [Brine shrimp lethality assay was done to study the cytotoxic activities of ectively . A simil32 µg/mL .C. spinosa is reported to be used in traditional medicine as well as in dietary regimens. Almost all parts of the plant have been used in traditional medicines such as leaves, fruits, stem, stem bark, fruit bark, and root bark. Fruits are also used in food. Various classes of compounds such as polyphenols, flavonoids, terpenoids, triterpenoids, and saponins are reported to be the main constituents found in this plant. The diverse pharmacological activities such as anti-inflammatory, antimicrobial, antidiabetic, antioxidant, and hepatoprotective activities have been investigated with promising results. Despite those promising results, many studies are based only on the in vitro models, and mechanisms of action are not well studied. Some of the research lacked the proper use of positive and negative control in the experiment setting. The in vivo studies were performed with very high doses of extracts and seem practically less efficient. Some ethnopharmacological reports show that this plant has been used to treat ulcer symptoms and acidity; however, pharmacological activities were not performed to evaluate these activities. Future research work must correlate the traditional knowledge with the isolation of active constituents and corresponding pharmacological significance. Along with this, the details of toxicological activities if studied can pave the way to develop new therapeutic agents.


This problem should be thoroughly investigated.The paper aimed to analyse the safety of drinking coffee by adult Poles in terms of Pb and Cd content. The degree to which Cd and Pb passed from coffee grounds into the coffee infusion was also examined. Twenty-three samples of natural coffee were examined. The content of metals was determined using the ICP method. On average, dry coffee contained ca. 0.004 μg Cd and 0.05 μg Pb per 1 g, and 95.5% Cd and 94% Pb passed into the infusion. Drinking coffee supplies these metals in the amount of less than 2% TWI (tolerable weekly intake) for Cd and BMDL (benchmark dose lower confidence limit) for Pb. In the presented studies, the values of CDI (chronic daily intake), THQ (target hazard quotient) and HI (hazard index) indicators were lower than 1, which means that the risk of developing diseases connected with chronic exposure to Cd and Pb consumed with coffee must be evaluated as very low. The content of Cd and Pb in the analysed coffee infusions was very low, so drinking coffee does not pose a risk for consumers in terms of the content of these metals. However, it must be remembered that no threshold limits for toxic metal consumption exist because these metals accumulate in the body for a long time. The studies presented here also showed a low ( Coffee, next to tea, is one of the most popular drinks in the world . It is a−1 of body weight per week [01—10.5 μg kg−1 of body weight per week—and BMDL10—4.4 μg kg−1 of body weight per week [Apart from antioxidants and other bioactive compounds, coffee contains carbohydrates, lipids, nitrogen compounds, vitamins and minerals, including toxic elements such as cadmium (Cd) and lead (Pb) , 14, 15.per week , whereasper week . The papTwenty-three samples of natural coffee were examined Table . The proGrained coffee was ground in a laboratory grinder with plastic blades. Ground coffee was mixed by hand. Coffee infusions were prepared as follows: 6 g of ground coffee was poured with 100 ml of drinking water with a temperature of 95–100 °C; after 10 min, the solutions were drained through the Whatman drain. The resulting coffee grounds were dried in a drier at a temperature of 65 °C for 24 h. Afterwards, they were pulverised in a laboratory grinder with plastic blades. The analyses covered both fresh ground coffee and coffee grounds remaining after coffee brewing.3 [−1; the solutions were prepared in 1% ultra-pure nitric acid (V).Cd: standard characterised by 99.999% purity used to prepare solutions with the concentration of 0.2; 0.4; 1; 2; 4; 10 μg of Cd L−1; the solutions were prepared in 1% ultra-pure nitric acid (V).Pb: standard characterised by 99.999% purity used to prepare solutions with the concentration of 0.1; 0.2; 0.5; 