Chenopodium botrys from Bulgaria. The polyphenols were fractionated with solvents of varying polarity . The fractions were analyzed by HPLC-PDA and UHPLC-MS. The ethyl acetate fraction contained mono- and di-glycosides of quercetin, di-glycosides of kaempferol, and isorhamnetin and monoglycosides of hispidulin and jaceosidine. We found quercetin triglycosides in the butanol fraction. The ethyl acetate and butanol fractions contained 168.82 mg/g Extr and 67.21 mg/g Extr of quercetin glycosides, respectively. The main components of the polyphenolic complex in C. botrys were 6-methoxyflavones (355.47 mg/g Extr), which were found in the chloroform fraction. The flavonoids pectolinarigenin, demethylnobiletin, and isosinensetin, and the glycosides of quercetin , kaempferol, isorhamnetin, hispidiulin, and jaceosidine, were discovered and reported in Chenopodium botrys for the first time. We used in vitro methods to assess the biological activity against oxidative stress (hydrogen peroxide scavenging activity (HPSA) and hydroxyl radical scavenging activity (HRSA)), nitrosative stress (nitric oxide scavenging activity (NOSA)), anti-inflammatory activity (IAD inhibition), and anti-tryptic activity (ATA). Quercetin mono- and di-glycosides exhibited greater HPSA and HRSA , while 6-methoxyflavones had a greater NOSA (IC50 = 146.59 µg/mL). The same components showed the highest ATA (IC50 ranging from 116.23 to 202.44 µg/mL).In the present work, we have investigated the polyphenolic composition of  Chenopodium (Family Chenopodiaceae) includes more than 200 species native to much of Europe, Asia, India, China and the Americas −. This ion loses a radical –CHO by producing [M–H–CH3–CHO]− or [M–2H–CH3–CO]− ions , and the resulting ion is –CH3 0.2A−, and the second pathway involves the cleavage of the C2–C1′ bond between rings C and B, yielding an ion [M–H–CH3–HCO–ring B]−. Which one is the most likely to be seen is difficult to determine, since the elemental composition of the two ions is the same, i.e., isomeric ions  in ring B. Therefore, in nepetin and hispidulin, ion m/z 163 is of low intensity due to the presence of OH groups. The presence of OCH3 groups increases the intensity of ion m/z 163. The same ion in jaceosidine, eupatilin and pectolinarigenin loses a neutral molecule CO, and an ion m/z 135 [–CH3 0.2A−–CO] or [M–H–CH3–HCO–ring B–CO]− is obtained. The ions m/z 164, m/z 163, m/z 136 and m/z 65 are specific to the fragmentation of nepetin in negative mode. They contribute to the identification of 6-MFs being characteristic for the substitution in the A ring. For 6-MFs, these ions characterize the substituents in the A ring. Significant differences in the fragmentation of 6-MFs appear only in the substitution in the B ring. The mass spectra of [M − H]− ions of 6-MFs are summarized in The intensity of ion R = 25.80) and an isomer of sinensetin (tR = 26.73)  demethylnobiletin (t= 26.73) A. Previoid forms . The samCO–CO2]− .3 groups, ring A regroups in a stable quinoid form (3 groups and the subsequent RDA cyclization (1/3) of ring C leads to the formation of ion m/z 168 ([–3CH3 1.3A+]), which systematically loses neutral molecules CO and leads to the receiving of the ions m/z 140 ([–3CH3 1.3A+–CO]) and m/z 112 ([–3CH3 1.3A+–2CO])  .m/z 147 ([–CH3 1.3B+]), which also loses CO, and the product fragment (m/z 119) corresponds to ([–CH3 1.3B+–CO])  . In the 3B+–CO]) . In this2. RDA cyclization yields the ions m/z 132 and m/z 133  were identified that have not been reported before now.As a consequence of this research, we can infer that in C. botrys is the least researched member of the Chenopodiaceae family, particularly because of its polyphenol content. Metabolism in various species of Chenopodiaceae is known to synthesize mainly glycosides of quercetin, kaempferol and isorhamnetin. The carbohydrates that are involved in the structure of glycosides are glucose, galactose, rhamnose and apiose −) that is related with the loss of the disaccharide rutinose (308 Da). In addition, we detected an ion with m/z 463, which, following fragmentation, produces an ion with m/z 301, but one carbohydrate unit of 162 Da  is lost. The ESI-MS/MS experiment and fragmentation of ion 301 showed the formation of ions with m/z 179 [1.2A−], m/z 151 [1.2A−–CO] and m/z 107 [1.2A−–CO–CO2], which are characteristic of the aglycone quercetin -β-D-glucopyranoside. In the MS/MS experiment, the molecular ion m/z 757 lost a pentose unit of 132 Da, and an ion m/z 625 ([M–H–132]−) was obtained. The same ion lost 162 Da, and the resulting ion corresponds to [M-H-132-162]− with m/z 463, which lost another 162 Da, and the resulting ion 301 fragmented into the characteristic quercetin ions m/z 179 [1.2A−], m/z 151 [1.2A−–CO] and m/z 107 [1.2A−–CO–CO2]. The derivatives of the aglycone quercetin were discovered to be peaks 2–4 . Furthermore, the glycoside is acylated with malonic acid. During the MS/MS experiment, the molecular ion sequentially lost two units of rhamnose (2 × 146 Da) and malonic acid, yielding a m/z 463 ion. The resulting ion from the MS/MS experiment was quercetin, and its specific fragment ions were m/z 179 [1.2A−], m/z 151 [1.2A−–CO] and m/z 107 [1.2A−–CO–CO2]. Peak 5 with tR 13.77 min . The molecular ion lost an acyl group, yielding the ion m/z 841 ([M–H–acyl]−). This indicates that the molecular ion was acylated with acetic and malonic acids. The same ion was structurally identical to the one before it, and fragments in the same way.Peak 2.11 min  was ideneaks 2–4 . All thr3.77 min  is molec8, 9, 15) in the polyphenol composition. Peak 8 (tR 12.31 min) was identified as Quercetin-O-(acyl)-hexoside-hexoside with molecular ion m/z 667 , which is an ion with two hexoses in its structure. The resulting ion gradually lost 162 Da, generating the ions m/z 463 ([M–H–acyl–162]−) and m/z 301 ([M–H–acyl–162–162]−). Fragment ions m/z 179 [1.2A−], m/z 151 [1.2A−–CO] and m/z 107 [1.2A−–CO–CO2] prove that the molecular ion m/z 667 is a quercetin glycoside. Peaks 9 and 15 are distinguished by the same molecular ion, m/z 709. We identified them as quercetin diacyl glycosides using MS/MS data. The molecular ion lost an acyl group, yielding an ion m/z 667 ([M–H–acyl]−), which then lost a neutral molecule CO, yielding an ion m/z 639 ([M–H–acyl–CO]−). Furthermore, the molecular ion lost 204 Da (acyl+hexose) and produced ion m/z 505 ([M–H–acyl–hexose]−). The same ion lost another acyl group, and the resulting ion was m/z 463 ([M–H–2acyl–hexose]−). The resulting ion from the MS/MS experiment was quercetin, and its characteristic ions were m/z 179 [1.2A−], m/z 151 [1.2A−–CO] and m/z 107 [1.2A−–CO–CO2].It lost an acyl group and produced the ion 18 (tR 14.85 min) (O-(acyl)-hexoside-O-(acyl)-hexoside with molecular ion m/z 693. It lost 204 Da immediately, yielding the m/z 489 ion, and then lost an acyl group, yielding the m/z 447 ion. The same ion lost 162 Da and produced an ion with m/z 285. Under ESI-MS/MS conditions, it fragmented to the ions m/z 151 [1.3A−], m/z 125 [1.4A−] and m/z 107 [1.3A−-CO2].Peak .85 min)  was iden10, 13 and 16  lost 162 Da and produced ion m/z 461 [M–H–162]−. The same ion lost rhamnose moiety, and the ion 315 [M–H–162–146]− corresponds to isorhamnetin. Glycoside 13 (Peak 13) successively lost 146 Da; the product ion 477 corresponded to [M–H–146]−, which then lost another 146 Da. The resulting ion [M–H–146–162]− was isorhamnetin. The characteristic ions for isorhamnetin are m/z 151[1.3A−], m/z 137 [–CH3 0.2B]−, m/z 125 [1.4A−] and m/z 107 [1.3A−–CO2].Peaks 3 and 16 , Table 316 was identified as Isorhamnetin 3-O-glucopyranoside with molecular ion m/z 477. The fragmented ion was 315. The characteristic ions for the same flavonol are m/z 151[1.3A−], m/z 137 [–CH3 0.2B]−, m/z 125 [1.4A−] and m/z 107 [1.3A−–CO2].Peak 14 and 19  with a stable quinoid form was produced during ESI-MS/MS. Hispidulin as 6-MF generated the characteristic ions m/z 164 [CH31.3A−–H], m/z 163 [CH30.2A−], m/z 136 [CH31.3A−–CO] and m/z 117 [1.3B−].Peak 17 , Table 32+ and Fe2+ ions take part as cofactors in a variety of enzymes and physiological processes in the human body. However, when they are present in their free state, they have a harmful impact, because Cu2+ ions accelerate the oxidation of ascorbic acid, which produces ROS such superoxide radicals (O2•−) and H2O2. Additionally, the Fenton reaction causes the Cu2+ and Fe2+ ions to interact with H2O2 and produce hydroxyl radicals (•OH) [The extremely important Culs (•OH) . Proteinls (•OH) .ROS. The most common cause of inflammation is the generation of superoxide anion radicals, which is linked to the creation of other ROS species such as H2O2. It also participates in reduction decomposition processes (known as the Haber–Weiss reaction) and organic hydroperoxides’ ROOH [In addition to the oxidative stress mechanisms, the inflammatory process promotes and increases the generation of es’ ROOH .As a result, the study of substances with high scavenging ability for oxygen free radicals is a relevant and essential topic of research.The antioxidant activity of the various fractions was compared to natural substances with recognized antioxidant characteristics, such as ascorbic acid and quercetin. The antioxidant activity values of the obtained fractions ranged from 39.18 µg/mL to 978.07 µg/mL A.HPSA of the fractions decreases in the following order: EtOAc > CHCl3 > n-Hexan > BuOH.Except for the ethyl acetate fraction, the produced fractions had low HPSA compared to ascorbic acid (24.84 g/mL) and quercetin (69.25 g/mL). The ethyl acetate fraction had much stronger antioxidant activity than the other fractions, and was almost twice as active as the quercetin standard A. The pr•OH), which is produced by ROS. It is produced by