S cuspicaudus in mitochondria (Mit-d), plasmatic membranes (Mem-d), and DNA fragmentation (DNA-f) with an extender composed of ethylene glycol , glucose (6%) and skim milk powder (5%). Sperm kinetics and damage in fresh, pre-frozen, and thawed semen were evaluated. In pre-frozen semen, sperm kinetics decreased, and Mit-d (11 to 13 times), Mem-d (2.4 to 4 times), and DNA-f (2.8 to 4 times) damages increased regarding fresh semen. In thawed semen, damage continued to rise in Mit-d (34 to 37 times), Mem-d (24.5 to 26.6 times), and DNA-f (13 to 18.5 times). Fertility (37–59%) and hatching (29–46%) rates of thawed-cryopreserved semen were reduced to half that recorded for fresh semen. In conclusion, the present study demonstrated that mitochondria, membrane, and DNA suffer considerable damage in pre-freezing and freezing–thawing, affecting their fertilizing capacity, which is reduced to half regarding fresh semen.The study aimed to evaluate the damage suffered by cryopreserved semen of Sorubim cuspicaudus sperm with ethylene glycol (EG) at different rates . Fresh, prefrozen, and post-thawed sperm quality as motility total, velocities, mitochondria damage (Mit-d), membrane damage (Mem-d), and DNA fragmentation (DNA-f), were examined. The Mit-d, Mem-d, and DNA-f were evaluated through flow cytometry. High motility (>95%) and a low percentage of Mem-d (1.0 ± 0.5%), Mit-d (1.4 ± 0.9%), and DNA-f (2.4 ± 0.8%) were recorded for fresh semen. Prefrozen semen increases in Mit-d and DNA-f were observed compared to fresh semen (p < 0.05). In thawed semen, increased Mit-d (2.6 to 3-fold), Mem-d (6 to 1-fold), and DNA-f (3.3 to 6.6-fold) compared to prefrozen was observed. Thawed semen showed Mit-d (34 to 37-fold), Mem-d (24.5 to 26.6-fold) and DNA-f (13 to 18.5-fold) increased high. In conclusion, the present study demonstrated that mitochondria, membrane, and DNA integrity undergo significant damage during both pre-freezing and freezing/thawing with EG inclusion percentages from 6 to 10% that affect its fertilizing capacity, which is reduced to half of that obtained with fresh semen. It is suggested that a cryoprotective solution composed of 6% EG, 6% glucose, and 5% skimmed milk powder is a useful protocol for the cryopreservation of S. cuspicaudus semen.The study aimed to evaluate cryo-injury during the cryopreservation in  Sorubim cuspicaudus is a Neotropical catfish belonging to the Pimelodidae distributed in the Magdalena-Cauca and Sinú basins in Colombia and the lake basin Maracaibo in Venezuela [The shovelnose white catfish enezuela . This caenezuela . It alsoenezuela . Low semenezuela . For thienezuela ,4.Sperm cryopreservation is a safe method for storing and preserving genetic material ,6 and a The plasma membrane is the first affected structure during cryopreservation due to chemical composition changes or environmental temperature. It is suggested that spermatozoa’s resistance to the freezing process and media toxicity is influenced by structural membrane lipid and protein chemical composition ,19. The Prochilodus magdalenae [Astrolebias minuano [S. cuspicaudus sperm during cryopreservation. Therefore, this study aimed to evaluate the injuries suffered by S. cuspicaudus spermatozoa at the DNA, mitochondria, and plasma membrane through the cryopreservation process (prefrozen semen and thawed semen) using EG as a cryoprotectant agent to different inclusion percentages .Studies that examine semen damage in the cryopreservation process of Neotropical fish are scarce, but the works on gdalenae  and Astr minuano  stand ouS. cuspicaudus breeders from the Fishculture Research Institute at the University of Córdoba  were kept in 400 m2 in earth ponds at a density of 0.5 kg/m2. The fish were fed twice daily using a commercial diet containing 45% net protein and supplemented with forage fish (Oreochromis niloticus juveniles) once a week.Three-year-old 3 useful volume tanks with steady water flow (5 L/min). The fishes were kept for 48 h to acclimate them to the experimental conditions and reduce the stress from handling and environmental changes.Selected males (n = 18) in the spermiation phase at the reproductive mid-season (July-August) had a total length of 47.9 ± 2.2 cm and weighed 416.9 ± 123.8 g; for females (n = 4) in the final maturation phase, a length of 47.5 ± 29.4 cm and a weight of 592.2 ± 258.7 g was recorded. Selected breeders were transferred to circular-shaped, 3 mSelected males were treated with a single dose of salmon GnRH analog (12 µg/kg of live weight) applied to the pectoral fin base. Twelve hours later, semen was collected into 2.0 mL Eppendorf vials by hand stripping. The urogenital papilla was dried using a blotting towel, and the initial ejaculate was discarded to avoid contamination with water, urine, and feces.®, Barcelona, Spain).For the motility duration, 0.25 µL of fresh semen was placed onto a Makler chamber  and was activated with 75 µL of distilled water (1:300 dilution). The duration time was measured from activated until movement stopped in the spermatozoa . Sperm c®, Barcelona, Spain) linked to a camera of high speed (50 frames/s). Sperm were analyzed no later than five seconds after motility activation. The percentage of rapid sperm (>100 µm/s), medium sperm (45 to 100 µm/s), slow sperm (10 to 45 µm/s) sperm velocities, as well as that non-motile sperm (static) were obtained. The curvilinear (VCL) and linear (VSL) velocities were also estimated. Duration of motility, sperm concentration, total motility, and sperm velocity was run in triplicate to obtain the average from each semen sample analyzed.Analysis of sperm kinetics was performed on fresh, prefrozen (fresh semen-diluted), and thawed semen. The activation of spermatozoa was carried out by distilled water (1:300 dilution), and the parameters total motility and motility types  were recorded by the CASA software   was used as a cryoprotectant agent (CPA) at three inclusion percentages  combined with 6% glucose and 5% skim milk powder. The semen was diluted at a ratio of 1:4 . A 1 mL aliquot was taken from this dilution after five minutes of exposure to cryoprotective solutions (pre-frozen semen) to assess damages and sperm kinetics. In all cases, the semen used in the trials was obtained from a mixture of six males (pool) with total motility greater than 90%. Motility and sperm velocity, damage to plasma membrane (Mem-d) and mitochondria (Mit-d), and DNA fragmentation (DNA-f) were evaluated for prefrozen and thawed semen. The damage was also analyzed in fresh semen (control) to identify possible effects of exposure to cryoprotectant, cryoprotectant inclusion level, and the freezing–thawing process.Diluted semen was packaged in 2.5 mL cryotubes  and frozen in nitrogen vapors into a dry shipping container of 4 L  for 30 min. The cooling rate was 27.3 °C/min from 28 to −20 °C, 29.9 °C/min from −20 to −100 °C, and 5.5 °C/min from −100 to −196 °C . Frozen The osmolarity of the seminal plasma and the cryoprotective solutions at the different EG inclusion rates  was measured with an osmometer .6(3)) (70 nM) and 2 µg/mL propidium iodide (PI) , incubating in the dark for 20 min, and then analyzing the samples by flow cytometry. DiOC6(3) staining allowed us to distinguish cells showing high DiOC6(3) uptake (viable cells) and low uptake . Propidium iodide staining showed sperm membrane alterations. A FACSCanto II flow cytometer  equipped with a 488 nm wavelength laser for excitation, 530/30 nm wavelength detectors for DIOC6(3) fluorescence detection, and 670 nm for PI were used.Mem-d and Mit-d were assessed in fresh, prefrozen, and thawed semen in 1, 10, and 10 µL aliquots, respectively. Sample staining was performed by adding 1 mL of a solution containing 3,3′-dihexyloxacarbocyanine iodide , and thawed sperm taking 10, 100, and 100 µL aliquots, respectively. Samples were fixed using 3 mL of 70% ethanol . Subsequently, they were incubated for 12 h at 20 °C, washed using calcium-free, ultrafiltered BD PBS, vortexed, and centrifuged at 2500 rpm for 10 min at 4 °C to separate semen from the cryoprotective solution. The supernatant was discarded, and the pellet was resuspended in 300 µL of propidium iodide (PI) + RNase A (20 U) for every million cells. The samples were then vortexed and incubated in the dark for 20 min at room temperature. The samples were then analyzed by flow cytometry. To discriminate sperm cell (haploid) from non-sperm cell events (diploid), chicken red blood cells and ram T-cells from the DNA QC commercially available kit  was used as system linearity controls and for the coefficient of variation. Samples were analyzed by a flow cytometer  with a 488 nm solid-phase excitation laser and fluorescence detection at 585/42 nm.In fresh semen samples, membrane (Mem-d) and mitochondria (Mit-d) damage, as well as DNA fragmentation (DNA-f), were assessed fifteen minutes after collection. In prefrozen sperm, parameters were evaluated five minutes after diluting the semen into the extender. For thawed semen, the evaluation was performed immediately after thawing. The results show the spermatozoa percentage exhibiting mitochondrial and membrane damage and haploid cells with fragmented DNA.6 spz/µL) of thawed semen from each treatment, activated using 10 mL of distilled water in 20 mL vials. Incubation was performed in 2 L-capacity experimental incubators with a 0.05 L/min upflow. Fresh semen was used as the control. At five-hour post-fertilization (hpf), when eggs were at the final phase of gastrulation (blastopore closure), a fertilization rate assessment from three aliquots (n = 80–100 eggs) of every incubator was performed. Eggs were placed in Petri dishes, and the viable embryo count was determined using an optical stereoscope  and image analysis software . The appearance of viable embryos was translucent or hyaline, with no detachment of cell material and a normal aspect. On the contrary, non-viable embryos were opaque or whitish [Fertilization and hatching rates were estimated using 1.0 g of oocytes (~1104 oocytes/g) obtained by the induction of females in the final maturation phase using a single dose of salmon GnRH analog (12 µg/kg of live weight). The oocytes were taken from a spawning mixture of two females (pool). Fertilization was performed using 400 μL  was estimated, taking three 80-to-100 embryo aliquots and counting the viable embryos  and non-viable (the opaque and whitish ones). The same equation to estimate the fertilization rate was used. The fertility and hatching test was performed twice. In each trial, the treatments were evaluated in triplicate.3/L), alkalinity (69.8 ± 8.8 mg CaCO3/L), and total ammonia (0.01 ± 0.004 mg/L) from water used for the incubation of eggs fertilized with fresh and thawed semen were within the range usually set for incubating Neotropical fish, such as S. cuspicaudus [Recorded values for dissolved oxygen (7.1 ± 0.7 mg/L), temperature (26.3 ± 0.2 °C), pH (7.5 ± 0.1), hardness (52.7 ± 4.9 mg CaCOpicaudus .p > 0.05) to determine the best treatment. The data were analyzed with the software package SAS ver. 9.1.All variables are presented as the mean ± SD, and previous to analysis, percentage data were standardized by square root transformation. All variables were subjected to one-way analysis of variance (one-way ANOVA) test and then followed by Tukey’s multiple range test (S. cuspicaudus (n = 18) recorded semen volume of 1.6 ± 0.4 mL, sperm concentration of 20,403.3 ± 6740 × 106 spz/mL, and motility duration of 44.3 ± 6.3 s. The seminal plasma osmolarity was 273.3 ± 7.2 mOsmol/kg, while the cryoprotective solutions were 1905 mOsmol/kg for 6% EG and greater than 2000 mOsmol/kg for 8 and 10% EG (equipment measurement limit).Fresh semen of p < 0.05). The total motility in prefrozen sperm decreased from 61 ± 15.3% (6% EG) to 33.8 ± 11.6% (10% EG) (p < 0.05). In thawed sperm, total motility ranged between 35 ± 5.7% (6% EG) and 31.9 ± 