1; 2; 5 μg of Pb LThe analysed material was manually mixed. Samples weighing ca. 3 g were weighed in 3 replications into previously heat sterilised china crucibles and then subjected to dry mineralisation in a muffle furnace at a temperature of 450 °C. The oxidant was hydrogen peroxide. The mineralisate was dissolved in 10 ml of 1 M HNO3 , 21. The3) and two certified reference materials (CRM): INCT-TL-1 Tea leaves (containing 0.030 mg Cd and 1.78 mg Pb per 1 kg) and INCT-MPH-2 Mixed Polish herbs (containing 0.199 mg Cd and 2.16 mg Pb per 1 kg).Each chemical analyses was repeated 3 times. The accuracy of determination was verified using a blind test (1 M HNO2O2 (30% pure) and nitric acid HNO3 (65% ultra-pure) were purchased from POCH S.A. (Poland). Deionised water used for dilution was made in our laboratory . The Cd and Pb standards were purchased from Merck (Germany). Certified reference materials INCT-TL-1 and INCT-MPH-2 were obtained from the Institute of Nuclear Chemistry and Technology .Hydrogen peroxide HBased on the difference in the content of Cd and Pb in coffee grounds, the degree (%) to which those metals passed into the infusion was calculated prior to after coffee brewing.Percent of tolerable dose:The safety of drinking coffee for adult Poles was estimated on the grounds of (1) calculation of the percentage of Cd and Pb intake in comparison with the acceptable level proposed by EFSA , 17, (2)Estimated weekly intake (EWI) of Cd and Pb was calculated according to the formula :\documenTolerable weekly intake % (TWI) was calculated according to the formula :\documen−1 of body weight per week [The value adopted for TWI was 2.5 μg Cd kgper week .Benchmark dose lower confidence limit % (BMDL) was calculated according to the formula :\documen01—10.5 μg Pb kg−1 of body weight per week—and BMDL10—4.4 μg Pb kg−1 of body weight per week [The value adopted for BMDL: two values suggested by the European Food Safety Authority (EFSA) were calculated per 1 week: BMDLper week .(2)Cancer risks parametersThe mean body weight was assumed as 70 kg.tot is the exposure duration (years)—since in Poland regular coffee drinkers are adults only, it was assumed that the time of exposure was calculated from 18 to 74 years of age (74 years—average life span in Poland), which is 56 years; AT is the period of exposure (365 per year).Chronic daily intake (CDI) of Cd or Pb was calculated according to the formula , 24:\doc−1 BW per day and is contaminant specific.(3)Non-carcinogenic risks parametersCSF is a cancer slope factor which is the risk produced by a lifetime average dose of 1 mg kgTarget hazard quotient (THQ) was calculated according to the formula :\documen−1 of body weight per day, whereas, for Pb, it is 3.5 μg kg−1 of body weight per day [RfD (reference dose) for Cd is 1 μg kg per day .When THQ is higher than 1, it is assumed that there is a significant risk of developing negative effects on health resulting from chronic exposure to Cd and/or Pb .Hazard index (HI) was calculated according to the formula :\documenP < 0.05) were computed by single factor analysis of variance (ANOVA), using the Duncan test. The correlation between the content of Cd and Pb in coffee and the degree (%) to which they passed into the infusion was calculated using Pearson’s method (Statistica 6.0 software).The mean content of Cd and Pb was calculated for each sample . A statistical analysis of the results  was carried out using Statistica 13.1 software. Statistically significant differences (−1). About 79 to 100% (on average 95.5%) of Cd present in the output material passed into the infusion; the infusion contained, on average 3.613 μg kg−1 (range 1.2–10.33 μg). A very low positive correlation r = 0.15 was identified between the content of Cd in coffee and the degree (%) to which Cd passed into the infusion , whereas coffee grounds < LOQ—10.2 μg kg−1. In 17% of coffee ground samples, the level of Pb was lower than determinable using the analytical method applied (LOQ = 0.03 μg kg−1). From nearly 79 to 100% (on average 94%) of Pb passed into the infusion, the infusion contained, on average, 46.86 μg kg−1 (range 16.66–80.06 μg). A low positive correlation r = 0.26 was identified between the content of Pb in coffee and the degree (%) to which Pb passed into the infusion , is presented in Table Cd and THQCd indicators is identical and it amounts to 0.022. The estimated weekly intake of Pb is 1.968 μg, which corresponds to ca. 0.27% BMDL01 and ca. 0.64% BMDL10. The value of CDIPb = 0.281, whereas that of THQPb = 0.08. The HI risk factor (Cd + Pb) is 0.103.The estimated weekly intake (EWI) of Cd with coffee infusion is 0.156 μg, which accounts for about 0.09% TWI. The value of CDICd and THQCd indicators is 0.045 each. EWI of Pb is ca. 3.94, which corresponds to 0.54% BMDL01 and nearly 1.3% BMDL10. The value of CDIPb = 0.56, whereas THQPb = 0.16. The HI risk factor equals 0.205.EWI of Cd with coffee is 0.312 μg, which accounts for 0.18% of TWI. The value of CDICd and THQCd indicators are 0.067 each. EWI of Pb was equal to 5.715 μg, which corresponds to about 0.78% BMDL01 and about 1.86% BMDL10. The value of CDIPb = 816, whereas THQPb = 233. The HI risk factor equals 0.3.EWI of Cd with infusion is less than 0.5 μg, which accounts for about 0.27% TWI. The values of CDIn = 4), Lebanon (n = 1), Brazil (n = 3) and in Bosnia and Herzegovina (n = 3). These studies showed that infusions contained from 0.615 (Polish coffee) to 1.24 μg (Bosnia and Herzegovina) Pb per 1 g. According to those authors, in Poland, a person drinking 2.4 kg of coffee on an annual basis simultaneously consumes 1.48–2.43 mg Pb, which is slightly more than 4 μg Pb per day, whereas, in Bosnia and Herzegovina, it is ca. 33 μg Pb per day (12 mg per year).In the presented studies of this author, dry coffee contained on average nearly 3.8 μg Cd and ca. 50 μg Pb per 1 kg of the natural product, which accounts for ca. 0.004 μg Cd and 0.05 μg Pb per 1 g. As 95.5% Cd and 94% Pb passed into the infusion, the infusion contained on average 0.0037 μg Cd and ca. 0.047 μg Pb per 1 g. Considering the consumption of coffee infusion , an adult Pole consumes less than 0.5 μg Cd and nearly 6 μg Pb per day. Nędzarek et al.  examined−1, LOD Pb—0.01 mg 100 g−1). Gebretsadik et al. [−1, whereas that of Pb < 0.04 μg g−1. Similarly, Ashu and Chandravanshi [n = 3), the level of Cd and Pb was lower than LOD. Studies in Brazil demonstrated that the level of Cd in roasted ground coffee (n = 15) was < 0.025 μg g−1, whereas the level of Pb ranged from 0.14 to 2.59 μg g−1 [−1 [−1). In addition, da Silva et al. [−1 Cd and 0.02–0.2 μg g−1 Pb [−1 [−1, whereas that of Pb < 2.6 μg g−1. Turkish studies showed that green coffee grains contained on average 0.005 μg g−1 Cd (0.003–0.006 μg g−1) and 0.12 μg g−1 Pb (0.06–0.3 μg g−1) [Grembecka et al.  in 120 sk et al.  in Ethioravanshi  found thffee n =  was < 0.ffee n =  was < 0. g−1 [−1 , ground a et al.  found thg g−1 Pb . Studies1 Pb [−1 . In turn1 Pb [−1  found th μg g−1) . Those a μg g−1)  also sho−1 of body weight), while the content of Cd in those coffees was lower than LOD.In the presented studies, the values of CDI, THQ and HI indicators for all the assumed patterns were lower than 1, which means that the risk of developing diseases connected with chronic exposure to Cd and Pb consumed with coffee must be evaluated as very low. Coffee drinking safety is also confirmed by the degree of coverage of the tolerable intake level of Cd and Pb recommended by EFSA , 17. AccPb close to 1. It must be taken into account that some authors found that the Pb level was higher than acceptable in 75% of the analysed samples [r = 0.26) but still, a positive correlation between the content of Pb in coffee and the degree (%) to which Pb passed into the infusion. This problem should be thoroughly investigated.To sum up, the content of Cd and Pb in the analysed coffee infusions was very low. However, it must be remembered that no threshold limits for toxic metal consumption exist because these metals accumulate in the body for a long time; in the case of Cd and Pb, it is even 30 years , whereas samples , 30. TheThe content of Cd and Pb in the analysed coffee infusions was very low, so drinking coffee does not pose a risk for consumers in terms of the content of these metals. However, it must be remembered that no threshold limits for toxic metal consumption exist because these metals accumulate in the body for a long time; in the case of Cd and Pb, it is even 30 years.


