Apis mellifera capensis. The bioactivity of Fynbos (FB1-FB6) honeys and Manuka, unique manuka factor 15+ (MAN UMF15+) honey subjected to simulated in vitro digestion, was compared. The effect of each phase of digestion on the antioxidant properties and nitric oxide- (NO-) associated immunomodulatory effects was determined. The total phenolic content of MAN (UMF15+) was higher than that of FB honeys, and following digestion, the percentage bioaccessibility (BA) was 68.6% and 87.1 ± 27.0%, respectively. With the Trolox equivalent antioxidant capacity assay, the activity of FB1 and FB6 was similar to MAN (UMF15+) but reduced for FB2, FB3, FB4, and FB5 with a %BA of 77.9% for MAN (UMF15+) and 78.2 ± 13.4% for FB. The oxygen radical absorbance capacity of MAN (UMF15+) and FB honeys was similar and unaltered with digestion. In a cellular environment, using colon adenocarcinoma (Caco-2) cells, both undigested and the gastric digested honey reduced 2,2′-azobis-(2-amidinopropane) dihydrochloride- (AAPH-) mediated peroxyl radical formation. In contrast, following gastroduodenal digestion, the formation of reactive oxygen species (ROS) was increased. In murine macrophage (RAW 264.7) cells, all honeys induced different levels of NO which was significantly increased with digestion for MAN (UMF15+) and FB1. In LPS/IFN-γ stimulated RAW 264.7 macrophages, only undigested MAN (UMF15+) effectively reduced NO levels, and with digestion, NO scavenging activity of MAN (UMF15+) was reduced but increased for FB5 and FB6. In a noncellular environment, MAN (UMF15+), FB1, FB2, and FB6 scavenged NO, and with digestion, this activity was maintained. This study has identified that undigested and gastric-digested FB honey has antioxidant properties with strong potential anticancer effects following gastroduodenal digestion, related to ROS formation. MAN (UMF15+) had anti-inflammatory effects which were lost postdigestion, and in contrast, FB5 and FB6 had anti-inflammatory effects postdigestion.The Fynbos biome, Western Cape Province, South Africa, produces a unique honey from  Honey iminerals . Polypheof honey , 4, 5.α-dicarbonyls, sugars, organic acids, and vitamin C, as well as the associated antibacterial, antioxidant, and anticancer activity. The researchers concluded that the minerals in honey are bioaccessible; however, more information on the bioaccessibility and bioavailability of other components of honey is required. The number of studies that investigate the effect of simulated digestion on the antioxidant properties of honey is few and is limited to studies on Brazilian bracatinga honeydew, manuka, and blossom honey of unknown origin [Seraglio et al.  have revn origin . In thesn origin  evaluaten origin . O'Sullin origin  reportedn origin . Few stun origin , and forn origin .α) levels in monocytic THP-1 cells [α and cyclooxygenase -2 (COX-2), associated with an increase in prostaglandin E2 (PGE2) in RAW 264.7 murine macrophages [β activity by PC-3 (human prostate cancer) cells [The effect of honey on pro- and anti-inflammatory cytokines has been reviewed by Navaei-Alipour et al. . Several-1 cells . Treatmerophages . In cancrophages , whereasr) cells  and IL-6r) cells . No studα, IL-6, NO, PGE2, inducible nitric oxide synthetase (iNOS), COX-2, and vascular endothelial growth factor (VEGF). In gastric ulcer models, honey reduced the gastric mucosal malondialdehyde (MDA), IL-1β, IL-6, and TNF-α [α, IL-1β, and IL-6 and an increase in IL-10 levels [Nevertheless, the anti-inflammatory effects of honey in animal models have been investigated and provide an indication of the consequence of digestion. In rat and mouse models, oral consumption of honey reduced the levels of TNF-nd TNF-α . In a fu0 levels .α expression [β and TNF-α [Helicobacter pylori infection can cause gastric luminal inflammatory changes [Although immunomodulatory effects of honey in the duodenum are unknown, in rat models of breast cancer, Tualang and manuka honey reduced TNF-pression , while ind TNF-α . This innd TNF-α , only fond TNF-α . In addi changes . InflammLeptospermum scoparium [Apis mellifera capensis (Cape honey bee) in the Fynbos biome of the Western Cape Province of South Africa. The Fynbos biome is a unique region with endemic flowering plant species, namely, the Cape reeds (Restionaceae), heath or Erica (Ericaceae) family, and the Protea (Proteaceae) family [Manuka honey is produced by bees, from the nectar of coparium . This hocoparium . In cont) family . Some re) family  and anti) family , 25 actiSix Fynbos (FB) honey samples  were purchased from local beekeepers and farm stores in the Western Cape Province, South Africa. The samples were transported in air-tight containers to the laboratory at the University of Pretoria, South Africa. In this study, manuka honey, MAN (UMF 15+), was used as a control and was purchased from a local health shop. The physiochemical properties of these honeys were determined as described previously by Serem and Bester .in vitro digestion that simulates human digestion was performed according to a modified method of Daglia et al. [3 were prepared. The honey samples were then diluted to 90% (v/v) to achieve fluidity and subjected to in vitro GIT digestion. For gastric digestion (GD), the pH of the honey solution was decreased to pH 2 followed by the addition of 5 μL of the prepared pepsin solution/mL honey. The gastric digest was then incubated in a water bath for 30 min at 37°C. Then, for further gastroduodenal digestion (GDD), the pH of the honey samples was adjusted to pH 7 followed by the addition of 5 μL of the prepared pancreatin stock solution/mL honey. The mixture was further incubated in the water bath for 60 min at 37°C. For each phase of digestion, pH controls (no enzymes added) were included. To inactivate the enzyme reactions, the samples were heated in a water bath at 95°C for 5 min. Throughout the digestion process, volumes of various solutions that were added were monitored to calculate final % honey concentrations. Assays were then performed with honey solutions at 1 or 10% honey (v/v).Static a et al. . InitialμL of a 10% (v/v) honey samples was added to the wells of a 96-well plate followed by 50 μL Folin-Ciocalteu reagent and 50 μL of 7.5% sodium carbonate. After incubation for 15 min at room temperature, absorbance was determined at 630 nm, and the data was expressed as mg gallic acid equivalents (mg GAE/100 g).The total polyphenolic content was determined using the Folin-Ciocalteu reagent according to Amin et al. . A volum+) was reacted with 3 mM potassium peroxodisulfate to 8 mM ABTS. After a 12 h incubation in the dark, 290 μL of this solution was mixed with 10 μL of a 10% (v/v) honey, pH controls, or digests. After 30 min, absorbance was read at 734 nm, and the antioxidant activity was expressed as μmol TE/g.The TEAC assay was based on a method by Awika et al.  where frμM fluorescein (16 mL) was mixed with 14.8 mM AAPH (4 mL) to prepare a working solution. A 5 μL volume of a 1% (v/v) honey, pH controls, digests, or Trolox (0–800 μM) was then mixed with 195 μL working solution. The samples were well mixed, and the fluorescence was read every 5 min for 4 h at an excitation and emission wavelengths of 485 nm and 520 nm, respectively. The ORAC values were calculated by using the Microocal Origin 6.0 which measured the total area under the decay curve (AUC), and the results were expressed as μmol TE/g.The ORAC assay was determined according to a modified method of Ou et al. . A conce3 cells/100 μL in a 96-well plate and cultured for 24 h at 37°C and 5% CO2. Thereafter, 50 μL of 75 μM DCFH-DA was added to each of the wells, and the plate was incubated for 1 h at 37°C. The medium was then removed from the wells, and the cells were washed once with 0.1 M PBS followed by the addition of 50 μL of 10% (v/v) honey. pH controls or digests and 50 μL of 7.5 mM of AAPH to the wells. Negative controls included cells exposed to PBS only, whereas positive controls included cells exposed to only AAPH and PBS. The change in fluorescence was read  every 2 min for 1 h at an excitation and emission wavelengths of 485 nm and 520 nm, respectively. The gradient of change in fluorescence was measured, and the percentage oxidative damage (%OD) was calculated as follows: %OD = (Sample–PBS)/(AAPH–PBS) × 100.To determine the effect of a 5% honey solution on ROS formation, a modified method of Blasa et al.  was usedγ added), was determined, and then, in a second experiment, the NO scavenging activity, an anti-inflammatory effect, was determined in LPS/IFN-γ-stimulated RAW 264.7 macrophages as described by Malan et al. [The ability of each honey sample to induce NO formation, a proinflammatory effect in RAW 264.7 macrophages . To determine the ability of the honey samples to induce NO formation, 10 μL of the 10% (v/v) honey, pH controls, or digests  was added to the wells containing the plated RAW 264.7 macrophages (no LPS/IFN-γ added). To determine the NO scavenging activity, the RAW 264.7 cells were stimulated with 10 μL of 1 μg/mL E. coli LPS  and 10 μL of 250 U/mL IFN-γ , and then 10 μL of the honey, pH controls, or digests was added, yielding a final volume of 100 μL.For both experiments, a 70 2, and then 50 μL of the supernatant was used to determine NO levels with 50 μL of the Griess reagent (1% (w/v) sulphanilamide and 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride (NED) (w/v) in 2.5% phosphoric acid), and all data was reported as μM nitrite equivalents (NE).For both experiments, exposure was for 24 h at 37°C and 5% COμL of 1 mg/mL MTT solution was added and incubated for 3 h at 37°C and 5% CO2. The formazan formed was solubilised with 25% DMSO in ethanol, and the absorbance was measured at 570 nm and reported as % cell viability.The viability of the attached RAW 264.7 macrophages was determined with the  (MTT) assay. To the remaining media, 5 μL volume of 5 mM sodium nitroprusside (SNP) in 0.1 M PBS that generates 22.91 μM NO, 20 μL of a 10% (v/v) honey solution was added. The levels of NO were measured after 1 h, by adding 100 μL Griess reagent as prepared above, and after 10 min, the absorbance was read at 550 nm and reported as μM NE.To determine if the observed effect in the RAW 264.7 cell line was due to direct NO scavenging, the NO scavenging assay was undertaken. In this assay, the ability of the undigested (UD), GD, and GDD to scavenge sodium nitroprusside- (SNP-) generated NO was determined. To an 80 p < 0.05.All experiments were done at least three times in triplicate yielding 9 data points and presented as mean ± SEM. Statistical analysis was first done by determining the normality of data using the D'Agostino Pearson test and confirmed using the Shapiro-Wilk test. Depending on the normality of the