Isolated coronary arteries and cardiomyocytes were assessed for vascular reactivity and cellular Ca2+ transport using a multiwire myography system and a cell-contraction-ion detection system, respectively. Allicin administration improved cardiac function and myocardial pathology, reduced myocardial enzyme levels, and increased H2S and H2S synthetase levels. Allicin administration resulted in concentration-dependent effects on coronary artery dilation, which were mediated by receptor-dependent Ca2+ channels, ATP-sensitive K+ channels, and sarcoplasmic reticulum (SR) Ca2+ release induced by the ryanodine receptor. Allicin administration improved Ca2+ homeostasis in cardiomyocytes by increasing cardiomyocyte contraction, Ca2+ transient amplitude, myofilament sensitivity, and SR Ca2+ content. Allicin also enhanced Ca2+ uptake via SR Ca2+-ATPase and Ca2+ removal via the Na+/Ca2+ exchanger, and it reduced SR Ca2+ leakage. Notably, the protective effects of allicin were partially attenuated by blockade of H2S production with PAG. Our findings provide novel evidence that allicin-induced production of H2S mediates coronary artery dilation and regulation of Ca2+ homeostasis in AMI. Our study presents a novel mechanistic insight into the anti-AMI effects of allicin and highlights the therapeutic potential of this compound.Acute myocardial infarction (AMI) is a condition with high morbidity and mortality, for which effective treatments are lacking. Allicin has been reported to exert therapeutic effects on AMI, but the underlying mechanisms of its action have not been fully elucidated. To investigate this, a rat model of AMI was generated by ligating the left anterior descending branch of the coronary artery. DL-propargylglycine (PAG), a specific hydrogen sulfide (H Acute myocardial infarction (AMI) is a condition with high morbidity and mortality . Despite2+ homeostasis, which is regulated by Ca2+-induced excitation-contraction coupling, is a critical determinant of cardiac contractile function. In fact, Ca2+ dyshomeostasis may lead to impaired systolic-diastolic function of cardiomyocytes after AMI  plays a crucial role in cardiovascular homeostasis. In the human body, H2S production is predominantly catalyzed by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). To date, most scholars have proposed that CSE is the primary H2S-producing enzyme in the cardiovascular system  was purchased from Tianjin Tanabe Pharmaceutical Co., Ltd. . Potassium chloride (KCl), 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1), tetraethylamine , 4-aminopyridine , barium chloride  and glibenclamide  were purchased from Sigma-Aldrich . All other reagents were of analytical purity.Allicin was provided by Xinjiang Ailexin Pharmaceutical Co., Ltd. . DL-propargylglycine  was purchased from Shanghai Yuanye Bio-Technology . Diltiazem  rats (weighing 200–220 g) were utilized in the present study.n = 14–16 per group) by a random number table: sham, model, diltiazem (8.1 mg/kg), allicin (14 mg/kg), allicin (7 mg/kg), and allicin (14 mg/kg) + PAG (32 mg/kg) groups (The AMI model was generated via left anterior descending coronary artery (LAD) ligation in SD rats. Briefly, rats were anesthetized with 1% pentobarbital sodium. After left thoracotomy, the heart was exteriorized, and the LAD was ligated approximately 2 mm below the left atrium with a 6–0 silk suture. AMI was confirmed by elevation of the ST segment on an electrocardiogram and bulging of the relevant segment of the left ventricle (LV). In the sham group, the suture was removed without tying, and no infarction was generated. After establishment of the AMI model, rats were divided into six groups  groups . All groCardiac function was assessed using a Vevo 3100 echocardiography system . Rats were anesthetized with 1.5–2% isoflurane via continuous inhalation and warmed on a heated pad (37°C). Ultrasound transmission gel was applied to the chest, and echocardiography (M-mode and B-mode imaging) was performed. The LV internal diameter and thickness of the anterior wall at end-diastole  and end-systole , as well as LV fractional shortening (FS), ejection fraction (EF), and stroke volume (SV) were measured in each rat in a blinded manner. All values were averaged using three to five cardiac cycles per rat. Rat hearts were harvested after 1% pentobarbital sodium overdose via intraperitoneal injection and sliced into five sections of 1-mm thickness across the left ventricular long axis under the ligature. To identify the infarction area, heart slices were incubated with nitro-blue tetrazolium chloride (Sigma-Aldrich) for 3 min at 22 ± 2°C. Infarction areas were measured using Image-Pro Plus software  and presented as a percentage of infarct area to ventricular area or total area.Before the rats were sacrificed, blood samples were collected from the abdominal aorta. Serum was incubated at 22 ± 2°C for 30 min and centrifuged at 975.87 ×g for 10 min. The supernatant was collected for determination of serum cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and CSE levels. Levels of serum cTnI, LDH, and CSE were separately quantified with commercially available cTnI , LDH , and CSE ELISA kits ; antibody and chromogenic agent were added according to the manufacturer’s instructions. Absorbance was measured at 450 nm, detected by a microplate tester. Levels of LDH, cTnI, and CSE were calculated according to the standard curve .The border zone of myocardial infarction tissues were fixed with 4% (v/v) paraformaldehyde and incubated with dimethylbenzene for 30 min before serum blocking for 60 min. Specimens were incubated with CSE antibody  for 24 h at 4°C prior to incubation with goat anti-rabbit IgG (H + L) fluorescein isothiocyanate-conjugated polyclonal antibody  in the dark at 37°C for 60 min. After washing specimens in phosphate buffer solution, nuclei were stained with 4′, 6-diamidino-2-phenylindole (Sigma-Aldrich). Images were obtained using an upright fluorescence microscope  under ×400 magnification.2S in serum and the border zone of myocardial infarction tissue were measured using methylene blue spectrophotometry at 665 nm according to manufacturer’s instructions .Levels of HHearts were harvested, weighed, washed in phosphate buffer solution, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Each paraffin-embedded heart was cut into 4-µm thick sections through the infarction area and stained with hematoxylin and eosin (H&E) for morphological observation. Specimens were stained with Masson’s trichrome stain to evaluate collagen volume. Sections were imaged using a stereomicroscope .2, 1.5; NaH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 15; Glucose, 5.6; pH 7.4]. RCARs were prepared and performed as previously described  of SD rats were then isolated and placed in a cold Krebs buffer [composition (mM): NaCl, 119; KCl, 4.6; CaClescribed .–2 M) until a plateau of contraction was reached. The rings were then divided equally into two groups (8 rings each): PAG or control. RCARs were incubated with PAG (10–2 M) or the same volume of saline, respectively, for 5 min. Both groups were treated with allicin (10−5–10–4.2 M) and cumulative concentration-response curves were obtained.RCARs were contracted with KCl (6 × 102+ channels (VDCCs), whereas U46619, 5-HT, and ET-1 mediate the opening of receptor-dependent Ca2+ channels (RDCCs). In this study, RCARs were divided into three groups (8 rings each): allicin, allicin + PAG, and control groups. Rings were incubated with allicin (10–4.8 M), allicin (10–4.8 M) + PAG (10–2 M), or the equivalent volume of saline, respectively, for 5 min. Cumulative concentration-response curves for KCl (10–1.54–10–1.42 M), U46619 (10−8–10–5 M), 5-HT (10−8–10–4 M), and ET-1 (10−9–10–6 M) were obtained.KCl mediates the opening of voltage-dependent Ca2+-sensitive potassium channels (KCa), voltage-dependent potassium channels (Kv), inwardly rectifying potassium channels (Kir), and ATP-sensitive potassium channels (KATP) to allicin-induced vasodilation, the corresponding inhibitors, TEA (10–3 M), 4-AP (10–3 M), BaCl2 (10–5 M) and Glib (10–5 M) were applied. RCARs were divided into two groups (8 rings each): control and inhibitor groups. The rings were incubated with the four respective inhibitors for each channel or the same volume of saline for 5 min. Cumulative concentration-response curves for allicin (10−5–10–4.2 M) were obtained.To investigate the contribution of Ca−5–10–4.2 M) were obtained as described above.If the maximum vasodilatory effect in the inhibitor group was lower than that in the control group, subsequent experiments were conducted, as follows. The rings were divided into three groups (8 rings each): inhibitor, PAG, and PAG + inhibitor groups. Cumulative concentration-response curves for allicin (102+ Channel-induced Contraction” section). Contraction-response curves for caffeine (3 × 10–2 M) were obtained.The grouping and intervention for RCARs were performed as described above , and placed in a cell chamber. Myocytes were stimulated to contract at a pacing frequency of 1 Hz with 4 ms of electrical stimulation. Myocytes were exposed to 340 or 380 nm excitation wavelengths, and the emitted fluorescent signal was detected at 510 nm. Sarcomere length and fluorescence intensity (a proxy of Ca2+ concentration) were synchronously recorded with a cell contraction-ion detection system . Contractility parameters including amplitude, peak time, systolic half-time of decay (T50), diastolic T50, and myofilament sensitivity were measured. Ca2+ transient parameters including amplitude, maximum ascending and descending velocity, and Ca2+ decline time constant were also recorded.Isolated cardiomyocytes were loaded with 2 μM Fura-2 AM (Sigma-Aldrich) in the dark for 30 min at 22 ± 2°C. Cells were washed, resuspended twice in Tyrode’s solution .SR Ca release . Short p2+ release and assess the contribution of the Na+/Ca2+ exchanger (NCX) and slow transport systems . The contribution of the slow transport system to Ca2+ removal is only 1% and is often overlooked  uptake and NCX Ca2+ efflux with superfusion of Tyrode’s solution. Based on these factors, the time of SERCA-mediated Ca2+ removal was calculated (Tau).Rapid and continuous application of 10 mM caffeine was employed to induce SR Caerlooked . With co2+ leakage levels were assessed by perfusing myocytes with 1 mM tetracaine and reperfusing in 10 mM Na-, Ca-free Tyrode’s solution (containing Li+ and EGTA instead of Na+ and Ca2+). Levels of SR Ca2+ leakage were calculated based on the difference in cytosolic Ca2+ concentration before and after tetracaine perfusion.Ca50) and maximum relaxation (Rmax) or contraction (Emax) were determined using concentration negative logarithm-effect curves. The differences in vasomotor responses to allicin, as well as in the levels of KCl, 5-HT, U46619, and ET-1 were compared by a two-way analysis of variance (ANOVA), with a post hoc Bonferroni test for group comparison. Statistical significance was determined through a one-way ANOVA with Dunnett’s test for multiple-group comparisons in other experiments. *p < 0.05, **p < 0.01 vs. sham group, △p < 0.05, △△p < 0.01 vs. AMI model group, #p < 0.05, ##p < 0.01 vs. allicin 14 mg/kg group in all figures. All data are presented as the mean ± S.E.M.The SPSS statistical software  was used for statistical analysis. Vasodilation and vasocontraction are expressed as the percentage of precontraction amplitude. The negative logarithms of the concentration that produced the half-maximal effect  and total heart area (ITT) were significantly lower in the sham group  than in the model group . ITV and ITT were significantly reduced by treatment with 14 mg/kg of allicin  and 8.1 mg/kg of diltiazem . However, in the allicin 14 mg/kg + PAG group, ITV and ITT were significantly higher  than in the allicin 14 mg/kg group, but significantly lower than in the model group . Hence, cTnI and LDH levels were significantly higher in the model group than in the sham group , indicating that the model successfully mimicked AMI conditions. Levels of cTnI and LDH were significantly lower in the diltiazem 8.1 mg/kg group , allicin 14 mg/kg group , allicin 7 mg/kg group , and allicin 14 mg/kg + PAG group  than in the model group. cTnI and LDH levels were significantly higher in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group. These data suggest that treatment with diltiazem or allicin significantly alleviated the changes in cardiac function induced by AMI, and PAG partially reversed this effect .H&E and Masson staining revealed that in the sham group, the structure of cardiomyocytes remained intact, the transverse and fiber striations of the myocardium were clear, and cells were arranged regularly. No degeneration, necrosis, hemorrhage, inflammatory cell infiltration, or collagen deposition was observed in the sham group. In the model group, the arrangement of myocardial fibers was disordered, swollen, and disjointed; the septum of the fiber bundles was widened; transverse lines of cells were absent, striations were disordered, normal cell structure was disrupted; and extensive cardiomyocyte necrosis, interstitial vascular hyperplasia, hyperemia, edema, inflammatory cell infiltration, and collagen deposition were observed. These features are typical of myocardial infarction. In the allicin 14 mg/kg and diltiazem 8.1 mg/kg groups, the arrangement of cardiomyocytes was slightly disordered, myocardial fibers were neatly arranged, the interstitium was broadened, the septum of the fiber bundles was not significantly widened, no cell necrosis was observed, and minimal cardiomyocyte edema and collagen deposition were noted. Hence, the degree of pathological damage observed in the allicin 7 mg/kg and allicin 14 mg/kg + PAG treatment groups was intermediate compared with that noted in the model and allicin 14 mg/kg groups .2S in serum and the border zone of the myocardial infarction tissues were significantly lower in the model group  than in the sham group  , allicin 14 mg/kg , allicin 7 mg/kg , and allicin 14 mg/kg + PAG groups  than in the model group, suggesting that these treatments alleviated myocardial infarction symptoms. High levels of allicin were more effective than lower levels, as H2S serum and tissue levels were significantly lower in the allicin 7 mg/kg and allicin 14 mg/kg + PAG groups than in the allicin 14 mg/kg group. Finally, the levels of H2S in the serum and myocardial tissue were significantly higher in the allicin 14 mg/kg group than in the model group.The levels of the Hctively) . On the 2S production.Immunofluorescence analysis revealed that the average optical density (AOD) of CSE in myocardial tissue was significantly lower in the model group (0.039 ± 0.014) than in the sham group (0.141 ± 0.028). CSE showed a significantly higher level in the diltiazem 8.1 mg/kg (0.091 ± 0.009), allicin 14 mg/kg (0.101 ± 0.008), allicin 7 mg/kg (0.077 ± 0.010), and allicin 14 mg/kg + PAG groups (0.073 ± 0.003) than in the model group. On the other hand, CSE levels tended to be lower in the allicin 7 mg/kg and allicin 14 mg/kg + PAG groups than in the allicin 14 mg/kg group, but this trend did not reach statistical significance . Similar50 between the two groups.Allicin-induced dilation in rat coronary arteries precontracted with KCl in a dose-dependent manner. The maximum vasodilation reached 85.11 ± 2.11% of the pre-contraction amplitude. After the application of PAG, the maximum relaxation induced by allicin was 49.37 ± 6.94%, which was significantly lower than that in the control group . The inhmax and pEC50 of KCl-induced concentration-contraction curves were observed after administration of allicin or allicin + PAG . Following administration of allicin + PAG, the Emax values of the concentration-contraction curves induced by 5-HT, U46619, and ET-1 were 133.91 ± 8.65%, 124.09 ± 6.64%, and 144.02 ± 5.22%. These values were significantly lower than those of the control group but were significantly higher than those of the allicin group , whereas the Rmax of allicin was significantly lower in the Glib group (46.98 ± 3.86%) than in the control group. No significant differences were observed in the Rmax of allicin among the PAG, PAG + Glib, and Glib groups. Altogether, these data suggest that the response to allicin is mediated by receptor-dependent, rather than voltage-dependent, Ca2+ channels (The Rchannels .–2 M) induced a transient and rapid contraction of rat coronary arteries in Ca2+-free solution. The contraction amplitude was 40.70 ± 10.09% of the contraction induced by KCl (6 × 10–2 M). After administration of allicin, the contraction amplitude induced by caffeine was 28.45 ± 5.42%, which was significantly lower than that in the control group. Caffeine-induced contraction amplitude was significantly higher in the allicin + PAG group (40.28 ± 6.05%) than in the allicin group (Caffeine (3 × 10in group .50 (0.056 ± 0.017 s), and diastolic T50 (0.128 ± 0.044 s) were significantly longer in the model group than in the sham group . Contraction amplitude in the allicin 14 mg/kg (6.76 ± 2.43%) and allicin 14 mg/kg + PAG groups (4.65 ± 1.53%) was higher, whereas peak time, systolic T50, and diastolic T50 in the allicin 14 mg/kg group  was significantly lower than that in the model group. The addition of PAG significantly reduced the contraction amplitude in the allicin 14 mg/kg + PAG group (4.65 ± 1.53%) compared with the allicin 14 mg/kg group. Similarly, peak time, systolic T50, and diastolic T50 were significantly longer in the allicin 14 mg/kg + PAG group  than in the allicin 14 mg/kg group, indicating that PAG significantly alleviated the effects of allicin.2+ transients in the model group  compared with the sham group . The Ca2+ decline time constant in the model group (0.35 ± 0.08 s) was significantly longer than that in the sham group (0.23 ± 0.07 s) as well. Ca2+ transient amplitude in the allicin 14 mg/kg group (0.15 ± 0.03 RU) and maximum ascending and descending velocities of Ca2+ transients in the allicin 14 mg/kg  and allicin 14 mg/kg + PAG groups  were significantly higher than in the model group. The Ca2+ decline time constants in the allicin 14 mg/kg (0.25 ± 0.10 s) and allicin 14 mg/kg + PAG groups (0.28 ± 0.08 s) were significantly shorter than in the model group. Compared with the allicin 14 mg/kg group, the allicin 14 mg/kg + PAG group exhibited significantly lower Ca2+ transient amplitude and maximum ascending and descending velocities of Ca2+ transients, as well as significantly longer Ca2+ decline time constant  was significantly lower than in the sham group (3.86 ± 1.76). By contrast, myofilament sensitivity in the allicin 14 mg/kg and allicin 14 mg/kg + PAG groups was significantly higher than that in the model group. Myofilament sensitivity was significantly lower in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group  than in the sham group . Compared with the model group, the TauNCX and TauSERCA of the allicin 14 mg/kg group  and allicin 14 mg/kg + PAG group  were both significantly decreased. No significant differences were observed in TauNCX and TauSERCA between the allicin 14 mg/kg and allicin 14 mg/kg + PAG groups  than in the sham group (1.51 ± 0.37 RU). On the other hand, the allicin 14 mg/kg (1.43 ± 0.18 RU) and allicin 14 mg/kg + PAG (1.17 ± 0.13 RU) treatments induced significantly higher SR Ca2+ content when compared with the model group. SR Ca2+ content was significantly lower in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group.The SR Ca2+ leakage was significantly higher in the model group (0.097 ± 0.033 RU) than in the sham group (0.052 ± 0.040 RU), whereas allicin 14 mg/kg (0.056 ± 0.05 RU) and allicin 14 mg/kg + PAG (0.073 ± 0.044 RU) administration significantly decreased leakage compared with the model group. Ca2+ leakage was significantly higher in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group , and ET-1, by binding to their corresponding G protein-coupled receptors, are the main activators of RDCCs, whereas VDCCs are predominantly activated by cations, including K+. We demonstrated that allicin significantly inhibited the contractions induced by 5-HT, U46619, and ET-1 in rat coronary arteries, but it did not alter contractions induced by KCl, suggesting that RDCCs, rather than VDCCs, mediated the extracellular Ca2+ influx that contributed to vasodilation. Receptors mediating SR Ca2+ release include the inositol triphosphate and ryanodine receptors (RyRs), of which, the latter mediates more than 90% of SR Ca2+ release. In order to study the effects of allicin on RyR-mediated calcium release, we used caffeine in Ca2+-free solution, which increased the intracellular Ca2+ concentration by completely activating the RyRs in the SR (2+ release mediated by RyRs.In AMI, coronary microvascular dysfunction results in the insufficient supply of blood and oxygen and may affect myocardial function at rest and during stress . Given tlar tone , we focum the SR . Ca2+ inn the SR . Our