one-electron reductions in molecular oxygen (O2) in cellular metabolism, and is the primary cause of cytotoxicity in aerobic species, including humans [The most potent oxidant is the hydroxyl radical   B, while radicals B. This i3 groups . The O-methylation of OH groups in ring B leads to a decrease in HRSA [HRSA compared to quercetin mono- and di-glycosides. This is due to the fact that the O-glycolation of the C-3 OH group and the increase in carbohydrate units leads to a decrease in antioxidant capacity [6-Methoxy flavones differ in the functional groups in ring B. They contain OH groups (hispidulin and nepetin) and OCH in HRSA ,43. Quercapacity ,41. The L-arginine by NO synthases (NOS), which are NADPH-dependent enzymes. NO is a bioregulatory molecule that is necessary for a variety of physiological activities, such as brain signal transmission, immunological response, vasodilation, blood pressure control, etc. By interacting with the NO molecule, the superoxide ion (O2−) generates intermediate products such as NO2, N2O4 and N3O4, and stable compounds such as nitrate and nitrite. It is the formation of reactive nitrogen species (RNS) known as nitrosative stress that is responsible for several pathological conditions, including cancer [50 64.82 µg/mL), the fractions had a lower NOSA. This demonstrates that the flavonoid core is more effective in quenching NO radicals. The IC50 values for the different fractions varied from 146.59 to 227.12 µg/mL  was observed in ethyl acetate, butanol, and chloroform fractions  correlate very well with the total phenolic content (−0.6916 and −0.9900) and with total tannins (−0.7183 and −0.9941) . In geneTPC, TTS, and the concentration of quercetin glycosides. They bind to the polar groups of the allosteric centers of albumin and the active center of trypsin because they are more polar than 6-methoxy flavones. The structure of biologically active components influences in vitro biological activity. There was only a modest relationship between 6-methoxy flavones and IAD and ATA. However, this aspect should not be overlooked, because there is evidence that methoxy flavones have anticancer and other properties. The in vitro techniques IAD and ATA showed a moderate correlation dependency (r = 0.6362). This can be explained by the fact that the two approaches serve the same purpose, which is to keep the albumin molecule intact. Albumin is protected against denaturation (IAD) in the first example. Polyphenols bind to albumin allosterically in this case. In the second situation, it is shielded from the action of the enzyme trypsin (ATA), since polyphenols inhibit the enzyme. These two mechanisms prevent plasma viscosity from changing during inflammation and the progression of arthritic disorders.The results of the study demonstrate that there is a strong relationship between in vitro biological activity, For HPLC analysis, chromatographic-grade methanol was used . A Millipore purifier  was used to obtain water for HPLC. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid, potassium chloride, quercetin, hispiduline, ascorbic acid, ibuprofen, ketoprofen, sodium chloride, hydrogen peroxide, trypsin, Tris-HCl buffer, sodium nitroprusside, sodium salicylate, ferrous sulfate, sulfanilamide, naphthylethylenediamine dihydrochloride and perchloric acid were purchased from Sigma-Aldrich, Taufkirchen, Germany. Human albumin 20%—BB, 200 g/L was ordered from BB-NCIPD Ltd., Sofia, Bulgaria.C. botrys were collected from regions of Plovdiv, village Rogosh. The plants were gathered in August consecutively, at the full flowering stage. The plant material was identified by means of voucher specimens (05269 and 05271), which were deposited in the herbarium of Agricultural University—Plovdiv by Assoc. Prof. Koicho Koev, Ph.D. . The aerial parts of the plants were dried at room temperature in the absence of direct sunlight. The dried material was further ground to powder, packed into multilayer paper bags, and stored in a dark room at ambient temperature.The specimens of The fractionation of polyphenols with different in polarity solvents was described by Bojilov and co-workers . Finely,2CO3) solution. The tested samples were incubated for 2 h in dark at room temperature. The absorbance of the samples was measured at 765 nm with a spectrophotometer , using a blank sample. The total phenolic content was expressed as mg gallic acid equivalent per gram extract (mg GAE/g Extr) based on the calibration curve.The total phenolic content in crude extracts was determined with the colorimetric method using Folin–Ciocalteu’s reagent  with sliTTC) in the different fractions was determined spectrophotometrically using the Folin–Ciocalteu method [2CO3) and the flask is topped up with distilled water up to the mark. Test samples were mixed and incubated for 30 min in the dark at room temperature. The absorbance of the samples was measured at 700 nm with a spectrophotometer , against a blank. Total tannin content is expressed as mg tannic acid equivalent per gram of extract (mg TAE/g Extr).The total tannin content . The analysis of the samples was carried out as follows: in test tubes, 0.6 mL H2O2 (43 mM), 1 mL sample/standard with different concentrations (20–1000 µg/mL), and 2.4 mL potassium phosphate buffer solution were mixed. The mixture was stirred and incubated in the dark for 10 min at 37 °C. Absorbance was measured at 230 nm with a spectrophotometer  against a blank solution containing phosphate buffer and H2O2 without the sample. Ascorbic acid and quercetin were used as standards. The percentage HPSA of the samples was evaluated by comparing with a blank sample and calculated using the following formula:blankA is the absorbance of the blank sample, CSA is the absorbance of the control sample, and TSA is the absorbance of the test sample.The Manolov et al. approach was used to evaluate the capacity to scavenge hydrogen peroxide . A 43 mMC. botrys were determined according to the method described by Guo [4 (1.5 mM), 1 mL sample/standard with different concentrations (20–1000 µg/mL), and 0.7 mL H2O2 (6 mM). They were mixed immediately, and then the reaction tubes were put in a 37 °C water bath for 1 h, and the absorbance of the mixture was recorded at 510 nm against a blank. We used standards with proven high antioxidant activity, such as ascorbic acid and quercetin. The hydroxyl radical scavenging ability was calculated as follows:blankA is the absorbance without samples and sampleA is the absorbance in the presence of the samples of different fractions from C. botrys.Hydroxyl radical scavenging activities of different fractions of d by Guo . The scablankA is the absorbance of a blank and sampleA is the absorbance of the test sample.NO inhibition was measured according to the method of Marcocci . Sodium IAD). The analysis was performed according to the Manolov method [IAD) was calculated against control. The control sample was albumin with the same concentration dissolved in distilled water.The in vitro anti-inflammatory activity was assessed via the inhibition of albumin denaturation (v method  with minv/v) was added. The mixture was incubated for an additional 20 min. To the mixture, 2 mL of 70% perchloric acid was added for termination of the reaction. The cloudy suspension was cooled and centrifuged at 5000 rpm for 20 min. The absorbance of the supernatant was measured at 280 nm with a spectrophotometer  against a control solution. The control solution was the sample/standard in methanol with different concentrations. Ibuprofen and ketoprofen were used as standards. The analysis was performed three times. The percentage of antitryptic activity (ATA) of the samples was evaluated by comparing with a blank sample. The blank sample was prepared as the test sample but with a small exception—perchloric acid was added before albumin.blankA is the absorbance of the blank sample, CSA is the absorbance of the control solution (test sample in different concentrations) and TSA is the absorbance of the test samples.This method is known also as in vitro anti-arthritic activity. The analysis was performed according to the method of Oyedapo and Femurewa  with minp < 0.05. The mean IC50 value was estimated based on three replicates by interpolating the graphical dependence of activity on concentration.All the analyses were made in triplicates. Data were expressed as mean ± SD. A statistical SPSS 19.0 software package  was used. The level of significance was set at C. botrys, which was fractionated with differences in polarity solvents and analyzed by HPLC-PDA and UHPLC-MS in negative mode. The flavonoids pectolinarigenin, demethylnobiletin and isosinensetin, and glycosides of quercetin , kaempferol, isorhamnetin, hispidulin, and jaceosidine, were identified and reported in Chenopodium botrys for the first time. By using HPLC analysis, we have discovered that the primary components of the glycoside group are quercetin glycosides in a small amount, while the content of 6-methoxy flavones is the highest. The biological activity of the individual fractions was investigated. The applied approach allows us to distinguish the polyphenolic components via their biological activity.The aim of this study was to investigate the polyphenolic complex of HPSA, HRSA, NOSA, IAD, and ATA). We may derive valuable knowledge on the characteristics of the physiologically active components present in C. botrys from the experimental results. Aerobic organisms can be shielded from the negative effects of ROS by quercetin mono- and di-glycosides, which are potent oxidants. 6-Methoxy flavones, on the other hand, are more effective in scavenging NO radicals.In general, glycosides and 6-methoxy flavones have high biological activity  function ,59.


