The antioxidant potential of the defatted meal methanolic extracts was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and metal ion-chelating activity (MIC) assays. The highest total phenolic content of 3.15 mg gallic acid equivalent/g dry weight was recorded in the defatted meal extract from seeds pre-treated with air frying at 190 °C for 15 min. Sinapine, sinapic acid and an unknown compound at a retention time (RT) of 26.6 min were the major sinapates identified in the defatted meal with the highest concentrations of 7572 ± 479.2 µg/g DW, 727 ± 43.45 µg/g DW and 1763 ± 73.5 µg/g DW, respectively, obtained at 160 °C for 5 min. Canolol (151.35 ± 7.65 µg/g DW) was detected after air frying at a temperature of 170 °C for 20 min. The FRAP and MIC correlated positively (r = 0.85) and generally decreased with increased air frying temperature-time conditions. The highest FRAP and MIC values of 0.53 mM and 80% were obtained at 160 °C for 5 and 20 min, respectively. The outcome of this study will contribute new knowledge that could improve the value addition and by-product utilization of canola seeds.This study investigated the efficacy of roasting pre-treatment by air frying to enhance the extraction and recovery of the predominant sinapic acid derivatives (SADs) from roasted canola meal and the antioxidant potential of the methanolic extracts. Canola meal was obtained by air frying canola seed at 160, 170, 180 or 190 °C for 5, 10, 15 or 20 min. Oil was extracted using the Soxhlet method, and the de-oiled meal fraction was air-dried. Phenolic compounds were isolated using ultrasound-assisted extraction with 70% ( Brassica napus L.) is among the leading oilseed crops globally [Canola  and 427.1 ± 7.12 μg/g DW at pre-treatment time-temperature regimens of 160 °C and 180 °C for 5 and 10 min, respectively, have been reported [Canolol, an important vinyl phenol derivative, has potent antioxidant, anticancer, and lipid-lowering properties, with a potential for the prevention and treatment of cardiovascular diseases ,12,13 wiWhile there have been previous studies using a variety of roasting techniques to improve the production of canolol and other major meal-derived phenolics, this is the first study on the application of air roasting techniques for generating canolol and other sinapates from canola meal. Therefore, the objective of this study was to valorize defatted canola meal using air frying of the seeds as a green pre-treatment method followed by an ultrasound-assisted extraction technique to produce canolol and other polyphenols as natural antioxidants. This combined approach will help to establish the optimum extraction efficiency of canolol and other sinapates from defatted canola meal while minimizing the environmental impact. This will also enhance the value-added processing and by-product utilization of defatted canola meals as an additional source of revenue for the canola industry.Canola seed roasting was carried out by the modified method of Fadairo et al. . Canola Defatting of the air-fried canola seed with hexane was performed following the method of Khattab et al.  with sliThe extraction of phenolics from defatted canola meal was done according to the method of Liang et al. .n = 11) concentration range with R2 = 0.9994 for sinapic acid, R2 = 0.9999 for canolol, and R2 = 0.9996 for sinapine. Sinapic acid (purity > 98%), gallic acid ≥ 98%, HPLC grade solvents and other analytical reagents were purchased from Fisher Scientific Canada Ltd . Sinapine (purity > 97%) and canolol (purity > 97%) were purchased from ChemFaces® Biochemical Co., Ltd. . A flow chart for the extraction of defatted canola meal-derived sinapates is shown in The changes in the phenolic composition of defatted canola meal methanolic fractions extracted by ultrasound-assisted extraction were evaluated by high-performance liquid chromatography (HPLC) as described by Nandasiri et al. . The majv/v) methanol. A calibration curve was prepared from a series of standard solutions (25–350 µg/mL) using the gallic acid stock solution with linearity (R2 = 0.9987).The TPC of defatted canola meal extracts was determined according to the Folin–Ciocàlteu method described by Cai et al.  with a fThe radical scavenging activity of the meal extracts was determined according to the method of Girgih et al.  with sliThe ferric ion reducing antioxidant power (FRAP) was carried out according to the method described by Benzie and Strain  with modThe metal ion chelating ability was evaluated according to the modified method described by Xie et al. .n = 3). The differences between mean values were determined by analysis of variance (ANOVA). The treatment conditions were temperature  and time , respectively. All data analysis and visualization were done in R 4.1.3  and R studio 2021.09.0. All tests were considered statistically significant at p < 0.05. Correlation analysis of the predominant sinapic acid derivatives and related antioxidant activity was further performed using R 4.1.3  and R studio 2021.09.0 to determine the relationship between the phenolic derivatives and the various antioxidant activities evaluated.Results were expressed as means ± SD  between them. Moreover, sinapine was also recovered (7193 ± 86 µg/g DW) after 20 min of roasting at 160 °C. Conversely, the lowest amount of defatted meal sinapine (5940.32 ± 115.14 µg/g DW) was obtained with a seed pre-treatment temperature of 170 °C for 20 min. Sinapine, sinapic acid and canolol are the major heat-labile sinapic acid derivatives (SADs) in the defatted canola meal, and their extractability and concentration have been reported to be dependent on the pre-treatment and processing conditions employed [Air frying conditions of 160 °C for 5 and 10 min, respectively, produced the highest sinapine content (7571.6 ± 479.2 and 7471 ± 197.5 SPE µg/g DW) with no significant difference  under steam explosion pretreatment. Khattab et al. [w/w) and a significantly lower sinapic acid content while Yang et al. [The highest sinapine value observed in the current study is similar to the results of Nandasiri et al. , who recb et al.  reportedg et al.  reportedg et al.  reportedg et al.  found thg et al.  indicatiBrassica napus rapeseed following steam explosion pre-treatment was 1210 mg/kg. In a study by Yang et al. [The changes in the sinapic acid concentration in canola meal fractions obtained by air frying pre-treatment of the seeds is shown in g et al. , sinapicg et al.  who repog et al.  also obtg et al. . Cai andg et al.  recordedg et al.  identifip > 0.05) differences between these temperatures (p < 0.05) lower than those reported by Nandasiri et al. [B. napus and Brassica rapa rapeseed varieties pre-treated using steam explosion. The authors recorded improved canolol content. However, there was a reduction in the quantity of other phenolic derivatives. This observation is consistent with the recovery of canolol and other SADs, as previously reported [The concentration of canolol in the defatted meal increased progressively with an increase in seed air frying temperature-time regimens and ranged from 84.92 ± 3.41 to 151.92 ± 2.73 µg/g DW . The maxeratures . These vi et al.  reportedi et al.  also expi et al. . Sorensei et al.  showed ti et al.  obtainedreported . Yang etreported  further Changes in the TPC of canola meal as affected by air frying are shown in p > 0.05) between these two times (p < 0.05). However, a significant (p < 0.05) and negative association was observed between TPC and sinapine (r = −0.56) as well as TPC and sinapic acid (r = −0.91)  . Zago etk et al. , the estp < 0.05) differences between the values. Moreover, a strong and significant (p < 0.05) correlation was found between the DPPH radical scavenging activity of the extracts and sinapine (r = 0.63), sinapic acid (r = 0.67), RT = 26.6 min compound (r = 0.62), and canolol (r = −0.63) (p < 0.05) and moderate negative correlation (r = −0.54) was found between DPPH and TPC of the extracts obtained at various air frying treatments /100 g) and rapeseed cake (4948 μmol TE/100 g).The impact of seed roasting by air frying on the DPPH free radical scavenging capacity of defatted canola meal methanolic extracts is shown in = −0.63) . There weatments . The abiorted by . The autorted by  reportedorted by  studied The ability of phenolic compounds to reduce ferric ions to form stable ferrous ions isused as an indicator of the ability to mitigate oxidative stress. The FRAP values of defatted canola meal methanolic extracts obtained from seeds roasted at various roasting conditions are shown in B. rapa defatted rapeseed cake and B. juncea, respectively. In the same study, they also reported a FRAP value (1366.5 μmol TE/100 g) in oil produced from pretreated B. napus rapeseed, which was greater than that of untreated B. napus rapeseed (0.51 μmol TE/100 g). It was concluded that steam explosion treatment significantly (p < 0.05) improved the antioxidant activity of B. napus rapeseed oil. Teh et al. [p < 0.05) negatively correlated with canolol (r = −0.83) (p < 0.05) correlation between FRAP and sinapine (r = 0.91), sinapic acid (r = 0.81), and the RT-26.6 min compound (r = 0.81) (p < 0.05) relationship also existed between FRAP and DPPH (r = 0.70) as well as FRAP and metal ion chelation (r = 0.81) activities of the extracts. However, FRAP was significantly (p < 0.05) negatively correlated with TPC  was observed in the defatted canola meal extract from seeds air fried at 160 °C for 5 min, while the lowest values (0.31 ± 0.01 and 0.32 ± 0.01) were observed in meal extracts from seeds subjected to air frying at 170 and 190 °C for 20 min, respectively . Generalh et al.  observed= −0.83) . The obs = 0.81) . It is swith TPC , as can The metal-chelating ability of a compound is important in the food industry as it helps to reduce the concentration of free metal ions, which are promoters of lipid peroxidation. Moreover, metal chelating agents are regarded as secondary antioxidants as they help to reduce redox potential, thereby stabilizing the oxidized metal ion . The chep < 0.05) and positive correlation existed between metal ion chelation properties and DPPH (r = 0.70) as well as FRAP (r = 0.85) for all the extracts (The highest activity (75%) was observed in meal extracts obtained from seeds that were air fried at 160 °C for 20 min, which is a strong indicator of iron-binding potential. Conversely, the least metal ion chelating activity (74%) was obtained in meal extracts from seeds pre-treated at 190 °C and 20 min . The effextracts . The strThe current study investigated the influence of air frying pre-treatment of canola seeds on the formation of canolol and other sinapates, which can be extracted from defatted canola meal. The findings demonstrated the potential valorization of residual canola meal via seed roasting by air frying as a green pre-treatment approach to generate the major sinapic acid derivatives and improve the antioxidative potential of the extracts. The highest contents of the major sinapates, i.e., sinapine, sinapic acid and the RT-26.6 min compound, were obtained by air-frying the seeds at 160 °C for 5 min, while the highest canolol concentration was from the 170 °C for 20 min air frying condition. The canola meal extracts obtained at various air frying conditions showed improved TPC and exhibited enhanced in vitro antioxidant potential with respect to free radical scavenging, ferric ion reduction to ferrous ion and chelation of metal ions, all of which are implicated in lipid peroxidation. For the optimum recovery of canolol, an air frying condition of 170 °C for 20 min is recommended, while for other sinapates viz- sinapine, and sinapic acid, an air frying condition of 160 °C for 5 min is recommended. The outcome of this study will further contribute to the canola industry’s quest for value-added processing of canola seeds to generate natural antioxidants with potential applications in the feed, cosmetics, and pharmaceutical industries.


