Amax) and light saturation point were decreased under drought conditions in both cultivars. The change in dark respiration rate (Rd) in HY73 was not significant, but was markedly different in HHZ. After rewatering, the photosynthetic parameters of HY73 completely returned to the initial state, while the indices in HHZ did not recover. The antioxidant enzyme activities and osmoregulatory substance levels increased with worsening drought conditions and decreased with rewatering duration. HY73 had higher peroxidase (POD) activity as well as proline levels, and lower catalase (CAT) activity, ascorbate peroxidase (APX) activity, malondialdehyde (MDA) level, and soluble protein (SP) content during all of the assessment periods compared with HHZ. In addition, Amax was markedly negatively correlated with superoxide dismutase (SOD), POD, CAT, and SP in HY73 (p < 0.001), while in HHZ, it was negatively correlated with SOD, CAT, APX, MDA, Pro, and SP, and positively correlated with Rd (p < 0.001). These results suggest that WDR has a more simplified adaptation mechanism to protect photosynthetic apparatus from damage in drought and rehydration compared with drought-sensitive cultivars. The high POD activity and great SP content would be considered as important physiological bases to maintain high photosynthetic production potential in WDR.Water-saving and drought-resistant rice (WDR) has high a yield potential in drought. However, the photosynthetic adaptation mechanisms of WDR to drought and rehydration have yet to be conclusively determined. Hanyou 73  and Huanghuazhan  rice cultivars were subjected to drought stress and rewatering when the soil water potential was −180 KPa in the booting stage. The leaf physiological characteristics were dynamically determined at 0 KPa, −30 KPa, −70 KPa, −180 KPa, the first, the fifth, and the tenth day after rewatering. It was found that the maximum net photosynthetic rate ( Due to global climate change, various climatic factors greatly impact agricultural production. Drought is one of the most serious abiotic stresses restricting agricultural development . Rice isIn recent years, water-saving and drought-resistant rice (WDR) varieties have been successfully cultivated. WDR, a new rice variety, is characterized by high yields, good quality, and the ability to save water and resist drought ,9. The h2− in plants to form H2O2, which is then eliminated by POD and CAT [2O2 into O2 and H2O, while APX is responsible for the removal of H2O2 in the water–water and ASH–GSH cycles [Antioxidant defense systems come into play when plants are under stress, scavenging reactive oxygen free radicals (ROS) and providing an intact cellular environment for the photosynthetic system ,11,12,13 and CAT ,23. As oH cycles ,24. UndeH cycles . HoweverH cycles ,26. Unde2 on photosystem I (PSI) or PSII to make it O2− [MDA is the final product of membrane lipid peroxidation, which can reflect the degree of plant cell damage by stress and drought resistance of various varieties. Photoinhibition-mediated injury is majorly associated with reactive oxygen species production . Excess e it O2− . Excess e it O2− ,30,31.When water stress occurs, plant cells actively accumulate soluble osmotic regulatory substances such as proline and soluble proteins, reduce osmotic and water potentials, maintain turgor pressure, and carry out osmotic regulation ,33. ThesAfter drought and rewatering, there is a need to elucidate the changes to the photosynthetic physiology, antioxidant enzyme activities, and osmotic regulation in WDR, and determine whether antioxidant enzymes and osmotic adjustment substances work in tandem to adapt to drought stress. We used the water-saving drought-resistant rice (HY73) and drought-sensitive rice (HHZ) varieties to study the changes in leaf physiological indices under drought stress and rewatering at the booting stage. This study aimed to: (1) reveal the dynamic characteristics of the photosynthetic production potential of WDR under drought and rewatering; (2) analyze the physiological basis of WDR’s response to drought and rewatering and its relationships with the photosynthetic production potential.The relative water content (RWC) of the rice leaves decreased with increasing water stress, and after the rewatering of both rice varieties, there was a gradual increase in the initial state across both cultivars . From RWAmax and Is decreased with increasing drought stress levels and gradually increased after rewatering, while Ic exhibited the opposite trend. The Amax of HY73 decreased from 21.57 μmol·m−2·s−1 at the RW stage to 13.76 μmol·m−2·s−1 at the ED stage, while that of HHZ decreased from 29.82 μmol·m−2·s−1 at the RW stage to 11.71 μmol·m−2·s−1 at the ED stage. Changes in Amax of HY73 at the HD stage were not significant, while the decrease in Amax of HHZ was 34.74% when compared with Amax at the RW stage.Under different water potentials at the booting stage, the changing trends of the photosynthetic performance of the two rice varieties were the same, but the amplitudes were different . At the Ic levels from RW to ED, those of HY 73 were increased from 16.71 μmol·m−2·s−1 to 38.89 μmol·m−2·s−1 with an increase of 132.73%, and decreased by 48.68% to 19.96 μmol·m−2·s−1 at the D1 stage when compared with the ED stage. For cultivar HHZ, the Ic levels were 15.87 μmol·m−2·s−1 and 30.20 μmol·m−2·s−1 at the RW and ED stages, respectively, and then decreased to 22.03 μmol·m−2·s−1 at a rate of 27.05%, 15.53%, and 3.0%, respectively, at the D1, D5, and D10 stages in cultivar HHZ. The Is levels in cultivar HY73 decreased from 1825.88 μmol·m−2·s−1 (RW) to 1283.71 μmol·m−2·s−1 (ED), while those of HHZ decreased from 1649.49 μmol·m−2·s−1 (RW) to 1110.74 μmol·m−2·s−1 (ED). At the D1 stage, the Is of HHZ were still gradually decreasing, while those of HY73 had recovered significantly to 1586.48 μmol·m−2·s−1. At D5 and D10, the Is of HHZ increased to 1325.30 μmol·m−2·s−1 and 1503.73 μmol·m−2·s−1, respectively.Regarding the Rd was small in cultivar HY 73, but was greater in HHZ. The highest and lowest Rd of HHZ were at RW (2.52 μmol·m−2·s−1) and D1 (0.96 μmol·m−2·s−1), respectively.The amplitude of the change of p ≤ 0.05). All of the above-mentioned parameters declined after rewatering. At the HD stage, the POD, CAT, and APX activities of HY73 increased by 35.90%, 21.48%, and 75.12%, respectively, while those of HHZ increased by 28.56%, 31.64%, and 51.25%, respectively, compared with the RW stage. At the ED stage, the POD, CAT, and APX activities of HY73 were respectively increased by 6.61%, 25.71%, and 52.09%, and in HHZ were reduced by 9.34% and increased by 38.60% and 26.81%, respectively, compared with the HD stage. At the D10 stage, the POD, CAT, and APX activities of HY 73 respectively decreased by 25.09%, 50.24%, and 51.95%, while those of HHZ decreased by 20.95%, 38.45%, and 32.61%, respectively, compared with the ED stage. In general, the activities of the antioxidant enzymes in the leaves of HY73 quickly responded under drought and decreased to the initial value after rewatering. However, the activities of SOD and CAT in the HHZ leaves reached their highest levels at ED, while the POD levels were at the highest level at the HD stage. There was always a certain gap between rehydration and the initial state as the initial state could not be restored.The SOD activities in the two varieties were markedly increased under drought and significantly decreased after rewatering . The SODThe MDA levels in the rice leaves were markedly increased with increasing drought at the booting stage and rapidly decreased to stable levels after rewatering. There were significant differences in the leaf MDA levels between the two rice varieties under each observed stage. The MDA content of HY73 showed a 45.88% increase, 4.95% decrease, 23.05% decrease, and 24.15% decrease in HD, ED, D1, and D10, respectively, while in HHZ, it was a 45.74% increase, 63.99% increase, 31.90% decrease, and 6.80% decrease, respectively .The osmotic adjustment substance levels of the WDR and drought-sensitive rice varieties exhibited the same change trend under different water potentials, which increased significantly with worsening drought stress and decreased with the extension of rewatering time. Under drought stress, the Pro accumulation levels and rates in HY73 were higher than those in HHZ; after rewatering, the Pro levels in HY73 rapidly decreased compared to those of HHZ. The changes in Pro levels in HY73 were 140.79% increased, 29.52% increased, 30.60% decreased, 28.73% decreased, and 41.08% decreased in HD, ED, D1, D5, and D10, respectively, while the Pro levels in HHZ were 40.09% increased, 42.12% increased, 9.16% decreased, 9.58% decreased, and 6.19% decreased, respectively. The Pro levels in HD and ED were 1.95 and 1.78 times higher in HY73 than HHZ, respectively. Under different water conditions, the SP levels of HY73 were 28.36% greater, 33.54% greater, 32.10% lower, 19.42% lower, and 6.63% lower, while the SP levels of HHZ were 34.14% greater, 19.95% greater, 8.50% lower, 6.91% lower, and 9.81% lower in HD, ED, D1, D5, and D10, respectively .Amax of HY73 was negatively correlated with the levels of SOD, CAT, and SP . No significant correlational relationship was observed between Amax and POD activity in HHZ (p > 0.05). These results indicate that HY73 has sufficient capacities to clear reactive oxygen species by enhancing POD activities. However, the drought-sensitive variety (HHZ) needs to activate more defense mechanisms such as CAT and APX approaches in addition to the SOD and POD pathways  that had been harvested from the 0–20 cm layer at the experimental station. The physical and chemical properties of the soil were: organic matter levels of 32.40 mg·kg−1, total N levels of 2.00 g·kg−1, organic phosphorus levels of 24.80 mg·kg−1, total K levels of 19.40 g·kg−1, pH of 5.9, and soil bulk level of 1.15 g·cm−3. Each barrel contained 20 kg of fine-sifted soil.This study was conducted from May to November 2021 at Wanzhong Comprehensive Experimental Station, Lujiang County, Hefei City, Anhui Province . The pots had a volume of 0.026 mThe rice varieties were HY73 and HHZ, of which HY73 is a three-line hybrid rice with high-yield, high-quality, water-saving, and drought-resistant properties, while HHZ is an indica conventional weakly drought-resistant rice variety. Drought stress treatments were performed during the gestation period, and they included regular watering , moderate drought stress , severe drought stress , and extreme drought stress , with rehydration one day after ED and the same water management before water control until the end of the physiological period. The relevant indices were measured and collected on the first (D1), fifth (D5) and tenth (D10) days after rehydration. Each process was repeated three times. Maintain a 2–3 cm layer of water before water control, remove water from buckets in the evening before the day water control, and stop watering after the start of stress. Soil water potential is measured twice daily depending on weather conditions, rehydrated when it approaches or exceeds the bottom