Rhipicephalus microplus is a major threat to the cattle industry worldwide. The intensive use of acaricides and repellents has resulted in drug resistance. Hence, effective and eco-friendly pest control alternatives are urgently needed, especially from natural plant resources. In this study, the acaricidal and repellent activities of nine herbs against the larvae and eggs of R. microplus were evaluated. The results showed that ethanol extracts of star anise (Illicium verum), chaulmoogra (Hydnocarpus anthelmintica), motherwart (Leonurus artemisia), mandarin orange peel , and stemona (Stemona sessilifolia) had good contact acaricidal activities of 100, 98, 94, 88 and 86%, respectively, whereas star anise and clove (Syzygium aromaticum) had good fumigant acaricidal activities of 98 and 96%, respectively. The hatching inhibition rate of star anise against R. microplus eggs was 100%. All nine herbs had good real-time repellent rates, but only castor bean and star anise had repellent effects after 48 h . This is the first report of the acaricidal and repellent activities of these medicinal herbs against R. microplus. Ethanol extracts of these herbs might be considered as potential alternatives to chemical acaricides for control of R. microplus. Rhipicephalus microplus is considered a major threat to the cattle industry, accounting for economic losses of approximately 30 billion US dollar annually worldwide, primarily due to decreased quality of meat, milk, and leather products , Babesia bigemina (causing babesiosis), and the obligate intracellular bacterium Anaplasma marginale (causing bovine anaplasmosis), resulting in increased morbidity and mortality of cattle  and star anise (Illicium verum) were purchased from Beijing Tong Ren Tang , shrubby sophora (Sophora flavescens) and motherwort (Leonurus cardiac) from Henan Zhangzhongjing Pharmacy , and mandarin orange peel , chaulmoogra (Hydnocarpus anthelmintica), stemona (Stemona sessilifolia), castor bean (Ricinus communis), and box bean (Entada phaseoloides) from online Wenzexuan Traditional Chinese Medicine Shop . As a positive control, 100 mg/mL ivermectin was obtained from Henan Anjin Biological Technology . Anhydrous ethanol  was acquired from Tianjin Fuyu Fine Chemical . As a negative control, 0.9% sodium chloride was purchased from Henan Kelun Pharmaceutical .Clove  for identification . Rhipicephalus microplus eggs (n = 10) in good condition were attached to white cardboard (15 × 25 mm) with double-sided tape, immersed in herbal extract for 1 min, removed, blotted dry, and transferred to a Petri dish without the herbal extract. Each sample was assayed in triplicate. NaCl solution was used as a blank control and 100 mg/mL ivermectin as a positive control. Treated eggs were incubated at a constant 28 °C and 90% RH. Eggs were observed for hatching every 24 h until the hatching rate of the blank control was > 80%, and then continuously observed for an additional 48 h.Ovicidal tests were performed with reference to ‘Pesticides guidelines for laboratory bioactivity tests. Part 5: The dipping test for insecticide ovicidal activity’  of uniform size were transferred into 2-mL centrifuge tubes. Upon observation of normal movements of ticks, the prepared cotton balls were added to the centrifuge tube and removed after 1 h. All treatments were repeated 5×. Distilled water was used as a blank control, 0.9% NaCl solution as a negative control, and ivermectin as a positive control. Treated ticks were incubated at a constant 28 °C and 90% RH. The mortality rate was recorded after 48 h. Larvae were considered dead if there was no response after continuous stimulation with a needle for 1 min.The fumigation method was performed with reference to Jian et al. . Cotton Rhipicephalus microplus larvae (n = 10), crawling normally, with limbs intact and of uniform size, were transferred to the Petri dishes containing herbal extract. After 1 h, the larvae were transferred to clean Petri dishes. Each treatment was replicated 5×. Distilled water was used as a blank control, 0.9% NaCl solution as a negative control, and 100 mg/mL ivermectin as a positive control. Petri dishes were incubated at a constant 28 °C and 90% RH. After 48 h, the mortality rate was recorded. Larvae were considered dead if there was no response after continuous stimulation with a needle for 1 min.A filter paper was placed on the bottom of a Petri dish (60 × 15 mm) and 1 mL of the herbal extract  was evenly dispersed, then exposed to air to dry naturally for 24 h. Rhipicephalus microplus larvae (n = 10) – crawling normally, limbs intact and uniform size – were transferred to a Petri dish containing 5 mL of the herbal extract . After being immersed for 1 min, larvae were transferred to a clean Petri dish. Each treatment was replicated 5×. Distilled water was used as a blank control, 0.9% NaCl solution as a negative control, and 100 mg/mL ivermectin as a positive control. The Petri dishes were incubated at a constant 28 °C and 90% RH. After 48 h, the mortality rate was recorded. Larvae were considered dead if there was no response after continuous stimulation with a needle for 1 min.Filter papers (5 cm diameter) were cut into two semicircles, which were soaked in the ethanol extracts  and 0.9% NaCl solution for 10 min, respectively, then exposed to air to dry naturally for 24 h, recombined, and placed in a larval tick repellent device  during an LC/MS experiment. In this mode, the acquisition software  continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. ESI source conditions were set as follows: sheath gas flow rate as 45 Arb, aux gas flow rate as 15 Arb, capillary temperature 320 ℃, full ms resolution as 70,000, MS/MS resolution as 17,500, collision energy as 20/40/60 eV in NCE model, ion spray voltage floating (ISVF) 3.8 or -3.1 kV in positive or negative modes, respectively. Identification of compounds was based on a comparison of mass spectra of each peak with those of authentic samples in a mass spectrum library. The percentages of compounds were calculated by the area normalization method.Based on the results of the ‘Impregnated filter paper’ method, alcoholic extracts of the herbs with the best acaricidal activity (star anise and chaulmoogra) were selected and sent to Biomarker Technologies  for compositional analysis. Based on the UHPLC-QE Orbitrap platform, the qualitative and quantitative compositional analysis of two ethanolic extract samples of the herb was performed. LC-MS/MS analyses were performed using an UHPLC system  with a UPLC HSS T3 column  coupled to Q Exactive .The mobile phase consisted of positive: 0.1% formic acid in water, and negative :5 mM ammonium acetate in water (A) and acetonitrile (B), carried with elution gradient as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 1% B; 12 min, 1% B, which was delivered at 0.5 mL minThe mortality rate (%) was calculated as [number of dead larvae / total number of larvae] × 100%. In addition, the corrected mortality rate (%) was calculated as [ / ] × 100%  and median repellent concentration (RC50) were calculated with the Probit algorithm. The coefficient of determination (R2) and regression equation were calculated by linear regression. Graphs were generated with GraphPad Prism v.8.0.2 .Mean (and SE) mortality rate, egg hatching inhibition rate, and repellent rate were calculated with IBM SPSS Statistics for Windows v.26.0 , analyzed with ANOVA and compared using least significant difference (LSD) tests (α = 0.05). The median lethal concentration , stemona (66.7%) and CRP (60%). For comparison, the rates of the positive, negative, and blank controls were 83.3, 3.3 and 0%, respectively  followed by clove (96%), whereas the other seven herbs had no acaricidal activity. For comparison, the acaricidal rates of the positive, negative, and blank controls were 100, 0, and 0%, respectively  was the most effective, followed by motherwort (0.247 and 0.549), CRP (0.312 and 0.836), star anise (0.367 and 0.912), and stemona (0.648 and 1.208) , motherwort 94%), CRP (88%), and stemona (86%). For comparison, the acaricidal rates of the positive, negative, and blank controls were 100, 0, and 0%, respectively  > CRP (66.4%) > motherwort (43.9%). The acaricidal rates of the other six ethanol extracts were < 20%. For comparison, the acaricidal rates of the positive, negative, and blank controls were 97.8, 0, and 0%, respectively (Table p < 0.01). Notably, repellent rates of the ethanol extracts of all nine herbs decreased with time, but to different degrees. After 48 h, only castor bean (81.3%), star anise (79.6%), chaulmoogra (66.3%), and motherwort (66.3%) maintained repellency rates > 50%. As shown in Fig. 50 = 0.562 g/mL), whereas the RC50 values of the ethanol extract of the other eight herbs were > 0.7 g/mL.At 1 g/mL, ethanolic extracts of all nine herbs showed high repellency at 0 h , chaulmoogra (98%), motherwort (94%), CRP (88%), and stemona (86%). This study is the first to report acaricidal activities of ethanol extracts of star anise, chaulmoogra, motherwort, and CRP against n et al.  in an inDermanyssus gallinae) with a LD50 of 0.159 g/mL. Star anise essential oil has a relatively high content of trans-anisidine and, thus, better insecticidal activities against the Indian meal moth (Plodia interpunctella) and the litter beetle (Alphitobius diaperinus) with insecticidal rates of > 90% at lower doses , impregnated filter paper method (100%), and ovicidal test (100%).Star anise has been reported as a broad-spectrum insecticide primarily because of the presence of trans-anethole, which can be used directly as an insecticide or synergistically as an adjunct to other insecticides , stemocurtisinol (LC50 = 39 ppm), and oxyprotostemonine (LC50 = 4 ppm) from the roots of Stemona curtisii had good larvicidal activity against the mosquito Anopheles minimus, which is the primary vector of malaria in India. Brem et al.  and D. gallinae. The main active ingredients of CRP include volatile oils and the citrus flavonoids hesperidin, neohesperidin, and naringenin that not only act alone, but also synergistically with other drugs  had a synergistic acaricidal effect against R. microplus.Motherwort is commonly used for treatment of epilepsy, menstrual disorders, arterial diseases, and gastrointestinal disorders. The antioxidant and anti-inflammatory effects of motherwort and CRP have been linked to various flavonoids, terpenes, and alkaloids , acetyl eugenol (7.3%), and ß-caryophyllene (9%). At 3 µg/m2, the ethanol extract of clove was 100% effective against D. gallinae  showed high acaricidal activity against With the dip method, only chaulmoogra (92.8%) demonstrated good acaricidal activity, probably because of the relatively short contact time between the ethanol extract of most of the tested herbs and the tick larvae. In contrast, with the impregnated filter paper method, the herbal extract was distributed uniformly, which increased the contact time, demonstrating that the impregnated filter paper method is more suitable for the tactile method.Cinnamomum cassia and (E)-cinnamaldehyde exhibited acaricidal activity, with LC50 values of 3.81 and 3.15 mg/mL, respectively, against the larvae of Haemaphysalis longicornis, and 21.31 and 16.93 mg/mL, respectively, against H. longicornis nymphs  against nymphs of Ixodes ricinus also reported the decrease in its long-term repellent properties. Indeed, El-Seedi et al.  against nymphs of the tick I. ricinus decreases over the long term, probably because of the high volatility of 1,8-cineole. It was confirmed that the