The output correction factors were calculated by Monte‐Carlo method using EGSnrc toolkit for six field sizes (from 0.5×0.5cm2 to 10×10cm2) for isocentric and constant source‐to‐surface distance (SSD) techniques. The decrease in the field size led to an increase in kQclin,Qreffclin,fref for ionization chambers, while for solid‐state detectors and radiochromic film, kQclin,Qreffclin,fref were less than unity at the smallest field size. A larger sensitive volume of ionization chamber corresponded to a stronger deviation of output correction factor from unity: 1.847 (125 mm3 PTW 31010) versus up to 1.183 (16 mm3 PTW 31016) at the smallest field of 10 MV beam. The calculated output correction factors were used to correct the output factors for PTW 60017, PTW 60019, and EBT3. The deviation of the corrected output factor from the results of Monte‐Carlo simulation did not exceed 3% in the fields from 1.0×1.0cm2 to 4.0×4.0cm2 for 10 and 18 MV beams. Thus, Diode E, microDiamond, and EBT3 film can be recommended for small‐field dosimetry of high‐energy photons.High‐energy accelerators are often used in oncological practice, but the information on the small‐field dosimetry for the photon beams with nominal energy above 10 MV is limited. The goal of the present work was to determine the values of the output correction factor ( For example, corrections for the dose profiles calculation as well as beam quality correction factors for the case of small fields of high bremsstrahlung energies were calculated in Refs.22.1fmsr. For most medical accelerators, the msr‐field matches with the standard reference field,fmsr≡fref=10×10cm2. For those accelerators, which cannot form a standard 10×10cm2 reference field, the closest to 10×10cm2 field is taken as the msr‐field. For relative dosimetry of small fclin‐beams, it is necessary to strictly apply the experimental definition of the output factor as the ratio of doses in fclin and fref fieldsΩQclin,Qreffclin,fref  is the output factor of the clinical field fclin relative to the reference field fref, Qclin and Qref are the corresponding beam qualities. The ratio of absorbed doses Dw,Qclinfclin and Dw,Qreffref (“w” indicates the absorbed dose in water) in Equation  and FWHM=1.4mm .In the present paper, the output correction factors were calculated by Monte‐Carlo method using EGSnrc software toolkit.d=10cm depth in the first case and on the surface of the water phantom in the second case were 0.5×0.5cm2, 1.0×1.0cm2, 2.0×2.0cm2, 3.0×3.0cm2,4.0×4.0cm2, and 10×10cm2. The number of particles in each PSF was at least 2×106photons/cm2.The PSFs were generated on the surface of a water phantom at the distance of 90 and 100 cm from the accelerator's target, which corresponds to isocentric irradiation and irradiation with fixed solid‐state detector, respectively. The collimation jaws were set so that the radiation field sizes at ECUT=0.7MeV, PCUT=0.01MeV; maximum energy‐loss fraction per step ESTEPE = 0.25; energy threshold ESAVE = 2.0 MeV. To increase the number of bremsstrahlung, the technique of their directional splitting in the target was used with the parameter value NBRSPL = 2000 for 15 and 18 MV photon beams and NBRSPL = 500 for 10 and 20 MV beams.The following values of the parameters in BEAMnrc user code were used to simulate the radiation transport through the accelerator head  and percentage depth doses (PDDs) were calculated using DOSXYZnrc toolkit and measured experimentally in the water phantom for different sizes of the small field at SSD=90cm. The PSFs calculated by the method described previously were used as the radiation sources for the calculation. The following values of the parameters in the user code were used: ECUT=0.7MeV, PCUT = 0.01 MeV, ESTEPE = 0.25, ESAVE = 2 MeV, and nsplit = 10. The absorbed dose was determined on a uniform grid in rectangular voxels with Δx=Δy values from 0.025 to 0.25 cm and Δz values from 0.1 to 0.25 cm depending on the field size. The stories number was selected so as to obtain a statistical uncertainty of no more than 1.0%.The dose profiles  detectors for 0.5×0.5cm2, 1.0×1.0cm2, 2.0×2.0cm2, and 3.0×3.0cm2 fields of 10 MV photon beam. Comparison of central‐axial dose distributions for 2.0×2.0cm2, 3.0×3.0cm2, and 10×10cm2 fields, measured with the same detectors and simulated by Monte‐Carlo method, is presented in Figure 3G, the mean distance‐to‐agreement was ≤ 0.15 mm for all field sizes. When comparing the PDDs, the discrepancies in the readings of the studied detectors and the data of Monte‐Carlo simulation did not exceed 2%. So, the measured and simulated data are in good agreement. The verification of PSFs was also carried out for 18 and 20 MV beams . The results were generally similar in accuracy to the case of 10 MV (data not shown).As an example, Figure 2.4x and FWHMy), as well as the equivalent square field size Sclin were determined in the water phantom at a 10 cm depth according to Ref.Sclin values corresponding to different beam energies and nominal field sizes (Scol) formed by the collimation jaws for SSD = 90 and 100 cm are presented in Table The values of full width at half maximum (FWHM) for in‐line and cross‐line directions of the dose profile  and 0.2 cm radius (r) was set at a depth of 10 cm for 10×10cm2 field. The cylinder of h=0.1cm and r=0.05cm was set for 0.5×0.5cm2 field, and the cylinders of h=0.2cm and r=0.1cm were used for the other field sizes. Note that the larger sizes of cylinder were used for larger field sizes to improve the statistics. The absorbed dose was scored in rectangular voxels with a side of 0.1–0.4 cm depending on the field size, centered in the point of interest on the beam axis.The detectors were placed in the s Figure , and theAE=AP=ECUT=ESAVE=10keV. The electron range rejection (ESAVE = 1.0 and 2.0 MeV) and the cross‐section enhancement (XCSE=32and64) in egs_chamber as well as NRCYCL = 10 in DOSXYZnrc were used to reduce a dispersion. To increase the number of photons passing through the sensitive volume of the detectors, a water shell of 1.0–1.5 cm thickness was distinguished around each detector in egs_chamber. The step value of the condensed path of electrons and positrons was set as ESTEPE = 0.25.The absorbed doses in water and in the detectors were calculated using DOSXYZnrc and egs_chamber applications, respectively. The following parameters were used in the simulation: When calculating the absorbed dose for ionization chambers, the type A statistical uncertainty was ≤0.3%, for solid‐state detectors and radiochromic film, it was ≤(0.2–0.9)%. The statistical uncertainty was <0.2% when calculating the absorbed dose in water. Moreover, despite the known sensitivity of the output factor simulation results to the size of the electron focal spot and the geometry of the accelerator head,2.60.5×0.5cm2, 1.0×1.0cm2, 2.0×2.0cm2, 4.0×4.0cm2, and 10×10cm2 were formed for SSD = 90 cm (the sizes of the fields were determined at SAD=100cm) using 10 MV  and 18 MV  photon beams with flattening filter. The fref=10×10cm2 and fcol=4.0×4.0cm2 fields were formed using the collimating jaws at both X and Y axes. The small fields fcol=1.0×1.0cm2 and fcol=2.0×2.0cm2 were formed using a multileaf collimator, wherein the collimating jaws were set at 2.0×2.0cm2 and 5.0×5.0cm2, respectively.The output factors for microDiamond, Dosimetry Diode E, PinPoint 3D Chamber, and EBT3 radiochromic film were determined experimentally for TrueBeam and Clinac IX accelerators . The fields of 3 Farmer ionization chamber  according to TRS‐398.The output factors were obtained by Equation . For thiΩQclin,Qreffclin,fref were determined as ratio of the doses in small water volumes for fclin and fref. The volumes were set as square cross‐section prisms with a side from 0.05 to 0.2 cm and a height from 0.2 to 0.5 cm depending on the field size and centered on the beams’ axis at a depth of 10 cm in the water phantom.The output factors were also calculated for the case of water using Monte‐Carlo method. The values of 33.1kQclin,Qreffclin,fref decreased. For 4.0×4.0cm2 radiation field, the calculated values of output correction factor were close to unity (<1%) for the entire set of studied detectors. At the same time, the deviation of kQclin,Qreffclin,fref from unity for solid‐state detectors and radiochromic film in small radiation fields was less than 4%, while the readings of ionization chambers differed more significantly. For 3.0×3.0cm2 and 2.0×2.0cm2 fields, the reading deviation was ≤4%, while for the fields less than 2.0×2.0cm2, the deviation was up to ≈85%. The readings of the ionization chambers in small fields correlated with their sensitive volumes: a larger sensitive volume corresponded to a stronger deviation from unity with decreasing field size. For example, the highest calculated value of kQclin,Qreffclin,fref under 10 MV exposure for 125 mm3 Semiflex Chamber was 1.847, while for 16 mm3 PinPoint 3D Chamber, the highest value was 1.183. Since the detectors with the deviation of the output correction factor from unity larger than 5% are not recommended for clinical dosimetry,The output correction factors for ionization chambers, solid‐state detectors, and radiochromic film upon exposure with 10, 15, 18, and 20 MV photon beams in different irradiation techniques calculated using Monte‐Carlo method are presented in Tables 3.2≈1.5%(1σ). The values of the output factors calculated by Monte‐Carlo method were in good agreement with the data obtained for Exradin A16 Ion Chamber  for the case of Varian machine in Ref.2.0×2.0cm2 and 4.0×4.0cm2 fields, respectively. For 10 MV beam, the values obtained in the present work turned out to be 2%–3% less: 0.789 versus 0.817 for 2.0×2.0cm2 field and 0.879 versus 0.900 for 4.0×4.0cm2 field, respectively.As an example, comparison of corrected values of the output factors of microDiamond, Dosimetry Diode E, PinPoint 3D Chamber, and EBT3 film with Monte‐Carlo simulation for 10 and 18 MV photon beams are presented in Tables 4kQclin,Qreffclin,fref on the size of the radiation field for the studied ionization chambers upon 10, 15, 18, and 20 MV irradiation were quite similar to the data for 10 MV beams from TRS‐483.3.0×3.0cm2). First, this is due to an increase in the size of the region of absence of transverse electronic equilibrium; second, due to the ionization chamber volume averaging effects; and moreover, due to an increase in the slope of the decay of the photon beam transverse profile and an increase in the radiation flux perturbations caused by the placing the detectors within the field.The dependencies of 2.0×2.0cm2 strongly depends on the sensitive volume of the ionization chambers. If, in accordance with TRS‐483, the difference of ≈5% is taken as the applicability limit of the ionization chamber for small‐field dosimetry,2.0×2.0cm2 and PinPoint 3D for the field sizes above 1.0×1.0cm2. These measurement results correspond to the recommendations of these ionization chambers manufacturer.As expected, the output correction factor for the field sizes less than 0.5×0.5cm2. For the other fields, the deviations were 0.3%–1.1%.The dependence of the output correction factors on the field size for the solid‐state detectors and the radiochromic films differs significantly from that obtained for the ionization chambers. For the Dosimetry Diode E, this can be explained, first, by the volumetric effect due to the extremely small size of the sensitive volume; second, by the close to the absolute visibility of the radiation source from the sensitive volume; and third, by the material of the sensitive volume of the