data, significance differences were determined using one-way ANOVA followed by the Tukey post hoc test for data with a normal data set or the Kruskal-Wallis ANOVA followed by Dunn's post hoc test for data with a skewed data set. Significant differences were determined at 2O2 levels, and colour were 3.92, 0.80, 27.79 mg/100 g, 75.69 mg/100 g, 0.97 μmol/mL, and 7.27 (AU). For the 6 FB honeys, the average pH, Fru : Glu ratio, protein and proline content, H2O2 levels, and colour were 4.28 ± 0.03, 0.75 ± 0.02, 35.37 ± 3.35 mg/100 g, 62.41 ± 2.38 mg/100 g, 1.08 ± 0.06 μmol/mL, and 3.47 ± 0.05 (AU). All parameters were similar except that the MAN (UMF15+) was darker indicating a higher polyphenol content, confirmed in Honey characteristics were determined according to Serem and Bester  and Magop < 0.05 to p < 0.00001. This trend was the same for GD, and following GDD, the TPC of FB1 and FB6 was similar to MAN (UMF15+). The TPC of UD compared with GD was unchanged for all honeys, but compared with GDD, it was reduced for FB2 (p < 0.01) and FB5 (p < 0.05). Differences between GD and GDD were significant for MAN (UMF 15+), FB1, and FB2 (p < 0.01). For each honey comparing undigested samples to samples adjusted to pH 2 and pH 7, no significant changes were observed.The TPC range of the FB honeys was 12.67 ± 2.07–74.49 ± 4.19 mg GAE/100 g  with MANμmol TE/g (μmol TE/g. Comparing undigested Fynbos honeys with undigested MAN (UMF15+) honey, the antioxidant activity of FB2 (p < 0.05), FB3 (p < 0.01), FB4 (p < 0.0001) and FB5 (p < 0.001) was significantly less. Following GD, the antioxidant activity of all FB honeys was similar to MAN (UMF15+) except for FB4 (p < 0.0001) which was considerably less. Following GDD, FB2 (p < 0.01), FB3 (p < 0.05), and FB4 (p < 0.0001) were significantly lower than MAN (UMF15+), while the activity of FB1, FB5, and FB6 was similar. For UD vs. GD, antioxidant activity was unchanged for all honeys and was also unchanged for all honey for UD vs. GDD, except for FB2 where the antioxidant activity was decreased (p < 0.01). For GD vs. GDD, activity was significantly reduced for FB2 (p < 0.01). Changes in pH had no effect on the antioxidant activity. For the ORAC assay, the range for the FB honeys was 20.74 ± 2.78–63.66 ± 8.22 μmol TE/g (μmol TE/g. No significant differences were found between MAN (UMF15+) and any of the FB honeys, for UD, GD, and GDD, and in addition, digestion did not alter the antioxidant measured with the ORAC assay.Antioxidant activity was measured with the TEAC and ORAC assays. For the TEAC assay, the range of the FB honeys was 5.46 ± 0.98–19.35 ± 1.23 mol TE/g  with MANmol TE/g , with MAFor FB honey, the correlation between the phase of digestion for each assay and between assays was strong, and for TPC with UD vs. GD, UD vs. GDD, and GD vs. GDD, the correlation was 0.867, 0.958, and 0.918, respectively. The TEAC assay for UD vs. GD, UD vs. GDD, and GD vs. GDD was 0.990, 0.878 and 0.913, respectively. Lastly, for the ORAC assay, UD vs. GD, UD vs. GDD, and GD vs. GDD were 0.837, 0.920 and 0.905, respectively. The average %BA of the FB honey was 87.1 ± 27.0%, 78.0 ± 13.4%, and 100.7 ± 28.4% for the TPC, TEAC, and ORAC assays, respectively.p values ranging from p < 0.05 to p < 0.0001. For GD, differences between MAN (UMF15+) compared with the FB honeys were only significant for FB1 (p < 0.05). Transition from a low pH to a neutral pH environment associated with GDD resulted in a significant loss of this ROS scavenging effect. Compared with the control, although some ROS scavenging was still observed for MAN (UMF 15+), for FB1, FB2, FB5, and FB6, the ROS formed was similar to AAPH (p > 0.05), and for FB3 and FB4, a strong prooxidant effect (higher than AAPH) was observed, p < 0.0001. For the GDD phase of digestion, the correlation for the assays, TPC vs. TEAC, was 0.978; TPC vs. ORAC was 0.821; TPC vs. CAA was -0.666; TEAC vs. ORAC was 0.780; TEAC vs. CAA was -0.644; and ORAC vs. CAA was -0.840.The effect of honey on ROS formation in a cellular model was determined in the Caco-2 cell line representative of the gastrointestinal tract. All UD honey samples caused a significant reduction in AAPH-mediated ROS formation from 100% to 4.28 ± 0.78% for MAN (UMF 15+) with a range of 19.50 ± 2.76–58.83 ± 5.70% for FB honey . Except γ added, did not produce NO, and the samples did not contain significant levels of nitrite measured with the Griess assay (p < 0.05) and FB1 following gastric (p < 0.01) and gastroduodenal (p < 0.05) digestion. The greatest induction of NO levels was for the FB1 and FB3 after gastric and gastroduodenal digestion. In these experiments, the levels of NO-induced are similar to the 14.34 μM, NO induced by 10 μL of 1 μg/mL E. coli LPS and 10 μL of 250 U/mL IFN-γ. Compared to this control (14.34 μM), MAN (UMF15+), FB5, FB6, and the undigested FB1, FB2, and FB3 have no proinflammatory effects. The GD and GDD samples of FB1, FB2, FB3, and FB4 have strong proinflammatory properties related to the induction of NO in RAW 264.7 murine macrophages.The pro- and anti-inflammatory effects related to NO induction or scavenging, respectively, were then determined. The ability of 1% honey, pH controls, and digests to induce NO was determined in RAW 264.7 cells following 24 h exposure. The RAW 264.7 murine macrophages alone, no LPS/IFN-γ model. Undigested, MAN (UMF15+) had the best anti-inflammatory activity, with the Fynbos honeys having similar activity. Solutions of 1% undigested samples of MAN (UMF15+) (p < 0.01) and the gastric and gastroduodenal digests of FB5  and FB6  effectively scavenged NO (p < 0.05) and FB5 after gastric digestion (p < 0.05). These results confirm the anti-inflammatory activity of undigested MAN (UMF15+) and identify FB5 and FB6 as honeys with strong anti-inflammatory activity postdigestion. The pH 2 and pH 7 samples had similar activity to the GD and GDD samples, respectively. In both the proinflammatory and anti-inflammatory experiments, the 1% samples were not cytotoxic as determined with the MTT assay (data not shown).Honey has well-described anti-inflammatory activity, and these effects were further evaluated in the RAW 264.7 murine macrophage/LPS/IFN–γ model can be the result of direct NO scavenging or iNOS inhibition. The ability of the samples to reduce NO formed from SNP as a measure of direct NO scavenging was then determined (p < 0.0001), FB1 (p < 0.01), FB2 (p < 0.05), and FB6 (p < 0.05); the gastric digests of MAN (UMF15+) (p < 0.0001), FB1 (p < 0.01), and FB6 (p < 0.01); and all the gastroduodenal digests effectively scavenged NO (p < 0.05–p < 0.001). The best NO scavenging activity was seen in the undigested MAN (UMF15+) and FB1 samples. Following GD, all honeys had similar scavenging NO activity that was increased following GDD. This confirms that the anti-inflammatory effects of MAN (UMF 15+) are due to NO scavenging but also identifies FB1, FB2, and FB6 as honeys with strong direct NO scavenging activity.Anti-inflammatory activity related to a reduction in NO levels in the RAW 264.7 murine macrophage/LPS/IFN–In this study, the effects of GIT digestion on the antioxidant and inflammatory properties of Fynbos honey were determined and compared with manuka honey. All parameters were similar except that MAN(UMF15+) was darker, and a higher polyphenol content was confirmed. The TPC of MAN (UMF15+) was 119.42 ± 13.94 mg GAE/100 g, within the range of 63.85 mg GAE/100 g, 103.99 mg GAE/100 g, 217 mg GAE/100 g, and 203 mg GAE/100 g reported for Comvita®, New Zealand , manuka in vitro studies are within range and still physiologically relevant.In the present study, gastric and duodenal digestion of 0.5 h and 1 h, respectively, significantly reduced the TPC of MAN (UMF15+) to a %BA of 68.8%. O'Sullivan et al.  reportedFor Fynbos honey subjected to simulated gastrointestinal digestion for the TPC, the %BA was 87.1 ± 27.0%, and differences compared with MAN (UMF15+) were significant for FB2, FB3, FB4, and FB5. In contrast, the reported TPC of Irish, Tesco, and Lidl honey was unchanged after gastrointestinal digestion [To understand the complex antioxidant profile of food and derived products, several techniques are often used , and forμmol TE/g and ORAC values of 24.82 ± 0.5 and 12.40 ± 0.3 μmol TE/g for MAN (UMF22+) and super manuka honey, respectively. In the present study for MAN (UMF15+), the antioxidant activity was similar and was 22.09 ± 1.29 μmol TE/g. In contrast, the ORAC value of 48.41 ± 14.11 μmol TE/g was higher. The antioxidant activity of FB1 and FB6 was similar to MAN (UMF15+) but was reduced for FB2, FB3, FB4, and FB5 when measured with the TEAC assay, while with the ORAC assay, the antioxidant activity of all FB honey was similar to MAN (UMF15+).Anand et al.  reportedWith digestion, the %BA and TEAC assays for MAN (UMF15+) were reduced to 77.9%. Other studies have also reported a reduction in the antioxidant activity of manuka honey following digestion . The antIn vitro, gastrointestinal digestion reduced the %BA of an organic Necta® Floral, phloem honey measured with the TEAC assay to 20.17% [The antioxidant activity of the FB honeys measured with the TEAC and ORAC assays following digestion resulted in a %BA of 78.2% and 100.7%, respectively. In the study by O'Sullivan et al. [The degree of polyphenol stability in the different phases of digestion is pH dependent. In general, polyphenols are stable at low pH associated with gastric digestion while some polyphenols are unstable at neutral or alkaline pH, associated with duodenal digestion. Under these conditions, susceptible polyphenols undergo degradation or transformation, forming molecules with altered chemical and biological properties .For MAN (UMF15+), following digestion, Cianciosi et al.  reportedIn the present study, pH alone had little effect on the TPC, and antioxidant activity measured with TEAC and ORAC assays. Only for pH 2 vs. GD for FB1 was there a significant increase in TPC, and for pH 7 vs. GDD for FB2, a significant decrease in TPC was found that did not translate into a significantly altered antioxidant activity. Like the study of Seraglio et al. [Mimosa scabrella Bentham honeydew, the concentration of individual polyphenols determined with LC-ESI-MS/MS was increased [μg/100 g for Urupema, Urubici, and Lages honey blends, respectively. The differences found between measured antioxidant activity, compared with LC-ESI-MS/MS analysis, were proposed to be due to the complexity of honey, where in addition to polyphenols, other compounds, such as vitamins and peptides, also contribute to the antioxidant properties of honey. pH effects and proteolysis can reduce the antioxidant activity of the antioxidant vitamins and peptides, respectively. For example, the antioxidant