CNP), was successfully prepared for the selective and sensitive recognition of Sn(II) in different commercially-available toothpaste and mouth wash samples. The binding mechanism of CNP for Sn2+ was confirmed by UV–Vis, 1H, and 13C NMR titrations. The proposed sensing mechanism was supported by quantum chemical calculations. Selective detection of Sn(II) in the nanomolar range (85 nM), among other interfering metal ions, makes it exclusive. Moreover, Sn2+ can be detected with a simple paper strip from toothpaste, which makes this method handy and easily accessible. The potential application of this system for monitoring Sn2+ can be used as an expedient tool for environmental and industrial purposes.An easily accessible colorimetric probe, a carbazole–naphthaldehyde conjugate ( Sn(II) as fluoride is present in several dental care products such as toothpaste and mouth rinse2. Sn2+ has been utilized in dentistry as a chemical adjunct to prevent dental caries since 19504. It was found to effectively inhibit Streptococcus mutants, which leads to tooth decay in human interproximal dental plaque and oral disease5. However, the wide utilization of toothpaste in our everyday life may increase the consumption of Sn2+ in the human body. The continuous use of Sn(II) becomes detrimental to our health and environment as well. The human can intake Sn(II) by breathing, through the skin, or by the consumption of food6. The accumulation of Sn(II) can induce acute and long-term effects in the human body such as eye irritations, heavy sweating, urination complications. Excess consumption of Sn(II) also finds severe immunotoxic and neurotoxic effects in humans causing symptoms such as disorders in the immune system, damage in liver functioning, chromosomal destruction, damage in the brain, and lack of red blood cells10. Recent studies revealed that the presence of an excess of Sn(II) in humans can easily be coordinated by white blood cells and enter into the cells by calcium channels, inducing DNA damage12. According to the guiding principles of the World Health Organization’s (WHO) for metals, the permissible limit of Sn(II) in drinking water and canned foods are 8.4 × 10−4–8.4 × 10−3 M and 2.105 × 10−6 M, respectively13. Due to the hazardous impacts of Sn(II) on humans, consumption of Sn(II) must be closely monitored.The utilization of tin has been established for thousands of years and the most commonly used for the manufacturing of bronze, which is a tin and copper alloy. In the recent era, Sn(II) is used in many fields in the industry such as aerospace, construction and home decor, electronics, jewelry manufacturing, telecommunications, paint/plastic industries, and agriculture via pesticides20, fluorimetric and colorimetric sensing method is one of the simplest and convenient sensing methods over other established detection techniques because it offers the advantage of ‘on spot’ real-time detection with naked eyes, low cost, portable, and wide applicability29. Hence, inspired with the requirement of active colorimetric probes, herein, we report the synthesis and sensing behavior of a novel colorimetric probe carbazole-naphthaldehyde conjugate (CNP) that exhibits high selectivity and sensitivity toward Sn2+ in the neutral aqueous medium . The structure of the synthesized probe CNP was confirmed by detailed NMR , HRMS, and X-ray analysis s Figure –S4, ESI†CNP with Sn2+, we have performed several experiments such as NMR titration, absorbance titration, pH titration, selectivity test and theoretical calculations.To explore the interaction pattern of the probe CNP at 3 °C. It crystallises in the monoclinic space group P21/n , followed by downfield shift of imine proton peak at 9.84 ppm. This phenomena indicates the formation of strong coordination between –OH, –N groups of CNP and