detector. An increase in the contribution of scattered radiation to the fluence caused by a decrease in the field size leads to a change in the ratio of the effective mass energy absorption coefficients of water and silicon. For Diode E, the largest discrepancy with TRS‐483Zeff=7.46, while effective atomic number of water is Zeff=7.42. In addition, the placing of the dosimetric film into water practically does not disturb the distribution of radiation fluxes due to its small thickness. According to the results of Casar et al.,kvol=1.002.The EBT3 radiochromic film had the closest to unity values of the output correction factor in small fields among all the detectors under study. This was largely due to the closeness of the effective atomic number and density of the active layer of the film to those of water parameters. According to Butson et al.,kQclin,Qreffclin,fref on the field size for small radiation fields with a side of ≥1 cm is observed for 60Co, 6 and 10 MV beams, whereas for smaller field sizes, the data differ significantly. In Ref.,kQclin,Qreffclin,fref were found less than unity, as in Refs.There are very contradictory data in the literature for the case of a diamond detector. As noted in Ref.,kQclin,Qreffclin,fref with decreasing field size for the diamond detector under 10 MV photon irradiation. The increase in the beam quality did not change the character of the dependence within the uncertainty of results. The values of output correction factor obtained for microDiamond in the present work match with the data by Casar et al.0.5×0.5cm2 field—in this case, the difference was ≈7.4%). For the photon beams ≥10 MV, the deviation in the values of the output correction factor from unity for a diamond detector in our case was ≈2%–4% for all field sizes. This correlates well with the data by Casar et al.ZC=6) is less than that of water, for studied photon energies, a higher dose response was observed relative to water. This effect may be caused by the fact that the secondary charged particles generated in the materials adjacent to the sensitive volume of the detector make the main contribution to ionization due to the extremely small sensitive volume of the diamond detector.In the present work, we observed a decrease in 52.0×2.0cm2), and PinPoint 3D can be used for the field size above 1.0×1.0cm2.In the present work, the values of the output correction factor for a set of detectors widely used in clinical small‐field dosimetry were calculated by Monte‐Carlo method with use of EGSnrc software toolkit in the little studied region of photon energy above 10 MV. The obtained data demonstrate the limitations of the studied ionization chambers  for small‐field dosimetry in the high‐energy region. It is found that in small fields formed by 10–20 MV photons, the dependence of the output correction factors on the field size for solid‐state detectors and radiochromic film differs crucially from that for the ionization chambers. The deviation of the output correction factors from unity for solid‐state detectors and EBT3 film was less than 5%. Also, for solid‐state detectors and EBT3 film, the experimental output factors, adjusted by the calculated values of the output correction factors, were in good agreement with the results of Monte‐Carlo simulation within the uncertainty. Thus, Diode E (PTW 60017), microDiamond (PTW 60019), and EBT3 film have shown acceptable results and can be recommended to small‐field dosimetry. All studied ionization chambers can be used in a more limited range of the field size (above The authors declare they have no conflicts of interest.Conceptualization: Vladimir A. Klimanov and Maria A. Kolyvanova. Methodology: Vladimir A. Klimanov. Software: Vladimir A. Klimanov, Zarina K. Serikbekova, Alexandr V. Belousov, and Grigorii A. Krusanov. Validation: Maria A. Kolyvanova, Yury S. Kirpichev, Gonzalo Walvyn‐Salas, and Vladimir N. Morozov. Formal analysis: Vladimir A. Klimanov, Alexandr V. Belousov, and Maria A. Kolyvanova. Investigation: Vladimir A. Klimanov, Yury S. Kirpichev, Zarina K. Serikbekova, Alexandr V. Belousov, Grigorii A. Krusanov, Gonzalo Walvyn‐Salas, Vladimir N. Morozov, and Maria A. Kolyvanova. Resources: Vladimir A. Klimanov, Vladimir N. Morozov, and Maria A. Kolyvanova. Writing—original draft preparation: Vladimir A. Klimanov, Vladimir N. Morozov, and Maria A. Kolyvanova. Writing—review and editing: Vladimir A. Klimanov, Alexandr V. Belousov, Vladimir N. Morozov, and Maria A. Kolyvanova. Visualization: Vladimir N. Morozov, Alexandr V. Belousov, and Maria A. Kolyvanova. Supervision: Maria A. Kolyvanova. Project administration: Vladimir A. Klimanov and Maria A. Kolyvanova. Funding acquisition: Vladimir A. Klimanov and Maria A. Kolyvanova.


Thysanoessa sp.) in sub-Arctic Kongsfjorden. Chlorophyll a (Chl-a) concentrations in the surface and middle-layer water were 0.44 μg L−1 (±0.17 μg L−1) and 0.63 μg L−1 (±0.25 μg L−1), respectively. Alloxanthin  was observed at all stations, and its mean values for surface and middle-layer water were 0.09 μg L−1 (±0.05 μg L−1) and 0.05 (±0.02 μg L−1), respectively. The mean MAA-to-Chl-a ratios at the surface (3.31 ± 2.58 μg (μg Chl-a)−1) were significantly higher than those in the middle-layer water (0.88 ± 0.49 μg (μg Chl-a)−1), suggesting that these compounds play an important role in reducing UV photodamage. In gut pigment levels of Northern krill, the most abundant accessory pigment was Allo , as was the accumulation of Chl-a (8.29 ± 1.13 μg g−1 d.w.). The average concentration of MAAs was 1.87 mg g−1 d.w. (±0.88 mg g−1 d.w.) in krill eyes, which was higher than that in all other body parts (0.99 ± 0.41 mg g−1 d.w.), except for the gut. Thysanoessa sp. was found to contain five identified MAAs  in the krill eye, whereas shinorine and porphyra-334 were only observed in the krill body, not the eyes and gut. These findings suggest that Northern krill accumulate MAAs of various compositions through the diet (mainly cryptophytes) and translocate them among their body parts as an adaptation for photoprotection and physiological demands.We investigated pigment and mycosporine-like amino acid (MAA) concentrations of phytoplankton and Northern krill ( The reduction in stratospheric ozone in the Arctic has led to enhanced ultraviolet radiation (UV-R), which affects marine organisms such as phytoplankton and macroalgae ,4,5,6,7.Generally, living organisms survive in extreme environments by expressing various light protection substances and reacting with control mechanisms after exposure to strong light or UV-R . LikewisThe presence of MAAs in zooplankton are mostly considered to be photoprotection function. Zooplankton are not capable of synthesizing MAAs; they can absorb MAAs from their diet (phytoplankton) ,33. AmonThysanoessa sp.  [euphausiids, the key species are T. inermis and T. raschii, accounting for 71% of the euphausiid biomass in Kongsfjorden [Thysanoessa sp. in Kongsfjorden. Here, we assumed that the Thysanoessa sp.  acquired UV-R- absorbing MAAs from dietary phytoplankton during the sampling period. In addition, we analyzed phytoplankton taxonomy using photosynthetic biomarker pigments. This tool was useful to assess ecological characteristics related to the possibility of identifying the diversity of phytoplankton groups and their reaction to various environmental conditions [In the fjords of Spitsbergen, the krill community is dominated by caudata) ,40. Amonsfjorden . This stnditions ,43,44,45nditions ,47. Ther−1 (standard deviation (SD) = ±0.01 μg L−1), 0.38 μg L−1 (SD = ±0.24 μg L−1), 0.07 μg L−1 (SD = ±0.04 μg L−1), and 0.5 μg L−1 (SD = ±0.2 μg L−1), respectively  and A4 (1.1 μg L−1), respectively. Overall, Chl-a concentrations for the surface layer at transect T adjacent land stations  were high compared with the middle-layer, whereas Chl-a at the remnant stations was, on average, twofold higher in the middle-layer than in the surface layer.Among the five pigments (including chlorophyll a), four pigments  were detected in the surface and middle-layer waters of Kongsfjorden, with mean values of 0.04 μg Lectively . Phytopl−1) than in the middle layer (0.05 ± 0.02 μg L−1), while the mean concentration of algal pigment Fuco at the surface (0.2 ± 0.1 μg L−1) was three times lower than that in the middle-layer (0.6 ± 0.2 μg L−1) waters, with a maximum value of 0.80 μg L−1 at A3 (30 m depth). Per was mainly distributed in the middle-layer water, and it was not observed in the surface water at transect A. As a result, the contribution of Per reached 66.3% of total accessory pigments at T3, accounting for 54.5% of total accessory pigments in the middle-layer water of adjacent land stations.The transects also showed differences in accessory pigment compositions between the surface layer and the middle layer. Indeed, Allo among the accessory pigments was found in the surface and middle-layer waters throughout the fjord, whereas Fuco was detected only for several stations . In the surface water, the Fuco concentration increased from the inner fjord to the outer fjord, whereas Allo showed a decreasing trend. . Allo shThe overall compositions within 30 m depth showed that Fuco alone accounted for 50% to 96.6% of the total accessory in transect A. Conversely, in transect T, the pigments were characterized by a larger contribution of Allo  and by a moderate proportion of Per .The concentrations of individual MAA compounds of phytoplankton varied with depth and sampling sites. In the surface water, five distinct MAAs  were detected in natural phytoplankton, whereas four  out of the five MAAs were isolated in the middle-layer samples .−1) and PA (0.48 ± 0.47 μg L−1) than those from the middle-layer water   (The surface-water phytoplankton community had significantly higher concentrations of SH (0.41 ± 0.32 μg L < 0.05) . AS cont < 0.05) .−1 d.w.) and 19′-hexanolyoxyfucoxanthin  were present in trace amounts, whereas Allo exhibited relatively high concentrations (2.791 ± 0.333 μg g−1 d.w.) (−1 d.w. (±1.13 μg g−1 d.w.) in the Northern krill body, which means it occurs by dietary (phytoplankton) accumulation through feeding.For the distribution of accessory pigments in Northern krill, Fuco (0.016 ± 0.002 μg g−1 d.w.) . Chl-a w−1 d.w., with a mean of 1.87 mg g−1 d.w. (SD = ±0.88 mg g−1 d.w.), which was approximately two times higher than that in the remaining dissected body parts except for the gut   A. In add−1 d.w.) B.