best insect repellent effect of most of the herbs studied in this study occurred only at the highest concentration and the shortest drying time assessed. Hence, future studies should focus on the main active ingredients and delay the volatilization rate of the active ingredients to increase the repellent time.In this study, all nine herbs had a real-time  repellency rate of > 80%, but the ethanol extracts of only four herbs had a repellency rate of > 50% after 48 h, which included castor bean (81.3%) and star anise (79.6%), possibly due to the high volatility of these major constituents (Pålsson et al. i et al.  suggesteNatural products or extracts may be ideal tick control agents since they may be able to reduce the development of resistance and are not harmful to the environment. However, some disadvantages of plant-based products include short duration of activity, the potential for skin sensitization and allergies, and many plant-based compounds are toxic to some animals Vigan . The draR. microplus, whereas the ethanol extracts of star anise and clove also had good fumigant acaricidal effects, and the hatching inhibition rate of R. microplus eggs by star anise reached 100%. Therefore, these medicinal Chinese herbs should be further investigated as potential alternative drugs for tick control. In addition, the results of this study showed that all nine herbs had good real-time repellent effects, but only castor bean and star anise had good delayed repellent effects. Therefore, delaying volatilization of the repellent components is an important direction for future repellent development. Hence, further studies are warranted to identify the effective acaricidal components of these herbs, determine the synergistic effects among different components, and establish appropriate delivery methods to develop safer and more effective acaricides and repellents.The ethanol extracts of star anise, CRP, chaulmoogra, stemona, and motherwort had strong acaricidal effects against


Chryseobacterium indologenes is one of the primary causative agents of root rot of Panax notoginseng, which significantly affected plant growth and caused economic losses. With the increasing incidence of antibiotic-resistant bacterial phytopathogens, phage therapy has been garnered renewed attention in treating pathogenic bacteria. However, the therapeutic potential of phage therapy on root rot of P. notoginseng has not been evaluated. In this study, we isolated a novel lytic phage MA9V-1 infecting C. indologenes MA9 from sewage and monitored the formation of clear and round plaques with a diameter of approximately 0.5–1.5 mm. Phage MA9V-1 exhibited rapid absorption (>75% in 8 min), a latency period of 20 min, and a burst size of 10 particles per cell. Transmission electron microscopy indicated that the phage MA9V-1 is a new myovirus hosting C. indologenes MA9. Sequencing of phage genomes revealed that phage MA9V-1 contained a linear double-stranded DNA genome of 213,507 bp with 263 predicted open reading frames, including phage structure, host lysing, and DNA polymerase/helicase but no genes of tRNA, virulence, and antibiotic resistance. Our proteomic tree and genomic analysis revealed that phage MA9V-1 shares identity with Sphingomonas phage PAU and Tenacibaculum phage PTm1; however, they also showed apparent differences. Further systemic evaluation using phage therapy experiments on P. notoginseng suggested that phage MA9V-1 can be a potential candidate for effectively controlling C. indologenes MA9 infection. Thus, we have presented a novel approach to solving root rot in P. notoginseng. Phages  of 0.01 . Similarmic loss . In additolaasii . HoweverC. indologenes-infecting phage MA9V-1 from sewage samples. We also compared the whole genome of phage MA9V-1 with others, determined that it is a new virus targeting C. indologenes, and evaluated its efficacy in preventing P. notoginseng root rot to provide a novel approach to solve this major challenge.In this study, we isolated and characterized the first 2.2.1.C. indologenes strain MA9 (accession no. CP075170), isolated from rotten roots of P. notoginseng, was provided by professor Ji of Yunnan Agricultural University and was used as a host in this study. Samples for screening phages were collected from different regions as follows: (1) The rhizospheric soils of healthy and rotten P. notoginseng were collected from Pingyuan Street, Yanshan County, Wenshan Prefecture, Yunnan Province in November 2020; (2) the rotten roots of P. notoginseng and P. notoginseng root soils were collected from Shilin Yi Autonomous Region, Kunming City, Yunnan Province in January 2021; (3) the soils of different vegetables and fruit trees were collected from the farmland near Kunming University of Science and Technology, Chenggong District, Kunming City, Yunnan Province in January 2021; and (4) tewage samples were collected from the First People’s Hospital of Yunnan Province in February 2021. C. indologenes MA9 was cultured in an optimized nutrient agar (NA) medium  at 160 rpm at 28°C.2.2.P. notoginseng infected with C. indologenes MA9; however, we could not find any phage. Subsequently, under the same culture conditions, we attempted to use other samples such as rotten roots, P. notoginseng root soils, and soils of various fruits and vegetables. However, the targeted phage was also not found. Finally, we isolated a C. indologenes strain MA9 phage from sewage samples of the First People’s Hospital of Yunnan Province, China, using the virus enrichment and double-layer agar plate methods  and kept static for 12 h. Subsequently, 40 mL suspension, 30 mL of SM buffer, 25 mL of NA liquid medium, and 2 mL of C. indologenes MA9 cultures were mixed and incubated overnight on a horizontal rotary shaker (160 rpm) at 28°C. The mixture was transferred to a sterile 50 mL centrifuge tube and centrifuged at 15,000 × g for 15 min at 4°C. Subsequently, the supernatant was filtered with 0.22-μm microporous membranes. In total, 5 mL filtrate was taken and mixed with 50 mL C. indologenes MA9 host cell cultures during the period of exponential growth phase [optical density at 600 nm (OD600) =0.6–0.8] and then repeated 2–3 times to enrich phage MA9V-1. Successively, 200 μL of phage stock was serially diluted with SM buffer, and different dilutions were mixed with 300 μL of C. indologenes MA9, which were added to 4.5 mL semi-solid NA medium after being incubated at 28°C for 20 min, and finally, the solution was finally poured onto the surface of the NA solid medium. A single clear plaque was picked from NA double-layer agar plate incubated overnight at 28° C, placed into 1 mL of SM buffer, and vortexed vigorously before filtering through a 0.22-μm membrane. The above steps were repeated 2–3 times to obtain pure phage MA9V-1 particles, and the phage MA9V-1 titer was measured to be 109 PFU/ml by the double-layer agar plate method. The isolates were stored at 4°C.First, we tried to isolate phages from the rhizospheric soil mixtures of healthy and decayed  methods . In brie2.3.−4, 10−5, and 10−6. Phage stock of known titer and host cells at the exponential stage were mixed with a gradient of MOI ranging from 0.001 to 10 and cultured in a shaker (180 rpm/min) at 28°C for 12 h. The enrichment was centrifuged at 15,000 × g at 4°C for 15 min and filtered through a 0.22-μm filter membrane, and the phage titer was determined by the double-layer agar plate method with three replicates.The optimal multiplicity of infection (MOI) is the ratio of phages to bacteria at the time of infection. The experimental method followed was according to the study mentioned in the reference . In brie2.4.C. indologenes MA9 cultures with absorbance of OD600 at 0.6–0.8 was infected with 1 mL phage dilution (106 PFU/ml) at multiplicity of infection of 0.01 and transferred to a sterile 50 mL conical flask to incubate at 28°C. Approximately 100 μL of culture was taken out at 4-min intervals  and added to sterile 2 mL tubes with 1.9 mL of pre-cooled NA medium, vortexed for 10–15 s, and then centrifuged at 15000 × g at 4°C for 15 min, to remove the cell-phage complexes. The titer of free phage in the supernatant was determined by the double-layer overlay method and was repeated three times , and 10 mL of culture was collected by centrifugation  for harvesting, and the precipitate was resuspended with 2 mL of fresh NA liquid medium. Phage was added to the suspension at MOI = 0.01 and incubated at 28°C for 15 min without shaking. The mixture was, then, centrifuged at 11,000 × g for 8 min to remove the free phage particles, and the pellet was suspended with fresh NA medium to culture in a shaker at 180 rpm at 28°C after washed twice with 10 mL nutrient agar medium. In total, 200 μL of culture was taken out every 10 min until 130 min and mixed with 800 μL SM buffer. Finally, the mixture was centrifuged at 15,000 × g for 3 min at 4°C. The titers of the samples were immediately measured by the double-layer agar method. The burst size was calculated according to the following definition: Burst size is the ratio between the number of phage particles at the end of lysis and the number of the host cells at the beginning of the infection  was treated under different temperatures from 30 to75°C at 5°C intervals for 1 h. Then, these treated samples checked the survival rate of phage MA9V-1 using the double-layer agar assay. Similarly, 100 μL of phage stock (1010 PFU/ml) was added to 900 μL different buffers of pH ranging from 3 to11  for incubation of 1 h at 30°C. After treatment, the titer of phage MA9V-1 was evaluated using the same procedure. All assays were performed in triplicate.For determining the thermal and pH stability of phage MA9V-1, the experimental protocol was performed as proposed with slight modification . In brie2.7.Chryseobacterium phage MA9V-1 was tested against 11 different bacterial strains using the standard spot testing assay, including Chryseobacterium indologenes (n = 7), Bacillus cereus (n = 1), Pseudomonas syringae (n = 1), and Escherichia coli (n = 1), of which Chryseobacterium indologenes ATCC 29897, Pseudomonas syringae CGMCC 1.3070, and Escherichia coli ATCC 11303 were type strains purchased from Strain Preservation Center, while the others were isolates. Each 300 μL of bacterial cells was mixed with 200 μL of 10-fold series gradient dilutions (10−1–10−7) of phage MA9V-1 suspension (1010 PFU/ml), then adding all mixtures to 4.5 mL semi-solid medium before pouring into the surface of double-layer agar plates, respectively, and incubated at 28°C overnight in triplicate.The host range of 2.8.The morphology and structure of pure phage particles were visualized using transmission electron microscopy (TEM). The phage particles purified with the density gradient centrifugation method were loaded on a carbon-coated copper grid to adsorb for 10 min, followed by negative staining with 1% phosphotungstic acid. The stained phage MA9V-1 was observed using transmission electron microscopy (Hitachi HT7820) at 120 kV .2.9.De novo assembly” option of SPAdes v3.5.0 software  and 0.5 μL RNase A (1 mg/mL) were added to 500 μL phage stock to remove the host cell nucleic acid, and further experimental operation was performed using the phenol–chloroform protocol, as previously described in the study mentioned in the reference . Nucleicsoftware .2 Subseqsoftware  to virussoftware .4 Functisoftware .5 The visoftware 6 and thesoftware .8 The phsoftware 9 and anasoftware .10 MEGA software 11 was ussoftware .122.10.9 PFU/ml purified by the polyethylene glycol (PEG) precipitation method was