Sn2+  at neutral pH value . In the UV–Vis absorption spectra, we get a characteristic absorbance peak of CNP at 400 nm which decreases upon incremental addition of Sn2+, with an enhancement at 454 nm followed by a rapid colour change from pale yellow to deep orange. Furthermore, a notable isosbestic point at 425 nm indicates possible stronger interaction between CNP and Sn2+ . As depicted in Figure 2+. The synthesized probe CNP did not show any notable color changes with the addition of other metal ions. The specific color change of CNP with Sn2+ was attributed to several electron transitions in the CNP–Sn2+ complex such as π–π*, d–d, ligand–metal charge transfer, and metal–ligand charge-transfer effects. Furthermore, the comparative spectrophotometric response of CNP was also studied with these metal ions which confirms that our probe CNP selectivity sense Sn2+ over other metal ions  solvent model was used for solvent (water) effect incorporation. In the optimized structure of CNP, the positions of carbazole and naphthaldehyde units were nearly in the same plane. The optimized structure of the CNP–Sn2+ complex showed the formation of coordination bonds of Sn2+ with –OH and –N groups of CNP, which enhanced the stability of the complex  which is very close to that experimentally observed value at 400 nm, responsible for the absorption of the carbazole moiety. Moreover, in CNP–Sn2+, the transition at 448 nm  indicates the π–π* electronic transition from the carbazole to naphthaldehyde moiety which is distinctly executed in the absorbance graph at 454 nm  Å) observed in the X-ray structure is characteristic of ketones rather than phenols, and the C–N bond (1.324(3) Å) is elongated relative to that of a typical iminetsFigure , ESI†.Inn2+ Fig. .CNP toward Sn2+, such as high sensitivity and selectivity at physiological pH encouraged us to further evaluate the potential of the probe for realistic approach. The specific and selective recognition of Sn2+ by the chemosensor CNP was also examined in three different toothpaste samples. In this work, we took commercially available toothpastes from three different brands . The details procedure of the preparation of toothpaste solutions was described in ESI†. Toothpaste solutions were then added to the CNP solution, a rapid orange color change resulted after few minutes , and Fig. CNP-coated paper strip after dipping in the toothpaste solutions T1, T2, T3 respectively.The excellent photophysical properties of the probe tes Fig. a. Furthe2+ was also quantified from these three different toothpaste samples . For this work, the above-mentioned toothpaste samples were subjected to colorimetric analysis at pH 7.0 (10 mM phosphate buffer) to quantify the amount of Sn2+ present therein. Sn2+ was quantified from these given samples by CNP (1 µM) by virtue of its selective and direct recognition properties. All estimations were done in triplicate. Concentrations of Sn2+ were estimated by comparison with the CNP–Sn2+ standard absorbance curve. From the standard curve it was found that the concentration of Sn2+ were 0.73 µM, 0.70 µM and 0.64 µM in 100 µL of T1, T2 and T3 samples, respectively  using above mentioned procedure and the respective values are 0.25 µM and 0.28 µM in 100 µL sample solution  form in its solid state. The sensing mechanism has been triggered by the strong coordination bonding of CNP-enol with Sn2+, which was confirmed by absorbance, 1H and 13C NMR spectroscopy as well as mass spectrometry (HRMS). Theoretical calculations were also performed to justify the binding mechanism and optical behavior of the sensor probe. CNP showed high selectivity and sensitivity for Sn2+ even in the presence of other metal ions. The detection limit of the probe for Sn2+ was calculated to be 85 nM, which is much lower than the WHO permissible