−1 were observed during the growing season [Spring Chl-a concentrations showed considerable variability in Kongsfjorden; peak values of up to 4.9 μg Lg season . Consideg season ,49,50. Ag season ,52,53.Phaeocystis spp. dominated. The differences in phytoplankton communities may be due to the influence of oceanographic conditions. According to Hodal et al. [Thalassiosira spp. and Chaetoceros spp. in May, when Arctic water masses prevail. Increases in warm water intrusion into fjords have induced changes in the phytoplankton distribution and regime shifts from diatom dominance to a Phaeocystis pouchetii [Phaeocystis sp. [Pigment analyses revealed that the phytoplankton community structure was markedly different between cross-sectional areas. In this study, Fuco and Allo were used to reflect diatoms and cryptophytes, respectively ,44; Per l et al. , the phyouchetii ,59. Basestis sp. ) in the Five individual MAA compounds, SH, PA, PR, MG, and AS, are commonly found in phytoplankton . Large v−1 with a mean of 1.3 μg L−1 in the surface layer compared with the middle-layer (0.2–1.2 μg L−1 with a mean of 0.5 μg L−1)  −1 (SD = ±2.6 μg (μg Chl-a)−1), which was approximately three times higher than that of the middle-layer water (mean ± SD = 0.9 ± 0.5 μg (μg Chl-a)−1) (−1) from Ha et al. [−1) similar to our results, and the observed MAA/Chl-a difference could be due to the different phytoplankton communities studied between both studies. Phaeocystis sp. was dominant for Ha et al. [13C labeling, they confirmed that Phaeocystis sp. produced higher MAA production rates than diatoms. This result mirrored the findings of [Phaeocystis than in diatoms.Overall, the measured total MAA concentration  ranged from 0.3 to 4.3 μg L < 0.05) . In parahl-a)−1) . Howevera et al.  in May ia et al.  reporteda et al. , whereasdings of ,64; theyHowever, we did not find an increase or decrease in the percentage of specific MAAs between two sections , despite our results that show significant differences in the spatial distribution of phytoplankton. For example, the presence of diatoms can produce more SH and PR . Karentz et al. [Euphausia superba as 1.4 mg g−1 d.w., which were lower than those observed in krill in our study. The total MAA levels observed in the present study were within the range found in krill and/or other zooplankton [The average total MAA concentration in whole krill varied twofold among the samples, ranging from a maximum of 5.8 mg gr et al.  found siz et al.  measuredplankton , which mN. australis had similar concentrations of MAAs throughout the body, ranging from 1.6 to 2.0 mg g−1 d.w., with the exception of the carapace. Higher MAA concentrations were found in the eggs than in other body areas for the copepod Cyclops abyssorum tatricus [Many studies have reported remarkable differences in the MAA concentrations and compositions of algae and animals in natural or experimental environments, which are affected by simulating abiotic factors  and biotic interactions  of Northern krill, which indicates the transport specificity fraction of MAAs. Carroll and Shick  found th−1 d.w.) than in other body parts in N. australis. The exoskeletons of crustacean shells mainly comprise chitin, which has a protective effect against UV-R and acidification [Most importantly, krill eyes have considerably more diverse MAAs and higher concentrations than other body parts, and these compounds help prevent UV damage. These results indicate that the eyes are the body part most sensitive to light, and relatively small amounts of MAAs appear to be present in the body, because body parts with shells in Northern krill mainly block harmful UV-R. Indeed, Riemer et al.  observedfication . Thus, dIn summary, in this study, the dietary source of Northern krill seems to be closely associated with close proximity to the glacier front rather than with water depth in our study area of Kongsfjorden, Svalbard. Several lines of evidence indicate that cryptophytes are the most primitive food source for Northern krill and contribute to MAAs, supporting our hypothesis. The relative contribution of the different compounds to the total MAA pool in Northern krill varied among the body parts. The eyes of Northern krill contain a high concentration and various types of MAAs , whereas SH and PR and lower concentrations of MAAs were only observed in the body (without the gut and eyes) of krill. However, MAA compositions in Northern krill are not dependent on the MAA proportion of phytoplankton on the transect T surface, which may be due to differences in the MAA-specific kinetics or the translocation of MAA concentrations or other biological processes such antioxidant defense mechanism, osmotic regulation, and many other cellular functions. Therefore, additional data such as stable isotopes or fatty acids to identify food sources for Northern krill and other environmental factors inducing MAA synthesis will be needed for further investigation, to enable a more in-depth discussion.Kongsfjorden is a sub-Arctic inlet on the west coast of Spitsbergen in Svalbard, which is 26 km long and 6–14 km wide. It is a key open fjord and has a large impact on biodiversity and animal populations with physical and biological variations between the Arctic and Atlantic waters  and refe. SamplinThysanoessa sp.) samples were collected from the sea-ice edge , flash-frozen in liquid nitrogen, and stored at −80 °C for the later extraction of MAAs and pigments.Water samples were collected from the surface and middle-layer (depth of 30 m) waters for the MAAs and the pigments of phytoplankton using a Niskin sampler. At T1, seawater samples were only obtained from the surface layer due to the shallow water. Triplicate 1 L water samples were filtered through a preignited Whatman GF/F filter (25 mm) and then immediately stored at −80 °C for later analysis. Northern krill  was applied; then, they were stored in a cool, dark place for 24 h. An internal standard (50 μL of apo-8-carotennoate) was used to compensate for the losses during extraction. Extracted pigments in the solvent were filtered (0.2 μm hydrophobic syringe filter), and then a 1 mL aliquot of the sample was taken and 300 μL of deionized water (DIW) was added. The chromatographic separation and quantification of pigments were conducted on an Agilent 1200 series HPLC  using a Waters symmetry C8 column   with mob−1. The standard material used for MAA analysis was provided by Professor Häder of Freiderich Alexender University in Germany.The bodies of the Northern krill were separated into two parts for the MAA analysis: the eyes and remaining body parts, except for the gut. Gut contents from the Northern krill were removed, including the entire digestive gland. For MAA extraction, 3 mL of 100% methanol was added to phytoplankton and Northern krill, which were sonicated using an ultrasonicator  and then left overnight at 4 °C. Extraction and filtration were performed using a 0.2 μm syringe filter . Subsequently, 2 mL of extraction solution was transferred to a microtube and then dried using a centrifugal evaporator . The completely dried sample was soaked in 500 μL of DIW, and then 100 μL of chloroform  was added. The supernatant (400 μL) was collected by centrifugation at 10,000 rpm for 10 min. MAA concentrations were determined using high-performance liquid chromatography (HPLC). The detector used an Agilent DAD , and the wavelength was 313 nm (250–750 nm scan). The MAAs were separated using a Waters 120DS-AP (5 μm) 150 mm × 4.6 column. The mobile phase of HPLC used 0.1% acetic acid in 100% DIW at a constant flow rate of 0.8 mL min


The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both.Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids , neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration–response curves for each insecticide–species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population  and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively.A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species  for either The online version contains supplementary material available at 10.1186/s13071-022-05554-7. Anopheles spp. vectors resistant to insecticides from at least one mode of action class were found in 78 countries with ongoing malaria transmission, with resistance to the pyrethroids being the most common  µg/bottle) and An. gambiae s.s (14 [2.2–21] µg/bottle). For some other insecticides, only a little change in estimated LC50 values was observed between mosquito species; for example the LC50 for transfluthrin ranged from 0.09 (range 0.078–0.094) µg/bottle for An. funestus s.s to 0.15 (0.013–0.18) µg/bottle for An. minimus s.s. In addition, LC50 and LC99 values showed little variability between Ae. aegypti and Ae. albopictus for any insecticide  µg/bottle in Brazil to 3.7 (3.5–3.8) µg/bottle in Malaysia. All estimates of variability by institution are shown in Additional file 50 values for clothianidin against An. stephensi were 0.63 (range 0.59–0.67) µg/bottle at NIMR Bengaluru (India), 0.70 (0.66–0.75) µg/bottle at VCRC, Puducherry (India) and 0.80 (0.76–0.85 )µg/bottle at NIMR, New Delhi (India). The trend was similar for LC99 values used by the WHO to establish DCs  µg/bottle for An. gambiae to 10 (0.74–71) µg/bottle for An. stephensi. However, greater variability and difference between species were seen at the top of the curve, with an average OI99 of 62 (range 1.5–110) µg/bottle and 800 (5–1.6e+05) µg/bottle for An. gambiae s.s and An. stephensi, respectively. The raw data extracted from the experimental bioassays showed that the OI100, which was the lowest concentration inhibiting 100% oviposition in all bioassays, was 100 and 75 µg/bottle for An. gambiae and An. stephensi, respectively, with these values being more consistent.The concentration–response curves for pyriproxyfen show broadly similar results although there are insufficient data to draw conclusion on the OI estimates for any species other than nsi Fig. b. The avAe. aegypti  and five major malaria vectors in several regions  were tested with the WHO bottle bioassays, making this, to our knowledge, the largest mosquito toxicological dataset ever produced. Our findings showed that the WHO bottle bioassay was adequate to perform concentration–response analysis and calculate the toxicity (i.e. LC50/OI50 and LC99/OI99) of various insecticide class groups, namely the pyrethroids metofluthrin, prallethrin and transfluthrin; the neonicotinoid clothianidin; the pyrrole chlorfenapyr; the juvenile hormone mimic pyriproxyfen; and the butenolide flupyradifurone.Overall, about 200,000 mosquitoes of two 50 and LC99 estimates was seen for some mosquito species-insecticide combinations. The variability was generally higher with transfluthrin and flupyradifurone than with any other insecticide tested, especially against Anopheles species. For transfluthrin, which is a volatile pyrethroid, the loss of insecticide content in coated bottles during the 24-h drying time might explain the variation in the test results, especially when low concentrations were tested. The median LC99 ranged from 0.44 µg/bottle for An. stephensi to 1.5 µg/bottle for Ae. aegypti  due to lack of consistency and repeatability within and between laboratories in bottle bioassays . We assuFinally, the research of additional surfactants for the WHO bottle bioassays is of high priority considering that MERO® is currently produced by a single manufacturer . In addition, due to its oily composition, MERO® may facilitate insecticide uptake by increasing cuticular penetration , potentiAe. aegypti, indicating that the WHO bottle bioassay is a robust and reproducible method and can be advocated to evaluate the susceptibility of new public health insecticides.In this study we also validated a new Bayesian binomial model using a 5-parameter logistic function developed at Imperial College London to analyse large and complex bioassay datasets. The model offers a flexible Bayesian alternative to common probit or log-probit analysis methods , allowin95, LC90 or LC80 values are used rather than LC99 values, irregardless of the type of insecticide or species  as this information may allow the method parameters to be refined to help to reduce uncertainty and increase precision in estimates. Preliminary investigation showed that the variation or uncertainty in mortality is lower when LCAedes spp. or Anopheles spp. . The plots represent the uncertainty (density of estimates) of LC80, LC90, LC95 and LC99 as shown by the Bayesian model. The larger the plot, the wider the variability of the estimate.Additional file 4: Table S3. Insecticide discriminating concentrations for Aedes and Anopheles species in WHO bottle assays (source [ (source ).