vitamin, vitamin C, is pH sensitive, and Costa et al. [In contrast to an unaltered or reduced antioxidant activity, for ncreased . Followia et al.  reportedTo further validate these results, the ability of the honey samples to protect Caco-2 cells from AAPH-generated peroxyl radical damage was determined. This cellular model provides a more physiologically relevant assessment of these properties . The DCF50 for the undigested and digested honey was 16.97 mg/mL  [β-catenin pathway.In contrast with duodenal digestion, a loss of peroxyl scavenging activity was observed, especially for FB3 and FB4, where ROS levels were increased. The latter is attributed to the pH-dependent autoxidation of flavonoids and is reported to play a role in the anticancer effects observed following honey digestion. In the Caco-2, colonic cancer cell line, a significant loss in cell viability was observed at 2.5 to 7.5 mg/mL undigested and 1 to 3 mg/mL for digested manuka honey, indicating that with digestion cytotoxicity is increased  compared47 g/mL)  and 14.347 g/mL)  with HCTBesides these studies, further anticancer properties of manuka honey have been extensively reviewed by El Senduny et al. . Manuka 2O2 formation [μM, 116.7 ± 3.3 μM, and 110.2 ± 2.0 μM H2O2 formed for 1 mM propyl gallate, epigallocatechin gallate (EGCG), and quercetin, respectively. In DMEM/F12, H2O2 levels were lower with the formation of 95.2 ± 1.9 μM, 90.2 ± 2.6 μM, and 76.4 ± 5.9 μM H2O2 for the same concentration of propyl gallate, EGCG and quercetin, respectively. The presence of amino acids, vitamins, and other molecules in cell culture media with antioxidant activity accounts for the lower H2O2 levels for DMEM/F12 compared with PBS. The kinetics of H2O2 formation was linear, 0–60 min reaching a plateau after 120 min. Concentrations of 80 μM propyl gallate, 100 μM EGCG, and 40 μM quercetin reduced the viability of DU-145 cells after 24 h exposure [2O2 via the Fenton reaction requires transition metals that catalyze the conversion of H2O2 to ROS that accumulates in cell culture causing cell death. Addition of iron chelators to the cell culture media reduced the effects of H2O2 [In neutral or alkaline solutions as well as cell culture media, susceptible flavonoids undergo autoxidation, a pH-dependent process that occurs rapidly at alkaline pH, and likewise in the intestines, pH would also contribute to autoxidation . Polypheormation . Grzesikormation  reported2O2 levels formed via glucose oxidase (GOx) mediated conversion of glucose to gluconolactone with the reduction of molecular oxygen to H2O2. Levels of H2O2 are increased following the dilution of honey due to the activation of the GOx system [2O2. Dźugan et al. [2O2 content of 50 ± 2 to 1100 ± 100 μM, had significant levels of antioxidant activity (DPPH and FRAP assays) with p-hydroxybenzoic acid, p-coumaric acid, and kaempferol being the most abundant. These researchers also concluded that the antibacterial activity of these honeys was due to the presence of these phenolic acids rather than the high H2O2 content. Likewise, the anticancer effect of honey is not limited only to H2O2 formation; in addition, specific polyphenols contribute significantly to this effect. Cianciosi et al. [2O2 formation, and have antioxidant activity better than that of the parent compound. Consequently, the H2O2 levels generated are dependent on honey type, polyphenol composition, including structure and concentration, transition metals, and the activity of the formed autoxidation products. For FB honey, pH 7 was associated with an increase in ROS formation for FB1, FB3, FB4, and FB6 but not FB2 and FB5, indicating that pH effects are complex and are a combination of H2O2 formation, activity of pH-stable phenolic acids, and the activity of the autoxidation products.Manuka honey contains MG, and although cytotoxic , findingx system . In an ax system  increasen et al.  found thi et al.  showed tHelicobacter pylori infections increase the expression of iNOS and, together with oxygen radicals, form the highly reactive peroxynitrite radical, that is cytotoxic and induces inflammation, associated with an increased risk for cancer [In the GIT, endogenous NO (eNOS) released from the gastric mucosa, vascular endothelium, and sensory neurons, together with the prostaglandins, maintain the integrity and microcirculation of the gastric mucosa. In addition, NO can increase the resistance of the GIT mucosa to injury, and the development of NO-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk for NSAID-induced ulcer development . A proinr cancer . Therefoα, IL-1β, and IL-6 induced in MM6 and human monocytes were unrelated to LPS levels. Subsequently, Tonks et al. [v/v) honey. Other immune modulatory proteins identified in honey that increased TNF-α in monocytes, macrophages, and keratinocytes were the peptides, apalbumin-1 (Apa1) and -2, major royal jelly, and apisimin [α levels in murine macrophages when compared with the other fragments, Apa1 and recombinant Apa1 [MAN(UMF15+) honey did not induce NO formation, but with digestion, NO levels were increased, indicating that with digestion, MAN (UMF15+) in the duodenum is proinflammatory. A similar effect was observed for only FB1, where with digestion, NO levels were increased. Tonks et al.  reporteds et al.  identifis et al.  also conapisimin . Partialant Apa1 .α, IL-1β, and LPS are the main inducers of iNOS, that catalyzes the formation of NO, a critical inflammatory response of the innate immunity [α, IL-1α, IL-1β, IL-6, IL-12, IL-23, and COX-2 [TNF-immunity . M1 macrnd COX-2 . These pnd COX-2  and the γ RAW 264.7 macrophage model, but with gastric digestion, NO scavenging was reduced, and with duodenal digestion, NO was lost. In contrast for FB5 and FB6, with digestion, NO scavenging was increased, with FB6 having the greatest NO scavenging effect. This effect is either due to the direct NO scavenging or iNOS modulation. Direct NO scavenging was then determined with the SNP assay. Undigested and gastric digested MAN (UMF15+), FB1, and FB6 honey directly scavenged NO. Following gastroduodenal digestion, all honey digests directly scavenged NO, although this effect was not observed in the LPS/IFN-γ RAW 264.7 macrophage model.MAN (UMF15+) effectively reduced NO levels in the LPS/IFN-γ, NO scavenging can occur. In the present study, findings were that MAN (UMF15+) is predominantly a honey with anti-inflammatory properties due to direct NO scavenging and supports the findings of previous studies [A finding not previously reported is that honey can have a pro- or anti-inflammatory effect depending on the honey and the physiological environment. In the RAW 264.7 macrophages, in the absence of proinflammatory molecules, NO can be induced and depending on levels may have either a protective or inflammatory effect. In contrast in the RAW 264.7 macrophages, where NO is induced by LPS/IFN- studies . Howeverin vivo studies have identified the ability of honey to reduce indomethacin as well as ethanol and acidified ASA-induced gastric lesions due to the anti-inflammatory activity of honey and the associated reduction of NO, ROS, and associated nitrosative species of honey [Indomethacin is widely used to induce gastric ulcers in experimental animal models . The proof honey . In thisIn the duodenal phase of digestion, the activity of MAN (UMF15+) is retained, although the cellular anti-inflammatory activity is lost, and some NO scavenging activity is retained. In contrast for FB honey, in the duodenal phase of digestion, the antioxidant effect is reduced, although not associated with significant changes in antioxidant activity when evaluated with noncellular assays. Increased ROS formation has been identified as one of the anticancer mechanisms of several types of honey , 8 and wFynbos honey has antioxidant activity that is unaffected with gastric digestion, but with gastroduodenal digestion, there is a variable loss of polyphenol content and antioxidant activity, TEAC assay, although activity measured with the ORAC assay is unaltered. Evaluation of CAA in the Caco-2 cell line revealed a strong prooxidant effect after gastroduodenal digestion, which was pH dependent for FB1, FB3, FB4, and FB6 but not FB2 and FB5. This identifies FB honey, especially FB3 and FB4, as honeys with potential anticancer properties, related to ROS production, better than MAN (UMF 15+) honey.γ-stimulated RAW 264.7 macrophages, only undigested MAN (UMF15+) effectively reduced NO levels, and with digestion, NO scavenging activity of MAN (UMF15+) was reduced but increased for FB5 and FB6. In a noncellular environment, MAN (UMF15+), FB1, FB2, and FB6 effectively scavenged NO to a variable degree following gastric digestion but strongly for all honeys after gastroduodenal digestion. This study has identified that undigested and gastric-digested FB honey has antioxidant properties with potential anticancer effects following gastroduodenal digestion, related to ROS formation. MAN (UMF15+) had anti-inflammatory effects which were lost postdigestion , and in contrast, FB5 and FB6 gained anti-inflammatory effects postdigestion. Although further elucidation of the mechanisms involved is required, this study shows that FB honey has potential health benefits when digested.In murine macrophage (RAW 264.7) cells, all honeys induced different levels of NO which was significantly increased with digestion for MAN (UMF15+) and FB1. In LPS/IFN-