ultracentrifuged with CsCl density gradient centrifugation  at 4°C at 150,000 × g for 8 h and extracted the separated layer of phage with a sterilized syringe. Then, 20 μL of ultra-purified phage particles was mixed with 2× SDS loading buffer, heated in boiling water for 15 min, and subjected to SDS-PAGE [12% (w/v) polyacrylamide]  of 0.001, 0.01, 0.1, 1, and 10, respectively, and incubated at 28°C for 10 min. In total, 100 μL of different MOI mixtures were was added to the root slices, sealed with tape, and cultured in an incubator at 26°C. This experiment was divided into seven groups with eight parallels in each group. Among them, the negative control was the C. indologenes MA9 reaching the exponential stage, whereas the positive control was the inactivated C. indologenes MA9 at the log phase after treating with the autoclave sterilization method. Under the same experimental condition, 100 μL of the mixture of different MOI was added to the experimental group at 24-h intervals, the negative control and positive control were added to the equal volume of treatment liquid, and the number of infected and uninfected P. notoginseng plants was counted after 7 days. In this study, one condition for determining root rot was the rotting and softening of the root interior. The disease incidence was calculated as the percentage of rotten roots to the total number of parallel experiments. The experiment was performed in triplicate.The experimental material was the root of a healthy 2-year-old 3.3.1.C. indologenes strain MA9 is the first phage to have bacteriolytic activity against C. indologenes MA9. The results suggested that the phage MA9V-1 can form clear and round plaques with a diameter of approximately 0.5–1.5 mm on lawns of C. indologenes MA9 of the double-layered plate  of the ed plate . The mored plate  revealed P2virus .3.2.10 PFU/ml at an MOI of 0.001. The absorption curve revealed that phage MA9V-1 was capable of rapidly adsorbing onto the surface of the host cell membrane. Within 8 min, >75% of the phage particles had adsorbed, and after 12 min, approximately 90% were adsorbed, indicating that phage MA9V-1 had the ability of high adsorption efficiency , was 213,507 bp in length and contained 35.99% of G + C content . The phaenes MA9 .3.4.Sphingomonas phage PAU (accession no.NC_019521) and was branched together with Tenacibaculum phage PTm1 (accession no.NC_049340) and Tenacibaculum phage PT24 (accession no.NC_049383)  . Moreove_049383) . The res_049383) .3.5.Structural protein expression analysis of purified phage particles by SDS-PAGE gel electrophoresis showed at least 15 bands of protein molecular weight ranging from 20 to 260 kDa. The protein bands were mainly distributed in 75–135, 45–65, and 25–35 kDa regions of SDS-PAGE gel, which were likely related to structural proteins, terminal family proteins, DNA replication proteins, and hypothetical proteins. According to SDS-PAGE results, we only obtained limited knowledge, and whole-genome analysis of phage MA9V-1 was required to better elucidate the role of all proteins .3.6.Sphingomonas phage PAU taking as “tree root” than others and grouped into a clade of an independent branch. Similar tree was also obtained using the deduced amino acid sequences of capsid assembly protein ORF149; we can observe that phage MA9V-1 was branched with Tenacibaculum phage pT24, which shared the similar results with above three proteins ,Based on the results of the proteomic tree, we constructed a phylogenetic tree analysis with four representative proteins and aimed to accurately validate the taxonomic classification of phage MA9V-1. Phylogenetic relationships were analyzed among phage MA9V-1 and other 15 members based on the amino acid sequences of four highly conserved proteins, namely terminase family protein (ORF262), DNA polymerase II (ORF135), capsid assembly protein (ORF149), and phage tail sheath protein (ORF001), respectively . The resproteins . Taken t3.7.C. indologenes MA9, we first determined the absorbance value at OD600 of C. indologenes MA9 with and without phage MA9V-1 for 13 h  , which can make phage MA9V-1 quickly release progeny virus in a life cycle  . Morpholobserved . Furtherfe cycle . Moreovefe cycle . Due to fe cycle . The groae Eisa9  and P. aage PPAY . We hypoenes MA9 . ImportaBacillus phage SPR encodes a methyltransferase that modifies three base sites to make phage DNA escape from degradation by multiple nucleases  can be used for phage therapy of bacterial pathogens because they can continuously complete the life cycle of adsorption, invasion, reproduction, assembling, and release . Thus, wucleases . Furtherucleases . Thus, wC. indologenes MA9, which has a potential preventive and biocontrol effect on P. notoginseng root rot caused by C. indologenes MA9. However, there are some limitations exposed in this study. For example, treating methods and culturing conditions were both conducted under the laboratory environment, which did not really simulate the microecology surroundings in which microorganisms interact with the rhizospheric soils of P. notoginseng, leading to deviations in the therapeutic results. When phage therapy applied in the agricultural environment, its functional mechanism is a complicated process. A reasonable insight is that the host undergoes self-evolutionary in response to phage infection, which leads to modification in the cell membrane structure, making it lose the ability to compete with other microbial populations. The weaker ability to compete for survival resources results in a reduction in the abundance of pathogenic bacterium, which enriched bacterial taxa that are highly antagonistic toward the pathogen and achieved the purpose of phage therapy  can be found in the article/HZ wrote the first draft of the manuscript and carried out experiments. YW defined the research theme and revised the manuscript. GJ, JZ, XC and YY provided experimental materials. HZ, YD, JS, JL, and CM designed the methods and experiments and analyzed the results and data. All authors contributed to the article and approved the submitted version.


Background: Ciprofol, a novel sedative–hypnotic drug, has been approved for its use in inducing and maintaining general anesthesia, as well as for providing sedation.Methods: In this phase I, single-center, parallel, controlled, open-label clinical trial, our objective was to analyze the pharmacokinetics (PK), pharmacodynamics (PD), and safety of ciprofol emulsion in 24 participants with mild and moderate renal impairment (n = 8 per group) and matched healthy participants (n = 8). An initial loading infusion of ciprofol was administered at 0.4 mg/kg for 1 min, followed by a maintenance infusion at a rate of 0.4 mg/kg/h for 30 min. We collected plasma and urine samples from the participants to assess the PK of ciprofol and its metabolite M4. The evaluation of PD involved using a modified observer’s alertness/sedation scale (MOAA/S) in combination with bispectral index (BIS) monitoring. Safety assessments were conducted throughout the trial process.Results: The plasma concentration–time curve of ciprofol in participants with renal impairment was similar to that in participants with normal kidney function. The area under the curve (AUC) and maximum concentration (Cmax) of total and unbound ciprofol in plasma for participants with renal impairment were only slightly higher (0.7–1.2-fold) than those in participants with normal renal function. In contrast, for the metabolite M4, AUC values were 1.3- and 2.1-fold greater in participants with mild and moderate renal impairment, respectively, than in healthy controls. However, renal impairment had no significant impact on the PD parameters. The study found that ciprofol was well-tolerated, with all adverse events (AEs) reported being mild or moderate in severity.Conclusion: Based on these findings, we can conclude that no dosage adjustment of ciprofol is necessary for patients with mild–moderate renal impairment who receive the injection.Clinical Trial Registration:https://clinicaltrials.gov, identifier NCT04142970. A) receptors, resulting in an influx of chloride ions (Ciprofol (HSK3486) is a sedative–hypnotic compound approved in 2020 for use in inducing and maintaining sedation, as well as providing general anesthesia. The primary mechanism of action of ciprofol is to enhance the activity of the ion channel mediated by gamma–aminobutyric acid type A  adhered to ethical guidelines, such as the Declaration of Helsinki principles and Good Clinical Practice rules.The study, conducted at the First Affiliated Hospital of Zhengzhou University in China between November 2019 and August 2020, received approval from an independent Ethics Committee. All participants provided written informed consent. This clinical trial ; medical instability; including psychiatric or neurological disorders; cardiovascular, endocrine, pulmonary, hepatic, gastrointestinal, or metabolic illnesses; or any other condition that could interfere with the assessment of the PK of the investigational drug or the completion of the trial. Potential participants underwent a pre-study screening, which involved reviewing their medical history, physical examinations, a 12-lead ECG, and monitoring of vital signs . Laboratory examinations, including coagulation, hematology, clinical chemistry, and urinalysis, were conducted 14 days prior to the study.Participants eligible for the trial were males and females aged between 18 and 65, with a body weight ≥45 kg and a body mass index (BMI) ranging from 18 to 28 kg/m−1 and administration rate of the maintenance dose (mL/h) = 0.4 mg/kg/h × baseline weight (kg) ÷ 2.5 mg/mL. The degree of anesthesia or sedation experienced by participants was evaluated using two methods: the modified observer’s alertness/sedation scale (MOAA/S) and bispectral index (BIS) monitoring. Vital signs, electrocardiograms, and other indicators were monitored using an electrocardiogram monitor. These observations continued until the participants were fully awakened and achieved an MOAA/S score of 5 for three consecutive assessments. MOAA/S scores  = 0.4 mg/kg × subject’s baseline weight (kg) ÷ 2.5 mg/mL × 60 hS scores   were collected at specific time intervals, including 30 min before administration; 1 min after the completion of the loading infusion; and at 5, 10, 20, and 30 min after the start of the maintenance infusion, as well as at 1, 2, 4, 8, 15, 30 min, and 1 h after the end of the infusion. Venous blood samples (3 mL in volume) were collected at 2, 3, 4, 6, 8, 12, 24, and 48 h after drug administration. In addition, each 5 mL of arterial blood was sampled to estimate the protein binding rate, 1 min after the start of administration and 1 min after the end of the infusion. Blood samples were collected in KUrine samples were collected over a 24-h period before ciprofol administration and at specific time intervals after administration: 0–4, 4–8, 8–12, 12–24, and 24–48 h. The urine samples (3 mL) were aliquoted into polypropylene tubes and cryopreserved at −80°C for future analysis.Once the final PK samples were collected and safety evaluations were completed, the participants were allowed to leave the clinical site on the third day.