2.1% (8% EG), showing no significant difference between these values, even with prefrozen semen to 10% EG (p > 0.05).The total motility, motility types , and sperm velocity  in fresh, prefrozen, and thawed sperm are shown in p < 0.05). In prefrozen semen, rapid motility ranged between 0.9 ± 1.2% (6% EG) and 0.2 ± 0.2% (10% EG) (p > 0.05); in thawed semen, no rapid motility was recorded sperm. Medium motility sperm in prefrozen semen decreased from 16.9 ± 7.2% (6% EG) to 1.7 ± 0.2% (10% EG), showing the difference between these two values (p < 0.05). In thawed semen, medium motility ranged from 1.9 ± 0.6% (6% EG) and 1.3 ± 0.7% (8% EG), and no difference between these values was found, even with prefrozen semen to 10% EG (p > 0.05).In fresh semen, the percentages of rapid (18.6 ± 5.9%) and medium motility sperm (58.0 ± 9.8%) showed a significant difference compared with prefrozen and thawed semen (p > 0.05). In prefrozen semen, slow motility increased (30.4 ± 9.4%–43.3 ± 9.6%), and similar values were observed in thawed semen (30.6 ± 1.6%–33.1 ± 5.2%); no significant difference was observed for slow motility between prefrozen and thawed semen to the different ethylene glycol inclusion percentages (p > 0.05). Static sperm in prefrozen semen increment from 39 ± 15.4% (6% EG) to 66.2 ± 11.6% (10% EG) (p < 0.05). The amount of non-motile sperm (65 ± 5.7%–68.1 ± 2.1%) in thawed semen did not show a significant difference in the different ethylene glycol inclusion percentages, even with prefrozen semen to 10% EG (p > 0.05).In fresh semen, the percentages of slow motility sperm (20.5 ± 5.3%) and static sperm (5.5 ± 3.3%) showed a significant difference with prefrozen and thawed semen (p < 0.05). The prefrozen semen VCL decreased from 34.5 ± 4.2 µm/s (6% EG) to 23.3 ± 3.2 µm/s (10% EG) (p < 0.05); whereas the thawed semen VCL ranged between 23.2 ± 2.6 µm/s (6% EG) and 21.1 ± 1.3 µm/s (10% EG), no significant difference was observed between these values, even with prefrozen semen to 10% EG (p > 0.05). The fresh semen VSL (55.9 ± 8.8 µm/s) showed a significant difference with prefrozen and thawed semen. In prefrozen semen, VSL decreased from 18.5 ± 5.8 µm/s (6% EG) to 7.7 ± 3.6 µm/s (10% EG) (p < 0.05), whereas in thawed semen, VSL ranged between 7.1 ± 1.2 µm/s (10% EG) and 6.9 ± 1.1 µm/s (8% EG), but no significant difference was observed for these values, even with 10% EG prefrozen semen (p > 0.05).The fresh semen VCL (72.9 ± 5.5 µm/s) showed a significant difference with prefrozen and thawed semen (p > 0.05), although there was a difference with the values obtained in the prefrozen semen (p < 0.05).Membrane damage (Mem-d), mitochondrial damage (Mit-d) and DNA fragmentation (DNA-f) in fresh, prefrozen, and thawed sperm are shown in p < 0.05). Mit-d of prefrozen semen increased concerning fresh semen, with values ranging between 16.1 ± 2% (6% EG) to 18.3 ± 5.7% (10% EG), with no showing significant difference for these values (p > 0.05). For thawed semen, however, Mit-d results continued to increase to values between 47.8 ± 5.4% (10% EG) and 51.4 ± 2.7% (8% EG), but no significant difference was observed for these values (p > 0.05). Nonetheless, there was a significant difference in Mit-d values compared to those recorded in prefrozen semen (p < 0.05).In fresh semen, lower percentages of Mit-d were recorded (1.4 ± 0.9%), showing a significant difference with prefrozen and thawed semen (p < 0.05). DNA-f for prefrozen semen ranged between 6.7 ± 2.5% (6% EG) and 9.5 ± 2.6% (10% EG), without a significant difference for these values (p > 0.05). In thawed semen, however, DNA-f values increased from 37.9 ± 2.8% (8% EG) to 44.4 ± 3.4% (6% EG), but no statistically significant difference for these values was shown (p > 0.05). However, there was a significant difference in prefrozen semen (p < 0.05).In fresh semen, lower percentages of DNA-f were recorded (2.4 ± 0.8%), with a significant difference with prefrozen and thawed semen (S. cuspicaudus using fresh semen and thawed semen are shown in p < 0.05). The fertilization rate using thawed semen ranged from 41.6 ± 8.7% (10% EG) to 25.9 ± 9.7% (8% EG), and no statistically significant difference was observed for these values (p > 0.05). Likewise, the hatching rate varied from 22.6 ± 0.7% (6% EG) to 14.1 ± 1.5% (8% EG), with no statistically significant difference observed for these values (p > 0.05).The fertilization and hatching rates of S. cuspicaudus fresh semen used in this study was collected at reproductive mid-season (July–August). Previous studies suggest an adequate semen quality of S. cuspicaudus when it shows total motility greater than 80%, duration of motility in the range between 36 and 46 s and sperm concentration greater than 14.000 million spz/mL [p < 0.05), showing that total motility and sperm velocities decrease as the EG inclusion percentage increases. Lower values for these parameters were achieved when 10% EG was used; however, rapid, medium, and low motility were affected, regardless of the EG inclusion percentage used. In thawed semen, total motility, types of motilities , and velocities  indicate that the sperm was affected by freezing and thawing processes. After thawing, no rapid spermatozoa were registered, the percentage of medium motility sperm decreased, slow and immotile sperm increased, and sperm velocity slowed .Total motility, motility types , and sperm velocity  in prefrozen semen (fresh semen-diluted) were lower compared to fresh semen . Cuevas-Uribe et al.  attributp < 0.05), which shows the harmful effects on semen quality during exposure to CPA. Of all structures assessed, mitochondria were the cell organelle most affected at the prefrozen phase. In this study, the equilibration time was five minutes, and during this time, Mit-d increased 11- to 13-fold compared to fresh semen; however, the Mit-d did not show significant differences (p < 0.05) in the different EG inclusion percentages in the assessed range (6 to 10%). On the other hand, the membrane was the less affected structure (less than 5%) during the prefrozen phase, increasing from 2.4 to 4-fold regarding fresh semen, although the membrane underwent greater damage when 10% EG was included compared to fresh semen (p < 0.05). Fragmentation of DNA in prefrozen semen increased 2.8- to 4-fold compared to fresh semen, regardless of the EG inclusion percentage (6 to 10%). The results suggest that of the cell structures analyzed, mitochondria are more sensitive to damage caused by CPA toxicity and osmotic shock in the prefrozen phase.An increase in Mem-d, Mit-d, and DNA-f was observed in prefrozen semen compared with fresh sperm between the damage of the mitochondria and the EG inclusion percentage tested . Oxidative stress is considered a major cause of mitochondrial damage because it promotes the release of reactive oxygen species (ROS), which affects mitochondrial respiration efficiency [In thawed semen, damage to mitochondria increased 2.6- to 3-fold relative to prefrozen semen and 34- to 37-fold compared to fresh semen, indicating that mitochondria also suffer significant injury due to freezing and thawing. In thawed semen, there was no significant difference . DNA fragmentation affects embryo development and increases early-stage embryo mortality ,40,41. Ip > 0.05), so it might be suggested that EG rate in the range of 6 to 10% shows similar fertilization rates if freezing and thawing conditions like those described in this study are used. Nevertheless, it has been found that an EG included above 10% affects the quality of S. cuspicaudus cryopreserved-thawed semen [Piaractus brachypomus, it was reported that the EG rate of 10% or higher causes a decrease in semen quality, affecting fertilizing capacity [Brycon amazonicus semen with EG above 10%, the quality of the thawed semen was affected [While the ethylene glycol inclusion percentage tested (6 to 10%) affected semen quality both at the pre-freezing and thawing phase, no significant differences were observed regarding the fertilizing capacity of thawed semen  affect its fertilizing capacity. Although sperm DNA-f may exhibit motion and have fertilizing capacity, the embryo would not be viable. However, according to Kopeika et al. , sperm cFertilization is achieved, although the percentage of non-motile spermatozoa in thawed sperm is high. This is possible through sperm–oocyte interactions and a micropylar apparatus in fish oocytes allowing non-motile sperm in the presence of vibration to penetrate the ooplasma, producing oocyte fertilization ,52. ThisS. cuspicaudus semen. Further studies are required to optimize semen cryopreservation methods and examine the injuries that semen suffers in this process, particularly long-term effects, such as larval viability, to increase cryopreservation success and for their potential use in conservation programs.In conclusion, the present study demonstrated that the mitochondria, plasma membrane, and DNA integrity suffer considerable damage both in pre-frozen and in freezing/thawed semen when it is cryopreserved with EG in inclusions between 6 and 10% that affect its fertilizing capacity, which is reduced to half of that obtained with fresh semen. In the pre-freezing phase, the Mit-d and DNA-f are independent of the inclusion level (6 to 10%) when the equilibration time is five minutes, but Mem-d is higher when EG is included at 10% in this phase. In freezing–thawing, the cryoprotectant inclusion percentage (6 to 10%) is independent of the damage it causes to membranes, mitochondria, and DNA; therefore, it is suggested that a cryoprotective solution composed of 6% EG, 6% glucose, and 5% skimmed milk powder is a useful protocol for cryopreservation of


Apis mellifera ligustica remains elusive.Fungi associated with insects represent one potentially rich source for the discovery of novel metabolites. However, a comprehensive understanding of the fungal communities of Apis mellifera ligustica, royal jelly, and honeycomb, which belonged to four classes and eleven different genera. Furthermore, 28 bacterial 16S rRNA gene sequences were obtained by PCR from the fungal metagenome. High-throughput sequencing analyses revealed that the fungal communities were more diverse, a total of 62 fungal genera were detected in the honeybee gut by culture-independent method, whereas only 4 genera were isolated by culture-dependent method. Similarly, 247 fungal genera were detected in the honeycomb, whereas only 4 genera were isolated. In addition, we assessed the antibacterial and antioxidant activities of fungal isolates. Most fungal crude extracts obtained from the cultivation supernatant exhibited antioxidant activities. Only two fungal crude extracts displayed moderate activity against Escherichia coli and Staphylococcus aureus. Chemical analysis of Chaetomium subaffine MFFC22 led to the discovery of three known compounds, including cochliodinol (1), emodin (2), chrysophanol (3). Among them, cochliodinol (1) showed intense DPPH radical scavenging activity with the 50% inhibitory concentration (IC50) of 3.06 μg/mL, which was comparable to that of the positive ascorbic acid (IC50 = 2.25 μg/mL). Compound 2 displayed weak inhibitory activities against Micrococcus tetragenus and S. aureus.Here, we investigated the phylogenetic diversity and community composition of honeybee-associated fungi using combination of culture-dependent and culture-independent approaches. A total of forty-five fungi were isolated and purified from the This research provided a fundamental clue for the complex interactions among honeybees, fungi, bacterial symbionts, and the effects on the honeybee. Furthermore, the diversity of honeybee-associated fungi had great potential in finding the resource of new species and antioxidants.The online version contains supplementary material available at 10.1186/s12866-022-02580-4. Insects are the most abundant group of animals on the Earth. Over one million