fin+ channels, which leads to cell membrane hyperpolarization by promoting intracellular K+ outflow, is a way to decrease intracellular Ca2+ concentration and induce vasodilation. To date, four types of K+ channels with different activation mechanisms have been identified: these are the KV, KCa, Kir, and KATP channels (2+ influx. Further, they increase intracellular Ca2+ efflux by stimulating the NCX, thus reducing intracellular Ca2+ and causing vasodilation. To investigate the involvement of K+ channels in allicin-induced vasodilation, we applied 4-AP, TEA, BaCl2, and Glib separately on coronary arteries to observe their effects on allicin-induced vasodilation. Only Glib application significantly inhibited allicin-induced vasodilation, suggesting that the vasodilatory effects of allicin on coronary arteries were at least partly attributed to KATP channel opening but did not involve other K+ channels.The opening of Kchannels . These c2+-mediated excitation-contraction coupling is a requirement for correct cardiomyocyte contraction and relaxation (2+ enters the cytoplasm through L-type Ca2+ channels, which triggers the activation of the RyRs in the SR and leads to extensive Ca2+ release from the SR. Intracellular Ca2+ binds to troponin to cause cell contraction (2+ can be rapidly recaptured by SERCA on the SR and transported out of cells through the NCX on the cell membrane, which causes cardiomyocytes to enter a diastolic state. Ca2+ transients reflect the rapid dynamic changes in cytoplasmic Ca2+ during cardiomyocyte contraction and relaxation, and the changes in velocity and amplitude represent changes in myocardial contractility. In addition, the sensitivity of cardiomyocyte myofilaments to Ca2+ affects the contractile function of cardiomyocytes. Under pathological conditions, there is a decrease in the amplitude and velocity of Ca2+ transients of cardiomyocytes, SR Ca2+ content, and myofilament sensitivity to Ca2+. This leads to a decrease in cardiomyocyte contraction and relaxation function (D.M. 2+ transients, and myofilament sensitivity of cardiomyocytes. Allicin also decreased the peak time, systolic T50, diastolic T50, and elimination constant of Ca2+ transient time. Collectively, these findings indicated that allicin significantly improved the contraction and relaxation function of myocardial cells.Cardiomyocyte dysfunction is a direct consequence of AMI and leads to a decrease in cardiomyocyte vitality, which eventually results in heart failure and even death. Calaxation . During traction . Intraceion D.M. . In this2+ is not reabsorbed by SERCA or expelled by the NCX in a timely manner at the end of cardiomyocyte contraction. Rather, intracellular Ca2+ accumulates in the cytoplasm, which hinders the separation of thin and thick myofilaments and leads to myocardial diastolic dysfunction. On the other hand, under physiological conditions, the elimination of Ca2+ transients is predominantly mediated by SERCA recapture, Ca2+ efflux by the NCX, and slow transport systems during end-diastole. In this regard, the contribution of slow transport systems is less than 1% (2+ content and Ca2+ efflux induced by the NCX, we continuously perfused coronary arteries with caffeine in a Ca2+-free environment, which completely opened the RyRs. This enabled all Ca2+ in the SR to be completely released and temporarily offset SERCA-induced Ca2+ reabsorption. Therefore, cytoplasmic Ca2+ concentration reflected the SR Ca2+ content, and the elimination of Ca2+ transients was considered to be predominantly accomplished by the NCX. We demonstrated that allicin significantly enhanced SERCA recapture and increased Ca2+ extrusion by the NCX, and increased the SR Ca2+ content. Furthermore, RyR dysfunction is known to cause Ca2+ leakage during diastole (2+ overload, SR Ca2+ content decrease, and eventually, abnormal cell contraction (2+ leakage, suggesting its ability to regulate Ca2+ homeostasis.In pathological states, intracellular Ca than 1% . In ordediastole , which ltraction . Our dat2S functions as a gasotransmitter, similar to nitric oxide and carbon monoxide. H2S exerts various cardiovascular effects, including vasodilation, blood pressure reduction, and myocardium protection. Endogenous H2S, a potent vasodilator, is synthesized via the metabolic breakdown of L-cysteine by CSE, CBS, and 3-MST (2S production in organs that express all three enzymes (2S. Furthermore, our previous research demonstrated that allicin reduced blood pressure by promoting H2S production. Here, we expand upon our previous findings by demonstrating that the anti-AMI effects of allicin are at least partially underpinned by the production of H2S mediated by CSE. In this regard, PAG, a CSE inhibitor, significantly attenuated, but did not completely abrogate, the positive effects that allicin exerts on AMI injury, coronary artery vasodilation, and calcium transport regulation in cardiomyocytes. In the present study, allicin increased the levels of H2S and CSE with or without PAG, but PAG reversed this effect, indicating that PAG partially impedes the allicin-induced production of H2S and CSE. Therefore, we speculate that allicin directly induces H2S production both directly and indirectly by increasing the levels of CSE. Several studies have shown that the mechanisms underlying H2S-induced vasorelaxation include the opening of KATP channels, closing of VDCC, and decreased concentration of intracellular Ca2+ (2S-donor, improved diabetic cardiomyopathy by regulating the Ca2+-handling system in the SR (2+ and K+ currents are strictly superimposable to those exhibited by H2S. These findings suggest that H2S is involved in the anti-AMI effects of allicin, but other mechanisms are involved. Unexpectedly, our results showed that PAG weakened the effect of allicin on CSE production, causing a decrease in CSE levels. However, we are puzzled as to why PAG, an inhibitor of CSE activity, also reduces CSE level. After preliminary literature research, we found that quite a few studies reported that PAG could decrease CSE level (Hnd 3-MST . Found pnd 3-MST , CSE con enzymes . In addi enzymes . Allicinlar Ca2+ . It has n the SR . ConsistSE level . The imp2S. The results showed that the CSE inhibitor significantly weakened the vasodilatory effects of allicin on isolated rat coronary arteries, indicating that CSE is indeed involved in allicin-induced vasodilation by mediating H2S production. However, a previous study on swine coronary arteries suggested that CBS was the most important enzyme for the production of H2S under hypoxic conditions, and that the vasodilatory contribution from CSE and 3-MST becomes apparent only upon inhibition of CBS (2S-producing enzymes in coronary arteries is still debatable. In this study, we only evaluated the role of CSE, but not that of CBS or 3-MST; hence, this study cannot fully elucidate the role of each enzyme in the rat coronary artery. Our results showed that the CSE inhibitor inhibited allicin-induced vasodilation in coronary arteries by 42.4%, indicating that a considerable amount of CSE exists in rat coronary arteries, and that its role in inducing H2S production cannot be neglected, as previously suggested in some studies (2S-producing enzyme in coronary arteries, four reasons could explain such discrepancies. First, the expression and activity of CSE, CBS, and 3-MST may vary across different species, and under different physiological or pathological conditions. Second, the levels of enzyme expression may not be proportional to the levels of activity, and indeed an inverse relationship may be present where lower expression results in higher activity. Third, enzyme activity may vary under different experimental conditions and methods, and lastly, the predominance of one H2S-producing enzyme over the others may differ with or without drug intervention. Therefore, further in-depth studies are needed to better understand the dominant role of CSE, CBS, and 3-MST in coronary arteries.An additional point worth noting is that most scholars believe that the CSE enzyme is mainly expressed in the cardiovascular system . Therefon of CBS . Anothern of CBS . Therefo studies . Regardi2+ homeostasis via H2S production. Allicin induced the vasodilation of coronary arteries via favoring H2S production by inhibiting the opening of RDCC, promoting the opening of KATP, and decreasing the Ca2+ release induced by the RyRs. Allicin regulated Ca2+ homeostasis by increasing cardiomyocyte contraction, Ca2+ transient amplitude, myofilament sensitivity, and SR Ca2+ content, and reducing SR Ca2+ leakage via H2S production. Moreover, allicin enhanced the Ca2+ uptake induced by SERCA and Ca2+ removal induced by the NCX, in which H2S is not involved. (Our study demonstrates that allicin may exert cardioprotective effects in a rat model of AMI injury by inducing coronary artery vasodilation and maintaining Canvolved.  This new


Age-related increases in chronic inflammation lead to reduced physical function via damage to muscle and joints and contribute to osteoarthritis (OA) risk. Obesity in older adults with OA further exacerbates inflammatory damage. Whether obesity reduction can lessen inflammation and improve OA is unknown; however, novel biomarkers may provide an answer. We completed a 6-mo. weight loss intervention , studying blood biomarkers of inflammation and cartilage damage along with physical function in obese older adults with  and without an OA diagnosis . Participants were aged > 60 yrs (mean = 70.2±6.0) and obese (BMI =34.6±4.7 kg/m2). At endpoint, weight loss was -6.3±4.0% and -5.8±4.1% in OA+ and OA-, respectively, with no group difference. Change scores for function for OA+ and OA- were: Short Physical Performance Battery score (+1.7±1.3 and +2.1±1.5), 8 ft up and go (-0.7±1.0 and -0.9±1.12 sec) and 6 min walk (+31.4±105.1 and +39.5±57.4 meters). All improved from baseline (p<0.05), with no group difference. Concerning blood biomarkers, there was a decrease (p<0.05) in cartilage oligomeric matrix protein (COMP: OA biomarker), indicating a potential benefit for OA. Change in COMP also differed between groups; OA- had a greater (p<0.05) reduction than OA+. Pooled results showed improved adiponectin (p<0.05), with no group difference. There were no changes for CRP, CTX-1, IL-6 and TNF-α. Our novel findings link early intervention with better reduction of OA risk and inflammation in obese older adults and also show important benefits for improved physical function regardless of OA status.