+ mass spectrometer and Analyst™ 1.6.3 software (both AB Sciex). Protein precipitation was performed as a pretreatment step for human plasma and urine samples. To monitor ciprofol and M4, multi-reaction monitoring was performed in negative mode. The compound HSK23287 served as the internal standard for assessing ciprofol, while nimesulide was used as the internal standard for M4. A quadratic regression analysis was conducted to determine the best-fit line for the calibration standards, with a weighing factor set to 1/x2. The linear range of ciprofol and M4 in plasma was 5–5,000 ng/mL, and in urine, it was 10–10,000 ng/mL.The concentrations of ciprofol and its metabolite M4 in plasma, as well as the concentration of M4 in urine, were evaluated using established high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques. The analytical instrument used for the analysis was an LC-30 AD system (Shimadzu), coupled to a Triple Quad 6500max) was directly obtained from the plasma concentration–time plot. The peak time (Tmax) was defined as the time at which Cmax was obtained. The area under the plasma concentration–time curve (AUC) from 0 to t (AUC0-t) was computed using the linear trapezoidal rule. AUC0-inf was calculated as follows: AUC0-inf = AUC0-t + Ct/ke, where Ct is the final determined concentration and ke is estimated by linear regression of the log-linear part of the plasma concentration–time curve. The terminal elimination half-life (t1/2) was calculated as ln2/ke, the total clearance (CL) as Dose/AUC0-inf, and the distribution volume (Vd) as Dose/(AUC0-inf* ke). Protein binding (PB) was measured with equilibrium dialysis. For each participant, the protein binding was calculated as the average following the loading infusion and 1 min after the infusion was discontinued. The fraction unbound (Fu) was calculated as follows: Fu (%) = 100%—PB (%). Unbound concentration was estimated using the measured total concentration at each time point multiplied by Fu, assumed to be constant. The unbound Cmax  of ciprofol was calculated as Cmax × Fu(%), and unbound AUC0-inf  of ciprofol was calculated using the linear trapezoidal rule, as previously described, from the unbound ciprofol concentrations.PK parameters were computed using a noncompartmental approach with the Phoenix WinNonlin 8.3.1 . The maximum plasma concentration (C0–48) and renal clearance (CLR) of M4. Ae0–48 was determined by summing the quantity of M4 excreted in urine between 0 and 48 h following dosing. CLR was computed as Ae0–48/AUC0-t.The PK parameters of urine were as follows: cumulative excretion . The PD parameters included the following: minimum BIS value (BISpeak), the time to reach BISpeak (TBISpeak), and BIS AUC0-t.PD parameters were calculated using Adopt SASFor safety assessment, the alterations in vital signs, ECG signals, laboratory findings, physical examinations, and assessments of injection pain were surveilled. Particular attention was given to adverse reactions associated with sedation, such as bradycardia, apnea, hypoxia, and hypotension.® 9.4  was used to statistically assess the PK and PD data. The impact of renal impairment versus the control group was investigated using analysis of variance (ANOVA), which included renal function as a fixed effect, and the corresponding 90% confidence intervals (CIs) around the geometric least-squares mean (GLSM) ratio of Cmax, AUC0-inf, Cmax,u, and AUC0-inf,u were calculated. Multiple regression analysis was conducted to assess the correlation between clinical variables  and PK parameters . The correlation was determined by multiple regression analysis, using PK parameters as the dependent variable and clinical variables as independent variables.Continuous variables were expressed as medians (maximum and minimum) or means (standard deviations). Adopt SAS2), moderately impaired function (eGFR: 30–59 mL/min/1.73 m2), and normal kidney function (eGFR: ≥90 mL/min/1.73 m2) (n = 8 for each group), were enrolled in the study (2) = 175 × −1.234 × (age)−0.179 × . The demographic attributes of the normal control participants closely resembled those of the participants with renal impairment  approximately 8–12 h after the administration was stopped.The mean plasma concentration–time graphs of ciprofol and its metabolite M4 are shown in max and a 15.67% increase in AUC0-inf of ciprofol. On the other hand, the individuals with moderate renal impairment showed a decline of 15.15% and 1.80% in Cmax and AUC0-inf of ciprofol, respectively  values amounting to 138.15% and 42.72%, respectively.The primary PK parameters of ciprofol are presented in ectively , 3. The The results of the protein binding assay indicated that ciprofol exhibits a high degree of binding to plasma proteins. The mean protein binding (%) for ciprofol was 99.10%, 99.15%, and 99.24% among the normal, mild renal impairment, and moderate renal impairment groups, respectively. The Fu (%) was 0.9%, 0.85%, and 0.76% for healthy participants and patients with mild and moderate renal impairment, respectively. As renal function worsened, decreased Fu was noted.max, u and a 10.39% increase in AUC0-inf, u for unbound ciprofol  among the examined groups, it was identified that the eGFR exhibited a significant correlation with CLolite M4 . In the The results revealed that the MOAA/S score–time curves were similar across the three groups, consistent with the PK profiles . All thrpeak and BIS AUC0-t were similar across the three groups.Mean BIS–time profiles and parameters are shown in In the current study, 14 participants (58.3%) encountered a total of 19 AEs, as detailed in Vital signs, which included diastolic and systolic blood pressures, mean arterial pressure, oxygen saturation, and respiratory and cardiac rates, exhibited a consistent pattern of change , with flmax of total and unbound ciprofol in plasma for participants with renal impairment were only slightly higher (0.7–1.2-fold) than those in participants with normal renal function. Linear regression analysis revealed that PK parameters for ciprofol did not exhibit a significant correlation with the eGFR. However, given the relatively small sample size and larger variability (geometric % coefficient of variation), the reason behind the observed difference should be further investigated. It is crucial to acknowledge that the variation in Cmax and AUC between healthy individuals and patients with renal impairment may be influenced by differences in infusion rates or distribution volume. Additionally, variations in Cmax,u and AUCu could also be linked to variances in protein binding , individuals with end-stage renal disease (ESRD) (eGFR<15 mL/min/1.73 m2), or those undergoing dialysis. Notably, ESRD patients may experience gastrointestinal absorption alterations, changes in distribution volumes, shifts in protein binding, and variations in drug metabolic clearance. Patients with ERSD may require higher propofol induction doses  to moderate (eGFR: 30–59 mL/min/1.73 m2) renal function impairment displayed no clinically significant alterations in the PK and PD of ciprofol. Moreover, ciprofol exhibited favorable tolerance and an acceptable safety profile across all renal function groups. Consequently, the adjustment of ciprofol dosages is deemed unnecessary for individuals with mild to moderately impaired renal function based on the aforementioned findings.In summary, patients with mild (eGFR: 60–89 mL/min/1.73 m


Moreover, the calculated capacity for hydrogen evolution reaction of these δ-IV–VI monolayers further implies their potential for applications in photovoltaic and nano-devices.Two-dimensional materials have been developed as novel photovoltaic and photocatalytic devices because of their excellent properties. In this work, four δ-IV–VI monolayers, GeS, GeSe, SiS and SiSe, are investigated as semiconductors with desirable bandgaps using the first-principles method. These δ-IV–VI monolayers exhibit exceptional toughness; in particular, the yield strength of the GeSe monolayer has no obvious deterioration at 30% strain. Interestingly, the GeSe monolayer also possesses ultrahigh electron mobility along the  Monolayered WSe2 possesses outstanding mechanical properties, and experiments have shown that when the layers of the WSe2 is increased to 2–4, the photoluminescence (PL) is significantly enhanced by 2% under tensile strain  program, which provides a number of functions for performing visualization, modeling and simulation. And all that simulations using DS-PAW software are integrated in Device Studio program [k-point grids of 11 × 11 × 1 and 17 × 17 × 1 in the first Brillouin zone (BZ) were used for the relaxation and self-consistent calculations, respectively. The spin–orbit coupling (SOC) effect is not considered in this work, because it has a negligible effect on the electron band structure of the studied materials. The energy cut-off was set as 550 eV. To avoid interaction between nearby layers, the vacuum space adopted was 20 Å. The convergence values for force and energy were set within 0.01 eV Å−1 and 0.01 meV, respectively.In our simulations, the calculations for the structural optimization, electronic property, carrier mobility and the HER performances were calculated by Device Studio [Hongzhiwei Technology, Device Studio, Version 2021A, China, 2021. Available online:  program . All the program . General program ,50. The  program ,51. Furt program . Monkhory direction, while the yield strength was calculated for the SiS and SiSe monolayers in the x direction. The GeS, GeSe, SiS and SiSe monolayers showed semiconductor characteristics with a bandgap larger than 1.23 eV for water splitting. For this application, an excellent carrier mobility was determined for all these δ-IV–VI monolayers; in particular, the GeSe monolayer demonstrates an ultrahigh electron mobility in the x direction of approximately 32,507 cm2·V−1·s−1. Furthermore, the Gibbs free energies of these GeS, GeSe, SiS and SiSe monolayers were obtained and imply their potential for usage as photocatalysts for water splitting.In summary, the mechanical, electronic and HER properties of the δ-IV–VI monolayers, namely, GeS, GeSe, SiS and SiSe, were systematically investigated using first-principles calculations. The strain–stress relationships of these δ-IV–VI monolayers present a novel toughness along the