species of insects have been named and widely distributed in various habitats , 2. The Insect-associated fungi are vital microbial sources of natural bioactive products. The insect-associated fungi isolated from arthropod cadavers, leaf-cutting ants, and stoneflies were contained new microbial species –13. InseMonascus inside the brood cell −, calculated for C32H30N2O4 505.2128; 1H NMR  δ: 8.42 , 8.14 , 7.60 , 7.44 , 7.34 ,7.09 , 5.40 , 3.48 , 1.76 , 1.74 ; 13C NMR : 134.3 s (C8´), 133.7 s (C5´), 131.6 s (C12´), 127.1 d(C2´), 126.2 s (C9´), 124.4 d (C11´), 123.4 d (C6´), 120.8 s (C4´),111.0 d (C7´), 110.7 s , 104.2 s (C3´), 34.6 t (C10´), 25.6 q (C14´), 17.8 q (C13´). Due to the resonance of the benzoquinone ring, the carbonyls resonances  of cochliodinol were not observed; these carbons can only be observed in THF-d8 or CDCl3 at -75 ℃ −, calculated for C15H9O5, 269.0450; 1H NMR  δ:12.21 , 12.08 , 10.15 , 7.59 , 7.28 , 7.16 , 6.68 , 2.48 ; 13C NMR : 191.6 (C9), 182.1(C10), 166.5 (C1), 166.3 (C3), 163.3 (C8), 148.8 (C6), 136.9 (C12), 134.5 (C14), 125.0 (C7), 121.7 (C5), 113.8 (C13), 109.8 (C11), 109.7 (C4), 109.0 (C2), 22.2 (CH3).Emodin (3): orange crystals; HR-ESI–MS: m/z: 253.0521 [M—H]−, calculated for C15H9O4, 253.0501; 1H NMR  δ: 12.12 , 12.02 , 7.83 , 7.68 , 7.66 , 7.30 , 7.11, 2.47 .Chrysophanol  of cochliodinol (1) on DPPH scavenging activity was 3.06 μg/mL, which was comparable to that of positive ascorbic acid (2.25 μg/mL).The antioxidant activities of compound A. mellifera ligustica, the leading research mainly focused on the honeycomb, honeybee gut, pollen, and bread [A. mellifera ligustica. Forty-five fungi were isolated and characterized by the dilution-plate method and molecular biological identification. Furthermore, three metabolites were purified and characterized from C. subaffine MFFC22. Consequently, honeybee-associated fungi can provide a resource for microbial diversity and natural products.Microbial community is extensive among insects. As a special kind of microbial community, insect-associated fungi have been a source of new microbial resources , 30 and nd bread , 33–36. I. lacteus was isolated and identified from honeybee gut, head, hypopharyngeal gland, larvae, and royal jelly in our study. It suggests that the fungus is widely distributed in A. mellifera ligustica. Some fungi isolated from honeybee larvae, hypopharyngeal gland, honeycomb, and royal jelly were wood-rotting fungi, such as T. hirsuta, S. commune, B. adusta, and L. lactinea [Arthrinium isolated from honeybee gut and larvae has been reported as a plant pathogen [I. lacteus and P. brasiliense were also isolated from termites, and F. solani was isolated from beetles [Aspergillus, Alternaria, Chaetomium, Fusarium, and Arthrinium genera have already been found in the honeybee. For example, Aspergillus, Chaetomium, Fusarium, and Alternaria sp. were isolated from honeybee gut, and Arthrinium sp. was isolated from pollen [T. hirsuta and S. commune from both honeycomb and royal jelly, I. lacteus from all parts of honeybee samples. Additionally, one rare fungus P. brasiliense was found in honeybee cuticle, which was reported previously in the gut of Acrida cinerea [The same fungus of lactinea –39. Besipathogen . Compare beetles , 42. Aspm pollen , 33–35.  cinerea .Kodamaea, Zygosaccharomyces, Wallemia, and Wickerhamomyces were predominant genera. Previous study based on pyrosequencing of ITS region of honeybee gut samples derived from Korea revealed that Saccharomyces and Zygosaccharomyces were dominant [Starmerella and Hanseniaspora had high relative abundance in honeybee gut samples collected from Italy and Saudi Arabia [Aspergillus in honeybee gut, Trametes, and Chaetomium in honeycomb. However, culture-independent approaches in general revealed higher microbial composition and diversity compared to culture-dependent method. For example, 247 fungal genera were detected in the honeycomb by culture-independent method, whereas only 4 genera were isolated. Interestingly, as the causative agent of chalkbrood disease in honeybee Reserved literature [Ascosphaera spp. was detected in honeybee gut and honeycomb by culture-independent approach, but it has not been isolated yet.A total of 62 fungal genera were discovered in honeybee gut by culture-independent method. Among them, dominant . Howeveri Arabia . Previouterature , AscosphC. subaffine MFFC22 isolated from honeycomb has shown notable antioxidant activity. Chaetomium genus is already known to have strong DPPH scavenging activity, such as C. nigricolor, C. globosum, C. cruentum [C. subaffine and its metabolites have not reported DPPH scavenging activity. The compound 1 from MFFC22 showed strong DPPH scavenging activity with the IC50 values of 3.06 μg/mL, comparable to the positive ascorbic acid (Vc). Comparing a commercial antioxidant BHT with IC50 of 95.7 μM, Dehghan et al. found that compounds 2 and 3 also exerted moderate antioxidant activities against DPPH with IC50 of 271.2 and 297.0 μM, respectively [1–3 may be attributed to their phenolic hydroxyl groups. Additionally, it was reported that antioxidant compounds produced by fungi could protect their hosts by enhancing tolerance to abiotic stresses [1–3 have similar effects deserves further exploration. In addition, these isolated compounds have been shown to have other bioactivities. For example, compound 1 was a common fungal metabolite with cytotoxic activities against the KB, MDA-MB-435 and MRC5 cell lines [2–3 were anthraquinone derivative and acted as an antimalarial and antiallergic agents [The DPPH radical scavenging assay indicated that most crude extracts from honeybee-associated fungi showed antioxidant activities. Notably, the crude extract of cruentum , 44, 45.ectively . The pheectively . Therefostresses . Whetherll lines . Compounc agents .Symbiotic bacteria have occurred in some fungi, which affected fungi function and subsequent host-fungus interactions. However, only a few studies have explored their effects in insect-associated fungi , 53. HerA. mellifera ligustica. The results show that several of these fungi have antibacterial and antioxidant activities, among which the fungus C. subaffine MFFC22 was the most prominent. Furthermore, the antioxidant activities of C. subaffine MFFC22 can be attributed to the identified phenolic compounds. Collectively, the culturable honeybee-associated fungi provide insight into the widespread insect symbionts, which have great potential in finding the resource of novel species and antioxidants.Here, the diversity of the honeybee-associated fungi was investigated using both culture-dependent and culture-independent analysis methods. This study expands our knowledge of honeybee-associated fungi and further raises the pool of fungal species from A. mellifera ligustica , honeycomb, and royal jelly were collected from the Institute of Apicultural Research, Anhui Agricultural University, Hefei, China  between April and July 2021. The honeybee larvae and adults starved for 24 h, and all samples were stored at 4 ℃. Initially, seven larvae, seven adults, and one gram honeycomb were placed separately into 10 mL sterile phosphate-buffered saline (PBS) buffer solution in an autoclaved 50 mL tube to obtain fungi from external isolation. Then, the same tube from the external was filled with 10 mL 75% ethanol for 2 min, followed by rinsing in sterilized 10 mL 1% bleach with 0.1% tween 20 for three times (30 s each). The supernatant was removed and replaced with 10 mL sterile PBS solution. Subsequently, sterile forceps were used to dissect samples of adult honeybee to get head, gut, and hypopharyngeal gland. Each body part was grounded in 10 mL sterile PBS. According to the earlier report, the honeybee larvae, one gram honeycomb, and one gram royal jelly were fully homogenized separately in 10 mL sterile PBS [−1, 10−2, 10−3), and aliquots of 100 µL from each dilution were spread onto nine isolation media  with the barcode. The reaction conditions were 98℃ for 1 min, followed by 30 cycles of 98℃ for 10 s, 50℃ for 30 s, and 72℃ for 30 s, finally 72℃ for 5 min. Mixture PCR products were purified using Qiagen Gel Extraction Kit . Sequencing library was generated using TruSeq® DNA PCR-Free Sample Preparation Kit  according to manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina NovaSeq platform using 250 bp pairedend reads.Raw reads were demultiplexed and quality-filtered using QIIME V1.9.1 . EffectiEach fungus was inoculated in PDA medium and incubated at 28 ± 0.5 ℃for 3–4 days. Then, the fresh mycelia of each fungus (9 plugs of 5 mm) were inoculated in a 250 mL Erlenmeyer flask containing 150 mL of ME liquid medium and incubated at 28 ± 0.5 ℃ for 7 days in a shaker rotating at 180 rpm. The culture was passed through four layers of cheesecloth to remove the fungal thallus, then the supernatant was exhaustively extracted three times with ethyl acetate . The fungal crude extracts were concentrated in a vacuum to yield culture extract for further experimental use.Escherichia coli (ATCC8739), Micrococcus tetragenus (ATCC35098), and Staphylococcus aureus (ATCC6538) were selected for the test and cultivated on tryptic soy blood agar (TSBA) medium in 37 ℃. Next, sterile filter paper discs (5 mm in diameter) were added to 5 µL of the tested crude extracts, which were dissolved separately in acetone to get a concentration of 18 mg/mL. The filter paper disc treated with acetone alone and gentamicin sulfate was served as a negative and positive control, respectively. The plates were prepared in triplicate and were cultivated at 37 ℃ for 24–36 h. Finally, the diameters of inhibition zone diameter (in mm) were measured for evaluating antimicrobial activity.The filter paper disc method was used to screen the antibacterial activity of crude extracts . Three bThe 2,2-diphenyl-1 picrylhydrazyl (DPPH) radical scavenging activity was conducted according to the previous method with some modifications . 1 mL ofb- As) /Ab*100%Scavenging rate (SR) (%) = , A2Cl2/MeOH  to give six fractions (Fr1-Fr6). Compound 1 (15 mg) was crystallized from the CH2Cl2 solution from the Fr1 . Fr1 was further fractionated on a silica gel column, eluting with  to give compounds 2 (0.9 mg) and 3 (0.7 mg).A total of 13 L of the culture broth of MFFC22 was filtered and extracted with EtOAc (3 × 13 L). The EtOAc phase was concentrated under reduced pressure to obtain crude extract (2 g). The crude extract was subjected to column chromatography (CC) using silica gel (100–200 mesh) column with a gradient of CH1H/13C-nuclear magnetic resonance (NMR) spectroscopies. HR-ESI–MS spectra were measured on a TripeTOF 4600 mass analyzer . 1H/13C NMR data were acquired using Agilent DD2 600 Hz spectrometer  with tetramethylsilane (TMS) as an internal standard, and chemical shifts (δ) were reported as parts per million (ppm) values.The structures of all compounds were primarily analyzed by High Resolution-Mass Spectrometry (HR-ESI–MS) and The raw data supporting the conclusions of this manuscript will be made available to the authors, without undue reservation, to any qualified researcher. Publicly available datasets were analyzed in this study. This data can be found here: The obtained ITS gene sequences were deposited in the GenBank database under accession numbers OK184563-OK184606 and OK285068. The obtained 16S rRNA gene sequences were deposited in the GenBank database under accession numbers OK147622-OK147645 and OK169608-OK169611. Data of high-throughput sequencing by paired-end Illumina technology of ITS 1 gene amplicons can be retrieved from the NCBI Short Read Archive under accession number PRJNA817087 and PRJNA817099.Additional file 1: Table S1. Taxonomic assignment of the OTUs obtained in the study and their relative abundance at each honeybee gut.Additional file 2: Table S2. Taxonomic assignment of the OTUs obtained in the study and their relative abundance at each honeycomb.