O-gallate (TFDG) had more remarkable anti-viral activities. EGCG, TSA, and TFDG at 1 mM, 40 µM, and 60 µM, respectively, which are comparable to the concentrations of these compounds in tea beverages, significantly reduced infectivity of the virus, viral RNA replication in cells, and secondary virus production from the cells. EGCG, TSA, and TFDG significantly inhibited interaction between recombinant ACE2 and RBD of S protein. These results suggest potential usefulness of tea in prevention of person-to-person transmission of the novel coronavirus.Potential effects of tea and its constituents on SARS-CoV-2 infection were assessed in vitro. Infectivity of SARS-CoV-2 was decreased to 1/100 to undetectable levels after a treatment with black tea, green tea, roasted green tea, or oolong tea for 1 min. An addition of (−) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while the same concentration of theasinensin A (TSA) and galloylated theaflavins including theaflavin 3,3′-di- The pandemic of novel coronavirus is expanding across the world with nearly 170 million confirmed cases, and global death toll from COVID-19 has exceeded 3.5 million. The SARS-CoV-2 is a Group IV single-stranded positive-sense RNA virus and requires RNA-dependent RNA polymerase for replication  was used to analyze 5 μL of sample solution at a flow rate of 0.5 mL/min. MS was performed with the following parameters: capillary voltage of 2.5 kV, desolvation temperature of 400 °C, and source temperature of 150 °C. Cone voltages were TF 45 V, TF3G 45 V, TF3′G 45 V, and TFDG 55 V, depending on the substance to be analyzed. Single-ion recording was performed in negative mode. To determine concentrations of EGCG dimers, LC-MSMS analysis was performed on ACQUITY UPLC coupled with an ACQUITY TQD, which were connected by an electrospray ionization (ESI) source. An InertSustain C18 Analytical Column   was used at 40 °C for the separation of compounds. Gradient elution was performed with 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B). Linear elution was performed as follows: 93% A/7% B at 0 min, 93% A/7% B at 1 min, 75% A/25% B at 6.5 min, 74% A/28% B at 9 min, 70% A/30% B at 10 min, 40% A/60% B at 12 min, and 93% A/7% B at 15 min. MS was performed using the following parameters: capillary voltage, 2.7 kV; probe temperature, 600 °C; source temperature, 150 °C; and desolvation gas flow, 600 L/h; cone voltage, 50 V. The mass spectrometer was operated in the negative ESI mode. The peak was detected using an MRM transition for theasinensin A (m/z. 913.50 > 591.20) in the MS/MS detection.HPLC analysis of catechins was performed using an Alliance 2695 Separations Module  equipped with 2487 Dual Wavelength Absorbance Detector (Waters). For the separation of compounds, J’sphere ODS-H80 Column   was used at 40 °C. A total of 10 μL of the sample solutions were analyzed using a gradient elution with a solvent A: ultra-pure water, solvent B: acetonitrile and solvent C: 0.1% phosphoric acid in water. The detection wavelength was set to UV 230 nm. To determine concentrations of theaflavins, LC-MS analysis was performed on ACQUITY UPLC  coupled with an ACQUITY TQD (Waters), which were connected by an electrospray ionization (ESI) source. An ACQUITY UPLC BEH Shield RP18 Column  was used at 40 °C for the separation of compounds. An isocratic elution method using [ultrapure water/acetonitrile/formic acid  and incubation at room temperature for 1 min. Immediately, the virus/tea/DMEM mixture was serially diluted 10-fold with MS in the 96-well-plates. Chilled on ice, 100 μL of each sample was added to the VeroE6/TMPRSS2 cells that had been seeded into 96-well-plates at 5 × 104/100 μL/well a day before. After culture for 4 days, cells were washed, fixed, and stained with crystal violet solution to estimate CPE as described [Freeze-dried powders of green tea, roasted green tea, black tea, and oolong tea was dissolved in sterilized distilled water at 78 °C for 10 min to prepare ×4 concentration of original tea. After chilling at room temperature, each solution was passed through a 0.45 μm filter, and placed into 96-well-plates at 50 μL/well in triplicate. Each sample was mixed with 50 μL of ×2 serum-free DMEM, followed by an addition of 100 μL of SARS-CoV-2 suspension  and incubation at room temperature for 1 min. Immediately, the virus/compound/DMEM mixture was serially diluted 10-fold with MS, and TCID50 assay and calculation of TCID50 values were performed as above.Solutions of EC, ECG, EGC, EGCG, TSA, TF, TF3G, TF3′G, and TFDG were diluted in MS (DMEM supplemented with 0.5% FBS) to concentrations of 2 mM, 400 μM, and 80 μM. The same volume of ×2 DMEM was added to each solution followed by an addition of SARS-CoV-2 suspension (1.5 × 106 TCID50/50 μL) or MS was added to 50 μL of each tea/DMEM mixture and incubated at room temperature for 1 min. Immediately, 50 μL of the virus/tea/DMEM mixture was added to the VeroE6/TMPRSS2 cells that had been seeded in 96-well-plates at a density of 5 × 104/100 μL/well a day before (MOI = 3 or 0). One hour later, culture supernatant was replaced by 100 μL of MS, followed by culture for 30 h. To measure cell viability, cells were washed with PBS, and WST-8 solution  diluted in phenol red-free DMEM was added to the wells at 50 μL/well. After culture for 45 min, 20 μL of 10% SDS was added to each well and OD at 450 nm was measured. Cell-free wells were regarded as a reference.Tea was diluted and mixed with the same volume of serum free DMEM as above. Five μL of SARS-CoV-2 suspension (1.5 × 105 TCID50/50 μL) or MS was added to each mixture and incubated at room temperature for 1 min. Immediately, 50 μL of the virus/compounds/DMEM mixture was added to the VeroE6/TMPRSS2 cells that had been seeded into 96-well-plates at 5 × 104/100 μL/well a day before (MOI = 5 or 0). One hour later, culture supernatant was replaced by fresh 100 μL MS, followed by culture for 27 h. Cell viability was measured as above.EGCG, TSA, and TFDG was diluted and mixed with the same volume of ×2 DMEM as above. SARS-CoV-2 suspension  to the wells for 1 h (MOI = 5 or 0). After replacement of the culture supernatant by 100 μL of MS, cells were cultured for 27 h, and cell viability was measured as above.Compounds/DMEM mixtures were prepared as above and added to the VeroE6/TMPRSS2 cells that had been seeded into 96-well-plates at 5 × 10® LS  and reverse-transcribed using ReverTra Ace® qPCR RT Master Mix . Quantitative real-time PCR was performed using a Step-One Plus Real-Time PCR system  and the following set of primers/probes specific for viral N gene. Forward primer, 5′-AAATTTTGGGGACCAGGAAC-3′; reverse primer, 5′-TGG-CAGCTGTGTAGGTCAAC-3′; and probe, 5′-(FAM) ATGTCGCGCATTGGCATGGA (BHQ)-3′. Ct value for each sample was calculated by StepOne Software . N gene RNA levels in cells were normalized with respect to the 18S rRNA level in each sample. Relative RNA levels (average ± SD) were expressed relative to the value for the control infected with untreated-virus (set to 1.0).RNA was extracted from culture supernatant using TRI ReagentNeutralizing assay was performed using SARS-CoV-2 Surrogate Virus Neutralization Test Kit  according to the manufacturer’s protocol. Briefly, samples diluted to various concentrations were mixed with a horseradish peroxidase (HRP)-conjugated recombinant RBD fragment at a volume ratio of 1:1 at 37 °C for 30 min. One hundred µL of the mixture was added to the wells pre-coated with human ACE2 protein. After incubation at 37 °C for 15 min, wells were washed and 100 μL of 3,3′,5,5′-tetramethyl-benzidene (TMB) solution was added. Following incubation in the dark at 20–25 °C for 15 min, absorbance at 450 nm was measured.t test , and galloylated theaflavins including TFDG. The EGCG, TSA and TFDG interacted with Spike protein RBD to prevent interaction between RBD and cellular ACE2. These data suggest the possibility that ingestion of and rinsing with tea may attenuate spread of SARS-CoV-2 .