Cucurbita ficifolia Bouché are sources of minerals, vitamins, and functional compounds with nutraceutical and preventive potential against cardiovascular diseases and diseases derived from eating disorders. C. ficifolia is native from Mesoamerica and is currently cultivated in temperate zones from Mexico to South America and Asia. This study evaluated the fruit mesocarps of C. ficifolia for physicochemical parameters, antioxidant activity, and phenolic compound contents in a collection of farmers’ landraces. Germplasm is cultivated by traditional farmers in the temperate zones of two municipalities from Oaxaca, Mexico. The results show that the content of soluble solid contents (SSC), pH, total sugars (TS), and flavonoids are influenced by the fruit geographical origin  and implicitly by their agroecological cultivation conditions . Among populations preserved by traditional farmers, significant differences, and wide variability were found for all parameters evaluated . Eight compounds were identified by UPLC-MS: L-phenylalanine, an amino acid that is regularly associated with proteins; vanillin, a phenolic aldehyde with its functional groups including aldehyde, hydroxyl, and ether; and six phenolic acids: 4-hydroxybenzoic acid, 4-hydroxyphenylacetic acid, vanillic acid, 4-coumaric acid, ferulic acid, and salicylic acid, all with potential health effects. The C. ficifolia fruit mesocarp has bioactive compounds with high antioxidant activity with the potential to both improve diet and to obtain other benefits against nontransmissible diseases derived from food and its associated risk factors.The fruits and seeds of  Less known horticultural plants have recently gained more popularity. They include high contents of nonnutritive, nutritive, and bioactive compounds such as flavonoids, phenolics, anthocyanins, phenolic acids, as well as, sugars, essential oils, carotenoids, vitamins, and minerals. Certain lesser-known fruits also have distinct flavors and tastes, excellent medicinal value, and health care functions –3.Cucurbita ficifolia Bouché belongs to the Cucurbitaceae family and is distributed from southern Mexico to Central and South America in temperate to humid temperate climates at altitudes of 1,000–3,000 m. Both the immature and ripe fruits are widely consumed by indigenous communities as part of typical cuisine, in traditional sweets and cool drinks or as fodder for domestic animals; the dark to creamy-white seeds are consumed as a condiment for dishes or as snacks. Fruits are globular, oval-elliptical, oblong, or ovoid globe with a polar diameter of 20–50 cm, with an epicarp or rigid shell and creamy-white, lemon green to dark green coloration with white longitudinal stripes toward the apex; the pulp or mesocarp and endocarp are white with a granular-fibrous texture and a slightly sweet taste. Fruits have been used in traditional medicine to relieve gastrointestinal disorders, hemorrhoids, and fever and receive different names from the original peoples of Mexico . A plant can produce up to 50 fruits with an average weight of 6 to 7 kg  and an increase in the level of high-density lipoprotein (HDL). Bayat et al.  and Santo Domingo Yanhuitlán (Yanhuitlán), Oaxaca, Mexico. Four to six fruits were collected in a mature state per population and farmer, harvested in the 2021 crop cycle and after transported to the laboratory. The municipalities have a sub-temperate to temperate climate with rainfall of 7,000–1,000 mm distributed from June to November, average annual temperatures of 14–18°C, and average altitudes of 2,200 m. Yanhuitlán is located at 17° 31′ 1″ LN and 97° 19′ 59″ LW, and Huamelúlpam is located at 17° 23′ 48″ LN and 97° 36′ 24″’ LW in southeastern Mexico within the region known as Mesoamerica and its communities identified with the Mixteco indigenous group. The C. ficifolia populations collected and evaluated were made up of 10 populations from Huamelúlpam (ID: 01–10 H) and 10 from Yanhuitlán (ID: 01–10 Y).Once the hard epicarp or shell was removed, the seedless fruit mesocarp was obtained to make five different extract types: Aqueous (5 mg with 10 mL of solvent), ethanolic (1 g in 30 mL of solvent), acetone (5 g in 10 mL of 70% solvent acidified to 1% with HCl), protein [1 g with 6 mg of PVPP (polyvinylpolypyrrolidone) in 25 mL of KCl buffer solution with pH 13] and juice (20 mL), small pieces of mesocarp were processed in an electric domestic extractor . The aqueous, acetone and protein extracts, and juice, were homogenized at 50 rpm for 1 min  and after centrifuged  at 4,000 rpm for 20 min. The mixture was filtered through two layers of cheesecloth, and the filtrate was used for further analysis. The ethanolic extract was left to stand for 24 h and was later centrifuged and filtered for analysis.Soluble solid contents (SSC) and pH: SSC content was analyzed in a digital refractometer  and expressed as °Brix from 0.1 mL of the aqueous extract : Total sugar content was evaluated by the phenol-sulfuric acid method described by Dubois et al. . Quantification was performed with reference to a standard calibration curve of anhydrous D-glucose . The results were expressed as mg of glucose per gram of sample on dry weight (mg G g–1 dw).s et al. . A 1:20 Protein: Protein content was estimated based on the method described by Bradford . The results were expressed as mg of bovine albumin (BA) per gram of sample on dry weight (mg BA g–1 dw).Bradford . A totalTotal polyphenols: Total polyphenol content was evaluated using the method described by Singleton and Rossi , and the results were expressed in mg gallic acid equivalents per gram of sample on dry weight (mg GAE g–1 dw).nd Rossi . First, Total flavonoids: The determination of total flavonoid contents was made based on the method set up by Zhishen et al. , and the results were reported in mg equivalents of catechin per gram of sample on dry weight (mg CE g–1 dw).n et al. . First, Antioxidant activity: The antioxidant activity was evaluated by the DPPH  method described by Brand-Williams et al. . In FRAP, the reaction of 100 μL of the acetone extract and 3 mL of FRAP reagent  was started. It was incubated for 30 min in a water bath at 37°C, and the absorbance at 593 nm was recorded with a spectrophotometer. Additionally, in this case, the reaction was estimated based on the Trolox standard curve . In both cases, the results were reported as μmol equivalents of Trolox on dry weight (μmol TE g–1 dw).s et al.  and by ts et al. . In the Accelerated extraction was performed using solvents  based on the method described by Juárez-Trujillo et al. . A 300.6–1, and the injection volume was 2 μL.Phenolic compound identification and quantification were performed with an ultrahigh resolution liquid chromatograph  coupled to a triple quadrupole mass spectrometer . Chromatographic analysis was performed with an Agilent Eclipse Plus C18 column  worked at 40°C. For the elution of compounds, water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) were used as mobile phases. The gradient conditions were as follows: 0 min 1% B, from 0.1 to 40 min linear gradient 1–40% B, 40.1–42 min linear gradient 40–90% B, 42.1–44 min isocratic 90% B isocratic, 44.1–46 min linear gradient 90–1% B, 46.1–47 min 1% B isocratic . The flow rate was 0.3 mL min−1 dw).The identification and quantification of phenolic compounds was determined according to the analytical conditions described by Juárez-Trujillo et al. , and thep < 0.05), all using the SAS statistical program  were found between the municipalities of origin of the samples in SSC, pH, total sugars, and flavonoid content. Among populations within the municipalities of origin, the differences were significant (p < 0.01) for all the variables evaluated. These results show that the variation in SSC, total sugars, and flavonoids among populations is greater than the variation between municipalities of origin of the populations, based on the variance estimated for each source of variation  to 5.79 (01 H), respectively. In protein, the variation was from 5.02 (05 Y) to 15.31 (08 Y) mg BA g–1, with significant differences between populations within each municipality of origin  to 8.97 (07 Y) °Brix, 0.40 (06 Y) to 0.70 (09 H) mg G gf origin . Greaterp > 0.05) between municipalities of origin of the evaluated populations; in flavonoids, the average of Yanhuitlán was higher than that of Huamelúlpam. Between populations, the variation in the total polyphenol and flavonoid concentrations ranged from 1.94 (05 Y) to 4.89 (06 Y) mg GAE g–1 and from 0.86 (03 Y) to 1.87 (08 Y) mg CE g–1, respectively. The antioxidant activity varied from 4.36 (08 H) to 10.63 (06 H) and from 4.57 (03 Y) to 12.59 (08 Y) μmol TE g–1 evaluated by DPPH and FRAP methods, respectively  to more than 7.2 kg in weight (>45 g after anthesis), and consequently, their external and internal composition changes, including the seed composition (C. moschata fruits (6.42 ± 2.26 °Brix) but slightly higher than the values reported by Martínez-Valdivieso et al. (C. pepo (3.4–4.7 °Brix) and lower than the values determined by Biesiada et al. (C. maxima (9.4–10.9 °Brix) under increased application of organic matter (manure). The results show that the sugars contained in the C. ficifolia fruits provide a certain palatability similar to that of other cucurbits used fresh (vegetable) or processed during the preparation of traditional dishes.In Mexico, Central and South America, Europe and Asia, the immature and ripe fresh fruits of consumed , 5, but z et al.  showed tposition . That isposition , where tposition  in C. moo et al.  in diffea et al.  in C. maC. ficifolia fruits was acidic since among populations, it varied from 4.81 to 5.79, slightly lower than the range reported by Martínez-Valdivieso et al. (C. pepo landraces (6.5–6.9) and for C. moschata (6.77 ± 0.7) registered by Jacobo-Valenzuela et al. (C. ficifolia (5.4–6.1). In this work, each population had its own patterns, and the pH showed a proportional relationship to the total sugar amount (0.2 ≤ r ≤ 0.7), for example, in the cases of 1 H, 03 H, 04 Y and 06 Y. In other cases, the relationship was similar in acidity or pH, and sugar content influenced the taste perception for C. ficifolia fruit consumers and could influence food preferences. The sugar content varied from 0.40 to 0.7 mg G g–1, slightly lower than that reported by Bressani  and C. maxima (182 mg of AB g–1) using the Kjeldahl method . The values reported here are significantly lower than those reported by Oyeleke et al. (C. maxima and C. mixta (30–45 mg GAE g–1 dw), while those determined by Hussain et al. (C. maxima (1.34 ± 0.2 mg GAE g–1 dw) are slightly higher, as are the records of Priori et al. (C. moschata (0.26–0.79 mg GAE g–1 fw). Similarities and differences in C. ficifolia polyphenols with respect to other cucurbits depend on distinct factors, such as the variety, stage of fruit development, plant growth conditions, laboratory methodologies, and their interactions. However, the food contribution of C. ficifolia fruit for consumers and its potential effects on health are relevant.In z et al. , who shoe et al.  for C. mn et al.  in C. mai et al.  in C. mo–1, lower values than those found by Oyeleke et al. (C. mixta and C. maxima (5–9 mg QE g–1) and by Mokhtar et al. (C. moschata (28.66 mg QE g–1). Although these values are reported in terms of quercetin equivalents (QE), they are similar to those reported in catechin equivalents (CE), such as the records of Jacobo-Valenzuela et al. (C. moschata (1.38 ± 0.54 mg CE g–1) and by Hussain et al. (C. maxima (0.77 mg CE g–1). In these and other cases, the laboratory protocols, reference standards and extraction solutions used influence the flavonoid estimations. For example, Enneb et al. (C. moschata pulp flavonoids comparing the use of methanol (close to 30 mg QE g–1) vs. hexane, chloroform, and acetate (5–12 mg QE g–1) with a quercetin standard base. Despite the differences in estimated values, C. ficifolia pulp can contribute flavonoids to the diet, which have an antioxidant effect on the health of consumers and have benefits against coronary heart disease prevention and the antimutagenicity of certain cancer cells and antiviral processes are still under study . These registered compounds differ from most compounds found by Enneb et al. , by Kulczynski et al. (C. moschata and C. pepo (0.48–1.50 μmol TE g–1 dw) and by Priori et al. (C. moschata (0.71–3.58 μmol TE g–1 fw), all evaluated by the DPPH method. By the FRAP method, Kulczynski et al. . In addition, eight compounds were identified by UPLC-MS: L-phenylalanine (amino acid), vanillin , and six phenolic acids , all with potential health effects. Specifically, L-phenylalanine and 4-hydroxybenzoic acid were the compounds with major concentrations, based on UPLCS-MS evaluation, and they have nutritional potential. Consequently, these results encourage us to recommend C. ficifolia fruit pulp consumption due to their potential effects on health as well as complement earlier studies about their composition and their effects on diabetes control.We found wide variation in physicochemical parameters , total polyphenols and flavonoids, and antioxidant activity  in fruits of 20 The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JA-J, EA-B, and JC-S: conceptualization and methodology. GM-Q, JA-J, and JC-S: investigation and writing. All authors read and agreed to the published version of the manuscript.