amount of Sn2+ in drinking water. We further demonstrated that CNP has been utilized as a colorimetric sensor to detect and quantify trace amounts of Sn2+ in different toothpaste and mouth wash samples. A handy and accessible paper strip method has been proposed for this purpose. Being a potential probe, it can be used as an expedient ‘in-field’ approach to estimate Sn2+ for environmental and industrial purposes for sustainable and environment-friendly industrial production.In conclusion, a new carbazole-naphthaldehyde based colorimetric probe 1H and 13C NMR spectra were recorded on a Bruker 400 MHz instrument. For NMR spectra, DMSO-d6 and for NMR titration DMSO-d6 and D2O were used as solvent using TMS as an internal standard. Chemical shifts are expressed in δ ppm units and 1H–1H and 1H–13C coupling constants in Hz. The mass spectrum (HRMS) was carried out using a micromass Q-TOF Micro™ instrument by using methanol as a solvent. UV spectra were recorded on a SHIMADZU UV-3101PC spectrophotometer. FT-IR data were recorded on Shimadzu IRAffinity-1S Fourier transform infrared spectrometer (Spectrum Two) by ATR technique. The following abbreviations are used to describe spin multiplicities in 1H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Single crystal X-ray data of CNP was measured using a dual-source Rigaku Super Nova diffractometer equipped with an Atlas detector and an Oxford Cryostream cooling system using mirror-monochromated Cu-Kα radiation (λ = 1.54184 Å). Data collection and reduction for both compounds were performed using the program CrysAlisPro35 and Gaussian face-index absorption correction method was applied35. The structures were solved with Direct Methods (SHELXS)38 and refined by full-matrix least-squares based on F2 using SHELXL-201538. Non-hydrogen atoms were assigned anisotropic displacement parameters unless stated otherwise. The hydrogen atom bonded to nitrogen was located from Fourier difference maps and refined with an N–H distance restraint of approximately 0.96 Å. Otherhydrogen atoms were placed in idealised positions and included as riding. Isotropic displacement parameters for all H atoms were constrained to multiples of the equivalent displacement parameters of their parent atoms with Uiso(H) = 1.2 Ueq(parent atom). The single crystal X-ray data, experimental details as well as CCDC number are given in the Supporting Information.All the reagents were purchased from Sigma-Aldrich Pvt. Ltd. (India). Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification. Solvents were dried according to standard procedures. Elix Millipore water was used in all respective experiments. 2SO4, the organic layer was evaporated completely to get the solid product. This product was purified by column chromatography with the eluent CHCl3:PET  to get the product CNP with 86% yield : δ (ppm) = 16.37 , 9.84 , 8.57–8.59 , 8.26–8.28 , 7.90–7.92 , 7.77–7.82 , 7.71–7.73 , 7.62–7.64 , 7.55–7.57 , 7.47–7.51 , 7.34–7.37 , 7.23–7.26 , 7.04–7.06 , 4.47–4.49 , 1.31–1.34 . 13C NMR : δ (ppm) = 169.46, 153.90, 140.30, 138.46, 135.94, 133.15, 129.04, 127.93, 126.67, 126.32, 123.28, 123.02, 122.26, 122.11, 120.92, 120.36, 119.55, 119.05, 112.08, 109.98, 109.52, 108.67, 37.19, 13.83. HRMS (TOF MS): : Calcd. for C25H20N2O: 364.1576. Found: m/z = 365.1283 (M + H+).In a 100 mL round bottom flask, 2-hydroxy naphthaldehyde  in 30 ml ethanol was vigorously stirred at ambient temperature for few minutes. Then, 3-amino-9-ethyl carbazole  was dissolved in ethanol (10 mL) and added dropwise to the solution. The reaction mixture was refluxed for 24 h at 83 °C. After completion of the reaction (monitored by TLC), the solvent was evaporated completely under reduced vapor pressure, then extracted with chloroform and water. After drying it over anhydrous NaSupplementary Information.