Presented here is a novel Ca-based MOF containing 2,3-dihyrdoxyterephthalate  linkers Ca(H2O) (SIMOF-4). The material undergoes a phase transformation on heating, which can be followed by variable temperature powder X-ray diffraction. The structure of the high temperature form was obtained using single-crystal X-ray diffraction. The electrochemical properties of SIMOF-4 were also investigated for use in a Na ion battery.The rapid growth in the field of metal-organic frameworks (MOFs) over recent years has highlighted their high potential in a variety of applications. For biological and environmental applications MOFs with low toxicity are vitally important to avoid any harmful effects. For this reason, Ca-based MOFs are highly desirable owing to their low cost and high biocompatibility. Useful Ca MOFs are still rare owing to the ionic character and large size of the Ca The intion site . All Ca-4 dhtp carboxylic acid groups , °, and two µ4 dhtp groups via carboxylic acid oxygens, a water molecule makes up the remaining coordination site , . The intion site . All Ca-This high connectivity produces an interconnected 3D network . SIMOF-4The powder X-ray diffraction (PXRD) data pattern of SIMOF-4 is shown in 3. This is backed up by PXRD analysis after a 480 °C heat treatment, which matches theoretical peaks for CaCO3  shows multiple mass losses . The 12 or CaCO3 . The finThe water loss and first degradation step at 300 °C were explored further by use of variable temperature X-ray diffraction (VTXRD) . The VTXThe phase transition at high temperature caused significant damage to the crystals; they underwent a change in morphology and became significantly less diffracting, this reduced their suitability for scXRD. This was true for all crystals we investigated. However, careful crystal selection and truncating the scXRD data did give diffraction that was good enough to solve, producing a plausible structure. This is not unusual in this type of science. As suggested by TGA, all water is lost, producing Ca (SIMOF-4-h), and this induces a significant change to the crystal structure. The unit cell remains triclinic but the unit cell parameters change, leading to a reduction in the unit cell volume by ≈18%. While we cannot infer any fine detail of the atomic arrangement and atomic displacement parameters, it is notable that this model does reproduce the experimental powder X-ray diffraction pattern well , and so ab-plane connected by further 2,3-dhtp linkers  was performed on SIMOF-4 between 0.01 and 2.5 V vs. Na ~0.56 V . No prom−1 (−1 was observed for the first discharge with a low coulombic efficiency (43%). This could be indicative of the contribution of SEI (solid-electrolyte interphase) layer formation . SIMOF-4-h crystals were analysed using a Rigaku MM-007HF High Brilliance RA generator/confocal optics with XtaLAB P100 diffractometer [Cu Kα radiation λ = 1.54187 Å)]. Data collection was performed using CrystalClear , data re87 Å]. Da8H8CaO8, M = 272.22, triclinic, a = 7.1339(3), b = 11.5312(5), c = 13.1000(8) Å, α = 66.744(5), β = 80.811(4), γ = 80.834(4)°, U = 971.86(9) Å3, T = 93 K, space group PZ = 4, 11,622 reflections measured, 4197 unique (Rint = 0.0215), which were used in all calculations. The final R1 [I > 2σ(I)] was 0.0403 and wR2  was 0.1186.Crystal data (SIMOF-4). C8H4CaO6, M = 236.19, triclinic, a = 7.350(4), b = 9.540(6), c = 12.734(6) Å, α = 73.55(5), β = 73.92(5), γ = 71.84(5)°, U = 796.1(8) Å3, T = 173 K, space group PZ = 4, 2055 reflections measured, 1300 unique (Rint = 0.0549), which were used in all calculations. The final R1 [I > 2σ(I)] was 0.1835 and wR2  was 0.4867.Crystal data (SIMOF-4-h). C−2.The working electrodes for SIMOF-4 were prepared in a mixture of active compound (55%), conducting carbon—Super C65 (35%) and binder—CMC . A slurry was prepared in water and cast on an aluminium foil  by a doctor blade. The electrodes were subsequently punched (~12 mm diameter) upon air drying and dried overnight in a vacuum oven at 110 °C and transferred to an argon-filled glovebox. The average mass loading of the active material per electrode was 1.54 mg cm6 in EC:DEC  was used as the electrolyte and the process was carried out in the glovebox with oxygen and water content <1 ppm. Electrochemical measurements were recorded at 30 °C on a Biologic BCS-805 modular battery testing system in a potential window of 0.01–2.5 V (vs. Na+/Na) and all data were analysed and processed using the BT-Lab software.Electrochemical testing was done using coin cells (CR2325), that were assembled using sodium (Sigma-Aldrich) as the counter electrode and a glass fiber separator (Whatman GF/F). NaPFIn this paper we have presented a new Ca MOF, SIMOF-4. The crystal structure of the as-made material has been determined using single-crystal X-ray diffraction, and its high temperature behaviour was studied using variable temperature PXRD and TGA. Despite significant damage to the single crystals on thermal treatment the structure of the high temperature phase was also determined. Initial electrochemistry experiments on the material’s suitability for use as an anode in sodium-ion batteries showed that SIMOF-4 has moderate discharge capacity. These results present two possible avenues for future work. First, improving the discharge capacity to improve stability and creating a non-toxic anode for Na-ion batteries that may be viable in this type of material. Second, the replacement of water within the pores of the MOF with small biologically active molecules such as NO may yield non-toxic materials of potential utility.