The results show that WF substitution with TNF increased bread brownness and color saturation and decreased lightness, showing the highest impact on Sb, followed by Tb and Bb. Additionally, bread-specific volume decreased significantly after 20% (Bb), 25% (Tb), and 30% (Sb) TNF substitution. Furthermore, substituting WF with 30% TNF increased crumb hardness from approx. 1.87 N to 3.64 N (Bb), 3.46 N to 8.14 N (Tb), and 6.71 N to 11.39 N (Sb) and caused significant increases to 17.80 N (Tb) and 21.08 N (Sb) after 3 d storage. Only a marginal effect on storage hardness (4.32 N) was observed for Bb. Substituting WF with 10% TNF for Bb or 25% TNF for Tb led to significantly higher consumer (N = 56) scores for all attributes and overall acceptability. However, no significant effect on the overall acceptability of Sb was observed. Flash profiling showed frequently used descriptors for Bb as firm, moist, buttery, smooth, and astringent. After 10% TNF substitution, descriptors were chewy, firm, sweet, porous, dry, and caramel, and that of 30% TNF were grainy, chocolate, brown, nutty, and flaky. Substituting WF with TNF increased the lipids, fiber, and minerals content but decreased the protein and carbohydrate contents of bread. TNF substitution led to different physical and sensory effects depending on bread type, showing that Bb with 10% or Tb with 25% TNF is more comparable with the overall acceptance quality of 100% WF. The study is relevant for utilizing tiger nuts as an ingredient in bread products.Tiger nut is a valuable source of fiber, lipids, minerals, and carbohydrates. However, avenues for incorporating tiger nuts into food remain underexplored, especially in several tropical countries where the plant grows well. The current study investigated the effects of partially substituting wheat flour (WF) with tiger nut flour (TNF) on the physical and sensory properties of different bread types to evaluate the more amenable system for tiger nut incorporation. The substitution was done at WF:TNF ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread (B Cyperus esculentus L) is a tuberous rhizome belonging to the Cyperaceae family of grass-like plants. The edible, almond-like tuber grows in temperate and several tropical regions and is rich in fiber and lipids comprising high content of mono- and polyunsaturated fatty acids, carbohydrates, and some proteins. The nut-like tuber is also rich in minerals, vitamins C and E, and several bioactive phytochemical compounds [Tiger nut , (Pride of the West) supplied by Iran brothers and others Ltd., Ghana, was obtained from the Kotokoraba Supermarket, Cape Coast, Ghana. Granulated sugar, commercially available bakery shortening (margarine), yeast, nutmeg, and salt commonly used for baking were obtained from the Kotokoraba Supermarket trading center at Cape Coast, Ghana.Tiger nuts were hand-picked to exclude discolored or broken pieces and stones and washed with water to eliminate dirt, adhering root hairs, and soil . To enhab), tea bread (Tb), and sugar bread (Sb) at five (5) levels, namely, 10%, 15%, 20%, 25%, or 30%. Then, the procedure for bread preparation reported by Aryeetey et al. [b and Sb with TNF compositions of 7.7%, 11.6%, 15.4%, 19.3%, and 23.2% and for Tb, 8.5%, 12.7%, 17.0%, 21.2%, and 25.4%. Bread prepared using only WF served as the control. For each type of bread, 1000 g dough was sliced, rolled, molded, and arranged in a baking pan and covered with a clean cloth in an enclosed room to allow for proofing at ambient temperature (28-32°C) for 60 min. Then, the dough was transferred to a Swiss baking oven maintained at 230°C for 30 min [For making tiger nut-substituted bread, a Minitab software was used for a centroid mixture design whereby WF was substituted with TNF for butter bread  using a hand-held portable colorimeter . The instrument was calibrated against the white and black tiles, and the mean values for lightness (L∗), red–green intensity (a∗), and yellow–blue intensity (b∗) were calculated from the color primaries. Chroma was computed using equation (Color properties of the bread crust and the crumb were measured based on the Hunter parameters (equation  accordin3 [v (cm3) is the volume and w (g) is the weight of the bread.The specific volume of bread was determined by measuring the weight and the volume by the displacement method according to the procedure by Ahemen et al.  with a f3 . Specifi3  using eqSurface firmness of bread crumb was measured on the first day of baking and at 24 h intervals for 3 d using a modified penetrometer  as described by Manohar and Rao  with slib, Bb, and Sb. Each type of bread had five WF:TNF substitution levels, namely, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30. Bread prepared with only WF (100 : 0) served as the control. About 30 g samples were given random 3-digit codes and served to a randomly recruited 56-member panel  in a counterbalanced manner.Consumer acceptability was determined by using a 9-point hedonic scale as described by Lawless and Heymann . Attribub with TNF of 10%) and its analogs was chosen and flash-profiled [Based on the results of the hedonic study, the bread that showed the highest overall consumer acceptance (Bprofiled  to deterb with WF:TNF substitution ratios as follows: 100 : 0 (Bb), 90 : 10 (T10Bb), 85 : 15 (T15Bb), 80 : 20 (T20Bb), 75 : 25 (T25Bb), and 70 : 30 (T30Bb). An identical sample from T20Bb was included to assess the discriminatory quality. Samples were given 3-digit random codes and served in small trays. A bottle of water (300 mL) was added to rinse the mouth between samples. Raw data on the attributes and the corresponding intensities were analyzed by principal component and Generalized Procrustes Analyses using the Senstools. Net software . Results on hedonic assessment and flash profiling of bread attributes are based on single and duplicate experiments, respectively.A panel of 12 members  was randomly recruited and presented B10Bb), tea bread with 25% tiger nut flour (T25Tb), and sugar bread with 30% tiger nut flour (T30Sb), were carried out. Additionally, the composition of TNF was analyzed to help determine its contribution to the different bread samples. Proximate analysis was carried out as explained previously [N × 6.25) using the Kjeldahl method. Fat was determined by acid digestion followed by Soxhlet extraction with diethyl ether. Total fiber content was analyzed using the sequential acid and alkaline digestion method. Ash content was analyzed by combusting in a muffle furnace, and carbohydrates were determined by difference [Compositional analyses of WF bread and the TNF-substituted analogs that showed the highest consumer acceptance , namely,eviously . Brieflyfference . Resultsp < 0.05.To determine the effects of partially substituting wheat flour with tiger nut flour at a ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread, tea bread, and sugar bread on the response parameters, a Minitab software was used. Data were evaluated using a one-way analysis of variance. Tukey HSD or Games-Howell post hoc analysis was used to compare the mean values where it is necessary. SPSS software package version 16.0 was used for performing the analysis . All significance statements refer to b) gave the highest browning index (BI) followed by that of tea bread (Tb) and butter bread (Bb). Different browning intensities of bread could be caused by the variations in sugar composition, which is known to caramelize and also react with amino acids in a Maillard process during baking, contributing golden brown color compounds to the product [r = 0.995 (Bb), r = 0.992 (Tb), and r = 0.989 , and a gradient or magnitude (BI/Unit) of 9.2 ± 0.04 (Bb), 12.5 ± 0.06 (Tb), and 15.8 ± 0.05 (Sb) for the bread crust, showing that tiger nut addition significantly impacts crust of Sb the most, followed by Tb and Bb. The crumb showed lower browning effects compared to the crust, and after adding 25% TNF, a more pronounced effect on the crumb brownness was observed. The results agree with the report according to Oke et al. [Figures  product . Substite et al. , which ie et al. . Here, wL∗) and Chroma of the bread crust and crumb in Figures L∗ and higher values for color saturation (Chroma) when compared to the other bread types without TNF. Crust and crumb lightness of Bb was comparable, whilst significantly lower values of crust lightness than crumb lightness were observed for Tb and Sb. TNF addition diminished L∗, showing more pronounced effects on the crust or the crumb after 20% TNF or 25% TNF addition, respectively. Additionally, TNF improved the redness (a∗) and yellowness (b∗) of bread, causing a more drastic effect on the color saturation of the crust than the crumb after 25% TNF addition. The effects of TNF substitution on the crust and crumb appearance could be relevant for modulating bread color properties. Browning and other color attributes play a major role in the consumer acceptability of bread, besides the contribution to flavour and aroma. However, the browning reactions can adversely affect nutritional value due to amino acid-simple sugar Maillard-type reactions and the possible formation of acrylamide-related compounds [Color primaries  was the highest followed by Tb (1.301 ± 0.003 cm3/g) and Sb (1.105 ± 1.002 cm3/g), which were higher than that reported by Oke et al. [b (5.74 ± 0.08 cm3/g) and Sb (4.14 ± 0.03 cm3/g) by Aryeetey et al. [b, Tb, or Sb, respectively, which was higher than that reported by Oke et al. [Specific volume is an intrinsic property of bread, which immensely influences consumers' choices .  