limit of the stress level, and remains at this drought stress level. The soil water potential was monitored using a tensiometer .2O5, and 3 g K2O. Moreover, they were top-dressed with 30% N, 50% K2O, and P2O5. The residual N rate was applied at the early tiller stage (30% of N application) and panicle differentiation stage (40% of N application). In addition, 50% K2O fertilizer was applied at the panicle differentiation stage.We used the completely randomized experimental study design. Twenty-one-day-old seedlings were removed from the seedling tray and transplanted into each pot. Each pot was supplemented with 2.4 g N, 1.2 g P1) were immediately taken after the leaves had been sliced from the base, after which the leaves for each treatment were cut into small sections, soaked in distilled water for 12 h, and weighed to obtain the saturated leaf weight (m2). Then, they were placed into the oven for 2 h at 105 °C, baked at 85 °C to a constant weight, and weighed to obtain the dry weight (m3). The formula for determining the relative water content of leaves W(%) was: (m1 − m3)/(m2 − m3) × 100%.Three replicates of six to eight leaves per treatment were obtained. The fresh leaf weights  [I = 0,A Li-6400  portable photosynthesis meter was used with its own red and blue light source to provide a constant light intensity of 1500 μmol·m. (2007) . The modIs was obtained using the Formula (1),Amax was obtained when I = Is,The light saturation point An (I) represents the net photosynthetic rate when the light intensity is I; α, β, and γ are the parameters independent of light intensity; Ic represents the light compensation point; and Rd represents the dark respiration rate.In the formula, −1 phosphate buffer (pH 7.0), ground on ice, the volume increased to 10 mL, centrifuged at 4 °C, 13,000 r·min−1 for 20 min, and the supernatant obtained as the crude enzyme extract. The crude enzyme extract was used for the determination of SOD, POD, CAT enzyme activities, and soluble protein levels. Superoxide dismutase (SOD) activities were determined using the nitrogen blue tetrazolium (NBT) method; catalase (CAT) activities were determined using the UV spectrophotometric method; peroxidase (POD) activities were determined by the guaiacol method.In this assay, 0.5 g of fresh sample was mixed with 5 mL of pre-chilled 50 mmol·L−1 AsA, 3 mmol·L−1 β-mercaptoethanol, 0.5 mmol·L−1 PMSF, 2% PVP, 1 mM EDTA). The extracts were ground with liquid nitrogen, centrifuged at 4 °C, 13,000 r·min−1 for 20 min, and the supernatant used for determination of enzyme activities. About 0.10 mL of the enzyme solution was mixed with 1.70 mL of PBS containing 0.1 mM EDTA-Na2 , 0.10 mL of 5 mM AsA, and finally with 0.10 mL of 20 mM H2O2. The optical densities were determined at OD290 and the enzyme activities determined.In this assay, 1 g of leaf tissue with main veins removed was weighed and mixed with 1.6 mL of pre-cooled phosphate buffer (pH 7.8) extract , placed in a boiling water bath for 15 min, rapidly cooled, and centrifuged at 4000× g for 15 min. Then, the absorbance values of the supernatants were determined at 532 nm, 600 nm, and 450 nm. The results were calculated as: concentration of MDA (μmol·mol−1) = 6.45 × (OD532 − OD600) − 0.56OD450.The malondialdehyde level determination was performed using the thiobarbituric acid method with about 0.5 g of cut and mixed rice leaves with the main veins removed in a mortar. The samples were mixed with a small amount of quartz sand and ground rapidly with liquid nitrogen to powder. Then, 2.5 mL of trichloroacetic acid (TCA) was added to the samples, ground into a homogenate, supplemented with 7.5 mL of TCA, and ground further. Then, they were transferred into test tubes, centrifuged at 4000× Determination of proline levels: In this assay, to 5 mL of 3% sulfosalicylic acid, a plug was added, boiled in a water bath for 10 min, 2 mL of extract solution was aspirated, and 2 mL of glacial acetic acid and 3 mL of acid ninhydrin were added, and boiled in a water bath for 1 h. After cooling, 4 mL of toluene was added to each tube, shaken for 30 s, left to stand for a while, and the upper red proline layer was gently aspirated for measurement of absorbance at 520 nm wavelength.Determination of soluble protein levels: here, 20 μL of extract solution (enzyme solution) was mixed with 80 μL, pH 7.8 phosphate buffer , supplemented with 2.9 mL of Thomas Blue solution, and the optical density measured at OD595 after 2 min of reaction (0.1 mL of buffer solution plus 2.9 mL of Thomas Blue was used as the control for zeroing).Amax, Rd, Ic, and Is, Pearson correlation analysis for correlation plots, as well as the figures were all operated by OriginPro2021.The test results are expressed as the average value and standard error of three replicate analyses. Differences between the averages of the parameters were compared by Fisher’s Least Significant Difference (LSD) test at the 5% level of significance in SPSS 26.0. Light response curve models for (i) Drought stress decreases photosynthetic rates and generally increase the activities of antioxidant enzymes and the content of osmoregulatory substances in both rice cultivars. WDR (HY73) can be rapidly recovered to its initial pre-drought state after rehydration, while the photosynthetic potential is unable to recover after rehydration for the drought-sensitive cultivar (HHZ).(ii) High photosynthetic potentials mainly rely on high POD activity to eliminate reactive oxygen species for the protection of cell membrane integrity in WDR. In addition, the high SP content in WDR has a positive role in maintaining a steady intracellular osmotic pressure. Both factors could be essential physiological bases for the rapid recovery of photosynthetic potential after rehydration in WDR.


Increasing light intensity increased RGR (by 67% and 76%) and RPY (by 50% and 89%) and decreased chlorophyll a (by 27% and 32%) for L. minor and W. hyalina, respectively. The spectral distributions had no significant impact on any investigated parameter. Wolffiella hyalina achieved higher values in all investigated parameters compared to L. minor. This investigation proved the successful cultivation of duckweed in a small-scale, re-circulating IVF with artificial lighting.Duckweeds can be potentially used in human and animal nutrition, biotechnology or wastewater treatment. To cultivate large quantities of a defined product quality, a standardized production process is needed. A small-scale, re-circulating indoor vertical farm (IVF) with artificial lighting and a nutrient control and dosing system was used for this purpose. The influence of different light intensities (50, 100 and 150 µmol m The term duckweed comprises 36 species ,2 of 5 gIn order to continuously produce large quantities of biomass with a defined quality , a standardized cultivation process is necessary. One possible solution in the future might be the cultivation of duckweed in re-circulating  indoor vertical farms (IVF) with artificial lighting. By stacking several layers of cultivation areas above each other, the land utilization efficiency is increased ,14. WhenHowever, closed hydroponic systems are already successfully used to cultivate different crops in large quantities. This includes tomatoes, cucumbers, peppers, different leafy greens, strawberries and even rice or maize . The advLemnaceae grow in sunny as well as in shaded habitats, but the latter habitats are favorable due to lower light intensities and less extreme temperatures [Wolffia arrhiza cultivated in steady-state conditions with blue light showed higher protein and chlorophyll contents compared to red light [Lemna gibba [Lemna aequinoctialis [−2 s−1 for L. minor [−2 s−1 for L. minor and Lemna minuta were observed [Landoltia punctata (formerly Spirodela punctata) reached light saturation between 600 and 1200 µmol m−2 s−1 at 30 °C [−2 s−1 was obtained for L. aequinoctialis [In order to also achieve an efficient system for duckweed cultivation, all necessary abiotic factors must be evaluated. Two of these abiotic factors are light intensity and the spectral light distribution. In nature, eratures . The plaeratures . Wolffiaed light . Increasna gibba  and Lemnoctialis . Very hiL. minor  and of 4observed , while Lat 30 °C . Consideoctialis .L. minor and W. hyalina when cultivated in a small-scale, re-circulating, aquatic IVF. Additionally, for both species, the chlorophyll a content was determined as a plant cultivation indicator. We selected clones of these two species because they showed good performance in earlier experiments concerning growth rates and protein contents [The aim of our research was to evaluate the influence of different light intensities and spectral distributions on the RGR, crude protein content (CPC) and relative protein yield (RPY) in the duckweeds contents .Lemna minor L.  and Wolffiella hyalina Delile Monod , were chosen for the experiments due to their fast growth rates and high protein contents [Two duckweed species, contents . The plaExperiments were carried out in a container (length × width × height: 5 × 3 × 3 m) at the campus of the University of Applied Sciences Osnabrück, Germany. Trials were conducted in a re-circulating, aquatic IVF .2 by applying this method. The unoccupied surface was covered with black PE in order to prevent algae growth in that area. The outlet solution from each of the two storage rack levels was led through UV-C clarifiers  in order to reduce the growth of ubiquitous algae and bacteria.It consisted of a 90 L reservoir for the nutrient solution connected to all duckweed cultivation vessels via flexible tubes. A submergible and adjustable pump  was installed at the bottom of the reservoir to create a continuous flow between reservoir and cultivation vessels. A nutrient control and dosing system  added the required liquid fertilizers from stock solutions to the tap water in the reservoir. A heating system  was installed at the bottom of the reservoir to keep a constant water temperature. The vessels (56 cm length × 37 cm width × 10 cm height) used for cultivation were positioned in a two-layer storage rack. On one side (width) of the cultivation vessel, the water inlet, a rectangular pipe leading the water inflow to the bottom of the vessel, was installed. On the opposite side, an outlet was located at 7 cm height. To guarantee no duckweed was lost from the vessel by flowing through the outlet, a wall was installed 7 cm before the outlet. The upper side of the wall was above water level, while the bottom side did not touch the ground of the vessel. This way, the nutrient solution could flow back into the reservoir, while the floating duckweed was hindered from passing the barrier. The net cultivation area per vessel decreased to 0.49 × 0.37 m = 0.1813 mAs light sources, dimmable LEDs with an adjustable spectrum  were installed 34 cm above the water surface in the vessels. The settings were adjusted with the VisuSpectrum 3.0 software .