Candidatus Aramenus sulfurataquae.” This study reveals novel genomic sequences of Sulfolobales archaea.A plethora of hot springs are found at the Los Azufres volcanic complex in Mexico, and studies are needed to determine their microbial genomic diversity. Here, we report a metagenome of hot spring sediments and a metagenome-assembled genome of “ Sulfolobales are commonly found in acidic high-temperature environments   for “Candidatus Aramenus sulfurataquae” strain AZ2 from thermal sediments within Los Azufres.Archaea from the order ronments , 2. A la65°C, pH .6 (3). Trecently . Interesde novo with SPAdes v.3.8.2 (Candidatus Aramenus sulfurataquae” was recovered by mapping the reads against the genome of “Candidatus Aramenus sulfurataquae” strain AZ1 (GenBank accession number ASRH00000000.1) using bowtie2 v.2.3.4.3  (–http://enve-omics.ce.gatech.edu/ani)  at Los Azufres in May 2016. DNA was isolated using the DNeasy UltraClean microbial kit . Library preparation was performed using a DNA preparation kit  according to the manufacturer's instructions. Sequencing was performed at the Laboratorio Nacional de Análisis y Síntesis Ecológica  on an Illumina NextSeq550 platform, producing 2 × 75-bp paired-end reads. Reads were assessed using FastQC v.0.11.8 (with default settings) ( v.3.8.2 . Metagen v.3.8.2 . The MAG.2.3.4.3  using k-ettings) –15. MAG ttings) –, using tedu/ani) .N50, 7,585 bp) with a minimum length of 1,000 bp, containing 11,279,257 bp. The metagenome revealed Sulfolobales sequences related to the Acidianus, Sulfolobus, and Aramenus genera. A 1,681,874-bp draft MAG comprising 379 contigs , with a GC content of 47.8% and genome coverage of 84.31×, could be obtained from the metagenome. The MAG was estimated to be 99.29% complete, with no detected contamination. MAG annotation predicted 2,103 genes and revealed a 1,498-bp 16S rRNA gene that shares 100% sequence identity with the gene from “Candidatus Aramenus sulfurataquae” strain AZ1. Genes involved in carbon fixation and sulfur metabolism could be detected. The MAG exhibited an ANI value of 97.91% against the “Candidatus Aramenus sulfurataquae” strain AZ1 genome, suggesting that they correspond to the same species. We identified a “Candidatus Aramenus sulfurataquae” population in a new niche within Los Azufres with higher temperature and lower pH.Metagenomic reads  were assembled into 1,718 contigs  under accession number


Nowadays, nitrogenous heterocyclic molecules have attracted a great deal of interest among medicinal chemists. Among these potential heterocyclic drugs, benzimidazole scaffolds are considerably prevalent. Due to their isostructural pharmacophore of naturally occurring active biomolecules, benzimidazole derivatives have significant importance as chemotherapeutic agents in diverse clinical conditions. Researchers have synthesized plenty of benzimidazole derivatives in the last decades, amidst a large share of these compounds exerted excellent bioactivity against many ailments with outstanding bioavailability, safety, and stability profiles. In this comprehensive review, we have summarized the bioactivity of the benzimidazole derivatives reported in recent literature (2012–2021) with their available structure-activity relationship. Compounds bearing benzimidazole nucleus possess broad-spectrum pharmacological properties ranging from common antibacterial effects to the world’s most virulent diseases. Several promising therapeutic candidates are undergoing human trials, and some of these are going to be approved for clinical use. However, notable challenges, such as drug resistance, costly and tedious synthetic methods, little structural information of receptors, lack of advanced software, and so on, are still viable to be overcome for further research. H-benzimidazole and 1,3-benzodiazole, consists of benzene ring fused with a five-membered imidazole ring, and is an important heterocyclic pharmacophore. Benzimidazole is regarded as a “privileged structure” in heterocyclic chemistry due to its association with a wide range of biological activities  and evaluated in vitro antimicrobial activity against some Gram-positive and Gram-negative bacteria and fungi using broth microdilution minimum inhibitory concentration (MIC) method. Compounds 1, 3 and 4  displayed prominent antibacterial activity against S. typhi compared to standards ampicillin, chloramphenicol and ciprofloxacin . Compounds 2 and 3 exhibited notable antifungal activity against C. albicans (MIC = 250 μg/ml) in comparison with standard griseofulvin (MIC = 500 μg/ml). The derivatives with 4-methoxy (1) and cyano (4) group at 2-position of indole nucleus were found to possess excellent inhibitory activity against most of the tested organisms. Birajdar et al. (5–8) by epoxide ring opening of 2-methyl benzimidazole with different substituted cyclic amines. Compounds 5–8 demonstrated moderate to good activity against Gram-positive (S. aureus) and Gram-negative (E. coli) pathogens in comparison with reference drugs ciprofloxacin and norfloxacin.Kathrotiya and Patel  synthesir et al.  synthesi9–14) were prepared by Kahveci et al.  and investigated in vitro antimicrobial potential against a number of bacterial and fungal strains using conventional broth dilution method. Compounds 15 and 18 (MIC = 12.5–25 µg/ml) showed better antibacterial activity, and 16 and 19 (MIC = 25–100 µg/ml) displayed comparable activity to standard chloramphenicol (MIC = 50 µg/ml). The presence of electron withdrawing groups, e.g. fluoro  and nitro  at the meta or para position might have contributed for their antimicrobial property. Compound 17 containing chloro group displayed the most remarkable antifungal activity, with MIC values in the range of 25–62.5 µg/ml against three fungal strains compared to standard ketoconazole (MIC = 50 µg/ml). A library of 1-methyl-N-imidazole-5-carbohydrazides derivatives among which compounds 28–31 displayed notable inhibitory effect against A. niger with MIC value of 3.12 μg/ml. Compound 28 demonstrated a MIC value of 3.12 μg/ml against most bacterial and fungal strains and appeared to be a potent antibacterial and antifungal agent.Luo et al.  synthesia et al.  synthesi32–34) and Chandrika et al. (35–37), where the compounds 32–34 showed prominent antibacterial activity against S. aureus strain and compounds 35–37 were found to be the most potent antifungal agents against azole-resistant fungal strain C. albicans ATCC 64124 (strain B). Another study reported that among the total 22 synthesized novel 2-substituted fluorinated benzimidazoles, compounds 38–40 showed antimicrobial activity. In contrast, compound 40 containing a trifluoromethyl substituent showed the highest antifungal activity against the fungus C. albicans ((41–43), where compounds 41 and 43 showed notable activity against S. aureus, and compound 42 was found to be the most effective against B. cereus. Compound 41 displayed the highest antifungal potential against C. albicans, whereas 43 exhibited prominent activity against A. fumigatus.A series of benzimidazole derivatives were synthesized and evaluated by Padalkar et al.  (32–34) a et al.  (35–37),albicans . Similaralbicans  synthesi44 was found to be the promising broad-spectrum antibacterial agent against P. aeruginosa, S. aureus, B. subtilis and P. vulgaris. A series of N-(substitutedbenzylidene)-4-(1-((dimethylamino)methyl)-1H-benzimidazol-2-yl)thiazol-2-amine derivatives were investigated for antimicrobial activity using agar streak dilution test. Compound 45 appeared to be the most potent among the series. Notably, the presence of an electron-withdrawing group might have contributed to the improved antimicrobial property of the compound , where compound 46 emerged as the most potent antibacterial agent against both Gram-positive and Gram-negative bacteria compared to standard cefadroxil. All derivatives exhibited better antifungal activity than the standard fluconazole, and compound 47 showed maximum activity against A. niger (MIC = 0.018 mM). Liu et al. -5-aryl-4-5dihydro-1H-pyrazol-1-yl)-2- ethanones (49–52), where the electron-withdrawing groups (compound 49 containing chloro group at ortho position and 50 with a nitro group at the para position) were the most effective against bacterial strains. On the contrary, electron releasing groups at the para position  contributed to the most promising antifungal activity against the tested organisms . Amongst a series of α-aminonitrile based benzimidazole derivatives (54–57), the compounds 55 and 56 were found to be the most potent antibacterial agents having MIC values ranging between 3.9 and 7.8 μg/ml against different bacterial species. All the compounds exerted illustrious antifungal activity against C. albicans (MIC = 3.9–7.8 µg/ml) compared to the reference drug fluconazole   .58–61 containing the 1,3,4-thiadiazole ring and azo moiety showed excellent activity against S. aureus, B. subtilis, E. coli, and p. aeruginosa compared to amoxicillin and ciprofloxacin (62–67 and novel Schiff bases of 2-(1-amino benzyl)-benzimidazole compounds 68–75 indicated moderate to high in vitro inhibition of both gram-positive (S. aureus) and gram-negative (E. coli) bacteria   . Besides16 μg/ml . Malasal16 μg/ml  reportedis H37Rv . Furtheractivity .The antiviral properties of benzimidazole derivatives have been tested against different viral strains; human immunodeficiency virus (HIV), hepatitis B and C virus (HBV and HCV), enteroviruses, respiratory syncytial virus (RSV), human cytomegalovirus (HCMV), bovine viral diarrhea virus (BVDV) and herpes simplex virus-1 (HSV-1) are some to mention . This se97–98 showed notable antiviral activity against laboratory-adapted strains HIV-1IIIB and HIV-1Ada5 (EC50 = 15.4–40 µM) and primary isolates of HIV-1UG07O and HIV-1VB59 strains (EC50 = 5.28–31.86 µM) (1-aryl-benzimidazol-2-one derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs) against HIV-1, where the compounds 99–100 were more potent than the standard drug nevirapine (IC50: 1.3 and 0.79 vs. 1.55 µM). The sulfone derivatives 101–102, synthesized by the same research group were also found to be potent HIV-1 NNRTIs with IC50 values of 47 and 50 nM, respectively. The substitution at C-4 position of the arylacetamide portion of the compounds might have contributed for their notable activity against HIV-1IIIB strain in cell-based assays  against HIV-1 replication, among them compounds 1.86 µM) . Besides1.86 µM)  synthesid assays . Finallyd assays  has rece104 inhibited the secretion of HBsAg and HBV virions indicated by EC50 values of 1.5 and 0.6 μM, respectively, along with half cytotoxicity concentration (CC50) value of 24.5 μM.The hepatitis B surface antigen (HBsAg), an HBV surface protein is an important mediator of HBV life cycle . Xu et a105–106 with EC50 values 3.0 and 5.5 nM, respectively, against hepatitis C virus (HCV), a prime cause of liver cirrhosis and hepatocellular carcinoma. Besides, Henderson et al. (107–109) and evaluated for their HCV non-structural 5A (NS5A) inhibitory effect with balanced Genotype 1a (G1a) and Genotype 1b (G1b) potency. Compounds 107 , 108  and 109  appeared to be most active compounds from the series. Substitution of methyl group (107–108) and geminal dimethyl group (109) at 4-position of pyrrolidine nucleus was likely to contribute for G1a and G1b potency of the compounds. In another study, a set of 2-thiobenzimidazole analogues (110–111) containing triazole moiety were reported as promising HCV inhibitor, where the substituent at position-2 of benzimidazole nucleus played the crucial role to enhance the antiviral potency  inhibition with the IC50 values of 1.76 and 1.08 μg/ml, respectively  and 2-amino-5,6-difluorobenzimidazole nucleosides (119) inhibited Herpes Simplex Virus-induced cytopathic effect (CPE). Impressively, the IC50 value of compound 119 was 104 μM, four times lower than that of ribavirin and eight times lower than that of maribavir.Two distinct series of benzimidazole derivatives were synthesized, where the compounds ectively . The ben capsids . In a pa capsids , 2017, K120 appeared to be the most effective antiviral agent against Bovine Viral Diarrhea virus  due to the presence of 2,4-dimethoxy group in the phenyl moiety (H-benzimidazole derivatives (121–124) bearing substitution of heterocyclic rings at position 1. Compounds 121 and 122 exhibited equal potency as standard doxorubicin against A-549, HCT-116, MCF-7 and human liver carcinoma HepG2 cell lines. Besides, compounds 122–124 showed great potential to be used as potent antiviral agents due to their inhibitory effect against rotavirus Wa strain. Finally, compound 125, identified by Dai and co-workers, was found to be potent antiviral agent against Lassa virus envelope glycoprotein (LASV GP) pseudotypes with EC50 value of 1.1 nM , enzymes involved in biosynthesis of important inflammatory mediators called prostaglandins . Apart f126–128 were found to be the most promising agents among the series displaying significant (p < 0.01) analgesic and anti-inflammatory effect at a dose level of 100 mg/kg p.o. Li et al. . Interestingly, the compound 129 also prevented 33.30 and 50.60% ear oedema in xylene-treated mice at doses of 4 and 12 mg/kg, respectively, compared to standard ibuprofen . Besides, compounds 130–132 showed the potentiality as gastroprotective lead compounds which can be developed into orally active analgesic and anti-inflammatory agents   .H-benzimidazol-1-yl]-4H-pyrazol-3-ol derivatives (136–137) and investigated for in vitro cyclooxygenase inhibitory effect using Cayman’s colorimetric COX (ovine) assay. Compounds 136–137 showed remarkable in vitro cyclooxygenase inhibition  and significant (p < 0.05) reduction of edema volume compared to that of standard diclofenac at all time intervals. Prajapat and Talesara (138–140) and Siddiqui et al. (H-benzo[d]imidazol-2-yl)methyl}-3-arylthioureas (141–143). All the compounds (138–143) exerted notable anti-inflammatory effect compared to standard diclofenac.Moneer et al.  synthesiTalesara  synthesii et al.  preparedH-benzoimidazol-2-ylimino)-methyl]-6-methoxy-phenol Schiff base ligands (144–145) were synthesized from condensation of 2-aminobenzimidazole and o-vanillin (144–145) at 100 mg/kg b.w. showed around 55% inhibition of inflammation compared to standard diclofenac (65.4% inhibition). Furthermore, Bukhari et al.  and Zn(II) complexes of a 2-imidazole derivatives (160–163), where the compound 162 showed maximum selectivity towards COX-2 enzyme among the series (% inhibition 78.68 ± 0.46 and selectivity ratio 3.71) and it might be due to the presence of N-phenyl piperzine moiety in the benzimidazole nucleus. Besides, compounds 160, 161 and 163 demonstrated notable activity against COX-2 enzyme .By applying Mannich reaction, Sethi et al.  preparedr et al.  synthesi164–165 compared to the standard indomethacin (45 vs. 48%). Brishty et al. . In continuation of the research by the group, Saha et al.  in comparison with standard aceclofenac (88.81%). Very recently, the group reported the pharmacological investigation of some of their previously synthesized benzimidazoles . Besides, 171, 172 and 173 displayed notable reduction in paw edema  comparable to standard aceclofenac (87.83)  .+/K+-ATPase inhibitory properties. During recent times, several new synthetic benzimidazole-based compounds were developed which exhibited similar or better antiulcerogenic potentials compared to the established market preparations. The benzimidazole derivatives with antiulcer activity are shown in Many benzimidazole derivatives are known to possess potent antiulcer activity and H174) and pyrimidylsulfinylmethyl benzimidazole derivatives (e.g. 175) were developed and screened for antiulcer activity. Compounds 174–175 significantly reduced gastric acid secretion, free acidity and gastric ulcers in the pylorus-ligated rats at 10 and 30 mg/kg doses, where the sulfinyl derivative (175) was found to be more effective than thio derivative (174)  (A series of pyrimidylthiomethyl benzimidazoles (e.g. ve (174) . In anoteprazole . Besideseprazole  prepared0 mg/kg) .in vivo antiulcer property. The 4-methoxy phenyl piperazine substituted benzimidazole derivative (183) appeared to be the most effective agent  benzimidazole) emerged as the most potent inhibitor of Helicobacter pylori growth and pathogenesis of host cells. The compound specifically inhibited H. pylori adhesion and invasion of gastric epithelial cells, as revealed by in vitro H. pylori infection model. Mathew et al. (185–187) and reported that derivatives 185–187 exerted remarkable protection of ulcer  at a dose of 50 mg/kg b.w compared to omeprazole . Amongst a series of substituted methoxybenzyl-sulfonyl-1H-benzo[d]imidazole derivatives, compounds 188–193 appeared to be the most potent H+/K+-ATPase inhibitors compared to omeprazole  compared to standard butylated hydroxytoluene . Among the different benzimidazole derivatives with heterocyclic moieties developed by Mentese et al.  free radical scavenging method  in comparison with standard BHT (IC50 = 51.56 μg/ml).Two series of benzimidazole compounds made through coupling of coumarin derivatives with benzimidazole nucleus either directly or through amide linkage at 2-position were evaluated for antioxidant property . Compoune et al. , compoung method . CompounN-substituted pyrazole-containing benzimidazoles, compounds 202–204 attributed prominent antioxidant activity in both DPPH and hydrogen peroxide assay supposed to the presence of benzyl substituent on imidazole nitrogen (205–206 using tert-butyl hydroperoxide (tert-BOOH) induced oxidative stress on rat hepatocytes, and reported that both compounds showed significant effect comparable to standard quercetin. Similarly, compounds 207–212 displayed notable cytoprotective effect on rat hepatocytes  and urease (IC50 = 13.04 ± 0.89 μg/ml), respectively (215 had the most potency in the series of 2-(aryl)-6-morpholin-4-yl(or 4-methylpiperazin-1-yl)-1H-benzimidazoles -1ectively . Furtheridazoles .1) of the benzimidazole nucleus was responsible for exhibiting medium to high potency against several free radicals. In contrast, the derivatives containing the 5-sulfonic acid group showed poor or no antioxidant properties. Unsubstituted 2-aromatic ring or OH, Cl, Br, 2-OH-napthyl or 4-OH-steryl substitutions enhanced activity. In another study, compound 216 showed 40–80% antioxidant potential at different concentrations (10–100 µM)  for DPPH assay: 40.4 ± 0.9 to 60.4 ± 1.9 vs. 70.8 ± 6.6)  comparable to standard propyl gallate (29.20 ± 1.25). Interestingly, compound 225 exhibited more inhibition (%) of DPPH-free radical than the standard antioxidant Trolox (73% ± 2.42 vs. 70% ± 0.35)    . Amine K8 ± 6.6) . Taha et8 ± 6.6)  synthesi ± 0.35) . Further4 μg/ml) . Finally9 μg/ml) .Among the anticancer drugs discovered in the recent years, different benzimidazole derivatives occupy an important place. The current review accounts the anticancer activity of benzimidazoles reported after 2013. The benzimidazole derivatives with anticancer activity are shown in in vitro anticancer activity in human lung adenocarcinoma A549 cell line at normoxic and hypoxic conditions. Compound 230 was found to be the most cytotoxic agent with hypoxia/normoxia cytotoxic coefficient of 4.75, compared to standard tirapazamine (5.59) (231–232 showed notable anticancer effect against human liver carcinoma cell line (HepG2: IC50 = 0.518 and 0.578 mM) and pheochromocytoma of the rat adrenal medulla cell line (PC12: IC50 = 0.309 and 0.298 mM) -3-phenylprop-2-enoyl]-1H-benzimidazole derivatives among which compound 235 displayed notable antiproliferative activity against nine tumor subpanels, indicated by its selectivity