We demonstrate that THOC binds nucleoplasmic TERRA, and that RNaseH1 loss, which increases telomeric R-loops, promotes THOC occupancy at telomeres. Additionally, we show that THOC counteracts lagging and mainly leading strand telomere fragility, suggesting that TERRA R-loops can interfere with replication fork progression. Finally, we observed that THOC suppresses telomeric sister-chromatid exchange and C-circle accumulation in ALT cancer cells, which maintain telomeres by recombination. Altogether, our findings reveal crucial roles of THOC in telomeric homeostasis through the co- and post-transcriptional regulation of TERRA R-loops.Telomeres are the nucleoprotein structures at the ends of linear chromosomes. Telomeres are transcribed into long non-coding Telomeric Repeat-Containing RNA (TERRA), whose functions rely on its ability to associate with telomeric chromatin. The conserved THO complex (THOC) was previously identified at human telomeres. It links transcription with RNA processing, decreasing the accumulation of co-transcriptional DNA:RNA hybrids throughout the genome. Here, we explore the role of THOC at human telomeres, as a regulator of TERRA localization to chromosome ends. We show that THOC counteracts TERRA association with telomeres via R-loops formed co-transcriptionally and also post-transcriptionally,   Telomeres are the nucleoprotein structures at the termini of linear chromosomes. They are crucial for genome stability and ensure that chromosome ends are not inappropriately recognized as sites of DNA damage. Telomere functions at mammalian telomeres are in part achieved by a telomere-specific protein complex termed shelterin. It includes the double strand (ds) DNA-binding proteins TRF1 and TRF2, POT1 which binds the single stranded telomeric overhang, TIN2 and TPP1 which connect POT1 to TRF1 and TRF2, and TRF2-bound Rap1 , termed R-loops ,13,16–18iginated . Severaliginated ). Among iginated . More reiginated ,22. On tiginated ,24, as wiginated —which retranscription defects of hpr1Δ mutants by overexpression) complex at human telomeres.R-loops have primarily been viewed as harmful by-products of transcription, posing an obstacle for the progression of DNA and RNA polymerases, thus jeopardizing genome stability. However, their involvement in a myriad of cellular processes has been demonstrated . At chroS. cerevisiae as a functional unit involved in transcriptional elongation of genes with high GC content or that are particularly long, suppressing hyperrecombination events (Transcription and Export) complex, together with the helicase and splicing factor UAP56/DDX39B and the RNA nuclear export protein Aly/ALYREF/THOC4  (The THO complex (THOC) was originally identified in n events . In human events . It facin events . In addifactors) . Remarkafactors) ,31,36. Tfactors) ,37–40. TS. cerevisiae) (All THOC subunits were previously detected in purified human telomeric chromatin by mass-spectrometry . In yeasevisiae) . On the evisiae) . At humain trans, via R-loops. Additionally, we find that the THO complex is recruited to telomeres when cells are depleted of the ribonuclease RNaseH1, which specifically degrades DNA:RNA hybrids. In addition, THOC interacts with nucleoplasmic TERRA. Therefore, we propose that THOC counteracts telomeric R-loops through several mechanisms. First, THOC may prevent the association of TERRA with telomeric DNA through R-loops during transcription. Second, THOC contributes to the resolution of DNA:RNA hybrid structures that have formed post-transcriptionally. Third, through binding of TERRA in the nucleoplasm, THOC may counteract TERRA association with telomeric DNA post-transcriptionally. In addition, we observe that loss of THOC subunits results in fragility at telomeres replicated by leading strand synthesis, and also to a lesser extent by lagging strand synthesis. Finally, in ALT cells—but not in telomerase-positive HeLa cells—loss of THOC leads to an increase in the frequency of telomeric sister-chromatid exchange events and increasing amounts of C-circles, likely as a consequence of accumulated R-loops at the telomeric tract in these cells. Thus, our data demonstrate that THOC promotes telomere stability through the regulation of the lncRNA TERRA.Here, we demonstrate that THOC subunits 1 and 2 counteract R-loops formed at human telomeres. Resorting to a previously developed system to detect the nuclear localization of ectopically-expressed TERRA molecules , we show2, at 37°C. Generation of rtTA+ PCP-GFP+ HeLa cells for PP7-15qTERRA expression was previously described  (Gibco), supplemented with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin. Cells were maintained in a controlled humidified atmosphere with 5% (v/v) COescribed . Generatescribed . Suspens180 000 cells/well were plated in six-well plates one day before siRNA transfection. The following day, growth medium was replaced by antibiotic-free DMEM supplemented with 10% FBS, and cells were transfected with 20 pmol siRNA see  using caHek293T cells were plated one day before transfection for lentivirus production. The following day, growth medium was replaced by Opti-MEM (Thermo Fisher Scientific). Cells were transfected with 1 μg pMD2.G plasmid, 3 μg pCMVR8.74 plasmid and 4 μg of lentivirus transfer plasmid see , using LCells were grown on round coverslips. Coverslips were washed twice in 1× PBS, fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and washed twice in 1× PBS. Coverslips were then incubated in detergent solution  for 5 min, and incubated in 2% BSA dissolved in PBS for 10 min. Incubation with primary antibody see  in block3 PNA probe (PNA Bio) in 15 μl hybridization mix per coverslip ), at 80°C for 3 min, followed by 3 h at room temperature, in a humidified chamber. Slides were washed twice with wash buffer 1  for 15 min/wash, and then washed three times with wash buffer 2  for 5 min/wash. DAPI was added to the second last wash at 0.1 μg/ml. Slides were dehydrated with 70%, 95% and 100% ethanol, air-dried and mounted with Vectashield.For FISH staining, hybridization was done with 100 nM Cy3-[CCCTAA]Images were acquired with an Upright Zeiss Axioplan equipped with a 100×/1.40 oil objective.3 PNA probe (PNA Bio) in 70 μl hybridization mix ), at 80°C for 3 min, followed by 3 h at room temperature, in a humidified chamber. Slides were washed twice with wash buffer 1  for 15 min/wash, and then washed three times with wash buffer 2  for 5 min/wash. DAPI was added to the second last wash at 0.1 μg/ml. Slides were dehydrated with 70%, 95% and 100% ethanol, air-dried and mounted with Vectashield.Cells were treated with 0.05 μg/ml demecolcine for 2 h before harvesting by trypsinization, resuspended in hypotonic solution (0.056 M KCl), and incubated at 37°C for 7 min. Cells were fixed in cold methanol:glacial acetic acid (3:1) solution overnight at 4°C. Fixed cells were dropped onto glass slides, incubated at 70°C for 1 min in a humidified oven, and air-dried overnight at room temperature. Slides were incubated in 4% formaldehyde in PBS for 5 min, washed three times in 1× PBS and dehydrated with 70%, 95% and 100% ethanol. Hybridization was done with 100 nM Cy3-[CCCTAA]Images were acquired with an Upright Zeiss Axioplan equipped with a 100×/1.40 oil objective, or with a Leica SP8 confocal microscope equipped with a 63×/1.40 oil objective and a DFC 7000 GT camera.2O was done after irradiation, followed by treatment with 10 U/μl Exonuclease III (New England Biolabs) for 1 h at 37°C. Slides were incubated in 4% formaldehyde in PBS for 3 min, washed three times in 1× PBS, and dehydrated with 70%, 95% and 100% ethanol. Following overnight air-drying of slides, hybridizations were done sequentially using 0.22 μM TYE563- or 6-FAM-labeled LNA probes (Qiagen)  indicate LNA nucleotides) in 60 μl hybridization mix per slide ), at room temperature for 2 h each. After each hybridization, slides were washed three times in 2× SCC for 3 min and dehydrated with 70%, 95% and 100% ethanol. After the last probe hybridization, DAPI was added to the second 2xSSC wash at 0.1 μg/ml. Slides were air-dried and mounted with ProLong Diamond Antifade mountant.CO-FISH staining was performed as previously described , with soImages were acquired with a Leica SP8 confocal microscope equipped with a 63×/1.40 oil objective and a DFC 7000 GT camera.Cells were collected by trypsinization, washed with 1× PBS and resuspended in 2× Laemmli buffer, to a final concentration of 10 000 cells/μl of Laemmli buffer. Samples were incubated at 95°C for 5 min. Proteins were separated on a 4–15% SDS-PAGE precast gel  and transferred onto a 0.2 μm nitrocellulose membrane (Amersham). Membranes were blocked with blocking solution (3% BSA (w/v) in 1× PBS with 0.1% Tween-20) for 1 h at room temperature and incubated with primary antibodies see  at 4°C oRNA was isolated from 3 to 5 million cells with the NucleoSpin RNA kit (Macherey-Nagel). Two on-column and one in-solution rDNase digestions (Macherey-Nagel) were performed.4, 1 mM EDTA, pH 8.0, 1% (w/v) BSA, 7% SDS) for at least 1 h at 50°C, and hybridized with a 32P-radiolabeled telomeric probe in Church buffer at 50°C overnight. The membrane was washed twice in 2x SSC, 0.5% SDS and twice in 1× SSC, 0.5% SDS, for 15 min/wash. The membrane was exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE). After signal detection, the membrane was stripped by incubation with boiled 0.1× SCC, 1% SDS at 55°C, three times for 30 min each, and blocked in Church buffer for at least 1 h at 55°C. A 32P-radiolabeled 18S rRNA oligonucleotide probe was used for hybridization at 55°C overnight. The membrane was then treated as described for the telomeric probe.For RNA dot blot, purified RNA was digested with RNase , as a control. All samples were denatured at 65°C for 3 min and blotted onto a Hybond-XL membrane (Amersham) using a dot-blot apparatus (Bio-Rad). RNA was UV-crosslinked to the membrane. The membrane was then blocked in Church buffer , GAPDH and TERRA reverse primers see . Reverse2, 0.5% NP-40, 1 mM dithiothreitol (DTT)), supplemented with 400 U/ml RNasin Plus (Promega) and a protease inhibitor cocktail  (100 × 106 cells/ml of RLN buffer). Lysates were homogenized with a Dounce homogenizer, incubated on a rotating wheel for 20 min at 4°C, and centrifuged at high-speed for 10 min. The supernatant was collected and pre-cleared with magnetic protein G Dynabeads (Thermo Fisher Scientific) on a rotating wheel for 1 h at 4°C (70 μl beads per 1 ml of extract). Pre-cleared extracts equivalent to 50 × 106 cells were used per immunoprecipitation with 6 μg of antibody ). Input samples were diluted appropriately to ensure that the amount of probed RNA in each immunoprecipitated sample lies within the linear dynamic range of the assay. RNA dot blot was done as described above.Cells were harvested by trypsinization (or collected by centrifugation in the case of suspension Hek293E cells), counted, washed with 1× PBS and placed on ice. Cells were then lysed in RLN buffer ), resuspend in 1 ml LB3 buffer, transferred to sonication vials with AFA fiber (Covaris), and sonicated with a Focused-Ultrasonicator  , to achieve fragments of < 500 bp. Sonicated samples were centrifuged at 21000 g for 15 min. Supernatants were collected and diluted 1:2 in IP dilution buffer ). Samples were pre-cleared with sepharose protein G beads (Cytiva) pre-blocked with yeast transfer RNA, on a rotating wheel for 1 h, at 4°C. Pre-cleared extracts equivalent to 2 × 106 cells were used per immunoprecipitation with 4 μg of antibody .Cells were harvested by trypsinization 72 h post-siRNA transfection, counted, and washed with cold 1× PBS. For Western blot analysis of depletions, ca. 0.5 × 10body see , and 20 6 cells/condition were collected. For DRIP, 1 × 107 cells/condition were resuspended in 175 μl of ice-cold RLN buffer , and 100 U/ml RNasin Plus (Promega)), incubated on ice for 5 min, and centrifuged at 300 g for 2 min at 4°C. Nuclei were brought to room temperature and lysed with 500 μl RA1 buffer  containing 1% 2-mercaptoethanol, and