Taxodium “Zhongshanshan” are a group of intraspecific Taxodium hybrids with superparental dominance and high ecological and economic value in southern China. Identifying the parentage of hybrids, especially the male parent, is critically important for genetic studies. However, the large nuclear genomes of members of the genus Taxodium pose a major challenge for the development of molecular markers. Here, we developed novel molecular markers by conducting a comparative analysis of the chloroplast genomes of four artificial Taxodium hybrids and their parents. The lengths of the whole chloroplast genome ranged from 131,942 to 132,128 bp, and the total guanine (GC) content of the chloroplast genomes ranged from 34.6% to 35.81%. A total of 120 unique genes were identified, including 83 protein-coding genes, 33 transfer RNAs, and four ribosomal RNAs. There were 69‐71 simple sequence repeats were detected in the four hybrids. Phylogenetic analysis revealed that these hybrids clustered with their paternal parents. Similar findings were obtained by analysis of the GC content of protein-coding genes. Molecular markers were developed using the highly variable regions of the chloroplast genomes, and polymerase chain reaction (PCR) assays revealed that these markers were effective for identifying the male parents of these hybrids. Our findings indicate for the first time that the chloroplast genomes of Taxodium are paternally inherited. Generally, these molecular markers could facilitate breeding and genetic studies of Taxodium. Taxodium, Taxodium distichum (L.) Rich , T. mucronatum Tenore (Montezuma cypress), and T. ascendens Brongn (pond cypress), are 30–40 m tall trees native to North America  and T. “Zhongshanshan 401” (T. ascendens × T. mucronatum) included their high growth rate, ability to grow well in alkaline and saline soils, and high ornamental value (T. “Zhongshanshan 118” (T. “Zhongshanshan 302” ×T. mucronatum) is higher than that of its parents; the salt, alkali, and flood resistance of T. “Zhongshanshan 118” is also higher than that of its parents. Because of the desirable characteristics of these varieties, they have become increasingly popular in 18 provinces and municipalities in China  is a critically important organelle in green plants; it contains the photosynthetic machinery that plays a key role in carbon fixation, as well as plant growth and development . The ratT. “Zhongshanshan” varieties and compared them with the cp genome sequences of their parents. Phylogenetic analysis and analysis of the GC content of protein-coding genes revealed that these hybrids were most closely related to their male parents. Analysis of hypervariable regions was used to generate molecular markers that could be used to aid Taxodium breeding.Here, we characterized the complete cp genomes of four T. “Zhongshanshan 302” (T. distichum × T. mucronatum), T. “Zhongshanshan 118” (T. “Zhongshanshan 302” × T. mucronatum), T. “Zhongshanshan 61” (T. mucronatum × T. ascendens), and T. “Zhongshanshan 401” (T. ascendens × T. mucronatum) were grown in a nursery at the Nanjing Botanical Garden , Jiangsu Province, China. Fresh leaves of these plant were collected for DNA extraction with a modified CTAB method  with 150 based on sequencing by synthesis. Approximately 43 Mb of high-quality, clean paired-end reads was generated, and the sequences with cp-like reads were assembled with NOVOPlasty Version 4.3.1 .http://ogdraw.mpimp-golm.mpg.de) (T. “Zhongshanshan” were deposited in the GenBank of NCBI (https://www.ncbi.nlm.nih.gov/) database under the accession numbers MW307789, MW307790, MW307791, and MW307792. The associated bioproject numbers are SRR13347061, SRR13347063, and SRR13347064.The genome was annotated using GeSeq software  and BLAS.mpg.de) . The cp 4 (25 mM), 1.5 µL per primers (10 µM), 1 µL KOD-Plus Neo enzyme, and 100 ng genomic DNA. Thermal cycling consisted of 94°C for 2 min followed by 35 cycles of 98°C for 20 s, 62°C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 7 min 2.5% agarose gels were used to visualize the PCR products after electrophoresis.The highly variable regions were amplified with the high-fidelity polymerase of KOD-PlusNeo . The PCR reactions were performed in a total volume of 50 µL with 5 µL 10 × KOD-plus Neo buffer, 5 µL dNTP (2 mM), 3 µL MgSOhttps://webblast.ipk-gatersleben.de/misa) software (https://bibiserv.Cebitec.uni-bielefeld.de/reputer) software with the best top 50 sequences (Simple sequence repeats (SSRs) were identified by MISA v2.1 (software  with theequences . The Codhttp://genome.lbl.gov/vista/mvista) (T. distichum (NC_034941), T. distichum (MN535013), T. “Zhongshanshan 61” (MW307790), T. ascendens (MN535012), T. “Zhongshanshan 118” (MW307791), T. “Zhongshanshan 302” (MW307792), T. “Zhongshanshan 401” (MW307789), and T. mucronatum (MN535011), in the Shuffle-LAGAN mode. BLAST Atlas on the GView server was used to assess the chloroplast genomes similarity with T. distichum (MN535013) genome as a reference  and overall GC content of the genomes (The mVISTA (/mvista)  program eference . DNaSP v genomes .Taxodium and an outgroup (Cryptomeria japonica) were downloaded from the National Center for Biotechnology Information (NCBI) database. MAFFT software (v7.450) was used to multiply sequence alignment of these cp genome sequences  and a small single-copy region (SSC), which is separated by two inverted repeat (IRs) regions. Previous studies have shown that IRs play key roles determining the stability of the genome and the conservation of genes . HoweverT. “Zhongshanshan” cp genomes contained 120 genes, including 83 protein-coding genes, 33 tRNAs, and four rRNAs; the distribution of these genes was consistent among all four “Zhongshansha” cp genomes examined. A total of 62 genes belonged to the self-replication category; 33 of these encoded tRNAs, 12 of these encoded small ribosomal subunit proteins, and nine of these encoded large ribosomal subunit proteins. Fifty of these genes were related to photosynthesis, including six genes that encoded ATP synthase subunits, 11 genes that encoded NADH-plastoquinone oxidoreductase subunits, six genes that encoded cytochrome b/f complex subunits, eight genes that encoded photosystem I subunits, 15 genes that encoded and PSII subunits, three genes that encoded photochlorophyllide reductase subunits, and one gene that encoded the large subunit of Rubisco. A total of six groups were in the biosynthesis category, and each group contained one gene  contained three exons. Two copies of the trnQ-UUG and trnI-CAU genes were detected in the cp genomes of T. “Zhongshanshan”  was conducted. 68–71 SSRs were detected in the cp genomes , followed by AC/GT  and AG/CT   .Taxodium species examined. Forward repeats were the most common type in the cp genomes, followed by palindromic repeats  and two complementary repeats , were identified in the other species , followed by sequence lengths of >91 bp, then 51–70 bp and 71–90 bp  were identified in the cp genomes of the seven  repeats . Reverse species . In addi71–90 bp . The disentified .Taxodium, which is consistent with the results of previous studies of the cp genomes of members of the genus Cupressus and genus Juniperus  in plant genomes, they are some of the most important molecular markers used in genetic analyses of plant populations, evolutionary studies, and breeding programs . In our uniperus . Reverseocumbens . This inTaxodium species. The total number of codons for protein-coding genes ranged from 24,739 to 24,823, and the patterns of codon usage in T. mucronatum, T. “Zhongshanshan 118”, T. “Zhongshanshan 302”, and T. “Zhongshanshan 401” were similar . Furthermore, the GC content of the ycf1 and ycf2 genes  in T. ascendens and its offspring T. “Zhongshanshan 61” differed significantly from that of other species, indicating that these two genes could be used as markers to distinguish between them. Cluster analysis based on heat maps of GC1s, GC2s, GC3s, and GCall revealed that the four offspring were more closely related to their paternal parents than their maternal parents  was determined. GC1, GC2, GC3, and GCall showed significant differences between genes but almost no differences between genomes  with Pi values were identified (>0.002), and all of these were located in non-coding regions  have been identified in seed plants cpDNA: uniparental  and biparental  . The mosjaponica . Other sanksiana , Pinus tus taeda , and Seqervirens . The mecTaxodium distichum was used as the reference sequence. First, a neighbor-joining (NJ) tree was built based on the amplified sequences of these four regions  were detected, and these regions could aid future genetic and phylogenetic studies. Phylogenetic analysis of the evolutionary relationships based on the GC content of protein-coding genes indicated that the four hybrids were most closely related to their parents. In addition, molecular markers were developed using four hypervariable regions. Neighbor-joining trees were constructed based on PCR-amplified regions, as well as gel electrophoresis of specific primers, which were developed based on the SNPs/indels in these regions. Our findings suggest that these markers could be used to identify the male parent of the Taxodium hybrids. To our knowledge, this is the first study to demonstrate that the cp genomes of Taxodium are paternally inherited. The molecular markers developed in our study will aid Taxodium breeding research.The complete cp genomes of four


Betula pendula, Plantago lanceolata, and Dactylis glomerata along an urbanization gradient in Ingolstadt, Germany. We compared pollen production with the potential influencing factors urbanization, air temperature, and the air pollutants nitrogen dioxide (NO2) and ozone (O3). While we measured air temperature in the field, we computed concentration levels of NO2 and O3 from a land use regression model. The results showed that average pollen production (in million pollen grains) was 1.2 ± 1.0 per catkin of Betula pendula, 5.0 ± 2.4 per inflorescence of Plantago lanceolata, and 0.7 ± 0.5 per spikelet of Dactylis glomerata. Pollen production was higher in rural compared to urban locations on average for B. pendula (+ 73%) and P. lanceolata (+ 31%), while the opposite was the case for D. glomerata (− 14%). We found that there was substantial heterogeneity across the three species with respect to the association of pollen production and environmental influences. Pollen production decreased for all species with increasing temperature and urbanization, while for increasing pollutant concentrations, decreases were observed for B. pendula, P. lanceolata, and increases for D. glomerata. Additionally, pollen production was found to be highly variable across species and within species—even at small spatial distances. Experiments should be conducted to further explore plant responses to altering environmental conditions.Pollen production is one plant characteristic that is considered to be altered by changes in environmental conditions. In this study, we investigated pollen production of the three anemophilous species  Along urbanization gradients, high variability of different environmental factors can be observed at short distances. The variability arises from varying degrees of anthropogenic influence, such as building density and traffic volume, which are related to the Urban Heat Island effect and emission of air pollutants  concentrations have been reported to lead to an increase in pollen production (Ziska and Caulfield 2) might reduce pollen production , 82 individuals of ribwort plantain (Plantago lanceolata L.), and 54 individuals of orchard grass (Dactylis glomerata L.) along an urbanization gradient in Ingolstadt, Germany. We analyzed the relationship between pollen production and the potential influencing factors urbanization, air temperature, NO2, and O3, separately for the three species. In this study, we assessed the pollen production of 24 birch trees  and surrounding areas, located in southern Germany Fig. . The aveB. pendula, P. lanceolata, and D. glomerata. were distributed along an urbanization gradient with a length of approx. 7.3 km  from different positions on the crown at approx. 