The terminal phenoxy group is a moiety of many drugs in use today. Numerous literature reports indicated its crucial importance for biological activity; thus, it is a privileged scaffold in medicinal chemistry. This review focuses on the latest achievements in the field of novel potential agents bearing a terminal phenoxy group in 2013–2022. The article provided information on neurological, anticancer, potential lymphoma agent, anti-HIV, antimicrobial, antiparasitic, analgesic, anti-diabetic as well as larvicidal, cholesterol esterase inhibitors, and antithrombotic or agonistic activities towards the adrenergic receptor. Additionally, for selected agents, the Structure–Activity–Relationship (SAR) is also discussed. Thus, this study may help the readers to better understand the nature of the phenoxy group, which will translate into rational drug design and the development of a more efficient drug. To the best of our knowledge, this is the first review devoted to an in-depth analysis of the various activities of compounds bearing terminal phenoxy moiety. In 2021, the Food and Drug Administration (FDA) approved 50 new drugs . The othOne method of optimizing the lead structure is to introduce new substituents, which resulted in different shape, conformation, and created interaction with the drug target ,9. It maThe phenoxy group is a key pharmacophore component for many drugs that are used today, such as viral drugs ,12, prosIn our review, we focused on the latest achievements in the field of novel potential agents bearing a terminal phenoxy group in 2013–2022. The first part is a compilation of the currently FDA-approved drugs bearing the terminal phenoxy group, the second part concerns the ingredients of cosmetics, and, in the final part, the most promising compounds from most recent studies are presented.In the previous section, we mentioned some classes of drugs bearing the phenoxy group. Now, we present a comprehensive list of drugs currently used in treatment, broken down into groups by biological activity. Drugs for treatment neurological disorder, antiviral, cardiac, analgesic, antimicrobial, anti-cholesterol, diuretic, anti-leukemia, and other drugs with various biological activities are presented in Cosmetics, vaccines, and pharmaceuticals contain preservatives. Therefore, they keep their properties and activity longer. Triclosan and 2-phenoxyethanol are commonly used as antimicrobial ingredients ,17. PresThe latest developments in the field of new potential terminal phenoxy agents in 2013–2022 are presented below. The paper presents information on neurological, anti-cancer, potential lymphoma, anti-HIV, anti-microbial, anti-parasitic, analgesic, anti-diabetic as well as larvicide, cholesterol esterase inhibitors, anticoagulants or adrenergic agonists. Additionally, for selected structures, the Structure–Activity–Relationship (SAR) is also discussed.In 2016, neurological disorders were the second most common cause of death . On the 1) exhibited the highest inhibitory activity against RAGE (83.1 ± 0.5%) with a half-maximal inhibitory concentration (IC50 = 1.9 µM). Studies of directed binding indicated that the modifications improved the binding strength with RAGE, which resulted in an increase in activity. The most active compound bore 4-trifluorophenoxy moiety ), identified crucial pharmacophore elements for inhibitors of Aβ aggregation . The KLVde chain . The comcompound .1A, 5-HT2A, 5-HT3, 5-HT6, and 5-HT7 in MDD or anxiety, as well as in antidepressant and anxiolytic effects GTP γS 5-HT1A pD2 = 9.41) and may be useful in the treatment of disorders related to this receptor. The authors noted the possibility of creating hydrogen bonds with the ether oxygen atom of the phenoxy group. Moreover, they stated that the function of the ether oxygen atom of the phenoxy moiety is presumably favoring the optimal arrangement of the phenyl ring at the binding site and is never involved in direct interactions in complexes with the open model. The most active compound bore 2-methoxy(metoxy)phenoxy substituent  pyrimidine-based dual EGFR/HER2 inhibitors . The epiy moiety .24) exhibited the highest activity against the A549 lung cancer cell, HepG2 hepatocellular carcinoma cell, A498 kidney cancer cell line, CaSki cellosaurus cell line, and SiHa squamous cell carcinoma with IC50 = 6.6 ± 0.6 μM; 6.9 ± 0.7 μM; 6.8 ± 0.8 μM; 7.5 ± 0.5 μM and 7.8 ± 0.4 μM, respectively, as compared to the reference standard—5-Fluorouracil with IC50 = 7.4 ± 0.5 μM, 8.3 ± 1.8 μM, 5.4 ± 0.7 μM, 7.3 ± 0.4 μM, and 8.3 ± 0.7 μM, respectively. Furthermore, the proposed compound (24) downregulates metalloproteinase 2 (MMP-2) and metalloproteinase 9 (MMP-9) and thereby impaired metastatic cancer cell migration and invasion. The most active compound bore 2,4-diisopropylophenoxy moiety pyrimidin-4-amine derivatives as novel anti-arthritic agents pyridine-3-carboxyamides . Compouny moiety .According to UNAIDS data, more than 36 million people have died from AIDS-related diseases since the epidemic began. In 2020 alone, over 1.5 million people became infected with HIV . This inThe dominance of the 4-substituted-2,6-dimethylphenoxy moiety is clearly observed, which may prove its key importance for anti-HIV activity. The novel potential anti-HIV agents bearing a phenoxy group are presented in 55) did not exert the highest activity against MT-4 cells infected by wild-type HIV-1 strain IIIB with an EC50 = 0.056 μM but possessed the best selectivity index (SI = 1251). It is about 2-fold higher than the highest activity compound from reported series. Even if it was not the most active, it still has significant activity compared to Nevirapine and Delaviridine with an EC50 = 0.23 μM and 0.51 μM, respectively. The activity of proposed compound (50) was also tested against wild-type HIV-1 reverse transcriptase (RT) with an IC50 = 6.9 μM, which is comparable to the reference standard—TMC125 with an IC50 = 6.5 μM. Reverse transcriptase enzyme is responsible for the catalytic synthesis of viral DNA from the viral ssRNA genome, which enables replication; therefore, it is a valuable molecular target for therapy pyrazine derivatives as HIV-1 non-nucleoside RT inhibitors . Compouny moiety .61) possessed the highest activity against wild-type HIV-1 with an EC50 = 35 nM, as compared to the reference standard—Delavirdine with an EC50 = 33 nM. Furthermore, antiviral activity of compound (62) against the K103N mutation was about 140-fold higher with an EC50 = 49 nM, as compared to the reference standard—Nevirapine with an EC50 = 6.78 µM. Authors stated that substituted phenoxy moiety is a crucial part of pharmacophore responsible for hydrophobic interaction. The most active compounds bore 2,4,6-trimethylphenoxy and 4-cyano-2,6-dimethylphenoxy moiety [Yang et al. obtained novel diarylpyridine derivatives targeting the entrance channel of HIV-1 non-nucleoside RT . Compouny moiety .63) exhibited the highest activity with an EC50 = 0.11 µM and 2.18 µM against HIV-1 IIIB and K103N/Y181C double mutant HIV-1 strain (RES056), respectively, as compared to the reference standard—Nevirapine with an EC50 = 0.28 ± 0.038 μM and >15.02 μM, respectively. Inhibitory activity of (63) against HIV-1 RT was over 100-fold more potent with an IC50 = 0.0727 μM, as compared to the reference standard—Nevirapine with an IC50 = 8.8 μM. The cyano group at the para position of the phenoxy group is crucial for high activity. Authors stated that phenoxy moiety is responsible for hydrophobic interaction. The most active compound bore 4-cyano-2,6-dimethylphenoxy moiety [Meng et al. obtained novel diarylpirimidine derivatives as a potent HIV-1 non-nucleoside RT inhibitor . Compouny moiety .Trypanosoma brucei. If left untreated, the disease is almost always fatal [The Anopheles mosquito transmits Plasmodium parasites that cause malaria. Estimates from 2017 showed that over 219 million people were affected by malaria, and over 400,000 died . Anotherys fatal . The nov64) turned out to be the most active against Chloroquine resistant strain P. falciparum with an IC50 = 0.09 µM and (65) against Chloroquine—sensitive strain P. falciparum with an IC50 = 0.03 µM, as compared to the reference standard—Chloroquine with an IC50 = 0.20 µM and 0.03 µM, respectively. Compound (65) also had the same high activity against the last target as (64). These results were confirmed by molecular docking to β-keto acyl −ACP synthase I/II (KAS I/II), which is responsible for the synthesis of fatty acids. They are crucial for the membrane biosynthesis, which is necessary for the formation of the invasive stage. Therefore, this enzyme represents a promising target for new antimalarial drugs. Furthermore, authors stated that the para-substituted phenoxy group is stabilized by hydrophobic interactions with active site residues of the enzyme. The fluorine atom in the para position of the phenoxy moiety is found in the most active compounds [Sainy and Sharma obtained novel thiolactone derivatives as a novel antimalarial agent . Compounompounds .66) exhibited anti-leishmanial activity against L. (V.) panamensis and T. cruzi with an EC50 = 3.82 ± 0.19 μM and 8.25 ± 1.21 μM on intracellular amastigotes, as compared to the reference standard—Triclosan and Benznidazole with an EC50 = 38.61 ± 2.38 μM and 40.3 ± 6.92 μM, respectively. The most active compound bore 2--5-chlorophenoxy moiety [Otero et al. obtained novel Triclosan-caffeic acid hybrids . Proposey moiety .67) had high activity against epimastigote and trypomastigote forms of T. cruzi (Dm28c strain) with an IC50 = 1.42 ± 0.07 μM and 0.65 ± 0.10 μM, respectively, as compared to the reference standard—Nifurtimox with an IC50 = 21.05 ± 0.90 μM and 10.00 ± 0.40 μM, respectively. Authors stated that phenoxyquinones can be considered as new anti trypanosomical agents with lower cytotoxicity against normal cells. The most active compound bore phenoxy moiety [López-Lira et al. obtained novel benzimidazolequinones as potent trypanosomicidal agents . Proposey moiety .68) exhibited the highest activity against the clinically relevant form of T. brucei with an IC50 = 0.38 μM. The introduction of electron donating groups such as NHAc, CH3, and the OCH3 to the phenoxy group resulted in