homogenized with a syringe with a 0.9 × 40 mm needle. Nucleic acid extracts were then loaded in Phase Lock Gel heavy (5PRIME) tubes, mixed with 250 μl H2O and 750 μl phenol-chloroform-isoamylalcohol (25:24:1) with a pH of 7.8 to 8.2, and centrifuged at 13 000 g for 5 min at room temperature. The aqueous phase was transferred into a new tube. 750 μl cold isopropanol and NaCl to 50 mM were added to the aqueous phase, mixed thoroughly, and incubated on ice for 30 min. Samples were centrifuged at 10 000 g for 30 min at 4°C to precipitate nucleic acids, followed by two washes with 70% cold ethanol. After air-drying, nucleic acids were dissolved in 130 μl of H2O, and sonicated with a Focused-Ultrasonicator  , to achieve fragments of 100–300 bp. The concentration of fragmented nucleic acids was determined by spectrophotometry with a NanoDrop (ThermoFisher). Appropriate amount (see below) of nucleic acids was digested with 10 μl RNaseH —as a negative control—or H2O, in 15 μl RNaseH buffer , in a total volume of 150 μl, and incubated at 37°C for 90 min. Digestion was stopped with 2 μl 0.5 M EDTA (pH 8.0) per sample. Samples were diluted 1:10 in DIP-1 buffer  and pre-cleared with 40 μl of sepharose protein G beads (Cytiva) for 1 h, on a rotating wheel, at 4°C. 30 μg of diluted nucleic acids from HeLa cells or 12.5 μg of diluted nucleic acids from U2OS cells were used per immunoprecipitation with 3 μg (HeLa) or 6 μg (U2OS) of S9.6 antibody (Kerafast) or mouse IgG antibody  containing 10 μg/ml RNase (DNase-free (Roche)). DNA was isolated with the QIAquick PCR Purification kit (Qiagen) and eluted in 100 μl H2O. Samples were then analysed by dot blot or qPCR (see below).DRIP was performed as previously described . Cells wbody see , and 20 2O up to 10 μl total reaction volume. Each input or immunoprecipitation sample was run in technical duplicate. qPCR was caried at 95°C for 10 min, followed by 95°C for 15 s, and annealing and extension at 60°C for 1 min for 40 cycles in a QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific). At least two serial dilutions (with dilution factors of 5 and 50) of each input sample were included, in order to perform a regression analysis to determine the equation of the standard curve of each input. Using each input equation, the corresponding S9.6 and IgG immunoprecipitations were calculated as percentage of input.Each qPCR comprised 1 μl of purified DNA (immunoprecipitation and diluted input samples—as aforementioned), 5 μl Power SYBR Green PCR Master Mix (Thermo Fisher Scientific), 1 μM forward and reverse primers see , and H2O4, 1 mM EDTA, pH 8.0, 1% (w/v) BSA, 7% SDS) for at least 1 h at 65°C. The membrane was hybridized with a 32P-radiolabeled TeloC probe in Church buffer at 65°C overnight. Washes were done three times for 30 min/wash at 65°C, in 1× SSC, 0.5% SDS. The membrane was then exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE).Input samples (ChIP and DRIP) were diluted appropriately to ensure that the amount of probed DNA in each immunoprecipitated sample was within the linear dynamic range of each assay. Purified DNA (diluted inputs and immunoprecipitated samples) was incubated at 95°C for 5 min and kept on ice. Samples were blotted onto a Hybond-XL membrane (Amersham) using a dot blot apparatus (Bio-Rad), and DNA was UV-crosslinked to the membrane. The membrane was denatured in 0.5 M NaOH, 1.5 M NaCl for 15 min on a shaker at room temperature, neutralized in 0.5 M Tris–Cl pH 7.0, 1.5 M NaCl for 10 min on a shaker at room temperature, and then blocked in Church buffer (0.5 M NaHPO− control reaction), in the presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Reaction products were blotted onto a Hybond-XL membrane (Amersham) using a dot blot apparatus (Bio-Rad), and DNA was UV-crosslinked to the membrane. The non-denatured membrane was blocked in Church buffer (described above) for at least 1 h at 42°C. The membrane was hybridized with a 32P-radiolabeled TeloC oligonucleotide probe in Church buffer at 42°C overnight. Washes were done three times for 30 min/wash at 42°C, in 1× SSC, 0.5% SDS. The membrane was then exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE). For detection of the Phi29− signal, the membrane was stripped and denatured as mentioned above, blocked in Church buffer for at least 1 h at 42°C, and re-probed with a 32P-radiolabeled TeloC oligonucleotide probe in Church buffer at 42°C overnight. Washes and signal detection were done as described.The C-circle assay was performed as previously described . Briefly32P-radiolabeled TeloC probe in Church buffer at 50°C overnight. Washes were done for 1 h/wash at 50°C, in 4× SSC, followed by 4× SSC 0.1% SDS and 2× SSC 0.1% SDS. The gel was then exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE).Genomic DNA (gDNA) was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and digested with HinfI and RsaI (NEB) in CutSmart buffer (NEB), overnight at 37°C. 0.5–2 μg of digested gDNA/lane were resolved by constant-field gel electrophoresis in 0.8% agarose gel in 1× TBE for 17 h at 70 V, or by pulsed-field gel electrophoresis in 1% agarose gel in 0.5x TBE for 16 h at 14°C, at 5.2 V/cm with 0.5 s initial switch and 6 s final switch, using a CHEF-DRII apparatus (Bio-Rad). The gel was dried for 4–5 h at 50°C, denatured in 0.5 M NaOH, 1.5 M NaCl for 15 min on a shaker at room temperature, neutralized in 0.5 M Tris-Cl pH 7.0, 1.5 M NaCl for 10 min on a shaker at room temperature, and then blocked in Church buffer (as described above) for at least 1 h at 50°C. The gel was hybridized with a Images were processed and analysed with ImageJ (2.0.0-rc-69/1.53k). Dot blots were analysed using Aida Image Analyzer (v. 5.1). Preparation of graphs and statistical analyses were performed using GraphPad Prism (v. 9.4.1 (458)). Illustrations were generated with BioRender.32P-radiolabeled [CCCTAA]3 probe and normalized the signal to 18S rRNA  before  hybrids  and H. I hybrids . When ex hybrids  and J. TOf note, no changes in the average telomere length were detected by terminal restriction fragment analysis on Southern blots, following THOC1 or THOC2 depletion in HeLa, U2OS or Saos2 cells for 72 h .Pseudomonas aeruginosa Phage 7 (PP7) stem-loops. The PP7 stem-loops are recognized and bound by GFP-tagged PP7-Coat Protein (PCP) proteins (in situ hybridization (FISH)  .in trans. Also, in cells with shorter average telomere length (ca. 3 kb), where PP7-15qTERRA was previously shown to be recruited with higher frequency to telomeres  transfected with the PP7-15qTERRA-coding plasmid and where THOC1 and -2, or RNaseH1 were downregulated , and quain trans, which take the form of direct DNA:RNA hybrids. Additionally, in cells with shorter telomeres, RNaseH1 overexpression could rescue the telomeric colocalizations induced by THOC siRNA transfection to the basal levels observed with a non-targeting siRNA  structures, that can form on the telomeric G-rich strand which serves as a template for lagging strand DNA synthesis, as well as R-loops, which can form on the telomeric C-rich strand upon base pairing with TERRA. The C-rich strand serves as a template for leading strand DNA synthesis ,17,18,55We set out to investigate whether loss of THOC subunits  . In thisThe CO-FISH protocol not only allows to examine telomeric strand-specific abnormalities, but to investigate telomeric recombination events with exchange of parental strand DNA. We analyzed both types of single sister-chromatid exchange events (single t-sce)—resulting in double leading or double lagging telomeric signals –, as well as reciprocal sister-chromatid exchange  . Depletin DNA circles, referred to as C-circles (− cell line)—depletion of THOC subunits did not trigger formation of such structures -circles . C-circl-circles . In U2OS-circles  led to a-circles  and F. T-circles . In contAiming to understand whether R-loops could play a role in the C-circle accumulation observed when THOC was downregulated in ALT cells, we transiently overexpressed RNaseH1 from a plasmid in U2OS cells , and evaTogether these data point to a role of the THO complex in counteracting C-circle accumulation in ALT cells, presumably through the regulation of R-loops.In this paper, we explore the roles of the THO complex at human telomeres. We show that THOC negatively regulates TERRA-mediated telomeric R-loops. This was most perceptible in U2OS ALT cells, which display a stronger accumulation of telomeric DNA:RNA hybrids when compared to telomerase-positive HeLa cells , renderiin cis , which render the chromatin less accessible . In addi6A modification of RNA was hypothesized to contribute to THOC recruitment (6A modification was recently found to be present on the subtelomeric region of TERRA (We show that THOC (subunits 1 and 2) binds nucleoplasmic endogenous TERRA in HeLa and Hek293E nuclear extracts Figure , suggestruitment . Over 90ruitment , and preruitment . On the ruitment , and theof TERRA . While tof TERRA . FurtherR-loops formed across the genome have been shown to pose an obstacle to the transcription machinery during elongation as well as an impairment to DNA replication, resulting in accumulation of DNA damage and genomic instability ,76–78. TBlm-deficient cells was proposed to result from G4 accumulation, followed by formation of double-strand breaks repaired by break-induced replication coupled with alternative non-homologous end joining (Telomeric fragility is commonly assumed to originate from replication defects and the ensuing repair processes of damaged replication forks . Therefo joining . WhetherTERRA levels are regulated throughout the cell cycle, reaching the lowest levels in late S phase when telomeres are replicated ,16, presFinally, despite the observed increase in TERRA R-loops and telomeric fragility, no changes were detected in the frequency of sister-chromatid exchange events in HeLa cells depleted of THOC subunits, compared to control siRNA. Thus, loss of THOC is not sufficient to induce this ALT-like hallmark phenotype in telomerase-positive cells. Additional events may be required to increase telomere recombination frequency, such as the modulation of the telomeric chromatin structure and telomerase inhibition (as observed upon depletion of the histone chaperones ASF1a + b in immortalized fibroblasts and HeLa cells . A conseFinally, analysis of extrachromosomal telomeric DNA in the form of C-circles in ALT cells depleted of THOC subunits revealed a role for THOC in limiting the accumulation of C-circles Figure . EctopicWhile TERRA was shown to be a crucial trigger/amplifier of the ALT pathway, inappropriate restriction of TERRA-induced ALT activity is thought to compromise telomere integrity . Therefogkad448_Supplemental_FileClick here for additional data file.