−2 s−1) were used for the experiments. All of the three spectral treatments contained 20% light at 6500 K (white light), and the remaining 80% were split according to the following red (660 nm)/blue (450 nm) ratio: 70/30, 50/50 and 30/70 (%). This resulted in eight different treatments  biomass was placed in each vessel.Pre-cultivation occurred for three days under above mentioned conditions. Experiments lasted for seven days and were conducted under non-axenic growth conditions. Vessels were placed in the storage rack based on a block design. This storage rack had eight compartments, each containing two LEDs and space for two experimental vessels. Eight treatments, with four replications for each of the two species, were investigated. In total, 16 vessels could be used at a time. Two replicates per light intensity and spectral distribution per species were investigated at the same time. To start with a similar surface coverage of ca. 80% in each vessel, 20 g of −1, which corresponds to a nutrient solution of 75-25/10 with the following composition and concentrations : NO3−-N: 0.76, NH4+-N: 0.25, PO43−: 0.1, K+: 0.91, Mg2+: 0.13, SO42−: 0.32, Ca+: 0.22, Cl−: 0.34, Fe3+: 0.0025, BO33−: 0.0005, Mn2+: 0.0013, Zn2+: 0.001 and Na+: 0.08 [The nutrient medium applied mainly consisted of commercially available fertilizers see . The nuta+: 0.08 . When th−1.The pH at the beginning of the experiments was 7.6. The heating system was set to a target value of 24 °C, and the pump was adjusted to a flow rate of 2 L minAt the end of the experiments, duckweeds were harvested with a metal sieve, rinsed with tap water, spin-dried for three minutes with a Top Spin Compact  to remove attached water and weighed.Dry weight (DW) was determined from FW via oven drying at 65 °C for 72 h. At time 0, four samples per species of the same FW as the starting material were used to determine the DW at the beginning of the experiments.−1).Relative growth rates (RGRs) per day were calculated according to Equation (1) , using tDried samples were ground and homogenized using a laboratory mill and stored for further analysis. The nitrogen content of the dried samples was determined using the Dumas method  using anThe relative weekly yield  was calculated from the RGR using Equations (2) and (3):−1 m−2) by multiplying it with the crude protein content (CPC) and extrapolating it to one square meter, according to Equation (4):2 is the cultivation area of the vessels used in the experiments.The RY was further used to calculate the relative protein yield ;ω665v: Absorption of the extract before acidification, measured at 665 nm;A750v: Absorption of the extract before acidification, measured at 750 nm (for the correction of phaeopigments);A665n: Absorption of the extract after acidification, measured at 665 nm;A750n: Absorption of the extract after acidification, measured at 750 nm (for the correction of phaeopigments);A665v/A665n for pure Chlorophyll-a; R = 1.7;R: Ratio of AE: Volume of the extract in milliliters (ml);VP: Net weight of the duckweed biomass sample (g);md: Thickness of the cuvette (cm); d = 1.The following modified Equation (5) was applied to calculate the chlorophyll a content in the fresh duckweed biomass :(5)ωChloAdditionally, the dry matter content of each sample was determined by drying plant material at 105 °C until it reached a constant weight. The chlorophyll a FW content was then multiplied with the dry matter content to calculate the chlorophyll a DW content.A nutrient solution sample was taken at the start (day 0) and the end (day 7) of the experiments from the reservoir, filtered  to remove particles and instantly frozen at −18 °C. Nitrate-N and ammonium-N concentrations in these samples were measured according to German standard methods ,33 with All data are based on four replicates and are given as mean ± standard deviations. The data were analyzed statistically using one-way ANOVA and Tukey’s post hoc test at 5% significance level, using the software program SPSS 25 .L. minor was reached at 150–70/30 (0.13 ± 0.013 d−1) and for W. hyalina at 150–50/50 (0.21 ± 0.01 d−1). The minimum values were obtained at 50–30/70 for L. minor with an RGR of 0.078 ± 0.012 d−1 and at 50–50/50 for W. hyalina with an RGR of 0.119 ± 0.003 d−1. The percentage increase from the lowest to the highest RGR was 67% for L. minor and 76% for W. hyalina. The results of all three L. minor treatments cultivated at 150 µmol m−2 s−1 were significantly higher compared to the 50 µmol m−2 s−1 treatments. W. hyalina cultivated at a light intensity of 150 µmol m−2 s−1 reached significantly higher RGRs than the 100 and 50 µmol m−2 s−1 treatments. The light spectrum showed no significant impact on the RGR in any treatment.L. minor and between 39.3 ± 1.0% and 40.0 ± 0.8% for W. hyalina for the different treatments. No significant differences in the CPC for the different light intensities and spectral distributions within a species were detected.The CPC, based on DW, varied in a narrow range between 31.8 ± 0.8% and 32.4 ± 1.2% for 2, based on DW, is presented in −1 m−2  for L. minor, while for W. hyalina, the range was from 5.01 ± 0.35 g week−1 m−2 at 50–30/70 to 9.48 ± 0.39 g week−1 m−2 at 150–50/50. The difference from the lowest to the highest value for L. minor was 50%, and for W. hyalina, it reached 89%. Higher light intensities resulted in higher relative protein yields. Overall, W. hyalina achieved higher RPYs in all treatments compared to L. minor. The higher the light intensity, the higher the difference between the species RPYs, meaning that at the highest light intensities (150 µmol m−2 s−1), W. hyalina yielded more protein compared to L. minor than at the two lower light intensities. The treatments 50–70/30 and 50–30/70 were significantly lower compared to all other L. minor treatments, except for 100–30/70. For W. hyalina, all treatments with a light intensity of 50 µmol m−2 s−1  were significantly lower compared to the other treatments with higher light intensities. No significant differences, in neither of the two duckweed species, were observed between the different spectral distributions.The RPY in grams per week and m−1 and 7.29 ± 0.39 mg g−1 for L. minor at 150–50/50 and 50–70/30, respectively  was obtained at 150–30/70. This corresponded to a decrease of 27% for L. minor and 32% for W. hyalina.The content of chlorophyll a for both species after seven days of experiments ranged between 5.32 ± 0.51 mg gectively . The max−2 s−1) and the two higher treatments (100 and 150 µmol m−2 s−1) can be observed for L. minor. For W. hyalina, the 150 µmol m−2 s−1 treatments were significantly lower compared to the 50 µmol m−2 s−1 treatments. Different light spectra had no significant impact on the chlorophyll a content of both species.A significant decline between the treatments of the lowest light intensity  and showed a minor increase for W. hyalina. Similar results were also observed for potassium. An increase in magnesium, sulfur and calcium occurred for both species.A strong reduction of more than 80% can be seen for ammonium-N, iron, manganese, zinc, and in case of L. minor experiments and 7.9 for the W. hyalina experiments.Compared to the start of experiments, the pH showed a minor increase with an average value of 7.8 for the −2 s−1 significantly increased the RGR of L. minor and W. hyalina. Our data agree with other published investigations. Paolacci et al. [−2 s−1 increased the RGR of L. minor and L. minuta cultivated in sterile growth rooms at 20 °C with a light:dark cycle of 16:8 h. At light intensities below 40 µmol m−2 s−1, no significant differences were detected between the RGR of both species, while above 90 µmol m−2 s−1, L. minuta had significantly higher RGRs than L. minor. The latter reached an RGR of 0.26 d−1 when grown at 150 µmol m−2 s−1. This was higher compared to our result, but cultivation conditions varied, which might provide a possible explanation for this difference.The RGR determined in our study differed between both investigated species and growth conditions. An increase in light intensity from 50 to 150 µmol mi et al.  reported−2 s−1, L. aequinoctialis reached an RGR of 0.19 d−1 at the highest light intensity, when cultivated in monoculture, while L. punctata and Spirodela polyrhiza reached 0.18 d−1 and 0.15 d−1 under the same growth conditions, respectively [L. aequinoctialis. Considering the costs for lighting, an optimum regarding those factors was reached at 110 µmol m−2 s−1 [−2 s−1) resulted in a 25% greater RGR of L. gibba [L. minor’s RGR increase of 67% and W. hyalina’s increase of 76% at a 200% greater light input in our study.At comparatively low light intensities between 30 to 105 µmol mectively . Increas m−2 s−1 . A sevenL. gibba . This in−1 for L. minor and 0.21 d−1 for W. hyalina in the presented study are lower compared to the highest achieved values of 0.42 d−1 and 0.52 d−1 for the same clones, respectively, grown under sterile conditions in batch cultures [−1 for W. hyalina. This is in exact agreement with the results of the current study.The maximum obtained RGRs of 0.13 dcultures . Howevercultures ,37. A hicultures , the samLemna minor grown on synthetic dairy wastewater did not increase with increasing light intensities between 50 and 850 µmol m−2 s−1 [Lemna gibba reached constant high growth rates under different light intensities between 50 and 1000 µmol m−2 s−1; however, higher intensities led to increasing zeaxanthin levels. This way, a large fraction of the absorbed light was dissipated non-photochemically [In contrast, other studies reported that different light intensities had no significant impact on the RGR of duckweed species. The RGR of  m−2 s−1 . Lemna gemically .The light spectra in the presented experiments had no significant impact on any investigated parameters for both species. However, it has to be kept in mind that in this study, pure red or blue light was never used. There was always a white light background of the light intensity of 20%, and the ratios between blue and red light were never higher than 70:30%.Landoltia punctata cultivated under fluorescent white light, blue LED and white LED at 110 µmol m−2 s−1 showed no significant RGR differences [S. polyrhiza when cultivated at 60 µmol m−2 s−1 using red and blue LEDs  [Spirodela polyrhiza cultured in eutrophic medium reached a significantly higher total biomass yield when a red:blue ratio of 2:1 or 4:1 at a light intensity of 110 µmol m−2 s−1 was applied compared to monochromatic  or fluorescent light sources at the same intensities.Up to now, only a few investigations concerning this parameter have been carried out regarding duckweed RGR. ferences . There wctively) , which ictively)  describeL. gibba, cultivated at 50 and 1000 µmol m−2 s−1, increased from 25% to 46%, respectively. A protein content increase from 1.5% to 2% (based on FW) was observed for L. minor when cultivated on synthetic dairy wastewater at a light intensity of 850 µmol m−2 s−1 compared to 50 µmol m−2 s−1. In C3 plants, such as duckweed, higher light intensities induce the increased production of Rubisco, a soluble protein [−2 s−1) only slightly increased the percentage of activated Rubisco in S. polyrhiza [−2 s−1. A more substantial increase in light intensity, as described above, will lead to rising protein contents.The