Figuree et al.  but lowey et al. . The spey et al. . Substity et al. . Signifib (1.87 ± 0.01 N), Tb (3.46 ± 0.0 2 N), and Sb (6.71 ± 0.02 N) significantly increased to 2.46 ± 0.01 N, 13.12 ± 0.02 N, and 16.40 ± 0.02 N, respectively, after 3 d storage. Although differences in bread types exist, a similar range of bread hardness and storage-related increases were reported by Oke et al. [b, 2.36-fold for Tb, and 1.70-fold for Sb, showing that TNF contributes to bread texture differently. Furthermore, whilst storing T30Bb for 3 d did not significantly affect the crumb hardness (4.23 ± 0.01 N), that of T30Tb and T30Sb significantly increased to 17.80 ± 0.02 N and 21.08 ± 0.02 N, respectively. This shows that although TNF increases bread hardness as reported by Oke et al. [b and its TNF substituted analogs. Bread hardness is relevant as it affects consumers' perception of anticipated chewing and judgment and strongly influences product acceptability [Plane bread showed different levels of crumb hardness, which varied according to bread type and storage duration . Hardnese et al. , purporte et al. . Substite et al. , the effe et al. , probable et al.  and impae et al. , thus, ctability .b showed reasonably high scores for all attributes with an overall acceptance rating of 6.50 ± 0.33  of the overall acceptance.The impact of substituting WF with TNF on the sensory scores for bread is shown in 0 ± 0.33 . Nonetheb) had low consumer scores for all attributes with an overall acceptance of 4.81 ± 0.27 , 25% TNF for Tb (T25Tb), and 30% TNF for Sb (T30Sb), are shown in b, 21.09 ± 0.13%, Tb, 26.02 ± 0.03%, and Sb, 26.70 ± 0.39%. The moisture values were comparable to that reported by Oke et al. [10Bb, 21.83 ± 0.07%; T25Tb, 26.87 ± 0.4%, and T30Sb, 25.73 ± 0.04%. The low moisture of Bb and T10Bb may be attributed to the high margarine content, which is known to decrease water binding because of the coating effects, rendering a higher propensity to bake-related moisture loss [Nutrient composition of Be et al. , althouge et al. , this sture loss . Low moiure loss .The nutrient composition of the bread was within the range reported by Maietti et al.  and Oke b), tea bread (Tb), and sugar bread (Sb) to determine the bread type that is more amenable to tiger nut incorporation. Generally, substituting WF with TNF increased the brownness and color saturation, decreased bread lightness with a greater influence on the crust than the crumb, and showed the greatest impact on Sb, followed by Tb and Bb. The incorporation of TNF in bread progressively decreased the specific volume. The amount of TNF that was required to significantly impact specific bread volume was the highest for Sb followed by Tb and Bb. Furthermore, bread hardness increased with TNF substitution, showing the greatest impacts on Tb, followed by Bb and Sb. Although ambient storage further increased bread crumb hardness of Tb and Sb, that of Bb did not significantly change. Regarding consumer evaluation, substituting Bb with 10%TNF gave the highest overall acceptance. Consumer scores for Tb were generally low; however, adding TNF significantly improved the scores for all attributes, and substitution using 25% TNF gave the highest scores for overall acceptance. Descriptors for Bb improved after substituting WF with TNF. Thus, WF substitution with TNF showed that Bb with 10% or Tb with 25% TNF is more comparable with the overall acceptance quality of 100% WF. The results are relevant for enhancing the utility of tiger nuts as an ingredient in bread processing to generate fiber-rich, palatable, and probably, more affordable bread alternatives. Future studies would assess the cost of tiger nut bread production and willingness-to-pay among bread consumers.The feasibility of tiger nut utilization for bread processing was experimented by partially substituting wheat flour (WF) with tiger nut flour (TNF) at a ratio of 100 : 0, 90 : 10, 85 : 15, 80 : 20, 75 : 25, and 70 : 30 for butter bread (B


Schima superba, a species in Theaceae that is a commercially important timer and fire-resistant tree, and revealed its LSI mechanism. Hormones, enzymes, transcriptomes, and proteins were compared between self-pollination (SP) and outcross pollination (OP) in the styles and ovaries from 0 to 120 h after pollination. The self-pollen tubes grew to the bottom of the style and entered the ovary within 48 h but failed to penetrate the ovule. Meanwhile, the hormone and peroxidase levels dramatically changed. Transcriptome and proteome analyses explored the molecular mechanisms of LSI and candidate genes related to LSI in S. superba. Overall, 586.71 million reads were obtained, and 79,642 (39.08%) unigenes were annotated. KEGG and GO analysis showed that there were 4531 differentially expressed genes (DEGs) and 82 differentially expressed proteins (DEPs) at 48 h in self- (SP) versus outcross pollination (OP). Among these, 160 DEGs and 33 DEPs were involved in pollen–pistil interactions. “Pollen–pistil interaction,” “signal recognition,” and “component of membrane” were downregulated in SP, whereas “cell wall and membrane biosynthetic process,” and “oxidoreductase activity” were upregulated. The DEGs involved with S-RNases and SCF during SP suggested that the LSI occurred at 48 h in the ovary and that the LSI in S. superba was under gametophyte control. Calcium ion increase and release, mitochondrial function loss, and ROS disruption further aggravated PCD progress and cell death. The LSI of S. superba, which happened 48 h after pollination, was a key time point. The incompatibility PT ceased growth in the ovary because of S-RNase recognition and PCD in this organ. This study highlights the LSI molecular mechanism in S. superba and provides a reference to other species in Theaceae.In angiosperms, self-incompatibility (SI) is a common and widespread mechanism for plant prevention of inbreeding, and late-acting self-incompatibility (LSI) may be ancestral in the group. In this work, we studied  SI systems are either sporophytic SI (SSI) or gametophytic (GSI)4. SSI always occurs in the stigma, and the pollen-specific S-locus cysteine-rich protein (SCR) and stigma-specific S-receptor kinase (SRK) both control pollen tube (PT) growth; the SSI pattern’s novel species is in Brassicaceae6. In GSI systems, the S-RNase  and S-locus F-box protein  interact to mediate the self-pollen tube rejection response, and this pattern is most common in the styles of Plantaginaceae, Solanaceae and Rosaceae9. In Papaveraceae, another kind of GSI is caused by the Ca2+ signaling cascade, leading to apoptosis and the termination of the pollen tube (PT) growth10. Additionally, ovarian SI (OSI) or late-acting SI (LSI) occurs when the PT ceases growth in the ovary11. LSI, an ancestrally conserved mechanism of SI, exists widely among Narcissus papyraceus, Clivia gardenia, Aloe maculate, Theobroma cacao, and Thryptomene calycina14.In angiosperms, self-incompatibility (SI) is a common and widespread mechanism to reduce the risk of inbreeding depression, and it is usually under genetic control16. LSI can be controlled by the SSI, GSI, or both3. Studies have shown that some growing molecular signals involved in PT attraction to the synergid cells and ovules affect self-PT growth differentially in LSI species20. For example, SCR/s-locus protein 11 (SP11), which is a synergid-derived cue (LURE) belonging to the supergene family CRPs in Brassica pollen, comprises a male signaling ligand determinant21. Growth of the self-pollination tube is halted in the style, whereas SCF and S-RNase genes expressed in Camellia sinensis show that LSI is under gametophytic control22. Sage predicted that the LSI system’s genetic basis is always gametophytic11. Diallel crosses with sibling progeny arrays suggest that multiple loci with multiple alleles control the LSI response24.Since abortion could occur before or after zygote formation, in addition to a much more complicated mechanism in postzygotic SI, the molecular mechanisms of LSI are poorly understoodSchima superba is a woody plant in the tribe Schimeae in Theaceae, which is well-known for the genus Camellia in the Theeae tribe. These two tribes have phylogenetic similarities26. Currently, research on SI has made great progress, and several metabolic pathways are regulated by multiple genes in C. sinensis27. Schima superba is widely distributed in southern China, and its function is mainly for biological fireproofing and valuable timber29. Its fluorescence occurs from the middle of May to the middle of July, and its flowers are small to moderate (lengthcorolla = 31.57 mm) , or GSI (SCF/S-RNase), or their combination control PT growth. The results explain the LSI mechanism in S. superba and refresh knowledge in Theaceae.To further confirm the PT growth inhibition time point, a quantitative analysis of hormone levels in SP and OP was revealed by HPLC–MS/MS. IAA and ZT biosynthesis were decreased in SP from 24 to 120 h Fig. A,B; howeChanges in the antioxidant system were important for determining the time point of self-pollinated PT growth inhibition. CAT, POD, and SOD activity increased from 24 to 84 h in both SP and OP , were sequenced with the Illumina HiSeq 2000 platform. A total of 586.71 million bases of raw reads and 569 million clean reads (Q30 > 92.86%) were generated. In total, 85.36 Gb of nucleotides, 203,767 unigenes, and 79,642 (39.08%) annotated unigenes were generated . There were 4531 DEGs identified in the SP versus OP ovaries, of which 2078 were upregulated and 2453 were downregulated , “cuticle development” (1), and “glycolipid metabolic process” (7) in BP, “host intracellular organelle and membrane” (10) in CC, and “oxidoreductase activity” (16), “ribonuclease T2 activity” (3), and “binding” (21) in MF. However, “pollen–pistil interaction” (9), “recognition of pollen” (9), “response to stress” (8), and “glucose metabolic process” (7) in BP, “signal recognition” (2) and “component of membrane” (32) in CC, “transferase activity” (28), “cellulose synthase activity” (7), and “pheromone activity” (3) in MF were downregulated in SP (S48 vs O48) , cell wall , pollination , oxidoreductase activity , auxin , oligosaccharide , and phosphors signal   was significantly upregulated; GRAS(c64954_g2), BBR/BPC (c102379_g1), HSF (c117518_g3), and MBF1 (c110410_g3) were specifically upregulated, and FAR1(c107861_g4|c55999_g2|c112693_g3), C2H2 (c148204_g1| c96967_g1), HB (c104947_g1), zf-HD (c95393_g1| c87920_g1), G2-like (c29149_g1), PLATZ (c118044_g7), RWP-RK (c116010_g1), TCP (c104077_g2), and TRAF (c110218_g1) were specifically down regulated in SP. Among these, FAR1, C2H2, GRAS, and TCP were closely related to pollen development.Of 62 TFs expressed specifically in the self-pollinated ovaries of 31, were significantly downregulated, and after this time, the ABA levels increased and exceeded the normal level , “pyruvate metabolism” (c114981_g1), and “phenylpropanoid biosynthesis” (c72974_g2), among others, were selected , and downregulated in “protein glutathionylation” , “glutamine biosynthesis” , “aspartate family amino acid catabolic process” , and “oxidation–reduction process”   belonged to an ancient taxon and that this branch should be an early node on the phylogenetic tree in Theaceae35. We found the ovary’s LSI in S. superba was not effective; during self-pollination, 11.2% still developed fruit30, and C. sinensis had only 1.1%27. This may be for an IPT abortion in the ovary, and this distance may not have been enough to encounter more lethal factors than that from the style to the ovule.Researchers have already partly revealed the SI mechanism in some species in Theaceae36. In plants, enzymatic and non-enzymatic antioxidants  act as ROS detoxicants. In our results, GST-related proteins, such as the lambda GSTs (GSTL| SS2206) and dehydroascorbate reductases (DHARs| SS9106), which belong to the outlying minor GST classes, lactoylglutathione lyase , glutathione (GSH| SS7412| SS8421| c112627_g1| c86003_g1| c90752_g1| c97760_g1), and SOD were all downregulated at 48 h in the ovary of SP. The reduction of detoxification factor levels in SP induces ROS accumulation and damage that can be lethal38.Reactive oxygen species (ROS) are the reactive products of oxygen that have the potential to damage living cells, and they play a key role in diverse development stages, such as self-incompatibility