Teucrium hyrcanicum L. (family Lamiaceae) is widely distributed in the North and Northwest of Iran. It has been used in the form of tea, tonic, and tincture for the treatment of various diseases such as cough, rheumatism, and fever.T. hyrcanicum were measured. Furthermore, the potential ability of T. hyrcanicum to protect against H2O2-induced oxidative stress was tested on the NIH3T3 cell line. Then, the isolation and structure elucidation of the compounds were performed on the most potent fractions. Finally, the quantification of isolated compounds in methanol extract (ME) was done by the HPLC method. Isolated phytochemicals were assessed for the cytotoxic and antioxidant activities.In this study, the total phenolic and flavonoid contents, antioxidant and cytotoxic activities of methanol extract and different fractions of 50 44.32 and 61.12 μg.ml−1, respectively). The best cytotoxicity was obtained by ethyl acetate fraction (EF) against A431 and MCF7 cell lines . The pretreatment with MF exerts the highest reduction in malondialdehyde (MDA) formation  compared to the H2O2 group (5.77 μM). Also, MF significantly inhibited H2O2-induced Glutathione (GSH) oxidation (p < 0.001). Furthermore, two phenolic compounds, acteoside and quercetin, were isolated and identified in MF and EF, respectively. The IC50 values of acteoside and quercetin in the DPPH assay were 7.19 and 5.56 µg.ml−1, respectively. Both quercetin and acteoside significantly reduced the MDA formation and inhibited GSH oxidation, which was comparable with BHA (as a standard antioxidant) (p < 0.05). Acteoside demonstrated significant cytotoxicity against all tested cell lines (IC50 = 32 to 145 μg.ml−1). The HPLC quantification of isolated compounds revealed that the quantity of acteoside and quercetin in ME were 93.31 and 16.87 μg.mg−1, respectively.The results indicated that the methanol fraction (MF) had the highest amount of phenolic and flavonoid contents (69.36 mg GAE/g extract and 68.95 mg QE/g extract). The highest radical scavenging activities were observed from MF and ME (IC2O2-induced oxidative stress which was comparable with BHA.The isolated compounds (quercetin and acteoside) had significant antioxidant activities and revealed a protective effect on HThe online version contains supplementary material available at 10.1186/s12906-023-04262-8. It wfunction –4. Thesefunction . Also, lfunction . The natfunction –10. The function . There afunction , 13.Teucrium (family Lamiaceae) is a widespread genus with about 340 different species [Teucrium hyrcanicum L. is wildly distributed in the North and Northwest of the country. Until now, anti-inflammatory, anti-nociceptive, antibacterial, and anti-acetylcholine esterase activities of T. hyrcanicum have been reported [T. hyrcanicum [The genus  species –16. Many species , 15, 17. species , 18. Mor species . This ge species , and amoreported , 21. Thercanicum , 23.T. hyrcanicum were measured by DPPH radical scavenging assay and phosphomolybdenum reduction test. Furthermore, the potential ability of T. hyrcanicum for protection against H2O2-induced oxidative stress was tested on NIH 3T3 cell line. Also, the total phenolic and flavonoid contents were measured. Likewise, the cytotoxicity was tested against three cancerous cell lines, including A431 (epidermoid carcinoma), MCF7 (breast cancer), HepG2 (liver hepatocellular carcinoma), as well as HU02 (Foreskin fibroblast) as a normal cell line using MTT assay. Also, the isolation and structural elucidation of compounds were reported from the fractions with the highest biological activities. The quantitative HPLC analysis of methanol extract was performed. Finally, two pure compounds were tested for their antioxidant and cytotoxic activities.To find natural antioxidants from edible plants, in this study, the antioxidant activities of methanol extract and different fractions of 3), Folin–Ciocalteu’s phenol reagent, ammonium molybdate tetrahydrate, thiobarbituric acid, dithionitrobenzoic acid, and sodium bicarbonate were purchased from Merck, Germany. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum were obtained from Gibco and Invitrogen, respectively. HPLC-grade acetonitrile and acetic acid were purchased from DUKSAN, Korea. The three cancerous cell lines  were purchased from the Iranian Biological Resource Center. NIH3T3 and Hu02 cell lines were purchased from the National Cell Bank of Pasteur Institute . Other used reagents and solvents were of analytical grade.Gallic acid, quercetin, 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), and BHA (Butylated hydroxyanisole) were obtained from Sigma chemical Company (USA). Aluminum trichloride (AlCl1H and 13 C-NMR spectra were acquired using a Bruker NMR spectrometer (500 MHz for 1H and 125 MHz for 13C) with tetramethylsilane as an internal standard, and chemical shifts are given in δ (ppm). FT-IR spectra were obtained with a Spectrum Two FT-IR spectrometer with UATR accessory  in the range of 400–4000 cm.−1Column chromatography was performed on Sephadex LH-20 . Silica gel 60 F254 precoated plates  were used for TLC. HPLC separations were conducted on a Knauer system , Eurospher column (250 × 20 mm i.d.) and a PDA detector (λ: 210–400 nm). HPLC quantitative analysis was performed on a Waters Alliance e2695 HPLC apparatus equipped with Waters 2998 photodiode array (PDA) detector, and C18 reversed-phase column . Data acquisition and quantitation were performed by Empower 3 software . T. hyrcanicum (500 g) were dried at room temperature, powdered, and extracted with methanol by percolation method (7 days). The methanol extract of T. hyrcanicum was dried by a rotary vacuum evaporator  to give 92 g dried extract. A portion of 90 g of dried methanol extract was subjected to the fractionation using the liquid–liquid method [Aerial parts of the plant were collected from Rostam Abad mountain, Guilan province, in July 2020. The plant collection complies with relevant guidelines and regulations of plant protection at Guilan University of Medical Sciences (GUMS). The plant was identified, and the scientific name was confirmed by Dr. Fatemeh Yousefbeyk, Associate Professor of Pharmacognosy, School of Pharmacy, GUMS. The voucher specimen (114-HGUM) was deposited in the herbarium of the School of Pharmacy, GUMS, where it is publicly available. The aerial parts of d method . The sol−1) to get 13 fractions (M1-M13). Fraction M2 (500 mg) was submitted to reversed phase semi-preparative HPLC. The mobile phase consisted of H2O/acetic acid (99:1) (solvent A) and acetonitrile (solvent B). The initial mobile phase, including 9:1 (A/B), was delivered at a flow rate 5 ml.min−1 for 10 min, before it was changed to 7:3 (A/B) until 20 min, and remained in this ratio for 10 min (30 min after starting point). Then, chromatography was carried on with the ratio 1:1 (A/B) for another 10 min (40 min after the starting point). Finally, a gradient elution was used to reach 100% B in 20 min (60 min after the starting point). It afforded compound 1 (Rt: 22.4 min). The separation elution curve is provided in Isolation of compounds was carried out on the most potent fractions, including ethyl acetate fraction (EF) and methanol fraction (MF). First, the methanol fraction (50 g) was dissolved in distilled water and extracted by n-BuOH . The butanol fraction (6.5 g) was fractionated using a Sephadex LH20 column using MeOH as an eluent  and 20 fractions were collected (E1-E20). The sub-fraction E4 (170 mg) was loaded on a Sephadex LH20 column  with MeOH for further isolation and the compound 2 was isolated.The ethyl acetate fraction (5 g) was subjected to a Sephadex LH20 column with MeOH as an eluent , including 20, 50, 75, 100, and 150 µg.ml−1 were prepared for plotting the calibration curve. All the tests were repeated three times. Total phenolic contents were expressed as mg of gallic acid equivalents (GAE)/g extract [The Folin-Ciocalteu colorimetric method was carried out to determine the concentration of total phenolics in methanol extract and fractions of this plant . To begi extract .3) (2% in methanol). After 10 min, the absorbance of the mixture was measured at 415 nm using a UV–Vis spectrophotometer. A standard curve was plotted for measuring the total flavonoid content using different concentrations of quercetin as the standard . The tests were repeated three times for each sample. Finally, total flavonoid content was expressed as mg of quercetin as equivalents (QE)/g extract [The concentration of the total flavonoids was determined using a method described by Saeidnia and Gohari , 28. Fir extract , 29.−1) was added to 1 ml of each sample with different concentrations. The samples were kept at room temperature for 30 min before the absorbance was measured at 517 nm. The tests were repeated three times for each concentration. BHA was used as the positive control. The control comprised samples (1 ml) and water (2 ml). The blank contained distilled water (1 ml) and DPPH solution (2 ml). The inhibition activity was determined by the following formula [The methanol extract, fractions, and isolated compounds were tested for their antioxidant activities using the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Briefly, 2 ml of DPPH solution  resulting in 50% inhibition and calculated from the graph-plotted of inhibition (%) against samples concentration.Where AR2 = 0.998. In this equation, Y is the absorbance at 695 nm, and X is the concentration of αTE (μg.ml−1).The antioxidant capacity of the methanol extract and fractions were determined by the phosphomolybdenum reduction assay (PRA) . General−1 penicillin and streptomycin, and 2 mM L-glutamine in a humidified atmosphere of 95% air, 5% CO2 at 37 °C [6 cells/well were seeded into 6-well cell culture plates. The cells were treated with methanol extract (ME), methanol fraction (MF), ethyl acetate fraction (EF), two isolated compounds, including quercetin and acteoside, and BHA as the standard antioxidant , for 24 h. Then, cells were exposed to H2O2 (100 µM) for 2 h [NIH3T3 fibroblast cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), supplemented with 100 U.mlat 37 °C , 33. The for 2 h . FinallyFor the evaluation of lipid peroxidation, the end product of the reaction, malondialdehyde (MDA), was assessed using thiobarbituric acid (TBA) as the indicator , 33. BriGSH concentration of the cell homogenates was determined by dithionitro benzoic acid (DTNB) . The met4 cells per well were seeded in a 96-well plate in complete DMEM and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO2. Next, non-adherent cells were removed, and adherent cells were treated with different concentrations of samples . The treated cell lines were incubated in the same condition for 48 h. Afterward, the MTT solution  and DMEM (180 µl) were added to seeded cells and incubated for 4 h. Then, the supernatants were removed, DMSO (150 µl) was added to each well, and the samples were shaken for 10 min to dissolve formazan crystals [The methanol extract, fractions, and isolated compounds were tested for cytotoxic activities against three cancerous cell lines  and the normal cell line (Hu02) by methyl thiazol tetrazolium (MTT) assay. Briefly, for each cell line, 10crystals , 37. Thecrystals . The sol50 of samples against each cell line was defined as the concentration in which only 50% of cells were alive and computed using GraphPad Prism  [ICre, USA) , 40.T. hyrcanicum using a Waters Alliance e2695 HPLC apparatus equipped with the Waters 2998 photodiode array (PDA) detector and C18 reversed-phase column . The absorbance changes were monitored at 210 nm to 400 nm. Data acquisition and quantitation were performed using Empower 3 software . The mobile phase consisted of H2O/acetic acid (99:1) (solvent A) and acetonitrile (solvent B). The flow rate of 1 ml.min−1 