presented crude protein contents for both species showed no significant difference between the tested light scenarios. This is in contrast to the results reported by Stewart et al. , who sho protein . A smallolyrhiza . This coW. hyalina. Appenroth et al. [W. hyalina and 25% for L. minor. These duckweeds were cultivated with a modified Schenk–Hildebrandt medium at 100 µmol m−2 s−1 continuous white light. In another experiment, the highest values for crude protein of the three species L. aequinoctialis, L. punctata and S. polyrhiza , were reached at 105 µmol m−2 s−1 using a one-tenth strength Hoagland solution [L. minor and 35.3% for W. hyalina using a stationary system with the same nutrient solution as applied in these experiments. Wheeler at al. [W. hyalina, cultivated in the re-circulating system compared to the stationary system.The crude protein contents in the presented experiments were rather high considering the low nutrient concentration and the low light intensities, especially regarding h et al.  reportedsolution . Petersesolution  reached r at al.  assumed S. polyrhiza [W. arrhiza, using irradiation with wavelengths corresponding to white, red and blue light, no significant differences in amino acid concentrations of the soluble protein were detected [A red:blue ratio of 1:2 can increase starch yield significantly, while a higher portion of the red spectrum under eutrophic conditions caused a strong inductive effect on turion formation in olyrhiza . This isolyrhiza , who detdetected . These rL. minor compared to W. hyalina. The species L. minor reached a maximum of 4.44 ± 0.55 g week−1 m−2 at 150–50/50 and W. hyalina of 9.48 ± 0.39 g week−1 m−2 for the same treatment. This extrapolates to 2.31 and 4.93 t of pure protein per year and hectare, respectively. In the literature, a wide range of productivities are reported. For L. minor and W. hyalina, 28.8 and 34.7 g week−1 m−2, respectively, were reached using the same nutrient solution in a stationary system with smaller vessels [−1 ha−1 (ca. 46 g week−1 m−2) for duckweeds. Chakrabarti et al. [−1 ha−1 (ca. 17.5 g week−1 m−2) for L. minor. Regarding protein content of 27.1% for duckweed grown on an inorganic fertilizer-based solution, the protein productivity resulted in 4.74 g week−1 m−2. Comparing these values to soybean with a yield of ca. 3 t year−1 ha−1 and a protein content of 40% [−1 ha−1 was considerably lower compared to any duckweed protein productivity projection.The protein productivity, given as RPY, was lower for  vessels  reportedi et al.  reached L. minor had higher total chlorophyll contents for all investigated light intensities (6 to 1000 µmol m−2 s−1) than L. minuta, reaching up to ca. 1.4 mg g−1 of fresh biomass at the lowest light intensity [Lemna gibba contained ca. 250 µmol m−2 of chlorophyll a and b at 50 µmol m−2 s−1 and ca. 300 µmol m−2 at 100 µmol m−2 s−1 [The chlorophyll a content for both species was investigated as a parameter to indicate a possible color changes in the plants at different light conditions. It decreased with increasing light intensity. This negative correlation was also found for other duckweed species ,38,39,49ntensity . Lemna g m−2 s−1 ,39. The  m−2 s−1 .S. polyrhiza, when cultivated under red, blue and white light. This missing effects of the light quality in our experiment might be also caused by the use of mixed light quality.The different investigated spectral distributions had no significant impact on both species’ chlorophyll content. This has also been shown by Zhong et al. , who obtThe duckweed cultivation system applied in our experiments was a small-scale, experimental prototype of a re-circulating, aquatic IVF and specifically designed and built for conducting scientific experiments. In the literature, only a theoretical approach , but no The applied system for nutrient control and dosing is based on EC values. When the actual EC values fell below the target EC, the dosing system pumped stock solution into the reservoir until the target value was reached again. This is a well-established system for nutrient dosing used in many different hydroponic applications ,51. HoweTo gain more data about the behavioral pattern of duckweed in re-circulating systems, longer-lasting experiments investigating a broad range of abiotic, and in the case of non-sterile experiments, also biotic, parameters are needed. Nonetheless, the findings and experiences of our study were already successfully implemented into the operation of a large scale, re-circulating, aquatic IVF for duckweed biomass cultivation .


TFW may be explored as potential and natural compounds in food and pharmacological applications.The present study was designed to evaluate the chemical extraction, chemical composition, and antioxidant and antibacterial properties of the total flavonoids in Willow Buds (TFW). We investigated the optimal extraction of TFW using response surface methodology (RSM). Chemical compounds were analyzed using Q-Orbitrap LC–MS/MS. The DPPH radical scavenging capacity, hydroxy radical inhibitory ability, and superoxide anion radical inhibitory ability were explored to determine the antioxidant properties of flavonoid extractions. The antibacterial effect was assessed via minimal inhibitory concentration. The results demonstrated that the optimal extraction conditions were an ethanol concentration of 50%, a time of 35 min, and a liquid/material ratio of 70:1 mL/g. Under these conditions, the yield of TFW was 7.57%. Eight flavonoids, a phenolic glycoside, and an alkaloid were enriched in the Willow Buds. The TFW exhibited significant antioxidant activity, with IC Flavonoids are an important class of secondary plant metabolites and a class of natural beneficial chemicals . FlavonoModern extraction technology has the advantages of a high extraction efficiency, purity, and product quality, as well as simple operation and less solvent residue than traditional extraction processes. In recent years, supercritical fluid extraction, ultra-micro-grinding technology, and ultrasonic extraction have been commonly used—especially ultrasonic extraction, which is easy to operate and has a short cycle. Therefore, in this study, we aimed to use ultrasound-assisted extraction to enrich flavonoids and optimize their extraction process conditions using response surface methodology (RSM).Salix babylonica L. is widely distributed all over the world, and its leaves are rich in various compounds such as flavonoids, terpenes and phenols −, MS/MS [M–H]−, [M+H]−, MS/MS [M+H]−, calculated mass, and formula of each component are listed in 1 and 2 were compared with previously reported data [m/z 138.1 and 463.1 could be assigned to trigonelline and isoquercitrin, respectively. Similarly, the fragments appearing at m/z 609.1 and 303.0 could be attributed to rutin (compound 3) [4) [m/z 289.1 and 287.1 matched those of catechin and kaempferol (compound 5 and 6), respectively [m/z 447.1 and 317.1 were considered to be characteristic fragments of astragalin and isorhamnetin (compound 7 and 8), respectively [m/z 304.1 and 271.1 belong to salicin (compound 9) [10), respectively [The chemical components of TFW were acquired with LC–MS and aligned to different databases, including mzCloud, mzVault, and ChemSpider, for characterization. In addition, to screen out the most abundant constituents for more investigations in the future, compounds with a spectral peak area of more than 1 × 10ted data ,22, and pound 3)  and querectively . Additioectively . The obspound 9)  and nariectively . Among tectively  and querectively .2–· free radical scavenging capacity, and a lipid peroxidation method. The present work was aimed to determine the potential antioxidant activity of TFW through the measurement of DPPH, O2–·, and ·OH. The linear fitting equations for the relationships between DPPH, O2–· or ·OH with TFW concentration are as follows: Y (DPPH) = –77.383x2+133.81x+22.652; Y (O2–·) = –203.3x2 + 189.86x + 21.264; Y (·OH) = –144.08x2 + 227.86x + 1.3191. As shown in 2–· and ·OH inhibition capacity in a concentration-dependent manner. The IC50 value (mg/mL) was used to represent the antioxidant capacity. According to the equations shown above, the IC50 values for DPPH, O2–· and ·OH were 0.20, 0.18, and 0.23 mg/mL, respectively, as shown in 2–· and ·OH. The antioxidant activities of the TFW extractives were consistent with those of previous studies regarding flavonoid extractions from Saussurea involucrate and Semen Oroxyli [2–· than DPPH radical scavenging and ·OH inhibition, which was similarly to the antioxidant properties of VC. This difference may have been due to the great difference in the DPPH radical scavenging ability and O2–· and ·OH inhibition abilities of the different components. Consistent with our results, the antioxidant capacity of flavonoids from tartary buckwheat bran was shown to have a stronger ability for DPPH radical scavenging than ·OH and O2–· [Researchers have developed several methods to determine in vitro antioxidant capacity , such as by using total oxygen radical scavenging capacity, reducibility methods, ABTS radical scavenging capacity, DPPH radical scavenging capacity, ·OH free radical scavenging capacity, O Oroxyli . Importaand O2–· . Altogetand O2–· ,31, our Salmonella enterica, Streptococcus pneumoniae, Escherichia coli and Staphylococcus aureus strain were used as they are ubiquitous in the natural environment and can cause infections under certain conditions. For instance, a Streptococcus pneumoniae infection can cause pneumonia, lower respiratory tract infection, and other diseases [Salmonella endangers food hygiene and safety [Staphylococcus aureus was 2.5 mg/mL and that of Salmonella enterica was 10 mg/mL. Recently, accumulating traditional Chinese medicines were found to possess antibacterial pharmacological activities, and these were gradually used to replace the usage of antibiotics due to the bacterial resistance. González-Alamilla et al. demonstrated that the chemical compounds of Salix Babylonica L. exhibited antibacterial capacities against Escherichia coli, Staphylococcus aureus and Listeria monocytogenes. The best antibacterial property was obtained with luteolin against Staphylococcus aureus [Salix tetrasperma ROXB. were also found to show evident antibacterial capacity [S. babylonica against Escherichia coli, Salmonella typhi, Salmonella choleraesuis, and P. aeruginosa and determined an MIC of 100 mg/mL, 25 mg/mL for S. aureus and L. monocytogenes, and 12.5 mg/mL for Bacillus subtilis [To evaluate the antibacterial effect of TFW extractives, the diseases ,33. Salmd safety . These ps aureus . The salcapacity . In 2020subtilis . Our resIn summary, the authors of the present study extracted flavonoids from Willow Buds using an ultrasonic-assisted extraction method for the first time. The optimized conditions for flavonoid extraction were a 50% ethanol concentration, 35 min of ultrasonic extraction time, and a 70:1 mL/g solvent-to-material ratio. The TFW extracts were determined to exhibit in vitro antioxidant and antibacterial activities. We also described the active components  present in Willow Buds.