increased stability of the derivatives. The methoxy substituent in the para position of the phenoxy moiety is found in the most active compound [Prati et al. obtained novel 2-phenoxy-1,4-anthraquinone derivatives . Compouncompound .Pain is one of the most common symptoms that patients seek medical attention for. Despite the breakthrough discovery of opioids, salicylic acid, etc. several decades ago, first-line drugs still do not have long-term efficacy, e.g., long-term opioid use results in hyperalgesia. The complexity of the pain pathomechanism is a major challenge for medical chemists, so it is imperative to continue looking for potential drug agents that will provide a response to this problem . The nov69) was selectively potent over a mentioned target with an IC50 = 4.6 nM. Authors stated that an orto- or meta- phenoxy group substituent capable of forming hydrogen bonds is essential for CSF1R activity. The methoxy group at the 4-position of the phenoxy group (69) formed a hydrogen bond with Lys612. The compound that bore 4-methoxyphenoxy moiety was the most active [Farag et al. obtained a novel colony-stimulating factor receptor (CSF1R) inhibitor based on a phenoxypyrimidine scaffold . CSF1R it active .70) possessed the most promising activity against COX-1 and COX-2 with an IC50 = 11.107 µM and 0.095 µM, respectively. Furthermore, it exhibited the highest COX-2 selectivity index (SI = 116.91). Authors stated that bromo substituent at the para position at phenoxy moiety improved selectivity index, and it was found in the most active compound. A molecular docking study of binding (70) with COX-2 revealed π–π interaction between the phenoxy group and TYR385 and TRP387 [Pallavi et al. obtained novel N-(2-phenoxyacetyl) isonicotinohydrazide derivatives as anti-inflammatory and analgesic agents . Cyclooxd TRP387  , which bore a 4-ethoxyphenoxy substituent, was found to be the most active with an IC50 = 28 nM. Furthermore, compound (71) was also tested in vivo in a Lipopolysaccharide endotoxic shock model in male BALB/c mice, which resulted in a decreasing level of IL-1β in plasma, brain, liver, and lung tissue [Carrasco et al. obtained novel 2,3,5-trisubstituted pyridine analogs as potent inhibitors of Interleukin-1β (IL-1β) . IL-1β ig tissue .72) exhibited 75.17 ± 4.37% and 72.29 ± 3.80% of inhibition anti-inflammatory activity after 3 and 4 h, respectively, and 67.85 ± 2.30% of inhibition of analgesic activity in the mouse model. The SAR study indicated that halogen substituents such as bromo or chloro atom at the para position of phenoxy moiety proved to be crucial for high biological activity and the selectivity index. Compound (73) exhibited the highest activity against COX-I with an IC50 = 232.68 µM and SI = 68.43. A molecular docking study revealed that phenoxy moiety is responsible for π–π stacking with TYR355 in COX-1 [Al-Ostoot et al. obtained novel caffeic acid analogous as potent anti-inflammatory agents . Derivatin COX-1 .Fatty acid amide hydrolase (FAAH) is an important enzyme in endocannabinoid metabolism that inactivates anandamide by splitting it into arachidonic acid and ethanolamine. Thus, it interrupts the anesthetic and anti-inflammatory effect, making FAAH inhibitors a new class of painkillers and anti-inflammatory agents .74) possessed the highest inhibitory potency against FAAH with an IC50 = 0.046 μM, as compared to the reference standard—URB597 with an IC50 = 0.06 μM. The most active compound bore unsubstituted phenoxy moiety [Dahlhaus et al. obtained piperidine carbamates as FAAH inhibitors . Compouny moiety .75) exhibited the highest activity against human FAAH with an IC50 = 1.0 ± 0.5 nM. The most active compound bore 4-trifluorophenoxy moiety [Keith et al. obtained a series of heteroaryl piperazinyl-urea as FAAH inhibitors . Compouny moiety .76) exhibited the highest activity against the FAAH receptor with an IC50 = 0.008 μM. Related to the metabolic study, the fenoxycarbamate moiety was supposed to prevent the compound from being quickly deactivated in the body. The most active compound bore 2-(phenoxy)phenoxy moiety [Sundermann et al. obtained novel 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of FAAH . Compouny moiety .77) exhibited the best inhibitory activity against FAAH with an IC50 = 0.055 μM. The most active compound was bore a 4-(phenoxy)phenoxy group [76) and (77) possessed significantly higher activity than other FAAH inhibitors previously presented, which may be related to phenoxyphenoxy moiety.Althaus et al. obtained novel 1-(5-carboxyindazol-1-yl)propan-2-ones as FAAH inhibitors . Compounxy group . CompounDiabetes is characterized by a disturbance in the secretion or action of insulin resulting in hyperglycemia. Uncontrolled diabetes can lead to a coma and even death from ketoacidosis . WHO rep78) exhibited the highest activity with an IC50 = 0.035 μM. In the diet induced obese (DIO) mice model, the proposed compound resulted in a significant improvement in insulin tolerance and a decrease in body weight. The most active compound bore 3,5-difluorophenoxy moiety [Desai et al. obtained a novel 1,3-difluorophenoxy derivative with an inhibitory activity of neutral amino acid transporter B0AT1 . B0AT1 iy moiety .79) exhibited superior activity in stimulating glucose consumption (GC = 151.8 ± 4.0%). Compounds with the phenoxy moiety were generally more active than the series with the 4-aminopiperidinyl moiety. Moreover, the proposed compound had no inhibitory activity against mitochondrial complex I and did not cause the cardiac hypertrophy characteristic of this group of compounds. This proves it has a higher safety profile. The most active compound bore 4-chlorophenoxy moiety [Sun et al. synthesized novel adenosine monophosphate-activated protein kinase (AMPK) activators as a potential therapeutic approach to metabolic diseases . AMPK paion GC = .8 ± 4.0%80) possessed the highest potency for recombinant human glucokinase activation with an EC50 = 0.034 ± 0.01 μM. Studies in mice indicated a beneficial effect on glucose metabolism with a reduced risk of hypoglycemia. The most active compound bore 2,4-difluorophenoxy moiety [Deshpande et al. obtained novel 2-phenoxy-acetamide derivatives as glucokinase activators . The gluy moiety .81) exhibited an IC50 = 0.487 μM against PTP1B. The authors noted that the hydrophobic tail of the phenoxy moiety allowed for high selectivity with good membrane permeability and provided novel class of non-insulin-dependent drugs in the treatment of type 2 diabetes. The most active compound bore 4-tert-butylphenoxy moiety [Li et al. obtained novel catechol derivatives as selective inhibitors of protein tyrosine phosphatases 1B (PTP1B) . PTP1B ay moiety .Phenoxy derivatives also had larvicidal and anti-creep activity. Within this group of derivatives, cholesterol esterase inhibitors and adrenergic receptor agonists are known. The agents with previously mentioned biological activity, bearing a phenoxy group, are presented in 82) possessed the highest larvicidal activity with a half-maximal lethal dose (LC50 = 5.8 μM/L). A double-chlorine compound, especially as a meta and para substituent of phenoxy moiety, significantly increased the activity [Da Silva et al. obtained a series of phenoxymethyl-thiosemicarbazones as potent larvicidal agents . Compounactivity .83) exhibited inhibitory effects against CEase with an IC50 = 0.36 μM. A molecular docking study revealed π–π interaction between phenoxy moiety and residues Trp227 and Phe324. The most active compound bore a 4-trifluoromethylphenoxy group [Zhao et al. obtained a selective cholesterol esterase (CEase) inhibitor . Proposexy group .1 receptor antagonists [1 and P2Y12 receptors and are involved in the regulation of hemostasis and thrombosis. Compound (84) turned out to possess the highest antagonist potency against P2Y1 with an IC50 = 0.21 μM. Authors stated that most of the structure optimization of potent P2Y1 receptor antagonist focuses on optimization of the phenoxy group. The most active compound bore 2-tert-butylphenoxy moiety [Peng et al. obtained novel 2-(phenoxyaryl)-3-urea derivatives as novel P2Yagonists . Adenosiagonists . Plateley moiety .H-thiochromene-1,1-dioxide derivatives as potent α1D adrenoceptor antagonists [90) possessed the highest affinity potency with Ki = 0.3 nM for an α1D receptor, as compared to the reference standard with Ki = 1.1 nM. The most active compound bore 2,5-difluorophenoxy moiety [Sakauchi et al. obtained novel 3,4-dihydro-2agonists . The auty moiety .The terminal phenoxy moiety is essential for the activity and pharmacological properties of a large group of drugs. In the case of potential agents for the treatment of neurodegenerative diseases, the phenoxy moiety can mimic amino acid residues, form hydrogen bonds thanks to the ether oxygen atom, which also allows a better fit to the binding pocket, or expand the hydrophobic interactions such as π–π stacking. π-stacking, H–π interactions, and increased selectivity, which translates into lower toxicity, are functions of the phenoxy moiety for agents with anti-cancer activity and BTK inhibitors. In the case of agents with potential antimicrobial activity, the 4-chlorophenoxy group clearly dominates, which is mainly responsible for hydrophobic interactions. 4-substituted-2,6-dimethylphenoxy moiety performs the same function for agents with anti-HIV activity. In the case of antiparasitic agents, the phenoxy moiety also extends the hydrophobic interactions, and its substitution with electron donating groups increases the stability of the new derivatives. Hydrogen bond, π–π stacking, or an increase in metabolic stability are the effects of the phenoxy group in anesthetic agents. Moreover, the presence of two phenoxy groups as a phenoxyphenoxy moiety significantly increases the activity of the compound. The hydrophobic tail of the phenoxy group allows for high selectivity with a simultaneous good membrane permeability, and these are the effects of the phenoxy group on antidiabetic agents. Thus, the terminal phenoxy group has enormous potential for the future of medical chemistry. Therefore, future syntheses of new agents should contain a phenoxy moiety, which could lead to the discovery of new drugs.