was used for a total run time of 60 min. Elution was performed using the following gradient program: 1–10 min: 10% B, 10–20 min: linear gradient from 10 to 30% B, 20–60 min: linear gradient from 30 to 100% B. The peak related to each compound was identified by comparison of DAD absorbance spectra with external standards. Acteoside and quercetin, as standard compounds, were used to prepare two different concentration levels (100 to 1000 μg.ml−1). The peak areas of each standard were plotted against the corresponding standard concentrations (μg.ml−1) using linear regression to create standard curves. The determination coefficient (R2) was calculated by means of the least-square analysis. Limits of detections (LODs) and limits of quantitations (LOQs) were determined using the expressions 3.3σ/s and 10σ/s, respectively, in which σ is the intercept standard deviation, and s is the slope of the calibration curve.The quantitative analysis of acteoside and quercetin, as major compounds, was performed on the methanol extract of p < 0.05.All the results were expressed as the mean ± SD (standard deviation of the mean). The statistical analyses were done using the SPSS software, version 13 . All the statistical significance was calculated using the one-way analysis of variance (ANOVA) test, followed by the post-hoc Tukey test. Statistical significance was set at R2 = 0.995). Moreover, the total flavonoid content was measured, by standard curve of flavonoid quercetin  and reported in the range of 68.95 ± 0.05 to 18.48 ± 0.01 mg QE/g extract. The MF and ME had the highest amount of flavonoids (68.95 ± 0.05 and 53.53 ± 0.01 mg of QE/g extract).The total phenolic content of methanol extract and fractions were measured by the Folin-Ciocalteu method. As is presented in Table Teucrium has been reported. In Table Teucrium species were summarized. As is presented, the phenolic content of T. hyrcanicum is in the range of previous studies.A literature survey showed that the total phenolic content of different species of T. hyrcanicum, respectively  with those published in the literature [The identification of compounds was performed by comparison of their spectral data : δ ppm 7.47, 7.04 , 6.99 , 6.77 , 6.64 , 6.64 , 6.51 , 6.21 , 5.04 , 4.36 , 3.6 , 2.7 , 0.97 . 13C NMR  δ ppm 166.18 (C9'''), 148.98 (C4'''), 146.05 (C3'''), 145.46 (C3), 145.46 (C7'''), 144.01 (C4), 129.60 (C1), 125.97 (C1'''), 121.92 (C6'''), 120.02 (C6), 116.78 (C2), 116.27 (C5'''), 115.94 (C5), 115.15 (C2'''), 114.02 (C8'''), 102.74 (C1'),101.69 (C1''), 79.56 (C3'), 74.98 (C2'), 74.96 (C5'), 72.13 (C4''), 70.99 (C2''), 70.85 (C3''), 70.73 (C8), 69.59 (C4'), 69.21(C5''), 61.20 (C6'), 35.66 (C7), 18.62 (C6'').Compound 1: FT-IR (νmax): 3290 (OH), 1659 (C = O), 1605 (Alkene C = C), 1442 and 1558 (aromatic C = C), 1158 (C–O–C); 1H NMR : δ ppm 12.5 , 10.70 , 9.34 , 7.69 , 7.55 , 6.9 , 6.41 , 6.19 . 13C NMR  δ ppm 176.3 (C4), 164.35 (C7), 161.19 (C9), 156.60 (C5), 148.16 (C2), 147.26 (C4´), 145.51 (C3´), 136.20 (C3), 122.42 (C6´ and C1´), 116.10 (C5´), 115.54 (C2´), 115.52 (C10), 98.64 (C6), 93.82 (C8).Compound 2: FT-IR (νmax): 3298 (OH), 1688 (C = O), 1606 (Alkene C = C), 1439 and 1576 (aromatic C = C), 1038 and 1266 ; Verbascum species, Buddleja brasiliensis, Striga asiatica, Olea europea, Paulownia tomentosa var. tomentosa, Lippia javanica, Lantana camara, Plantago lanceolate, and Cistanche deserticola [T. hyrcanicum. Previously, Sadeghi et al. reported the presence of phenylethanoid glycosides such as verbascoside and rabinosyl-verbascoside in T. chamaedrys and Teucrium sub spinosum, respectively [.Acteoside, also named verbascoside, is a phenylethanoid glycoside, which is among the most prevalent disaccharide caffeoyl esters . It comperticola , 51–53. erticola , 54, 55.ectively .The flavonol quercetin  is among the most abundant flavonoids . It exisT. hyrcanicum were investigated for antioxidant activities by the DPPH radical scavenging assay. The IC50 results of DPPH assay varied from 44.32 to 205.40 µg.ml−1 . The IC50 values of two isolated compounds, acteoside and quercetin, were 7.19 ± 0.8 and 5.56 ± 0.9 µg.ml−1, respectively. BHA, the standard antioxidant, showed the IC50 value of 7.81 µg.ml−1.The methanol extract, different fractions, and isolated compounds from Furthermore, the total antioxidant capacity was determined by phosphomolybdate reduction assay (PRA) Table . The resTeucrium species have been investigated for their antioxidant activities. Sharififar et al. indicated that the antioxidant activities of different fractions prepared from T. polium, growing in Kerman, Iran, were between 20.1 µg.ml−1 (methanol fraction) and 85.4 µg.ml−1 (chloroform fraction) [T. chamaerdys L. var. glanduliferum varied from 341.08 µg.ml−1 (petroleum ether extract) to 29.46 µg.ml−1 (methanol extract) [T. montanum var. nontanum and obtained results ranged from 29.41 (water extract) to 1388.03 µg.ml−1 (petroleum ether extract) [T. trifidum were 17 and 12 µg.ml−1, respectively [T. chamaedrys had an IC50 of 26.70 µg.ml−1 [T. stocksianum exerted high antioxidant activity (IC50 = 12.5 µg.ml−1) [T. barbeyanum were tested and the IC50 ranged from 5.39 to 77 µg.ml−1. The best antioxidant activity was reported from ethyl acetate fraction [T. hyrcanicum can potentially be used as a source of these natural antioxidants.Until now, different extract) . In anotµg.ml−1) . Also, tfraction . The resp < 0.001) in MDA concentration (5.77 μM) was observed in NIH3T3 fibroblast cells treated with 100 mM H2O2, after 24 h, compared to the control group (1.8 μM)  prevented MDA elevation in NIH3T3 fibroblast cells treated with H2O2, for 24 h. BHA, as a standard antioxidant, showed potential antioxidant activity by decreasing the MDA formation . Moreover, methanol fraction, quercetin, and acteoside exert the highest reduction in MDA formation , compared to the ME and EF.A significant increase . MDA is a crucial degradation product of lipid peroxidation and acts as an indicator for measuring the degree of lipid peroxidation (42). In this study, the MF, quercetin, and acteoside showed better protective activities than BHA. They showed significant antioxidant properties, which lead to a lower degree of lipid peroxidation and higher hydroxyl radical scavenging abilities.2O2 leads to a significant decrease in GSH level compared to the control group (p < 0.001). BHA showed significant antioxidant activity by elevating GSH level . The results showed that pretreatment with MF, quercetin, and acteoside considerably inhibited the H2O2-induced GSH oxidation in NIH3T3 fibroblast cells . Pretreatment with ME and EF could not significantly increase the GSH level related to H2O2 group.As shown in Fig. 2O2, are regularly produced intracellularly because of usual metabolic activities, and can cause cellular oxidative damage [T. hyrcanicum can be considered a significant antioxidant, even more potent than BHA in increasing GSH level (169.69 μM).The GSH is a vital antioxidant defense agent against free radicals. GSH (ɣ-glutamylcysteinylglycine) is a ubiquitous low molecular weight thiol in eukaryotes and is the most abundant antioxidant within all cells. It acts to remove ROS and therefore, reduce the lifetime of the oxidative signals , 63. Reae damage . In thise damage . Accordi50s were demonstrated in Table −1 for 48 h, which reduced the viability of these cell lines.The anti-proliferative activities of methanol extract, different fractions, and isolated compounds were evaluated on three cancerous cell lines, including the human breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and epidermoid carcinoma A431) cell lines as well as a normal cell line (Hu02) by MTT assay and IC31 cell l−1, respectively). Also, ME had cytotoxic activity on MCF7 with IC50 of 392 μg.ml−1. Other fractions exhibited lower cytotoxicity on tested cell lines (> 500 μg.ml−1). The isolated compound, acteoside, demonstrated significant cytotoxicity against all tested cell lines (ranging from 32 to 194.6 μg.ml−1). Quercetin represented its best antiproliferative activity against A431 (132.32 μg.ml−1).Among different fractions, the best cytotoxicity was achieved by EF against A431 and MCF7  with the IC50 of 253.39, 190.07, 183.15, and 75.73 µg.ml−1, respectively [So far, the genus ompounds . A literll lines . In anot µg.ml−1 . The metectively .50 of 30 μM [50 of 32.49 μg.ml−1 [50 values of this compound on HEP-2 (human larynx epidermoid carcinoma) and RD (human rhabdomyosarcoma) cells were 55.6 and 36.24μg.ml−1, respectively [So far, the extracts or isolated compounds have been investigated for anti-tumor activities. These diverse and complicated structures can trigger anti-inflammatory, anti-tumor, and/or anti-metastatic reactions and reduce multidrug resistance , 68. In of 30 μM . Acteosiof 30 μM . Acteosiof 30 μM . This co μg.ml−1 . The IC5ectively .2●) and quinone-semiquinones complex. These compounds can cause DNA damage and apoptosis in normal and cancer cells [Former studies proved that polyphenol antioxidants can cause antiproliferative effects on specific cancer cells by acting as prooxidant agents , 74, 75.50 of 132.32 μg.ml−1).Flavonoids have recently attracted a lot of attention due to their potential role in cancer prevention and cancer therapy. They can regulate cell proliferation and pathways leading to cancer . QuercetT. hyrcanicum were performed by developing an HPLC method. Figure T. hyrcanicum methanol extract. The acteoside and quercetin were detected at Rt 15 and 54 min, respectively. The quantities of these compounds in ME were 93.31 and 16.87 μg.mg−1 extract.In this study, the quantitative analyses of acteoside and quercetin in methanol extract of −1, with R2 = 0.999 and 0.986 for quercetin and acteoside, respectively.The results of method validation for analysis of quercetin and acteoside according to linearity, LOD, and LOQ revealed that the developed method was valid Table . OutstanT. hyrcanicum (family Lamiaceae). Among prepared samples, MF showed the highest amount of phenolic and flavonoid contents, and the best antioxidant activity. Also, MF significantly protected the NIH3T3 cell line against H2O2-induced oxidative stress by reducing MDA formation and inhibition of GSH oxidation. The results of the MTT assay demonstrated that EF had the highest cytotoxic activity. The phytochemical analysis resulted in the isolation and identification of two phenolic compounds (acteoside and quercetin). The isolated compounds had significant antioxidant activities and revealed protective effects on H2O2-induced oxidative stress, which was comparable with BHA (standard antioxidant). The acteoside had the best cytotoxicity against the A431 cancerous cell line. The HPLC quantification revealed that the methanol extract of this plant contained considerable amounts of acteoside and quercetin.This study focused on the investigation of total phenolic and flavonoid contents, antioxidant, and cytotoxic activities of methanol extract and different fractions from Additional file 1: Figure S1. The separation elution curve for compound 1(acteoside): compound 1 was separated in Rt=22.4 min, using a semi-preparative HPLC. Figure S2-1. H-NMR spectra of compound 1 (acteoside). Figure S2-2. The expanded H-NMR spectra of compound 1 (acteoside). Figure S2-3. The expanded H-NMR spectra of compound 1 (acteoside). Figure S3-1.13C-NMR spectra of compound 1 (acteoside). Figure S3-2. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-3. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-4. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-5. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S4. FT-IR spectra of compound 1 (acteoside). Figure S5. H-NMR spectra of compound 2 (quercetin). Figure S6. C-NMR spectra of compound 2 (quercetin). Figure S7: FT-IR spectra of compound 2 (quercetin).