The greatest values for oleic acid (74.13%) and TPC (350.6 mg·kg−1) were observed in Gemlik and Domat oils, respectively (p < 0.05). Edincik oil showed the maximum hydroxytyrosol content (48.022 mg·kg−1) and TAC value (515.36 mg TE·kg−1) (p < 0.05). The Edincik, Domat, and Uslu oils were significantly not different for the total content of C6 compounds derived by lipoxygenase, which are the main volatiles responsible for the typical aroma of EVOOs (p > 0.05). Domat oil also exhibited the highest scores for bitterness and pungency perceptions (p < 0.05). The fruitiness scores of the oil samples  were close to each other, even if they were statistically different (p < 0.05). Principal component analysis (PCA) indicated that the Ayvalık oil was separated from the others due to its poor-quality characteristics. As a result, it can be stated that Domat olive oil has better quality than the others.Extra virgin olive oils (EVOOs) obtained from five Turkish olive cultivars widely produced in the Aegean and Marmara regions were investigated based on their total antioxidant capacity (TAC), total phenolic content (TPC), pigment contents, fatty acid (FA) profiles, phenolic compounds (PC), volatile compounds (VC), and sensory properties. The results showed that all properties of EVOO samples were significantly affected by the olive cultivar used. The pigment contents in Ayvalık (9.90 mg·kg Olea europaea L.) and is designated as the highest quality in the classification of olive oils (OO) [Extra virgin olive oil (EVOO) is obtained only via mechanical or physical processes from the olive fruit (ils (OO) . The maiils (OO) . In partNowadays, some comprehensive components, especially the profiles of volatile, phenolic, and pigment constituents, are used in the quality assessment of EVOO. In recent years, these components have been widely used to determine the characterizations ,4 and auThe positive effects of EVOO on health are mainly attributed to its fatty acid (FA) fractions and PC . It is rTurkey is one of the countries where olive farming has been carried out for a long time. According to a hypothesis that is generally accepted, the origin of the wild olive tree is a certain region of Turkey (as known Anatolia or Minor Asya), and olive cultivation spread from Syria to Greece and other Mediterranean countries through this area, which includes the Aegean region ,13. AddiNumerous comparative studies have been conducted on the main properties of different monovarietal EVOO. However, many of them have investigated only a certain class of components or routine quality characteristics of EVOO. Diraman et al. 2010)  studied 10  studiIn the literature, studies on the comprehensive characterization and comparison of monovarietal Turkish EVOO are limited. Although there have been various studies on some features of the country EVOO, these mostly focused on only one of their component groups, such as PC, VC, and FA fractions. In this sense, this study aimed to determine and compare the VC, PC, and FA profiles, TAC, pigment contents, and sensory properties of the EVOO of Turkish olive cultivars widely grown in the Aegean and Marmara regions.232, and K270 are the basic parameters used in the classification of olive oils. Among these, FFA is a measure of the enzymatic hydrolysis of lipids, while the others are indicators for determining the level of oxidation in lipids [232, and K270 of the oils ranged from 0.05 ± 0.00 to 0.12 ± 0.01%, 3.50 ± 0.27 to 6.33 ± 0.72 meq O2·kg−1, 1.64 ± 0.01 to 1.99 ± 0.02, and 0.13 ± 0.00 to 0.18 ± 0.02, respectively .The concentration of chlorophyll and carotenoid in OOs directly affects their color . The OOs mg·kg−1 . Both ch−1, respectively, as a wide range. The pigment contents in the current study also showed wide ranges. In a previous study by Jolayemi et al. (2016) [−1 and in Ayvalık oils were 1.28–2.57 mg·kg−1 and 1.01–1.61 mg·kg−1, respectively. These values reported for Ayvalık oil are lower than those found for the same variety in the present study.The amount of these pigments in OOs can vary widely depending on the primarily variety and harvest time  and also. (2016) , it was p < 0.05). A rich oleic acid content in oils is not only desirable for a long shelf life concerning oxidative stability but is also necessary for health benefits [Fatty acid compositions of the EVOO samples are given in benefits . In factbenefits . The valThe composition of fatty acids in OOs is mainly affected by genetic variations, geographical conditions , ripening degree, season, and production conditions ,34. The −1), followed by Gemlik (321.905 ± 6.185 mg·kg−1), Edincik (294.090 ± 13.810 mg·kg−1), Uslu (291.145 ± 5.485 mg·kg−1), and Ayvalık (234.71 ± 1.490 mg·kg−1) olives. These values were higher than the TPC levels reported for five different EVOO obtained from the Chemlali variety (117.64 ± 1.23–151.7 ± 1.97 mg GAE·kg−1) grown in different locations [−1. However, the TPC determined by these authors for EVOO from Uslu (114.22 ± 3.44 mg GAE·mg−1) was lower than that of this study. These differences could mostly be due to the variety [The PCs in EVOO are phenolic alcohols  and their secoiridoid derivatives, phenolic acids, flavonoids, and lignans . Among tocations . TPC in ocations , who rep variety , maturitp < 0.05). Many previous studies have also reported that the cultivar affects the phenolic compound content of OOs [−1 (Edincik), while caffeic acid had the lowest content (0.009 ± 0.001 to 0.184 ± 0.002 mg·kg−1). The phenolic acid contents determined in all samples are in agreement with those reported in many previous studies [A total of 11 phenolic compounds were identified and quantified in the oil samples . The cont of OOs ,29. Phent of OOs ,38. Amon studies ,39.−1, except in Ayvalık oil, which contained the greatest amount for pinoresinol (15.247 ± 0.524 mg·kg−1) within the PC. The amount of hydroxytyrosol in Edincik oil (48.022 ± 1.186 mg·kg−1) was significantly higher than that in other oils (p < 0.05). The level of hydroxtyrosol in the EVOO from Ayvalık olive (11.621 ± 0.420 mg·kg−1) was greater than that reported by Jolayemi et al. (2016) [−1, respectively, by Negro et al. (2019) [p < 0.001), but tyrosol was not interestingly correlated with TAC (p > 0.05).Hydroxytyrosol was the predominant PC in the samples, with an amount in the range of 11.621 ± 0.420 to 48.022 ± 1.186 mg·kg. (2019) . On the . (2019) . Additio. (2019) . In fact−1 (Gemlik) and from 1.640 ± 0.089 (Edincik) to 2.658 ± 0.080 mg·kg−1 , respectively. The apigenin contents of the five EVOOs were more than that of two fresh monovarietal EVOOs from Portuguese olive varieties (0.71–1.00 mg·kg−1) reported by Klisović et al. (2022) [−1). Moreover, the value of luteolin found in the EVOO from Ayvalık (2.658 ± 0.080 mg·kg−1) was partially less than the range (3.65–5.24 mg·kg−1) reported by Jolayemi et al. (2016) [−1 and from 1.00 ± 0.04 to 1.65 ± 0.11 mg·kg−1, respectively. These contents are lower than those of the oils from same cultivars investigated in the current study.Regarding flavonoids, EVOOs from Domat and Gemlik olives provided higher apigenin contents compared to the other cultivars. Apigenin and luteolin concentrations in the oils ranged from 2.409 ± 0.030 (Edincik) to 12.824 ± 0.010 mg·kg. 2022) , but the , but th. (2016) . In a pr. (2016) , the amoAs a noticeable point, although the amounts of major secoiridoids (hydroxytyrosol and tyrosol derivatives) in the samples based on their HPLC peaks have not been quantified, it is estimated that most of the TPC measured by spectrophotometry consisted of amounts of these compounds . Essenti−1. It can be seen that the TAC of Edincik and Domat oils were notably higher than those of the other three oils.Certain PCs in OOs, such as oleuropein derivatives, exhibit strong antioxidant activities based on their radical scavenging abilities, as well as some other useful biological functions. The antioxidant effects of these PCs also contribute to the extended shelf life of OOs against lipid oxidation in comparison with those of other vegetable oils . The TACp > 0.05), agreeing with the case between the EVOOs of Ayvalık and Gemklik olives. The TAC of Edincik and Ayvalık oils was higher than that reported by Uluata et al. (2021) [−1. They also reported the TAC of Uslu oil was 123.67 ± 6.23 mg TE·kg−1, lower than that of EVOO from the same variety investigated in this study. Previously, Borges et al. (2019) [−1 and 177.70 and 380.44 mg TE·kg−1, respectively, in agreement with those found in this study. Reboredo-Rodríguez et al. (2018) [−1. This value is similar to the TAC of the Edincik oil but higher than that of oils from other varieties.In a recent study, Comlekcioglu et al. (2022)  reported. (2021) , who fou. (2019)  studied . (2018)  found th−1), Domat (85.077 mg·kg−1), Uslu (87.987 mg·kg−1), and Gemlik (85.044 mg·kg−1) oils were not different significantly from each other (p > 0.05) but were higher than that of Ayvalık (52.368 mg·kg−1) oil (p < 0.05).The flavor of OO is a remarkable feature to evaluate their quality and is associated with VCs. These VCs are mostly formed during oil extraction and also develop during harvest and storage processes. The positive attributes and organoleptic defects in sensory perception of OO are associated with VCs . Table 4The predominant VCs in the samples were C6 and C5 volatiles derived by lipoxygenase (LOX) pathway from linoleic and linolenic acids as unsaturated fatty acids. These VCs identified in oils were as follows: C6 aldehydes -2-hexenal and 2,4-hexanedienal), C6 alcohols -3-hexen-1-ol and (E)-2-hexen-1-ol), C5 aldehydes -2-pentenal) and C5 alcohols -2-penten-1-ol). These compounds formed by LOX have been also reported previously as the most common volatiles in EVOO and the most responsible for their characteristic aroma . In addi−1, respectively) than those of the others, while the Ayvalık oil showed the lowest ones (36.886 mg·kg−1)  ,44. It wmg·kg−1) . SimilarWithin C6 compounds, (E)-2-hexenal  was the major compound found in all EVOO samples. (E)-2-hexenal and 3-hexenal have been reported to significantly influence the sensory properties of OO, providing its freshness and pleasant aroma ,55. (E)-−1) than that of others (ranging from 0 to 8.797 mg·kg−1). In a previous study [The amount of 1-penten-3-ol, as an aroma-active compound , was higher in Gemlik oil . The highest scores for the perception for bitterness and pungency were observed for Domat oil (p < 0.05). Concerning the relationships between the positive attributes, as expected, all three perceptions strongly correlated with each other . Furthermore, significant correlations were found between the positive attributes and both TPC and the total concentration of the VC group synthesized by the LOX pathway. These correlations are supported by the findings of Klisović et al. (2022) [p < 0.001) and the total concentration of VC derived by LOX . Additionally, there was also a good correlation between these VC contents  and fruitiness . These results are in agreement with those discussed in several previous studies [The results of the sensory evaluation of EVOO samples are depicted in . (2022) . Similar. (2022)  also rep studies ,44. It w studies .PCA was applied to all data sets (except for sensory properties) to visually reveal the relationships between the variables and the samples. The results of PCA are illustrated in t-ferulic acid, caffeic acid, pinoresinol, etc.) contents , Ayvalık , Domat , Uslu  and Gemlik (synonym Trilye)  : Marmara)  The morpMarmara) . About 3p-qumaric acid, caffeic acid, syringic acid, vanillic acid, ferulic acid, gallic acid, luteolin, apigenin, isobutyl acetate, DPPH, Trolox and fatty acid methyl esters (FAMEs) mixture were purchased from Sigma-Aldrich .Sodium hydroxide, sodium carbonate, potassium chromate, potassium iodide, phosphoric