In this work, the chemical constituents in ZGC and its metabolic profile in rats were both identified by high ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The network pharmacology was applied to predict the potential detoxifying ingredients of ZGC. As a result, a total of 115 chemical compounds were identified or tentatively characterized in ZGC aqueous extract, and 232 xenobiotics (70 prototypes and 162 metabolites) were identified in serum, heart, liver, kidneys, feces, and urine. Furthermore, 41 compounds absorbed in serum, heart, liver, and kidneys were employed for exploring the detoxification of ZGC by network pharmacology. Ultimately, 13 compounds  and nine targets were anticipated to be potential mediums regulating detoxification actions. The network pharmacology analysis had shown that the ZGC could detoxify mainly through regulating the related targets of cytochrome P450 and glutathione. In summary, this study would help reveal potential active ingredients in vivo for detoxification of ZGC and provided practical evidence for explaining the theory of traditional Chinese medicine with modern technology.Licorice is well known for its ability to reduce the toxicity of the whole prescription in traditional Chinese medicine theory. However, honey-fired licorice (ZGC for short), which is made of licorice after being stir-fried with honey water, is more commonly used for clinical practice. The metabolism  Glycyrrhizae Radix et Rhizoma, also named Gancao in Chinese or licorice in English, is the dried root and rhizoma of Glycyrrhiza uralensis Fisch. or G. glabra L. or G. inflata Bat. from the family Leguminosae . Next, the prototypes and phase Ⅰ metabolites identified in serum, heart, liver and kidneys were screened for network pharmacology to explore the potential ingredients of detoxification in ZGC. The workflow was shown in In the present study, a fast and selective ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was applied to characterize the chemical compounds of ZGC aqueous extract and metabolic profile ZGC (batch number: 181201191) was obtained from Kangmei Pharmaceutical Limited Corporation Co., Ltd . All ZGC samples were authenticated by associated professor Haibo Huang .The reference standards for liquiritin (batch number: RP200207), liquiritin apioside (batch number: RP200107), ononin (batch number: RP200209), isoliquiritin (batch number: RP190308), liquiritigenin (batch number: RP200702), isoliquiritigenin (batch number: RP210115), glycyrrhizic acid (batch number: RP190517), glycyrrhizic acid ammonium salt (batch number: RP201104), and glycyrrhetinic acid (batch number: RP190512) were obtained from Chengdu Madsen Technology Co., Ltd. Chromatographic grade methanol (batch number: I1077207008), chmatographic acetonitrile (lot number: JB079830) and chromatformic acid (batch number: H158A) were purchased from Honeywell Trading Co., Ltd . Purified water was supplied by Watsons . Oasis HLB solid-phase extraction (SPE) cartridge  was brought from Waters Technologies Co., Ltd. .All the individual reference standard stock solutions in methanol were prepared with appropriate concentration, Then, the stock standard solutions were further diluted appropriately by methanol to prepare the mixed standard solution. The concentrations of liquiritin, liquiritin apioside, ononin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhizic acid ammonium salt, and glycyrrhetinic acid were 54.2, 99.8, 102.1, 87.3, 89.0, 56.3, 106.3, 79.3, and 102.3 μg/ml, respectively.ZGC (30 g) was weighed and soaked in 300 ml water for 30 min at room temperature. After boiling for 2 h, the extract solution was filtered and concentrated to 50 ml. Finally, the ZGC aqueous extract at a concentration of 0.6 g/ml was prepared.Twelve healthy male rats weighing 220–250 g were obtained from the Experimental Animal Center of Guangzhou University of Chinese Medicine . This study was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. 20190926003). The animals were bred in the Class SPF Experimental Animal Room of Guangzhou University of Chinese Medicine for a week. All of the rats were kept in an environment at a temperature of 20 ± 2°C with the dark-light for 12 h. Food and water were provided spontaneously to rats for 2 weeks. Before the experiment, these animals were fasted with free access to water in cages separately overnight. Twelve rats were randomized into two groups: blank and ZGC-dosed group. ZGC aqueous extract was given to rats at a dosage of 7.2 g/kg twice a day  for thren = 3) (The blood samples of rats (n = 3)  were coln = 3) was sacrificed at the last time point. The heart, liver, and kidneys were removed and washed with normal saline. The tissue samples (0.1 g) were homogenized in 1 ml normal saline under the condition of ice bath. The mixture was then centrifuged at 12,000 rpm for 10 min at 4°C to obtain the supernatant. Then, the 4-fold volume of methanol was added to the supernatant solution obtained to precipitate protein, followed by vortexing for 2 min and centrifuging at 12,000 rpm for 10 min. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 200 μl of 50% acetonitrile and vortexed for 1 min and centrifuged at 12,000 rpm for 10 min. Subsequently, 1 μl supernatant was injected into the UPLC-Q-TOF-MS system for analysis.Each group of rats (n = 3) were collected from 0 to 12 h and 12–24 h after administration. The pooled urine sample (6 ml) was centrifuged at 4,000 rpm for 10 min. Then the 6 ml supernatant urine samples were added to pre-treated SPE columns and eluted with 0.1% formic acid and methanol, and washed with pure water once which would be discarded then. Then 0.1% formic acid and methanol were applied as the eluent for elution, with the further elution solution being collected. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 200 μl of 50% acetonitrile, vortexed for 1 min, and centrifuged at 14,000 rpm for 10 min. Finally, a 2 μl supernatant was injected into UPLC-Q-TOF-MS for analysis.The urine samples of rats (n = 3) were collected from 0 to 12 h and 12–24 h after administration. All feces samples of the two periods were mixed and dried. The feces samples (1 g) were extracted with methanol, on which the ultrasonic was performed for 30 min under the condition of 500 W and 40 kHz, before being centrifuged at 14,000 rpm for 10 min. Through vacuum centrifugal concentrator on AL mode vent at 30°C, the supernatant was dried. The residues were reconstituted with 50% acetonitrile, vortexed for 1 min, and centrifuged at 14,000 rpm for 10 min. Finally, a 2 μl supernatant was injected into UPLC-Q-TOF-MS for analysis.The feces samples of rats (+/ESI−), and declustering potential were set at 550°C, 5500/−4500 V and 100/−100 V, respectively. For the IDA experiments, the collision energies were set at −45 and 45 eV, and the collision energy spread was set at 10/15 eV. TOF-MS spectra were obtained from 100 to 2,000 Da.All samples were analyzed on an AB Sciex 5600 Triple-TOF™ mass spectrometer  coupled with a Shimadzu UPLC LC-30AD system  and controlled with the Analyst TF 1.7 software (AB Sciex). Chromatographic separation was carried out at 30°C using a Hypersil GOLD aQ C18 column . The mobile phase consisted of acetonitrile (A) and water (B) , and the flow rate was 0.4 ml/min. The gradient elution was performed as follows: 95% B at 0.0–1.0 min, 95–90% B at 1.0–2.0 min, 90–85% B at 2.0–4.0 min, 85–80% B at 4.0–6.0 min, 80–75% B at 6.0–8.0 min,75–65%B at 8.0–15.0 min, 65–55% B at 15.0–20.0 min, 55–5% B at 20.0–24.0 min, 5–0% B at 24.0–25.0 min, 0% B at 25.0–26.0 min. The mass spectrometer was coupled with an electrospray ionization (ESI) source, acquiring TOF-MS and TOF-MS-IDA-MS/MS spectra in the dynamic background subtraction by running in negative ion and high sensitivity mode. In addition, the automated calibration delivery system could acquire precise mass measurements. The operating parameters were as follows: Behind optimization, nitrogen was used as a source of gas, and the nebulizer gas, curtain gas, and heater gas were set at 55, 35 and 55 psi, respectively. The source temperature, ion spray voltage  were gained. Meanwhile, compounds were identified by the fragmentation information obtained. Finally, LC-MS data file in positive and negative ion modes was applied to characterize ZGC-related xenobiotics (prototypes and metabolites) for biosamples from rats being administrated with ZGC. The representative prototypes were imported into MetabolitePilot 2.0.4 to search and identify the metabolites along with their metabolic pathways. The useful clogP values of compounds calculated by ChemDraw Ultra 19.0 was introduced to distinguish between structural isomers. In general, compounds had a longer retention time when the values of clogP were higher in reversed-phase liquid chromatography systems .http://www.lilab-ecust.cn/pharmmapper/) (http://www.swisstargetprediction.ch/) (http://www.uniprot.org/). “Detoxify” were imported into GeneCards database (https://www.genecards.org/) to collect the detoxification-related targets.The compounds (prototypes and phase Ⅰ metabolites) of ZGC identified from serum, heart, liver, and kidneys were imported into PharmMapper   and Swision.ch/)  to prediThe potential detoxification targets of ZGC were selected using Protein–Protein Interaction (PPI) with a high-combining score of more than 0.900 . The keyhttps://www.omicshare.com/) was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The cut-off criterion of those GO and pathway terms was p-value < 0.05  and glycyrrhizic acid (33.88 mg/ml).m/z values of quasimolecular ion, characteristic fragment ion, mass fragmentation rules from available databases and by comparing mass data with those from authentic standards and previous studies  and neutral small molecules. Compound 4  was identified as liquiritigenin-in-7,4′-diglucoside, which exhibited characteristic ions at m/z 625.1783 [M + COOH]-, 417.1193 [M + COOH-HCOOH-C6H10O5]-, 255.0660 [M + COOH-HCOOH-2C6H10O5]-, with the further RDA fragmentation creating fragment pathway involved RDA with fragment ions at m/z 135.0088 [M + COOH-HCOOH-2C6H10O5-C8H8O]-. Compound 11  showed [M + H]+ ion at m/z 257.0803 and obvious fragment ion at m/z 137.0235, which was due to RDA cleavage. The fragment ions at m/z 119.0498 and 109.0293 by the elimination of a H2O moiety or CO moiety of m/z 137.0235, suggested that it was tentatively characterized as pinocembrin. Compound 10  gave [M-H]- ion at m/z 549.1626, which was 162.0528 Da (glu) and 132.0423 Da (apiose) more than the fragment ion (m/z 255.0654) of aglycone. Compound 13  gave the mother ion at m/z 549.1654, which was 132.0420 Da more than the fragment ion (m/z 417.1215) of aglycone. Compound 13 was characterized as an isomer of compound 10 because of their identical structural formulas. Moreover, the clogP values of compounds 10 and 13 were −0.52 and −0.27, respectively. And compounds 10 and 13 were characterized as liguiritigenin-7-O-D-apiosyl-4′-O-D-glucoside (16H12O4), giving [M-H]- ion at m/z 267.0691 which could spill into m/z 252.0453 and 135.0103, while the fragment ion at m/z 104.0330 was produced by loss of C9H7O3. These evidences