during pollination to induce PCD in incompatible pollen37. Receptor-like kinases (RLKs) mediate signaling pathways to control ovular guidance39. In our results, 16 G-type lectin S-receptor-like serine/threonine-protein kinases, which included 5 RLK1 (c126066_g2| c100047_g1| c100047_g3| c103929_g1| c126606_g1) and one RKS1 (c106281_g1), in addition to one PT reception gene at rapid alkalinization factor (RALF), c86998_g1, were significantly downregulated at 48 h in the SP ovary.PT guidance is divided into two processes: pre-ovular and ovular guidance, both of which occur in the ovary40. In our study, three S-RNase genes, which contain the catalytic domain of T2-type ribonuclease (PF00445), c110815_g1, c115031_g1(RNS1) and c94788_g1(RNS3), were significantly upregulated at 48 h in the ovary of SP. Comparing these three genes with other S-RNases in Rosaceae, Plantaginaceae, Rutaceae, Solanaceae, and Rubiaceae  and F-box LRR-repeat protein (c120445_g3| c103038_g1| c51202_g1| c110213_g1). Phylogenetic analyses of S. superba SFB-like genes together with Rosaceae, Solanaceae and Plantaginaceae SFB and SKP genes, shown in Supplementary Figure Prunus SFB gene. Interestingly, c51202_g1 had 5 polymorphic SNP loci in the F-box domain, tightly linked to the RHV region (second intron) of c110815_g1 , as in Prunus, and c41648_g1 and c110213_g1 may function as SLF45. Further experiments should be conducted to clarify these proteins’ interactions and verify this presumption. qRT-PCR showed that c110815_g1 was mainly expressed in the ovary and increased in SP ovaries from 24 to 72 h, and c51202_g1, c41648_g1, and c110213_g1 were significantly upregulated at 48 h in the ovary of SP 48. Indirect progress related to Ca2+ signaling is transduced by protein phosphorylation, and this signal transduction mechanism relates to mitogen-activated protein kinase (MAPK) cascades. The Ca2+ concentration in the cytosol can also trigger downstream sensors to form stimulus-specific information. In S. superba, one MAPK gene c120765_g5 was significantly upregulated, and the “Ca2+ channel activity”, “calcium ion transmembrane transporter activity,” and “calcium ion transport” pathway-related genes and proteins were also upregulated.Apoptosis or PCD is a highly conserved mechanism that removes unwanted cells in eukaryotes49. Then, some soluble proteins, such as cyt c, diffuse from the intermembrane space (IMS) to cytosol51. GO analysis of DEGs showed that 30 GO class 14 DEGs related to mitochondria, such as inner mitochondrial membrane protein complex, mitochondrial membrane organization, and mitochondrial proton-transporting ATP synthase complex, were downregulated, and none of these were upregulated at 48 h in the ovary of SP (Supplementary Table c), which are released from mitochondria52. The Bcl-2-associated athanogene-like protein gene c122719_g2, which contained the BAG domain, was 2.5-fold more highly expressed at 48 h in the ovary of SP , and the mitochondria system loses its functionc leakage from the mitochondria into the cytosol is an initial marker for PCD48. In our study, the concentrations of cytochrome oxidase COX (SS204) and ATP synthase (SS2015) were 9.7- and 5.8-fold higher, respectively, at 48 h in the ovary of SP. COX is the enzyme that catalyzes the oxidation of reduced cyt c by molecular oxygen. A higher COX concentration at 48 h in the ovary of SP indicates that the plants have already started the self-protection mechanism due to higher levels of cyt c. If the COX concentration exceeds the critical value, the activity is limited by either the cyt c or ascorbate concentration, and PCD occurs59. These results are consistent with the results of the disordered oxidation–reduction system shown previously.Cyt S. superba and identified several candidate genes. The time point 48 h after pollination in the ovary’s LSI in S. superba was different from 24 h after pollination in style’s SI in C. sinensis. High expression levels of 2 S-RNase genes and 3 SLF-related genes suggest that LSI in S. superba is under gametophyte control. Self S-RNase induced PCD of IPT could occur by indirect transduction of Ca2+ signaling and release of cyt c. Mitochondrial fusion and function loss caused the PCD; in addition, oxidation–reduction system disorder promoted cell death. These results revealed the LSI molecular mechanism in S. superba and provided a reference for other plants in the Theaceae family.In this study, the analysis of PT growth characteristics, internal physiology changes, transcriptome, and proteome of SP and OP ovaries revealed the LSI mechanism in This study was conducted by the Forestry Genetic and Breeding Lab at the Research Institute of Subtropical Forestry, CAF, State Forestry Administration, China (RISF-CAF). The State Forestry Administration is responsible for national parks and other protected areas. No specific permission was required for these locations/activities, as they were based on a non-destructive collection of plant material. The species is not endangered or protected, and the locations are not privately owned or protected by law.S. superba from Jianou and Youxi in Fujian; they were then grafted on local stock and maintained in Lanxi until 2013. The collection of plant material complied with institutional, national, and international guidelines. Field studies were conducted in accordance with local legislation.Two cultivars, ‘JO59’ and ‘YX1’, grown in Lanxi nursery, Zhejiang province, China, were used in this study. These cultivars were identified, bred, and preserved by our institution. The scions were collected from selected trees of the natural forest of In May 2019, at 9:00–11:00 am on sunny days, we performed pollination . The styles and ovaries were collected at different intervals .Then, the styles and ovaries of each sample with three replications were frozen in liquid nitrogen and stored at − 80 °C for enzyme activity, hormone, amino acid, protein, and RNA-seq analysis.55. SOD activity was measured using inhibition in the photoreduction of nitro blue tetrazolium (NBT)56. CAT activity was measured spectrophotometrically by monitoring the decrease in absorbance at 240 nm57.Three defense-related enzymes, peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), were analyzed using SP and OP ovaries from 24 to 120 h. POD activity was determined using the guaiacol–catalase activity method58. The SP and OP ovaries (0.1 g fresh weight), with three biological replicates from 24 to 120 h, were selected to extract the hormone, and HPLC–MS/MS analysis was performed (Agilent 1290). HPLC analysis was carried out using a poroshell 120 SB-C18 column . The parameters were set as follows: spray voltage, + 4500 V; automizing temperature, 400 °C; the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70 psi, respectively. SAS v8.0  was used to analyze the data.Using HPLC–MS/MS, the levels of auxin (IAA), abscisic acid (ABA), zeatin (ZT) were determined by Zoonbio Biotechnology Co., Ltd. 59. The functions of the unigenes were annotated using Blastx searches with 1e-5 against the protein databases. Blast2GO software60 was used for gene Ontology (GO) term analysis. The plant Transcription Factor Database (PlnTFDB) (version 3.0) was used to determine the transcription factors (TFs)61. FPKM was used to estimate the expression of each gene62, and the DESeq R package 1.10.1 was used to estimate the differential expression between the two treatments63. DEGs were determined with an adjusted p value < 0.05 determined by DESeq. The R-seq data were uploaded to the sequence read archive (accession no. SUB6596208).The total RNA from the S48 and O48 ovaries was extracted using an RNAprep pure Plant Kit . RNA quality and quantity were verified using 1% agarose gels and Agilent 2100 . The mRNA was enriched and purified by magnetic beads with oligo T (dT), cleaved and synthesized first-strand cDNA using random hexamers. The double-strand cDNA libraries were then purified using the AMPure XP system . Transcriptome sequencing was performed using the Illumina HiSeq™ 2000 sequencing platform in Novogene . The adaptor and low-quality sequences (Q < 20 or less than 35 bp) were removed in raw reads. Then the clean reads were assembled using Trinity assembler64. The SP and OP ovaries at 48 h were frozen and ground to power, and 1 g of the sample was used for protein extraction65. Then, 10 μg samples were run on 12% SDS-PAGE gel and visualized by CBB stain66. Gel images with 300 dots per inch were scanned using an image scanner . A total of 450 μl solution with 1500 μg protein sample was used to finish isoelectric focusing with the following parameters: 50uA per strip, rehydration at 50 V for 8 h, 100 V for 1 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 1000–10,000 V (gradient) for 1 h, 10,000 V for 13 h, 500 V for 12 h, temperature, 20 °C. The Ettan-DALT-Six system was run for 45 min at 100 V and then at 300 V for 6–8 h. Using PDquest 8.0 software, all gel images were processed in three steps: spot detection, volumetric quantification, and matching. The differential protein spots were selected using two thresholds . An ABI 5800 MALDI-TOF/TOF Plus mass spectrometer  was used for peptide MS and MS/MS detection, and CalMix5 was used to calibrate the instrument . GPS Explorer V3.6 software  with default parameters was used for data integration, and the proteins were identified using the MASCOT V2.3 search engine .With some modification, protein extraction was performed following Isaacsonhttp://wego.genomics.org.cn/). Clustering heat map analysis of GO/KEGG function enrichment was conducted using the fold change (taken as log2) of DEPs identified using the proteome and corresponding transcriptome genes. The drawing process was implemented using ComplexHeatmap.The corresponding transcripts were identified using the gene ID of the proteome, and the corresponding relationship between the protein and the transcript was determined. A series of association analyses were then perfomed. Correlation analysis was conducted on the fold change (taken as log2) of genes (proteins) identified by transcriptome and proteome analyses in the two omics stydies. The drawing process was implemented using Python. The different multiples of the corresponding transcriptome genes were determined in the two omics studies. The difference multiples were taken as log2 and then plotted using the ComplexHeatmap of the R package. GO annotation of the transcriptome and proteome was used for GO function enrichment using the tool wego (GADPH. Three biological and technical replicates were performed and the gene expression relative quantitation was calculated using the 2−ΔΔCt method67.Using specific primers designed by Beacon Designer 7.90 (Premier Biosoft International, Palo Alto, CA, Supplementary Table All field studies were performed in accordance with the local legislation in China and complied with the convention on trade in endangered species.Supplementary Figures.Supplementary Tables.