acid, methanol, ethanol, acetic acid , diethyl ether, phenolphthalein, chloroform, sodium thiosulfate, cyclohexane, acetonitrile, Folin–Ciocalteu reagent and standards of oleuropein, hydroxytyrosol, tyrosol, pinoresinol, 270 and K270) of the samples were determined according to the Turkish Official Methods (2014) [2·kg−1, respectively.The FFA, PV, and extinction coefficients (Ks (2014) . FFA and−1) prepared in methanol:water  were mixed and shaken in a 12 mL tube. Then, 3 mL of methanol:water was added and vortexed for 30 s. Thereafter, this mixture was centrifuged at 500 rmp at 4 °C for 5 min. The lower phase was transferred to another tube and the extraction was repeated two more times. Then, the collected methanolic phases were washed with 1.5 hexane two times to remove the oil residues. The lower phase was taken and used as the phenolic extract for the analysis of TPC, TAC, and PC.The extraction of PC in the samples was carried out based on the method of IOC (2017) , as desc−1 of pheophytin and lutein, respectively.470 and Abs670 are the absorbance values read at these wavelengths, and d is the length of the optical path (1 cm).The total amounts of chlorophyll and carotenoid in samples were determined according to the method of Mínguez-Mosquera et al. (1991) . BrieflyThe TPCs of samples were determined by using the Folin–Ciocalteu method, as adopted by Capanoglu et al. (2013) . Measurev/v) was used instead of the phenolic extract. The percentage of DPPH inhibition of each phenolic extract of samples was calculated following Equation (3):control and Abssample were absorbances recorded at 517 nm for the control and phenolic extract of the sample, respectively.The TAC of the samples was determined using the DPPH radical scavenging capacity of the metabolic extracts based on the method reported by Osei et al. (2022) , with soThe value of IC50 of each sample, which corresponds to the concentration of extract reducing half of the amount of DPPH radical, was calculated from the regression curve obtained using five different phenolic extract solutions diluted by methanol:water solution. Trolox was used as standard and the results were expressed as mg Trolox equivalent (TE) per kg sample.The analysis of FA composition in samples was carried out by a gas chromatography and flame ionization detector (GC-FID) system  equipped with an Rtx-2330 capillary column  . Approxi−1. Details of the analysis are presented in the SM.The analysis of phenolic fractions in samples was carried out using a Water Alliance e2695 HPLC  system, consisting of a photodiode array detector (PDA)  and an inertSustain C18 . The phenolic extract was filtered through a 0.45 μm polyvinylidine fluoride (PVDF) syringe filter before the injection into the system. The operational procedures of the HPLC were performed as described by Veneziani et al. (2018) [The VCs in samples were isolated by solid phase micro-extraction (SPME) and analyzed with a gas chromatography-mass detector (GC-MS)  system coupled to an autosampler , according to Genovese et al. (2015)  and KorkThe sensory properties of samples were evaluated by a trained sensory panel of ten assessors from the Central Laboratory of Mardin Artuklu University  according to the Turkish Official Methods (2014)  adapted p < 0.05). Principal component analysis (PCA) as a multivariate analysis was also performed to compare the sample based on their investigated properties. All statistical analyses were carried out using the SPSS  software package.The significance of differences among the values of all parameters of samples was determined by one-way analysis of variance (ANOVA) followed by Duncan’s multi-comparison test  were close to each other. This was also confirmed by the relationship between their total content of VCs derived from LOX. The result of PCA showed that the EVOO from Ayvalık olives is distinct from the oils from other varieties due to its lower values for many quality attributes. Based on this study, it can be concluded that the EVOO from Domat olives had the best characteristics sensory quality parameters and chemical properties affecting them. In the future, studies should be conducted on the storage stability of these oils.


Nasal obstruction is one of the most frequent otolaryngologic complaints; and the collapse of the internal nasal valve is one the main causes of the nasal air flow obstruction.We aimed to evaluate internal nasal valve by using reformatted CT-scans pre- and post- rhinoplasty at 3 months and to assess its correlation to symptomatic improvement of nasal obstruction using the NOSE scale.A prospective observational study was conducted between March 2017–May 2018 in a tertiary care otorhinolaryngology center. We included patients suffering from nasal obstruction secondary to internal nasal angle collapse and nasal deformity. Patients with sinusitis, nasal polyposis, and nasal masses were excluded.p < 0.0001), which was absent in CT-scan results.Twenty consecutive patients underwent rhinoplasty, with a mean age (22.2 ± 2.8), with majority of males . There was no significant correlation between pre- and post- CT-scans of the internal nasal valve angle/area and NOSE scores. A high significant difference was detected between mean pre- and post- NOSE scores (Reformatted CT-scans measurements of internal nasal valve area and angle were not of value. NOSE scores pre- and post- rhinoplasty had a significant value to determine degree of obstructive symptom improvement. Nasal obstruction is one of the most common complaints of patients in Otorhinolaryngology (ORL) medicine.10The INV is a remarkably significant area for ORL surgeons to correctly evaluate before rhinoplasty and/or septoplasty surgery for patients with nasal obstruction. Different surgical techniques are used to correct INV collapse during functional rhinoplasty, which may include partial inferior turbinectomy, septoplasty, and placement of spreader grafts, which is one of the most effective methods of correcting nasal obstruction.Stewart et al. developed and validated the Nose Obstruction Symptom Evaluation (NOSE) scale as a symptomatic improvement quality of life scale, to be used in evaluation of INV obstruction.We performed a prospective observational cohort study of twenty consecutive patients from March 2017 till May 2018, who underwent rhinoplasty surgery at the Otorhinolaryngology department in Ain Shams University Hospital, Cairo, Egypt. Institutional Review Board approval was received from Vice Dean of Medical Ethics Committee of Faculty of Medicine Ain Shams University for Graduate Studies and Research. All patients signed informed written consent without receiving any stipend, and they were informed about study requirements according to World Medical Association Declaration of Helsinki (version 2002). All patients agreed to undergo a CT-scan of the nose pre-rhinoplasty and three months post-rhinoplasty. Complete ORL examination and endoscopic assessment of the nose was completed in all patients prior to surgery. Inclusion criteria were as follows: male and female patients with range 20–50 years old, nasal obstruction secondary to internal nasal valve collapse, nasal deformity, and/or inferior turbinate hypertrophy, and/or deviated nasal septum. Exclusion criteria were as follows: sinusitis, nasal polyposis, and nasal masses.Reformatted coronal CT-scans of INV area/angle were requested at a plane perpendicular to the anterior aspect of acoustic axis. Acoustic axis is determined on a sagittal reformatted image based on results of Cakmak study,We measured the INV cross-sectional area and valve angle through a standardized section 19The statistician who performed statistical analysis was blinded and uninvolved with clinical record or radiologic analysis. All data were collected, coded, tabulated and analyzed using SPSS version 24.0 software .p-value > 0.05)Data were normally distributed, therefore, mean and Standard Deviation (SD) were calculated for parametric numerical data, while frequency and percentage for non-numerical data. A Shapiro–Wilk’s test  was used to assess strength of association between INV angle and area measures from each nares and average of both sides with NOSE scores measurements to define the strength and direction of the linear relationship between them. Student Stewart et al developed and validated NOSE scale as a disease-specific QOL instrument to be used in nasal obstruction problems (Supplementary Table S1).19p = 0.78), respectively.Comparing between males and female postoperative nasal scores showed no significant difference between them, where the mean ± SD was 39.3 ± 7.6 and 38.3 ± 4.1 (p < 0.0001), with the most difference between pre- and post-operative NOSE scores was 50 (80 vs. 30)  scale and showed that the mean preoperative (NOSE) score was 67.5 ± 7.9 and mean postoperative (NOSE) score was 39 ± 6.6 with a mean change of (NOSE) score 28.5 ± 9.3, indicating a highly statistically significant difference between pre and postoperative NOSE scores ( vs. 30) .Table 2RThe INV cross-sectional area showed that mean preoperative INV area on the left side, right side, and average of both were 130.3 ± 44.9, 138.6 ± 60.6, and 134.5 ± 52.8, respectively. As well, it showed that the mean postoperative INV area on the left side, right side and average of both were 125.2 ± 30.4, 130.9 ± 30.7 and 128.1 ± 30.5, respectively. There was no statistically radiologically significant difference between either means of pre- and post-operative INV area on left, right, or their average .p = 0.5), , , respectively. There was a statistically significant negative correlation between postoperative NOSE scores and postoperative INV cross-sectional area on left side    or average of both sides   .The INV angle showed that mean preoperative INV angle on the left side, right side, and average of both were 9.4 ± 4.6, 9.15 ± 5.2 and 9.3 ± 4.9, respectively. As well, it showed that the mean postoperative INV angle on the left side, right side and average of both were 9.9 ± 3.2, 10.8 ± 3.7 and 10.3 ± 3.5, respectively. Results revealed that there was no statistically radiologically significant difference between either means of pre- and post-operative INV area on left, right, or their average .p = 0.111), , , respectively. Postoperatively there was no statistically significant correlation between postoperative NOSE scores and postoperative INV angles either on left side, right side, or an average of both , , , respectively .In correlation between cases age and postoperative NOSE scores, there was no statistically significant correlation detected , , , , respectively  score. Thus, there was no correlation between CT scans analysis of the INV and the (NOSE) scale, which agrees with Veron et al.In spite of our attempts to discover an objective measurement, the study of 20 patients as a sample size may not serve as a true normal population. However, patients who had any significant nasal and sinus disorders that could affect nasal obstruction average improvement time were excluded.Radiological measurements of cross-sectional area and angle of INV by reformatted CT scans were not of value in evaluation of INV pre and post-rhinoplasty. On the contrary, there was a highly significant difference between NOSE scores pre and post-rhinoplasty, in which it was found to be of significant value to determine the degree of symptomatic improvement pre and post rhinoplasty. Therefore for better clinical symptomatic evaluation pre- and post-operative in rhinoplasty; using NOSE scale assessment is preferable to using routine CT scan commonly requested by ORL surgeons for some decision making after physical examination. Besides, NOSE scale would be a useful follow-up measurement tool for assessment of the degree of symptomatic improvement.The authors declare no conflicts of interest.