allowed the tentative characterization of compound 18 as formononetin (16H12O5) had [M + H]+ ion at m/z 285.0748, It produced m/z 270.0523 and 253.0483 fragments due to the elimination of the CH3 and CH4O, and the m/z 225.0538 was produced by the loss of CO. Thus, compound 27 was characterized as prunetin.In the present study, a total of 57 flavonoids had been observed in ZGC aqueous extract. For aglycones, the characteristic fragment pathways were mainly Retro-Diels-Alder reaction (RDA), and the loss of radicals , the remaining aglycones fundamentally had some neutral small molecule loss such as -CH42H62O17) displayed [M-H]- ion at m/z 837.3988 which cracked into m/z 661.3639 and 485.3227. Compound 58  gave [M-H]- ion at m/z 837.3989 which exhibited fragment ions at m/z 661.3646 [M-H-C6H8O6]-, 351.0577 [M-H-C30H46O5]-, 193.0356 [M-H-C30H46O5-C6H8O5]- and 113.0253 [M-H-C30H46O5-C6H8O5-2H2O-CO2]-. Besides, they were defined as a pair of isomers sharing the same structural formula. Furthermore, Compounds 30 and 58 were assigned as macedenosin A and licoricesaponin G2 (48H74O19) had [M+H]+ ion at m/z 955.4890. It produced m/z 779.4555 due to the elimination of C6H8O6. Then, the fragment could further eliminate C6H8O6 to produce m/z 603.4245, while the fragment ion at m/z 585.4128 was produced by the loss of H2O. Thus, compound 38 was characterized as uralsaponin T. Compound 43  generated [M + H]+ ion at m/z 837.3823 and produced m/z 661.3575 fragment due to the cleavage of C6H8O6. While the fragment ions at m/z 643.3477 and 485.3245 by the elimination of H2O or C6H8O6, suggested that it was tentatively characterized as 3-O-[β-D-glu-curonopyranosyl-(1→2)-β-D-glucuronopyra-nosyl]-24-hydroxy-glabrolide. Compound 45  had [M-H]- ion at m/z 879.4096, which produced m/z 703.3753 by eliminating the moiety of C6H8O6. Then, the fragment could further wipe off C2H4O2 (60 Da) to yield m/z 643.3522, indicating the presence of an acetyl group. Thus, compound 45 was deduced to be 22β-acetoxyl-glycyrrhizin (41H62O14) showed [M + H]+ at m/z 779.3824. It produced [M + H-C11H16O10]+ ion at m/z 471.3460 due to the elimination of gluA and arabinose. With fragment ions at m/z 471.3460 and 217.1573, which were consistent with those of glycyrrhetinic acid, compound 69 was thus inferred as araboglycyrrhizin.Compound 30  presented an [M-H]- ion at m/z 367.1182. Its typical fragment ion at m/z 337.0745 was originated from the loss of CH2O, while the fragment ion at m/z 309.0405 was produced by the loss of C3H5 and OH. Hence, compound 78 was identified as glycycoumarin (21H18O6) exhibited the precursor ion [M-H]- ion at m/z 365.1040. It had the characteristic dominant fragment ion at m/z 335.0556 and 307.0247, and was thus identified as glycyrol  produced the fragments ions of [M-H-CH3]- and [M-H-2CH3]- at m/z 324.1014 and 309.0763, respectively, which indicated continuous loss of two molecules of CH3. Then, the remaining residue lost two carbonic oxides. It was determined as glycybridin A.The [M-H]According to the retention time and MS fragments of chemical compounds in ZGC aqueous extract, a total of 70 prototypes were identified or tentatively characterized after oral administration of ZGC aqueous extract. Of these, 20 prototypes were identified in serum, 8 in heart, 6 in liver, 13 in kidneys, 57 in feces, and 23 in urine. The structures of these prototypes were mainly flavonoids and pentacyclic triterpenoid saponins. The data information of prototypes in rat biosamples after oral administration of ZGC by UPLC-Q-TOF-MS was listed in A total of 162 metabolites  were ide21H22O12S) and M5  were identified as the sulfated product and glucuronidated product of liquirtin, with the higher weight 80 Da (SO3) and 176 Da (gluA) than liquiritin (22H24O9) showed an adduct [M + COOH]- ion at m/z 477.1401, which was 14 Da (CH2) higher than that of liquiritin, giving rise to product ions at m/z 253, 135 and 117. It was supposed as the methylated product. Likewise, M49  was defined as the methylated product of hydroxylated liquiritin. M32  was supposed as the hydrogenated product of liquiritin because the molecular weight was 2 Da (2H) higher than that of liquiritin. Similarly, M79  presented the same molecular formula as M32 and was thus assumed to be isomer. M9  showed the [M + H]+ ions at m/z 597.2146, which was 176 Da (gluA) higher than that of M32 and M79. It is suggested as a hydrogenated product of glucuronidated liquiritin.M4  and conjugation of C2H2O (42 Da), and coinciding with a mass weight of 26 Da more than that of liquiritigenin. The clogP values of M31 and M61 were 2.82 and 2.93, relatively. Therefore, M31 and M61 were conjecturally characterized as liquiritin-Glu + C2H2O-O and its isomer, respectively. M34 , M39  and M40  were all identified as hydroxylation and hydrogenated product of liquiritigenin, respectively, because they were one more O (16 Da) and 2H (2 Da) than liquiritigenin. M15  was speculated to be methylated product of glucuronidated liquiritigenin based on the characteristic fragment ion of [M + COOH-GluA-CO-CH2]- at m/z 273.0757.M31 (Rt = 7.83 min) and M61 (Rt = 9.79 min) were the acetylated and dehydroxylated metabolites of liquiritigenin, whose molecular formula was Cin vivo.Our study demonstrated that liquiritin and liquiritigenin mainly experienced glucuronidation, sulfation, hydroxylation, hydrogenation, and methylation 23H24O10) was generated by acetylation of isoliquiritin because the metabolite had an additional acetyl group of isoliquiritin. M105  displayed [M + H]+ ion at m/z 369.1317 due to the loss of H2O and 2O from isoliquiritin, Thus, M105 was supposed as dehydroxylated product of dehydrated isoliquiritin. M60  showed [M + H]+ at m/z 435.1266, which was 16 Da higher than isoliquiritin. Thus, M60 was characterized as the hydroxylated product of isoliquiritin. M47  was characterized as the hydroxylated product of M60. M38  provided [M + H]+ ion at m/z 449.2785, which was 192 Da higher than isoliquiritigenin, and was supposed as glucuronidated product of hydroxylated isoliquiritigenin  gave an adduct [M + COOH]- ion at m/z 283.0635, which was 18 Da (H2O) lower than isoliquiritigenin. M91 was identified as dehydrated product of isomerized isoliquiritigenin.M54  and M8  were identified as the glucuronidated and sulfated product of liquiritin apioside (3) higher than liquiritin apioside. M51  presented the same molecular formular as liquiritin apioside, and thus was characterized as its isomer. M16  produced a deprotonated molecular [M-H]- at m/z 593.1522, which was 176 Da (gluA) higher than liquiritin. The product ion at m/z 255.0665 [M-H-gluA-C6H11O5]- suggested that it might be the glucuronidated product of liquiritin and further underwent isomerization. M2  showed a deprotonated molecular [M-H]- ion at m/z 593.1515, which was 176 Da (gluA) higher than liquiritin suggesting it as a glucuronidated product of liquiritin. M114  provided a protonated molecular [M + H]+ ion at m/z 299.0911, with successive loss of glu (162 Da) and conjugation of C2H2O (42 Da), coinciding with a mass weight of 120 Da less than liquiritin. It was thus supposed as acetylated product of deglucosed liquiritin with further undergoing isomerization. M17  and M20  gave a deprotonated molecular [M-H]- at m/z 431.0994 and 431.0993, which were produced from liquiritin by deglycosylation and glucuronidation, and this coincided with a mass weight of 14 Da lower than liquiritin. The characteristic fragment ions at m/z 255, 135, and 119 hinted that they might be glucuronidated product of deglucosed liquiritin. In addition, the clogP values of M17 and M20 were 0.27 and 0.56, respectively. Therefore, M17 was characterized as liquiritin apioside-apiose-Glu + gluA, and M20 as its isomer.M3  and M127  were identified as the sulfation product and acetylation product of glycyrrhizic acid, separately, because the molecular weights were 80 Da (SO3) and 42 Da (C2H2O) higher than glycyrrhizic acid. Besides, M135  and M140  gave the [M + H]+ at m/z 837.3865 and 837.4226, and the molecular weight was 14 Da (CH2) higher than glycyrrhizic acid. Thus, they might be the methylated metabolites of glycyrrhizic acid. The glycyrrhizic acid could lose one or two molecules of glucuronic acid to produce glycyrrhizic acid-GluA    and glyc42H64O15) and M130  exhibited similar fragment ions. In addition, M125 and M130 had the deprotonated molecule ions at m/z 807.4224 and 807.4217, respectively, which both weighed 162 Da (C6H10O5) higher than M146. So, they were considered to be the glucose conjugates of M146. The clogP values of M125 and M130 were 3.51 and 3.67, respectively. Therefore, M125 and M130 were tentatively characterized as glycyrrhizic acid-GluA + Glu and its isomer, respectively. Likewise, M148  was characterized as glucose conjugates of glycyrrhetinic acid. M126 (Rt = 16.63 min) showed the [M-H]- at m/z 689.3887, equalling C38H56O11 with a weight of 42 Da (C2H2O) greater than M146. Thus, M126 was characterized as the acetylated product of M146. Besides, M144  was defined as dehydroxylated product of M146 due to a weight of 16 Da (O) less than M146. Similarly, M136  weighing 16 Da (O) more than glycyrrhetinic acid, was defined as hydroxylated product of glycyrrhetinic (32H48O4) was deemed to be acetylated product of dehydroxylated glycyrrhetinic acid.The MS/MS spectra of M125  by RDA. Furthermore, segments of CO and H2O were usually featured by a neutral loss of 28 and 18 Da, correspondingly. Similarly, pentacyclic triterpenoid saponins experienced deglycosylation easily. The cleavage of allyl (between C-7 and C-8) along with characteristic fragment ion at m/z 317 caused by McLafferty rearrangement were prone to occur under positive ion mode. Additionally, RDA was occurred to yield characteristic fragment ion at m/z 263  and GSH-related . CYP450s are important phase Ⅰ DMEs (drug-metabolizing enzymes) in detoxification and exist a large amount in the gastrointestinal tract, liver, and kidneys . CYP1, 2in vitro and in vivo.This analytical strategy systematically investigated the potential compounds associated with detoxification based on metabolic analysis and network pharmacology analysis. However, it is a pity that the active compounds and targets predicted by network pharmacology have not been confirmed and validated by experiments In this work, UPLC-Q-TOF-MS was used to analyze the chemical and metabolic profiles of ZGC. 115 chemical compounds of ZGC aqueous extract and 232 xenobiotics (70 prototypes and 162 metabolites) in rats were identified. Furthermore, 26 prototypes and 15 metabolites in serum, heart, liver, and kidneys were used to construct network pharmacology explaining the detoxification of ZGC. The results have shown that the effects of 13 potential active compounds (5 prototypes and 8 metabolites) on the nine potential targets  were associated with CYP450s and GSH for detoxification of ZGC. Based on it, the potential active compounds, 5 prototypes  and 8 phase Ⅰ metabolites , could be detoxification essence of ZGC. Together, this study tried to clarify the potential active ingredients of detoxification and the possible mechanism of detoxification on ZGC.