Cryptocaryon irritans, a common parasite in tropical and subtropical marine teleost fish, has caused serious harm to the marine aquaculture industry. Honokiol was proven to induce C. irritans tomont cytoplasm shrinkage and death in our previous study, but the mechanism by which it works remains unknown.2+]i) concentration, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF) and caspase activities were detected by Fluo-3 staining, JC-1 staining, DCFH-DA staining, Tunel method and caspase activity assay kit. The effects of honokiol on mRNA expression levels of 61 apoptosis-related genes in tomonts of C. irritans were detected by real-time PCR.In this study, the changes of apoptotic morphology and apoptotic ratio were detected by microscopic observation and AnnexinV-FITC/PI staining. The effects of honokiol on intracellular calcium (i concentration, ROS, the activities of caspase-3/9 and the lowest necrosis ratio (NER) were obtained at a concentration of 1 μg/ml, which was considered the most suitable for inducing C. irritans tomont apoptosis-like death. When C. irritans tomonts were treated with 1 μg/ml honokiol, the [Ca2+]i concentration began to increase significantly at 1 h. Following this, the ROS, QDF and activities of caspase-3/9 began to increase significantly, and the ΔΨm began to decrease significantly at 2 h; the highest PADR was obtained at 4 h. The mRNA expression of 14 genes was significantly upregulated during honokiol treatment. Of these genes, itpr2, capn1, mc, actg1, actb, parp2, traf2 and fos were enriched in the pathway related to apoptosis induced by endoplasmic reticulum (ER) stress.The results of the study on the effects of honokiol concentration on C. irritans tomont apoptosis-like death. These results suggest that honokiol may disrupt [Ca2+]i homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway, which might represent a novel therapeutic intervention for C. irritans infection.This article shows that honokiol can induce The online version contains supplementary material available at 10.1186/s13071-023-05910-1. Cryptocaryon irritans, a common protozoan parasite of marine teleost fish, causes “white spot” disease i concentration), mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS), quantity of DNA fragmentations (QDF), caspase activities, and mRNA expression of apoptosis-related genes of C. irritans tomont were investigated to uncover the mechanism of honokiol for inducing C. irritans tomont apoptosis-like death.In this article, Annexin V-FITC/PI staining was used to determine whether honokiol induces Trachinotus ovatus, weighing 80.0 ± 10.6 g, were purchased from a marine cage culture, Lingshui County, Hainan Province, P.R. China. The fish were soaked in seawater containing 1 ml/l formaldehyde for 10 min and acclimated in 2000 l aquaria for 21 days  equipped with aerators and a flow-through water system . The fish were fed marine fish feed  at 4% body weight three times  every day.Honokiol with a high-performance liquid chromatography (HPLC) purity ≥ 98% was purchased from Century Aoke Biological Technology Co., Ltd. (P.R. China). An amount of 250 mg of honokiol was dissolved in 25 ml 50% (v/v) ethanol aqueous solution and diluted to 800, 400, 200, 100, 60, and 0 (control sample) μg/ml with 50% (v/v) ethanol aqueous solution.Cryptocaryon irritans used in this study were isolated from T. ovatus with white spot disease, which were obtained from a marine cage culture in Lingao County, Hainan Province, P.R. China, and passaged and propagated in T. ovatus i concentration, ΔΨm, ROS, QDF, and caspase activities of the above-treated C. irritans tomonts were analyzed using a direct microscopic observation method, an Annexin V-FITC/PI apoptosis detection kit, a Fluo-3 AM calcium concentration detection kit, a mitochondrial membrane potential assay kit, a reactive oxygen species assay kit, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit, and a caspase-3/8/9 activity assay kit, respectively. Cryptocaryon irritans tomonts were treated with honokiol at the optimum concentration according to the results of the above experiments for 0, 1, 2, 4, 8, and 16 h. Their morphologies, NOR, PADR, AADR, NER, [Ca2+]i concentration, ΔΨm, ROS, QDF, and caspase activities were also analyzed using the same methods as mentioned above. Cryptocaryon irritans tomonts were treated with honokiol at the conditions with the highest apoptosis rate of tomonts according to the results of the above experiments, and then the inhibition rate of tomonts hatching was observed according to the method described by Zhong et al.  as the abscissa axis and a logarithm transformed PI fluorescence intensity [LG (AEx/Em = 488/630 nm)] as the ordinate axis. Normal (in the I quadrant), prophase apoptosis-like death (in the II quadrant), anaphase apoptosis-like death (in the III quadrant), and necrosis (in the IV quadrant) C. irritans tomonts were identified according to the four-quadrant apoptosis diagrams, and their numbers were recorded i concentrations in C. irritans tomonts were determined using a Fluo-3 AM calcium concentration detection kit . The C. irritans tomonts, treated as described in “C. irritans tomonts.The i concentration and the ΔΨm, ROS, QDF, and caspase-3/8/9 activities in the C. irritans tomonts treated with honokiol at various concentrations are given in Fig. 2+]i concentration increased to a level significantly higher than that of the control sample at 0.6 μg/ml. It reached the highest level at 1 μg/ml, returned to the level of the control sample at 2.0 μg/ml, and then decreased to a level significantly lower than that of the control sample when the honokiol concentration increased > 4.0 μg/ml. As shown in Fig. Ψm decreased to a level significantly lower than that of the control sample when the honokiol concentration was > 0.6 μg/ml. As shown in Fig. The [Ca2+]i concentration, ΔΨm, ROS, QDF, and caspase-3/8/9 activities” showed that C. irritans tomonts had the highest PADR, [Ca2+]i concentration, ROS, caspase-3/9 activities, and higher QDF, and the lowest NER when the honokiol concentration was 1.0 μg/ml. Therefore, 1.0 μg/ml was considered the optimum honokiol concentration for inducing C. irritans tomont apoptosis-like death. Together with the extension of the treatment time, the changes in the NOR, PADR, AADR, NER, [Ca2+]i concentration, ΔΨm, ROS, QDF, and the caspase-3/8/9 activities for the C. irritans tomonts are given in Fig. 2+]i concentration and PADR began to increase significantly at 1 h, followed by ROS and QDF. The caspase-3/9 activities began to increase significantly, and the ΔΨm began to decrease significantly at 2 h. The [Ca2+]i concentration, PADR, ROS, QDF, and caspase-3/9 activities reached the highest levels at 4 h and then slightly decreased but remained at levels significantly higher than those at 0 h, while the ΔΨm continued to decrease at 16 h, and the AADR began to significantly increase at 8 h. The NER and the activity of caspase-8 showed nonsignificant changes at 16 h. These results suggest that the optimum treatment time for 1.0 μg/ml honokiol inducing C. irritans tomonts apoptosis-like death is 4 h. The inhibition rate of tomont hatching was 52.38% after treatment with 1.0 μg/ml honokiol for 4 h.The results found in “Effect of honokiol on  homeostasis in ER. Among the eight genes, fos was significantly upregulated at 4 h, while the other genes were significantly upregulated within 2 h. Gene gzmb, enriched in the Granzyme B pathway, was significantly upregulated at 16 h, and gene tuba1c, enriched downstream of the Granzyme B pathway, was significantly upregulated at 2 h. Genes hras and raf1, enriched in the MAPK signaling pathway, were significantly upregulated at 2 h, and gene hras was also significantly upregulated at 4 h. Gene akt1, enriched in the PI3K-Akt signaling pathway, was significantly upregulated at 2 h. Gene atm, the upstream regulatory of the p53 signaling pathway, was significantly upregulated at 2, 4, and 16 h.Among the 61 investigated apoptosis-related genes, 14 were significantly upregulated  resembles the apoptosis of metazoans i release and increase [Ca2+]i concentration by inhibiting the activity of endoplasmic reticulum protein 44 (ERP44), which has inhibitory activity on the calcium channel IP3Rs [2+]i concentration first significantly increased in C. irritans tomonts in this study. The increase of Ca2+ can cause two significant reactions, Reaction I and Reaction II. In Reaction I, the increase of Ca2+ can provoke the cascade reaction of caspases, including calpains, caspase-12, and caspase-3 i can also decrease the ΔΨm of mitochondria and then promote ROS production and release from mitochondria [2+]I release and increase QDF, and then the mitochondria sequentially activate caspase-9 and caspase-3 via apoptotic protease-activating factor 1 (Apaf1) combined with cytochrome c (CytC) i, disrupt [Ca2+]i homeostasis in ER, and then induce C. irritans tomont apoptosis-like death by caspase cascade or mitochondrial pathway. However, further studies such as the transcriptomic or proteomic analyses are needed to confirm this suggestion.Honokiol, as one of the main active components of  pathway , 36–39.  pathway , 40–42. mulation –47. Cho mulation  reportedaspase-3 –52. Coin metazoa , 53, 54.chondria , 56. An c (CytC) , 57. In  (PARPs) –60. Acti (PARPs) –63, whil (PARPs) –66. In s the QDF –69. Besi induced –47 (Fig. induced . The act induced –47. AlthC. irritans tomont apoptosis-like death and suggested that honokiol may disrupt [Ca2+]i homeostasis in ER and then induce C. irritans tomont apoptosis-like death by caspase cascade and mitochondrial pathway. This might represent a novel therapeutic intervention for C. irritans infection. Next, to further research on safe and efficient anti-C. irritans drugs, the intracellular target of honokiol in C. irritans needs to be verified.This article showed that honokiol can induce Additional file 1: Table S1. The 61 apoptosis-related genes and their primers used in this study.Additional file 2: Fig. S1. The fluorescence quantitative melting curves of the 14 significantly differentially expressed genes.