Salvadora oleoides Decne.) fruit is well known for its nutritional and medicinal values. The current study analyzed the chemical composition of Salvadora oleoides fruit. Fresh Peelu fruits were harvested, and physicochemical properties, proximate composition, macro- and micronutrients, and phytochemical properties were determined. Moreover, ethanol and methanol fruit extract was analyzed for physicochemical properties. The Peelu fruit seemed to be a potential source of essential macro- ((nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)) and micronutrients (zinc (Zn), manganese (Mn), iron (Fe), and copper (Cu)). The fruit had significant biochemical properties , total acidity (TA), and TSS : TA ratio) with appreciable moisture, crude fiber, and ash contents. The fruit extracts demonstrated significantly higher antioxidants and phenolics, ascorbic acid contents, and carotenoids. Phytochemical screening of fruit revealed the presence of coumarins, flavonoids, phlobatannins, tannins, and terpenoids. Physicochemical and sensory evaluation of extracts indicated its potential for further in vivo study trials. The Peelu fruit was found to be a good source of mineral nutrients, proximate contents, vitamins (ascorbic acid and carotenoid), phytochemicals , and pharmaceutically important metabolites that can be used as functional drink.The Peelu ( Salvadora oleoides Decne.) is a member of the Salvedoraceae family comprising of three genera, i.e., Azima, Dobera, and Salvadora. Ten (10) species of this family are mainly distributed in tropical and subtropical regions of Africa and Asia [S. persica and S. oleoides [S. alii has also been reported and described from Sindh province of the country [Salvadora species can grow in saline, arid, and semiarid regions [Peelu  by considering size uniformity and free from disease infestation. The characterization of nutritional composition was performed at Postharvest Laboratory, MNS-University of Agriculture, Multan, Pakistan under 30 ± 5°C temperature and 60-65% and relative humidity. Furthermore, proximate composition, mineral quantification, and phytochemical composition were explored. Hundred (100) grams of Peelu fruits were used as an experimental unit and replicated four times.The Peelu fruits were harvested from a commercial field , potassium sulfate (K2SO4), and ferrous sulfate (Fe2SO4) at the ratio of 5 : 94 : 1, and 25-30 mL sulfuric acid was added. The flask was placed on a burner and heated until 2-3 mL of the solution was left. The sample was then diluted up to 50 mL. Afterwards, 10 mL of the sample and 10 mL of NaOH (40%) were added in the reaction flask of the Kjeldahl apparatus. The 10 mL of boric acid (4%) was added to the receiving beaker of the Kjeldahl apparatus. The distillation continued until the volume of boric acid reached 20-22 mL and turned yellow. It was titrated with 0.1 N H2SO4 and N was computed according to equation  till simmering (70-80°C). Afterwards, the sample was filtered with a linen cloth in the fluted funnel. In the second step, the acid-treated sample was subjected to alkaline medium of NaOH (1.25%) followed by heating, simmering, and filtration. In the third step, the treated sample was subjected to moisture removal according to the standard protocol. The weight of the sample obtained after moisture removal was denoted as The ash (%) content was estimated by Muffle Furnace . The 3 g dry sample was incinerated after charring in Muffle Furnace according to Method 08-01 AACC  at 550°CThe fresh fruits were washed with distilled water and dried in a hot air oven at 105°C till constant weight. The dried fruits were ground proceeded by acid digestion. The resultant solution was used to determine various mineral nutrients as nitrogen (N) by the micro-Kjeldahl method, phosphorus (P) by vanadomolybdo analysis, and potassium (K) by flame photometer as elaborated by Ullah et al. . Additioμg·100 g−1·DW. Total carotenoids were determined and expressed as μg·g−1 of β-carotene equivalent [−1. The antioxidant activity was measured through 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) by spectrophotometer in ELX800 Microplate Reader  as outlined by Mimica-Dukić et al. [The ascorbic acid was determined by following the protocol of Kumar et al.  using truivalent . Total puivalent . The conć et al.  and was where AB is the absorption of a blank sample and AA is the absorbance of the tested sample.3 (0.1%) was added to the filtrate and a color change was noticed as blue-black or brownish-green as evidence of tannin presence. For the determination of coumarins, a fruit sample (300 g) was added to a test tube covered with a filter paper soaked in NaOH (1 N). The sample containing the test tube was placed in a hot water bath. The filter paper was removed and the sample was observed under UV light. The appearance of a yellow color indicated the presence of coumarins. In a small test tube, 300 mg of fruit was covered with filter paper moistened with 1 N NaOH. The test tube was placed in a boiling water bath for few minutes. After removing the filter paper, it was examined under UV light, yellow fluorescence indicated the presence of coumarins. The Peelu fruit was boiled in diluted hydrochloric acid (1%). The resultant deposition of red-colored precipitates indicated the occurrence of phlobatannins. The fruit sample (50 mg) was added in 100 mL of distilled water to obtain a filtrate. In the prepared filtrate, 5 mL of dilute ammonia solution plus few drops of concentrated H2SO4. The flavonoids were confirmed by the appearance of the yellow color [−1, mixed with 2 mL chloroform and 3 mL of concentrated H2SO4. The reddish-brown color indicated the presence of terpenoids as described by Saeed et al. [The fruit pulp (50 mg) was boiled in distilled water (20 mL) followed by filtration. After boiling, FeClow color . The detd et al. .The total soluble solids (TSS) were measured by a digital refractometer  and expressed as °Brix. The total acidity (TA) was assessed via the acid-base titration method. The juice sample was titrated against NaOH (0.1 N) by using a phenolphthalein indicator. The pH of juice was estimated through a digital pH meter .T0 (placebo), T1 , and T2 .Functional drink (Nutra-1) was developed by using various ingredients include citric acid, carboxymethylcellulose (CMC), aspartame, sodium benzoate, flavor, and food-grade color along with propolis extract of following treatments at the dose rate of 400 mg/500 mL of the functional drink. The treatment planning of drinks was designed as The functional drinks were analyzed for their physicochemical attributes including TSS, TA, and pH. Sensory evaluation  was done by using the 9-point hedonic scale  as specified by Meilgaard et al.  during 1The experiment was carried out according to completely randomized design (CRD) with four replications. The data regarding different studied attributes were analyzed by descriptive statistics and presented. Statistix® statistical software was used for the data analysis . The datSalvadora persica had significant amounts of moisture (73.8 ± 5.0%), protein (5.9 ± 0.6%), ash (9.3 ± 0.6%), and fiber (10.4 ± 1.9%) which was slightly higher than our results. Variation in proximate composition of the Peelu fruit may be due to different species, climatic conditions, soil type, or growing environment. The studied fruit was harvested from Southern Punjab, Pakistan, which had comparative lesser relative humidity which can be a major contributing factor in the low moisture content of fruit under investigation as compared to the results of Kumari et al. [The compositional profiling of fruit elucidated that it contained significant moisture content (63.2%), crude fat (7.1%), crude protein (4.2%), crude fiber (8.7%), and ash (7.8%) as indicated in i et al. .−1·DW, phosphorus (P) 85.0 mg·100 g−1·DW, potassium (K) 631.5 mg·100 g−1·DW, calcium (Ca) 507.5 mg·100 g−1·DW, and magnesium (Mg) 113.7 mg·100 g−1·DW  is a crucial mineral of the human diet that facilitates various biological functions of the body as differentiation, neuronal activities, muscles contraction, and immune responses that leads to apoptosis [+ which is important for various biological processes [The mineral profiling revealed the presence of a sufficient amount of macronutrients, i.e., nitrogen (N) 209.3 mg·100 g0 g−1·DW . Our resuineense . The K ia+ ratio . Calciumpoptosis . The Peepoptosis . The Peerocesses . The Mg rocesses . Our resrocesses –31. Henc−1·DW, respectively  has been reported for S. persica fruit [Carotenoids are a good source of antioxidants and considered fat-soluble pigments in plants. Moreover, color development is also dependent on carotenoids synthesis during maturity and ripening of the fruits. The carotenoid content in Peelu were 6.5 ± 1.4 ·100 g−1 . Caroten·100 g−1 . Higher ca fruit .Averrhoa carambola, Morus alba, and Syzygium cumini [Grewia subinaequalis, and lesser than Prunus persica and Prunus armeniaca [Data regarding biochemical profiling of the Peelu fruit indicated that TSS in fruit juice were 8.9 ± 1.3°Brix . The perm cumini , 42, comrmeniaca  suggestiData regarding various physicochemical attributes (pH and acidity) showed a significant effect of treatments and storage duration on functional drinks prepared from Peelu. However, the interaction between treatments and storage duration was nonsignificant for pH and acidity of the functional drinks. Moreover, a nonsignificant effect of treatment and storage period, as well as their interaction, was recorded for TSS .The highest concentration of TSS (1.6°Brix) was observed in functional drink with ethanol extract of Peelu as compared to placebo and functional drink with methanol extract of Peelu (1.58°Brix and 1.58°Brix). As far as the storage period was concerned, an increasing trend was observed for TSS for the storage period. The highest concentration of TA (1.67%) was obtained after 30 days of storage period as compared to 0 days (1.5%) . An incrPreviously, the average TSS documented was about 10.4°Brix in a diet drink of apple prepared with artificial sweetener. Similarly, the results of the current investigation are in