At the same time, the free HAs content increased from 53.52 ng·g−1, at 50 °C, to 127.16 ng·g−1, at 60 °C, and then decreased continuously. The content of the free HAs was the highest at 60 °C. For the protein-bound HAs, their content was found to generally decrease with the increase in smoking duration and temperature. However, at 5 h, the content of protein-bound HAs slightly increased to 984.2 ng·g−1. Meanwhile, at 90 °C, it increased to 1643.53 ng·g−1. Additionally, a total of 16 volatile organic compounds (VOCs) were found in all of the meat samples, of which 10 VOCs  were significantly related to the formation of free HAs. These findings showed that all the different types of HAs were produced under low-temperature processing, which provided scientific insights into the potential generation of HAs during meat smoking processes and could be used as a reference to minimize the risks of cancer related to the consumption of smoked meat products.In this study, UPLC-MS/MS was used to study the effects of smoking duration and temperature on the formation of heterocyclic amines (HAs) in smoke-processed meat patties. Four kinds of free HAs—including F-7,8-DiMeIQx; F-MeAαC; F-Harman and F-Norharman—and six kinds of protein-bound HAs—including B-AαC; B-7,8-DiMeIQx; B-Glu-p-1; B-MeAαC; B-Harman and B-Norharman—were detected and quantified. Among the free HAs, we observed a 23-fold content increase ( Food smoking is one of the oldest food preservation techniques and belongs to a typical thermal processing food treatment method. During the smoking of meat products, the process flow generally involves curing, air drying, ripening and smoking . With inHAs are heterocyclic aromatic hydrocarbons with known carcinogenic and mutagenic properties. Presently, 30 HAs have been isolated and identified . The IntConsidering that exploring the influence of different smoking processing methods on the formation of HAs in smoked meat could provide a basis for food quality control in the smoking process stage—which could be adaptable in the industrialized production of smoked food—the objectives of this study were to analyze the formation of HAs during different meat smoking conditions, including different temperatures  and durations , and to study the VOCs in meat patties under different processing conditions. The relationship between each VOC and HAs’ formation was ascertained.−1). UPLC-MS/MS-grade methanol, acetonitrile and formic acids were obtained from Thermo Fisher Scientific . Analytical-grade diatomaceous earth was purchased from Sinopharm Chemical Reagent Co., Ltd. . Oasis MCX cartridges  were purchased from Waters . Pork and salt were purchased from a local supermarket .HAs’ standards—including 2-amino-9H-pyridoindole (AαC); 2-amino-3-methyl-9H-pyridoindole (MeAαC); 1-methyl-9H-pyridoindole (Harman); 9H-pyridoindole (Norharman); 2-amino-3-methylimidazoquinoline (IQ); 2-amino-3,4-dimethylimidazoquinoline (MeIQ); 2-amino-1-methylimidazoquinoline ; 2-amino-1-methyl-6-phenylimidazopyridine (PhIP); 2-amino-1,6-dimethylimidazopyridine (DMIP); 2-amino-1,5,6-trimethylimidazopyridine ; 2-amino-3-methyl-3H-imidazoquinoxaline (IQx); 2-amino-3,8-dimethylimidazoquinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazoquinoxaline ; 2-amino-3,7,8-trimethyl-3H-imidazoquinoxaline ; 2-amino-3,4,7,8-tetramethyl-3H-imidazoquinoxaline ; 2-amino-5-phenylpyridine (Phe-P-1); and 2-amino-6-methyldipyridoimidazole (Glu-P-1)—were purchased from Santa Cruz Biotechnology, Inc. . All standards had a purity level >99.9%. All 17 HAs were diluted with chromatographic grade methanol to a standard solution (125 mg·LPork (tenderloin) was thoroughly ground using a meat mincer . The meat tendons were removed, followed by adding 1.5% NaCl, rolling the mixture for 1 h in a vacuum curing machine  and pickling under vacuum refrigeration at 4 °C, for 18–20 h. Lastly, the minced meat (80 g) was pressed into 10 cm diameter meat patties, using a meat patty forming machine .The meat patties were dried for 0.5 h in the Self-Cooking Center of RATIONAL SCC 61 , whose oven was pre-heated to 70 °C. The measurement was conducted in triplicate. The meat patties were then naturally cooled at room temperature. After cooling, they were separated into the following two treatment groups: Group 1, in which the meat patties were smoked for 0, 1, 2, 3, 4 and 5 h, at 70 °C; and Group 2, in which the meat patties were smoked for 3 h at ambient temperatures (AMB), 50, 60, 70, 80 and 90 °C. As each sample took a long time to be smoked, it took 2–3 days to complete the total processing of each group. Therefore, the meat of the two groups was purchased separately. In all treatments, the smoke was produced by VarioSmoker 61-201 . VarioSmoker was used together with Self-Cooking Center. In the experiment, the weight of woodchips used for each fumigation was 120 g, and the VarioSmoker was put in the Self-Cooking Center, as shown in L*), redness (a*) and yellowness (b*) of the sample were recorded. Each sample was analyzed in triplicate.The surface color of the smoked meat patties was determined according to the method of Zhang et al. , but we The water activity  and cooking loss were detected according to the method described by Zhang et al. .The changes in meat pH  were determined using a previously described assay .−1 pre-test speed, 2 mm·s−1 test speed, 3 mm·s−1 post-test speed, 50% strain, 5 s dwell time interval and a trigger force of 5 g.The texture properties of the pork patties were measured by TA. XT plus texture analyzer  was used according to the previously described methods, with some modifications . A flat-−1 in methanol) was added to each vial before SPME fiber exposure. The SPME fibers used were 1.0 cm in length with DVB/CAR/PDMS coating . Before exposure to the sample bottle, the SPME fiber was pre-heated at 270 °C, for 30 min, in the GC inlet. The sample bottles were equilibrated to 50 °C, for 30 min, during exposure. The samples were then manually injected at a depth of 5.0 cm into a split-less GC inlet, at 250 °C, onto an SH-WAX column , with a constant flow rate of 0.8 mL·min−1 of helium. The condition of GC-MS was set as described by Rizzo et al. [−1 to 100 °C, then accelerated to 10 °C·min−1 to 230 °C, which was held for 5 min. The purge time was set for 1 min. The MS transfer line was held at 250 °C, with the ion source held at 200 °C.The VOCs in the pork patties were identified by GC-MS , according to the methods described by Rizzo et al. [−1) solution was added and homogenized  . Then, 13 g of diatomite was added to the homogenate and mixed, followed by adding and mixing 50 mL ethyl acetate and ultrasonic  treatment, for 30 min. The extraction procedure was repeated twice, followed by centrifugation  to obtain a supernatant, which was concentrated with nitrogen to 10 mL. Furthermore, the filtrate was collected and added to a 48 mL thick-walled, pressure-resistant bottle, followed by the addition of 40 mL of 6 mol·L−1 HCl to the bottle, bubbling nitrogen through for 1 min and heating at 110 °C, for 24 h. Next, they were centrifuged and the supernatant collected was diluted with ultrapure water to 100 mL, from which 10 mL was used for solid phase extraction (SPE).Free HAs in smoked meat were extracted and concentrated according to the method described by Zeng et al. , with so−1 HCl. Ten milliliters (10 mL) of extract were added to the SPE column, the effluent was discarded and they were rinsed with 6 mL 0.1 M HCl and 6 mL methanol. The retained HAs were eluted with 6 mL methanol-ammonia mixture , at a flow rate of 0.6 mL·min−1 [For free HAs, Oasis MCX cartridges were activated in advance with 6 mL water, 6 mL methanol and 6 mL ethyl acetate, by automatic SPE . For protein-bound HAs, Oasis MCX cartridges were activated in advance with 6 mL methanol, 6 mL water and 6 mL 0.1 mol·LmL·min−1 . The elu−1 and the injection volume was 2 μL. The mass spectrometry detection was conducted in multiple-reaction monitoring (MRM) scan mode, under positive electrospray ionization (ESI), as described by Li et al. [−1 flow rate; 3.5 kV capillary voltage; 50 L·h−1 cone flow (nitrogen); 0.15 mL·min−1 flow rate of collision gas (argon); and 2-2000 Da scanning range. According to the peak shape and intensity of different treatments, the channel reaction, dwell time, cone voltage, collision energy and delay time of 17 HAs in the MRM mode of this study were optimized . Acquity BEH C18 column  was used. The mobile phases used consisted of A: 100% acetonitrile and B: ultrapure water + 0.1% formic acid. The elution gradient at a 45 °C column temperature was 2% A (0–2 min), 20% A (12 min), 100% A (14 min), 2% (17 min) and 2% A (20 min) . The floi et al. , with soptimized .p < 0.05) among the treatments. Statistical analysis was performed using the general linear model program of the Statistic software v9.0 . HAs’ data were imported into SIMCA 14.1  for principal component analysis (PCA). Spearman rank-order correlations were assessed using the Origin Pro 2021b software . The Origin Pro 2021b and Anaconda Jupyter Notebook v6.4.8  software were used for drawing.The UPLC-MS/MS results were analyzed using the Masslynx v4.1 software (Waters). Data were reported as means ± standard deviation and analyzed using the one-way analysis of variance to determine significant differences  in all the pork meat patties, which were associated with the changes in the water, protein and lipids’ constituents. The heating of the meat was associated with the thermal denaturation of the proteins and a simultaneous change in the binding of the water and oxidation of the lipids, which were reflected by the changes in the gel properties [L* value decreased from 71.26% to 65.23%, from 0.987 to 0.979, from 6.66 to 5.36 and from 61.84 to 52.34, respectively (p < 0.05) [The physicochemical properties of the smoked meat patties are presented in  pathway . These o pathway . The pH  pathway . Moreoveoperties . These aoperties . Further < 0.05) .−1); F-MeAαC (0–1 ng·g−1); F-Harman (0.2–3.6 ng·g−1) and F-Norharman (1.15–123.99 ng·g−1)—were detected in the smoked meat patties  (4.24 ng·g−1). During the smoking and heating process, water was gradually lost and acted as a reaction and transmission medium for several precursor molecules on the surface of the meat patties. On the surface, the precursors were exposed to higher temperatures, resulting in the formation of HAs [−1 to 47.82 ng·g−1. Among the four kinds of HAs, the content of F-Norharman was the highest, while the content of the other HAs changed little with the increase in the temperature. The amount of total free HAs decreased gradually, primarily due to a decrease in F-Norharman. A similar trend was observed with the formation of HAs in trout fillets that were cooked by sous-vide, under different cooking temperature conditions [−1); B-7,8-DiMeIQx (1.33–5.63 ng·g−1); B-Glu-p-1 (14.55–66.65 ng·g−1); B-MeAαC (54.37–191.3 ng·g−1); B-Harman (139.57–571.53 ng·g−1) and B-Norharman (333–1140 ng·g−1)—were detected in the smoked meat patties (−1), while the content of B-Harman decreased gradually . An increase in the smoking duration from 1 h to 5 h was associated with the 63, 74, 72, 73 and 60 kinds of VOCs contained in the smoked pork patties, respectively . Zhenba p ≤ 0.001), including five methoxyphenols. In principle, this confirmed that a darker color was associated with a higher content of HAs, which might be related to some phenolic compounds in smoking that promoted the formation of HAs. For instance, smoking-associated carbonyl compounds might provide intermediate compounds that promote the formation of HAs [−1, at 0 h, to 2935.41 μg·g−1, at 5 h; and (ii) Phenol,2-methoxy-, which increased from 11.72 μg·g−1, at 0 h, to 2789.87 μg·g−1, at 5 h. The content increased by 322 and 238 times, respectively, and gradually increased with the increase in the smoking duration . Furthermore, 2-Methoxy-5-methylphenol; p-Cresol; Phenol; Phenol,2-methoxy-; Phenol,2-methoxy-4-propyl-; Phenol,4-ethyl-2-methoxy-; and trans-Isoeugenol showed a strong positive correlation with the free HAs, with correlation coefficients of 0.63, 0.68, 0.71, 0.61, 0.66, 0.64 and 0.57, respectively (p ≤ 0.001) (p < 0.05). Among them, tetradecane deserved special attention as it had a positive correlation with F-Harman  and a strong negative correlation with B-Glu-P-1 and B-Harman . In general, the smoking process significantly promoted the formation of HAs in meat products, due to the involvement of the smoking ingredients in producing HAs in meat products [The Wayne mapping showed that there were 16 VOCs, including one acid, one alcohol, four aldehydes, two alkanes, one ketone and seven phenols, under different durations and temperatures A—specifin of HAs . Mixed pn of HAs . It was n of HAs . Among tduration . With that 90 °C . All of at 90 °C C. At 60 ≤ 0.001) B. Howeveproducts .−1 (p < 0.05), in which the Harman content was 0.85 ng·g−1 and the Norharman content was 5.81 ng·g−1. The TPA indexes were all concentrated in the third quadrant, with the same quadrant of 80 °C samples representing a higher processing temperature or 4 h and 5 h of a longer processing duration in the loading scatter plot. An increase in the smoking duration and temperature made the texture of the smoked meat patties hard and significantly deteriorated their chewiness, elasticity and resilience.Two principal components were extracted, representing 65% total variance, with component one (PC1) and component two (PC2) contributing 42.1 and 22.9% of the variance, respectively. As shown in the score scatter plot A, clear A low temperature for meat processing significantly increased the generation of various kinds of HAs. During the smoking of meat, some components may induce the production of HAs in the smoked meat. The 16 VOCs that existed under different conditions deserved special attention. In this study, the concentration of various HAs varied with the changes in the smoking durations and temperatures. In particular, VOCs with a high correlation with free HAs were concentrated in the second quadrant, corresponding to the position of 60 °C in the scores scatter plot of PCA. This also coincided with the situation that 60 °C was the most different from the other smoking temperature conditions in the cluster analysis. Our results showed that the smoking process significantly affected the formation of HAs in meat products. Therefore, the smoking of meat should be carefully controlled to minimize cancer risks.