2 m a.g.l. Catkins were harvested in February 2020, which ensured that anthesis had not started and no pollen had been released yet, following the recommendation of Damialis et al. . We determined the number of flowers of each spikelet and per cm. The extraction of pollen grains and the preparation of microscope slides were performed according to the procedure used for P. lanceolata inflorescences.We selected 54 individuals of All microscope slides were stored horizontally and counted at × 100 magnification using a light microscope .The count of each microscope slide was then used to determine pollen production at different levels, depending on the species Table .Table 1OB. pendula pollen production was estimated per catkin Pca . The periods considered were June 01 to August 30, 2019, for B. pendula  and two weather stations  across the study area  for the locations where the plant material was sampled. The models are based on additive regression smoothers of spatial and structural explanatory variables and reflect the intra-urban variability  and 2020  at the different locations. The data used to fit the models were compiled following the description in Fritsch and Behm  models of Fritsch and Behm  to obtaiand Behm . We empland Behm , land usand Behm , German and Behm , and theand Behm . Germanyand Behm  and highWe employed land use data including the classes forest, low vegetation, water, built-up, bare soil, and agriculture, which are based on automatically processed Sentinel-2 satellite data and available at a resolution of 10 m  and investigated if there are differences in pollen production at urban and rural locations for the three plant species via a Wilcoxon rank sum test. We tested the null hypothesis that the correlation (difference) is zero for rs (Wilcoxon rank sum test) and considered the correlations (differences) to be statistically significant when p ≤ 0.05. We additionally investigated component plus residual plots for the different environmental influences urban index, temperature , NO2, and O3 on pollen production. The plots consider the effect of one explanatory variable Xj on pollen production P at once, while controlling for all other explanatory variables X(j) based on a linear regression. Each plot shows Xj (abscissa) and the partial residuals (ordinate). The latter is based on a linear regression of P on all environmental influences X and result from subtracting the partial effect of all environmental influences but the considered one X(j)β(j) from P. The implied partial effect is illustrated by a regression line and a univariate spline function.We analyzed the relationships between pollen production and temperature, urban index, and air pollutants by calculating Spearman’s correlation coefficient (We used R (4.2.2) with the packages ggplot2 Wickham , dplyr , mgcv W, terra , sf Peb, stars , npreg  and car Pca of the studied B. pendula trees averaged at 1.2 ± 1.0 million pollen grains and varied between 83,000 and 3.7 million pollen grains. The number of flowers per catkin averaged was at 134 ± 20, the mean length of catkins was 33.2 ± 6.4 mm, and the mean diameter was 3.7 ± 0.3 mm. Mean Pinfl of P. lanceolata was 5.0 ± 2.4 million pollen grains with a range of 1.5 to 12.1 million pollen grains. We counted 73 ± 29 flowers per inflorescence on average; the mean length of the inflorescence was 13.8 ± 3.6 mm and the diameter was 6.2 ± 0.9 mm. For D. glomerata, the average Psp was 0.7 ± 0.5 million pollen grains and ranged from 79,450 to 2.4 million pollen grains. The mean number of flowers per spikelet was 16 ± 5 .Using the urban index to distinguish between urban and rural locations resulted in 10 rural and 14 urban locations for p < 0.001). In 2019, during the considered period from June to August, the mean temperature at the rural location was 13.7 °C, at urban locations it was 2.1 °C higher with a mean of 15.9 °C. In the considered period in 2020 (April 02 to June 30), the temperature at rural locations averaged 13.7 °C, and at urban locations, it was 1.2 °C higher with 14.9 °C.Overall, daily mean temperature at urban locations was higher compared to rural locations, with statistically significant differences (2 concentrations ranged from 13.5 to 21.4 µg/m3 at the plant locations in 2019 with higher concentrations at urban (mean = 20.8 µg/m3) compared to rural locations (mean = 17.4 µg/m3). NO2 concentrations in 2020 were about one-third lower on average and ranged from 10.1 to 15.1 µg/m3 with higher concentrations in urban (mean = 13.5 µg/m3) compared to rural locations (mean = 12.2 µg/m3). In both years, the differences between the locations were statistically significant (p < 0.001). This also applies to O3 concentrations, for which urban locations averaged at 45.9 µg/m3 in 2019 and 44.4 µg/m3 in 2020, while rural locations averaged at 47.7 µg/m3 in 2019 and 45.0 µg/m3 in 2020. The maps derived from the LUR model are included in the We found different pollutant concentrations between the years and locations. While NOB. pendula: mean Pca was 0.9 ± 0.9 (urban) vs. 1.6 ± 1.1  million pollen grains. This difference was statistically significant (p = 0.016). The mean number of flowers per catkin was 134 ± 24 (urban) vs. 135 ± 14  .As illustrated in Fig. P. lanceolata, mean Pinfl was lower at urban locations (4.4 ± 2.4 million pollen grains) compared to rural locations (5.8 ± 2.2 million pollen grains). The mean number of flowers per inflorescence was higher at rural locations with 85 ± 31 flowers compared to 64 ± 24 flowers at urban locations. Both differences were statistically significant (p ≤ 0.003).For D. glomerata, mean Psp were quite similar with an average of 0.7 ± 0.6 at urban and 0.6 ± 0.4 million pollen grains at rural locations. The mean number of flowers per spikelet was 17 ± 5 flowers at urban and 15 ± 6 flowers at rural sites (p = 0.033).For rs of pollen production of the three analyzed species with urban index, air temperature variables, and air pollutants. While we observed negative correlations of pollen production with the urban index and all temperature variables, the strength of correlations varied across the three species. For B. pendula, the correlations of Tmean, Tmin and accTmean were significantly different from zero. For P. lanceolata, this was the case for all considered correlations (for Pinfl) and Tmean (for Pfl), while none of the correlations were statistically significant for D. glomerata. For the air pollutants, correlations were negative for NO2 for all species besides Pfl for P. lanceolata and Psp for D. glomerata. For O3, negative correlations were obtained for P. lanceolata and positive ones for B. pendula and D. glomerata. None of the correlations was different from zero at the considered significance level.Table P and the j-th considered environmental influence, while controlling for all other influences via a linear regression. In the first line of the plot, for example, the effect of the urban index on P was investigated for each species, while temperature , NO2, and O3 are used as controls. Overall, the figure mostly confirms the findings reported in Table B. pendula and negative effects on P. lanceolata and D. glomerata. For the air pollutants, negative effects are indicated for B. pendula and P. lanceolata and positive ones for D. glomerata. When considering the other available temperature variables for the three plant species , the results remain qualitatively identical. The plots also indicate that a linear regression line approximates the relationship between pollen production and environmental influences reasonably well, as the spline function and the regression line largely coincide.As correlation coefficients are only suitable to assess bivariate relationships between variables, we also used the component plus residual plots displayed in Fig. B. pendula, P. lanceolata, and D. glomerata and its connection with environmental influences. The chosen study area reflected urban–rural differences, as indicated by the differences between the sites regarding temperature and air pollutant concentrations. Urban–rural differences in temperature varied between 1.2 and 2.1°C. NO2 concentrations differed between the years, illustrating the effect of COVID-19-related lockdowns on air pollution, which was documented for Germany . Other recent studies examining Pca of B. pendula reported 1.7 ± 1.3 million pollen grains . González-Parrado et al. . Tormo-Molina et al.  and P. lanceolata (Pinfl) compared to urban locations. In addition, we found a higher number of flowers per inflorescence of P. lanceolata and of flowers per spikelet of D. glomerata. All three described differences were statistically significant. There are only few studies that have analyzed differences in pollen production in regards to urbanization, while there are a few more that examined relationships between pollen production and environmental factors. Jochner et al. . In Augsburg, Germany, pollen production of B. pendula was investigated by Kolek . The relationship between the air pollutants NO2 and O3 and pollen production was found to be negative for B. pendula and P. lanceolata, while the opposite was the case for D. glomerata. This result implies that these air pollutants affect pollen production – even when accounting for the other considered environmental influences. In general, our results indicate that the effect of environmental influences on pollen production is species-specific. Jochner et al.  of the previous year. In addition, there were no significant correlations between Pca and O3 or NO2. In our study, correlations between Pca, Pfl, and the cumulative temperature were, however, significant and negative. Darbah et al.  model to generate data on the air pollutants NOBy studying pollen production along an urbanization gradient, we made use of the space-for-time approach. However, investigations along with other gradients should be considered for future studies. One approach was recommended by Tito et al. , who sugP. lanceolata, the species with higher pollen production at rural sites, was growing in areas close to agricultural land. In addition, some sites in urban areas did not seem favorable for plant growth due to the vicinity to roads and possible exposure to waste and high nitrogen input (Allen et al. D. glomerata in rural areas. This might indicate that this grass species is not benefitting as much from the mentioned site conditions as P. lanceolata. Furthermore, a study examining Juniperis communis pollen reported that nutrient availability had an impact on the development of pollen grains (Pers-Kamczyc et al. Besides temperature and air pollution, other site conditions might modify plant growth, which was also suggested by González-Parrado et al. . These sP. lanceolata and D. glomerata. Similar results were found in the case of Brassica rapa (Rivkin et al. Our results revealed differences in the number of flowers between urban and rural locations for Betula it has been observed to be every two (Latałowa et al. While our findings give new insights into the possible effects of changing temperatures and air pollution on pollen production, shortcomings have to be accounted for, such as the pollen production data that were based on one year. Conducting this investigation over more years would lead to more robust results, and in addition, influences on pollen production such as masting would be considered. Masting is the phenomenon of trees producing a high number of flowers and seeds in one year, which is then followed by a period of lower seed production (Herrera et al. B. pendula and P. lanceolata, and positive relationships with NO2 and O3 for D. glomerata. Further studies should inter alia focus on the physiological performance of trees growing in urban areas, which might give a hint for explaining their behavior related to pollen production. Furthermore, additional locations representing semi-urban sites would support the continuous representation along the gradient. This is essential for drawing conclusions and may allow more profound predictions related to the future effects of climate change on pollen production. In order to identify single influencing factors, a combination of experiments in a controlled environment with field research should be considered.In this study, we showed variations in pollen production of three allergenic species along an urbanization gradient. Pollen production for two species was overall higher in rural compared to urban locations and we found negative relationships with temperature for