corroboration with González-Molina et al. , who repT2 (6.51 ± 0.07) as compared to T1 and T0  (T1) showed a better flavor score (7.14), as compared to drinks prepared with methanol extract (7.09) and drink without extract (6.94). Average values documented for sweetness and sourness at 0-day were 7.20 and 7.68, 7.60 and 7.79, and 7.36 and 7.5, respectively, whereas mean values for sweetness and sourness after 30 days of storage were 7.08 and 7.38, 7.22 and 7.5, and 7.18 and 7.39 for T0, T1, and T2 treated drink accordingly. The overall acceptability means a score of functional drinks was recorded about 7.77 during storage.Treatments, storage period, and interaction were nonsignificant for sensory parameters of functional drinks. Color of the product/physical appearance is considered a prime factor for selection/rejection of the product by the consumer. With the increase in storage duration color score of the functional drinks was significantly reduced. The color score of the drinks was more at 0-day (6.6), while rescued to 6.1 after 30 days of storage. Among the treatment, the highest color score was obtained from ctively) . The flactively) . As far Our results were in line with Ahmed  who repoin vivo study. Conclusively, Peelu exhibit high nutritional potential and could be included in the food chain with value addition.The Peelu fruit is a good source of macro- and micronutrients and has a sufficient quantity of biochemical contents . Like other fruits, the juice of the Peelu fruit is a rich source of phenolic and antioxidants, including ascorbic acid and caroteniods. However, the sensory attributes of the functional drinks were in corroboration with control representing its potential for further


This pilot study aimed to assess the practicability and effectiveness of percutaneous nephrolithotomy (PCNL) with vacuum-assisted nephrostomy sheaths for patients under modified local anesthesia (m-LA).PCNL with a vacuum-assisted nephrostomy sheath under m-LA was performed in 83 patients between November 2020 and May 2021. An 18F or 20F ClearPetra Nephrostomy Sheath connected vacuum aspiration was used in surgery to keep low pressure in the renal pelvis. For LA, lidocaine and ropivacaine hydrochloride were 1:1 mixed and instilled under ultrasound guidance through the percutaneous nephrolithotomy channel directed toward the design calix. Demographic characteristics, stone characteristics, visual analogue scale (VAS) score, vital signs, operation time, complications, and stone clear rate were recorded and analyzed.All operations were completed. The mean VAS score was 3.9 ± 1.0. The mean operation time was 55.1 ± 23.6 min. The changes for systolic blood pressure, diastolic blood pressure, and heart rate were 3 ± 21 mmHg, 1 ± 14 mmHg, and −6 ± 14 beats/min, respectively. The change for hemoglobin was −10.7 ± 10.9 g/L. The change for C-reactive protein was 5.39 ± 43.1 mg/L. The total stone-free rate was 69.9% (93.8% for simple stones and 54.9% for complex stones).Performing PCNL with vacuum-assisted nephrostomy sheaths under modified local anesthesia under ultrasound guidance was found to be strongly practical and effective. Percutaneous nephrolithotomy (PCNL) is a minimally invasive treatment for some indicated renal stones and upper ureteral calculi. Guidelines recommend PCNL as the preferential treatment modality for all renal stones >20 mm owing to the increased stone-free rate and reduced re-treatment sessions . Mini trGenerally, PCNL is performed under general anesthesia (GA); however, comorbidities such as coronary heart disease and chronic obstructive pulmonary disease increase the risk of anesthesia . GA also2, and (5) local anesthesia drug allergies. Patients routinely received urinalysis, urine culture, routine blood test, serum creatinine, and electrolyte test. Upper urinary calculi were diagnosed by ultrasound, kidney, ureter, bladder x-ray, and CT scans. Simple stones refer to single renal or ureteral stones, and complex stones refer to multiple stones, except in patients with anatomical abnormalities. In addition, complex stone included staghorn calculi.Between November 2020 and May 2021, 83 cases that met the criteria were enrolled in the study. Inclusion criteria were (1) renal stone larger than 2 cm, (2) upper ureteral calculi larger than 1 cm, and (3) the patient was willing to receive local anesthesia. Exclusion criteria were (1) concomitant middle or lower ureteral calculi, (2) stone secondary to ureteral stricture, (3) second-stage operation, (4) BMI >30 kg/mAntibiotics including latamoxef or etimicin were administered to patients 30 min before the operation. The patient was in the prone position, and precise local anesthesia in the percutaneous nephrolithotomy channel was performed under ultrasonic guidance; the key points were (1) 1% lidocaine 10 ml and 1% ropivacaine hydrochloride 10 ml were mixed for local injection, lidocaine had rapid onset, but short effectiveness, ropivacaine had a long-term effect, and the mixture giving full play of merits. (2) A 20GA 1.88IN venous indwelling needle [Singapore Becton Dickinson Medical (S) Pte Limited] with a 20 ml syringe was used for drugs injection, with the fine needle relieving the injection pain. (3) The injection of anesthetic was along the percutaneous nephrolithotomy channel under ultrasonic guidance , and anesthesia reached the renal fascia. (4) Using an 18F or 20F ClearPetra Nephrostomy Sheath  in surgery to keep low pressure in the renal pelvis can avoid distending pain in the kidney. (5) No ureteral catheter was inserted in any of the patients; it can avoid the pain caused by inserting a ureteral catheter, and furosemide (20 mg) would be given by injection into a vein to form artificial hydronephrosis, which was convenient for puncture.Other procedures were routinely performed as previously reported , and somThe demographic characteristics that included sex, age, size of the stone, location of the stone , and comorbidity  of the patients were recorded. The surgery data that included the tract number, duration of the operation, systolic blood pressure (SBP), heart rate (HR), and VAS scores were evaluated. Postoperative evaluations were drop in hemoglobin, fever, perirenal hematoma, pleural effusion, residual stone, length of hospital stay, medical cost, and Barthel index. An x-ray of KUB was taken after one day after surgery. We defined stone-free rate as the radiological absence of stone in asymptomatic patients with residual stone fragments <3 mm . Blood p2. Patients with ASA score I–III were 51, 17, and 15, respectively. The mean hemoglobin was 130 ± 20 g/L, and the mean C-reactive protein (CRP) was 15.3 ± 32.9 mg/L. A total of 32 cases were simple stones, and 51 cases were complex stones. The size of the stone was 19.4 ± 8.1 mm.The characteristics of included patients are listed in The mean VAS score was 3.9 ± 1.0. The mean operation time was 55.1 ± 23.6 min. The postoperative hospital stay was 4.1 ± 1.2 days. The changes for SBP, DBP, and heart rate were 3 ± 21 mmHg, 1 ± 14 mmHg, and −6 ± 14 beats/min, respectively. The change for hemoglobin was −10.7 ± 10.9 g/L. The change for C-reactive protein was 5.39 ± 43.1 mg/L. There were 64, 17, and 2 patients who received 1, 2, and 3 tracts, respectively. It was noted that 55, 26, and 2 patients retained 0, 1, and 2 nephrostomy tubes. One patient received a blood transfusion and renal arterial embolization, and there was no Clavien grade IV–V complications. The total stone-free rate was 69.9%, which was 93.8% for simple stones and 54.9% for complex stones. The main clinical results of the patients are listed in Subgroup analysis was performed according to ASA status; the patient's perioperative status is recorded in Local anesthesia broadens the indications for PCNL. It hedged risks caused by deformity (chest and spine), and poor heart and lung function; meanwhile, the renal function may be maximum preserved , 14. OthPrevious reports about LA in PCNL were improvable , 18, 19.In our study, m-LA in PCNL achieved satisfactory results. All operations were completed successfully, and none of m-LA was converted to GA; we continuously communicated with patients during the operation to monitor pain, and local anesthetics can be added for analgesia during the operation. For some stones with large angles, the stone clearing rate may be sacrificed to complete the operation. No serious cardiovascular incident occurred, no death occurred, and postoperative rehabilitation was satisfactory. The pain was evaluated using a VAS score. The mean VAS score was 3.9 ± 1.0, and the pain levels during the operation were mild and could be tolerated, which was similar to previous reports , 10. OurOther parameters also evaluated the effect of surgery. Operation times were 55.1 ± 23.6 min. One patient felt discomfort and discontinued surgery because of keeping a long-time prone position; due to the lack of preoperative preparation of jejunitas, the patient was not converted to GA. He received a II-stage operation under m-LA on the second day, and the stone was removed. The total stone-free rate was 69.9%, which was 93.8% for simple stones and 54.9% for complex stones. It seems that the total stone-free rate was inferior to what was previously reported ; the reaP = 0.309). The swing of the sheath was restricted and increased pain. Second, patients with renal stones in the polar calix were not included for the same reason. Third, we recommended two-step puncture techniques for PCNL (Our study did not simply duplicate the work of our predecessors, and the key points for our study are listed as follows. First, although obesity was not a contraindication, obese patients were not encouraged for PCNL under m-LA, although data indicated that BMI was not associated with VAS score (the Pearson coefficient was 0.113, for PCNL . It reduThe main limitation of our study was the limited population. Some previous reports about LA contained hundreds or thousands of patients , 9, and Performing PCNL with a vacuum-assisted nephrostomy sheath under modified local anesthesia under ultrasound guidance was found to be strongly practical and effective.