ratios within the range of 0.79–1.53 and 0.47 to 1.69 at the GI50 (growth inhibitory 50%) and TGI level, respectively  . The ben.298 mM) . CompounK0106023 . Kalalbaectively . Similar 1.05 µM .239 showed notable inhibition of PI3K-AKT-mTOR pathway having GI50 values in the range of 0.07–0.41 μmol/L against most of the tested cancer cell lines, signifying its potential to be used as anticancer agent. Sontakke and co-workers (240–242) bearing hydrogen, benzoyl and carboxyl substituents, respectively at 5th position. Compounds 241 and 242 showed anticancer potency against MCF-7 cell lines  and HL-60 cell lines  followed by compound 240 (MCF-7 IC50 = 20.48 µM and HL-60 IC50 = 23.23 µM). In another study, compound 243 displayed most notable activity against HeLa cell lines (IC50 = 05.34 ± 1.2 µM) compared to standards doxorubicin and 5-flurouracil . On the other hand, compound 244 showed marked activity against Hep3B cell line with an IC50 value of 11.10 ± 1.1 µM  among which compounds 246–248 demonstrated inhibition against K-562 and MCF-7 cell lines in micromolar range. Compounds 246 and 248 were reported as activators of caspases, which play important role in apoptosis of cancerous cells. Bramhananda Reddy et al. designed and synthesized a library of benzimidazole fused ellipticine derivatives (249–253) and delineated antiproliferative potential against human cancer cell lines Zr-75–1, HeLa, MCF-7 and A-549 with GI50 values of <0.1–34.6 µM, compared to standard etopoxide (GI50 = 0.2–3.08 µM) (254–256) towards PC-3, HeLa, A549 and HT1080 cancer cell lines with IC50 values of 0.096–0.63 µM. In a different study, Compound 257 exhibited potent antiproliferative effect against MFC cells IC50 (mean ± SD) value of 25.72 ± 3.95 μM (H-benzimidazole derivatives 258–259 bearing carboxyl substituent manifested the most prominent inhibitory effect against protein kinase 2 (CK2) with binding affinity value in the range of 1.96–0.91 µM , lung (NCl H-522), liver (HepG2), colon (HCT-15) and ovary (PA-1) with IC50 (mean ± SD) value of 7.5 ± 0.3 to 14.6 ± 0.4 μM (50 (1.08 µM) of derivative 265 against A549 cell line. Compounds 266–268 displayed prominent cytotoxic activity against A549 and MCF-7 cancer cell lines in comparison with standard drug cisplatin with IC50 values of 0.03–0.06 μM. The presence of 2,4-dichlorobenzylidene (266), 2-nitrobenzylidene (267) and 2-methoxybenzylidene moiety (268) contributed for notable cytotoxic activity of these compounds   and colorectal (IC50 10.97 ± 0.09 µM against HCT116) cancers  . Sharma 3.08 µM)  designed 3.95 μM . Triazol–0.91 µM . Compoun± 0.4 μM . Upon as± 0.4 μM  enumeratompounds . The oxe–3.8 µM) . Besides cancers .271–274 and narrated potential anti-proliferative property of these derivatives against two breast cancer (T47-D and MDA-MB-231) and one prostate cancer cell line (PC3) compared to standard Doxorubicin. It is mentionable here that the chloro substituent 274 at the chalcone ring proliferated the anticancer effects. In a distinct research, a total of 24 new molecules containing benzimidazole group, arene, and alkyl chain-bearing cyclic moieties were synthesized, where the compound 275 impeded the growth of MCF-7 and human ovarian carcinoma (OVCAR-3) cell lines manifesting superior effects to standard cisplatin : 8.91 ± 0.07 vs. 11.7 ± 0.12 and 10.76 ± 0.12 vs. 16.04 ± 0.74, respectively) (276) of benzimidazole derivatives showed excellent potency at 72 h post treatment against prostate cancer cell line (DU145) with IC50 10 µM (in vitro potency against human prostate (DU-145) and human ovarian (A-2780) cancer cell lines. Notably, compounds 277–280 at a concentration of 0.1 µM exhibited superior activity against A-2780 cell line in comparison with standard docetaxel. Jian-Song et al. . Notably, compound 281 inhibited predominately of all the three cancer cell lines compared to 5 Fluorouracil : 1.02 ± 0.03 vs. 6.82 ± 1.17, 3.34 ± 0.09 vs. 18.42 ± 1.73, and 5.40 ± 0.51 vs. 17.11 ± 2.94, respectively). The structural modifications of the compound 281 might significantly influence its anti-proliferative property that has been illustrated in 282) significantly suppressed sorafenib-resistant cell lines growth in xenograft model by inhibiting the phosphorylation of AKT, p70S6 and the downstream molecule RPS6, has unlocked another milestone in the treatment of hepatocellular carcinoma. Yeong et al. . Among them, compound 284 elicited significant inhibition of SIRT1-3 compared to tenovin-6 : 7.7 ± 1.4 vs. 42.10, 5.6 ± 1.3 vs. 25.6, and 9.8 ± 2.0 vs. 82.65). Moreover, among 37 synthesized molecules, compound 288 exerted the most inhibition of angiogenesis (79%), and HUVEC and HepG2 cell lines , and VEGFR-2 kinase inhibition  . A coppe50 10 µM . Besides50 10 µM . In anot50 10 µM , 18 compg et al.  synthesig et al.  synthesihibition .289–291 and reported the most promising anti-breast cancer feature of compound 291 compared to standard cisplatin : 1.26 ± 0.85 vs. 5.77 ± 0.40). In another study, Atmaca et al. (292 against breast cancer (MCF-7), prostate cancer (DU-145), and lung cancer (H69AR) with IC50 values of 17.8 ± 0.24, 10.2 ± 1.4 and 49.9 ± 0.22 μg/ml, respectively, compared to 5-Fluorouracil. Compounds 293–294 showed stronger anticancer property against HepG2  and DLD-1 cells  than cisplatin (IC50: 30.38 and 60.79 µM) (295–296 demonstrated potent anti-breast cancer effect by obstructing MCF-7 cell growth compared to standard cisplatin (IC50: 0.016 ± 0.001 and 0.018 ± 0.001 vs. 0.020 ± 0.009 µM). Besides, compound 295 displayed efficiency for impeding estrogen-dependent breast cancer by inhibiting aromatase enzyme with IC50 0.032 ± 0.042 µM, compared to IC50 0.024 ± 0.001 µM for letrozole (297 blocked neddylation process with superior anticancer property compared to candesartan cilexetil (IC50 5.51 vs 16.43 mM) : 1.69 ± 0.90 and 3.20 ± 0.50, respectively). Apart from, the compound 298 significantly suppressed the breast cancer cell lines MCF-7 and MDA-MB-23 with IC50 (μM) values, comparable to 5-Fluorouracil   . Particuetrozole . Further6.43 mM) . Notablyctively) .299–300 exhibited superior inhibition of EGFR, HER2, PDGFR-β and VEGFR2, in comparison to erlotinib that opened several promising fighting tools against cervical cancer. In a distinct study, compounds 301–304 strongly prevented breast cancer cell lines manifesting IC50 values of 5.70 9.55, 5.58 and 6.84 μg/ml, respectively compared to standard doxurubucin (IC50 at 4.17 μg/ml) , lung , colon , and ovarian  cancers. Similarly, Srour et al. . Besides, compounds 312, 314, 316, 317 and 318 showed significant cytotoxicity against epidermal growth factor receptor tyrosine kinase with IC50 values of 71.67–152.59 nM compared to IC50 of standard erlotinib 152.59 nM. Finally, Yamani et al. (319 selectively inhibited FGFR (1–4) with IC50 values of 0.75, 0.50, 3.05, and 87.90 nM, respectively. Due to acceptable safety and pharmacokinetic profiles along with in vivo anti-tumor potency, the compound 319 is now undergoing with an open-label, multicenter, dose-escalation phase I clinical trial for assessing the safety and tolerability against the adults patients with bladder, gastric, and squamous cell lung cancers (NCT04149691)  . Further7 μg/ml)  synthesir et al.  synthesii et al.  applied 4149691) .Compounds containing heterocyclic moieties, such as pyrrole, imidazole and benzimidazole have been reported to demonstrate excellent antitubercular properties . Benzimi320–321 appeared to be the most promising antitubercular agents among the series with minimum inhibitory concentration (MIC) value of 25 μg/ml against Mycobacterium tuberculosis H37Rv strain. Compound 322 also displayed good activity against same strain under aerobic conditions, as indicated by its IC90 and MIC values   . In a dictively) , compoun329–330) were synthesized and evaluated for in vitro antimycobacterial property against pathogenic M. tuberculosis H37Rv strain (ATCC 27294) using MABA method. Compounds 329–330 exhibited notable antitubercular activity against H37Rv strain and their activity was contributed by the incorporation of methylenedioxyphenyl moiety at 2- and 6-position of benzimidazole ring (331–333) using condensation of o-phenylenediamine with different aliphatic, aromatic, fatty acids, and amino acids, and depicted remarkable antitubercular property against M. tuberculosis H37Rv strain compared to reference drugs pyrazinamide, streptomycin and ciprofloxacin. In addition, Yeong et al.  by conventional and microwave-assisted methods and evaluated for in vitro antitubercular activity against M. tuberculosis H37Rv strain. The derivatives 337–339 showed prominent antitubercular activity with MIC values in the range of 3.125–6.25 μg/ml. Compound 338 was the most potent among all (MIC = 3.125 μg/ml) which was likely due to the presence of nitro group at ortho position of phenyl ring. Compound 340 displayed notable activity (MIC = 0.05 μg/ml) and emerged as a promising antitubercular agent among the reported series   . Gobis e6 mg/ml)  have pre.6 μg/ml .The exploration of benzimidazole nucleus to discover new structural features required for the optimization of novel antiprotozoal agents is of utmost importance. Benzimidazole derivatives with antileishmanial, antimalarial, and antiprotozoal activities against different species are shown in 343–344 against axenic amastigotes of Leishmania donovani. Tonelli and co-workers  antileishmanial activity against the amastigote of L. mexicana and L. braziliensis with IC50 values of 2.62 and 3.21 µM, respectively, and their activity was 5.8 and 4.8 times better than standard miltefosine (IC50 = 15.34 µM)   . Upon evectively . Recentl ± 0 μM) .Plasmodium species responsible for human malaria, P. falciparum has already started to show resistance to available antimalarial drugs, thus causing the urgency to develop drugs with novel drug targets and new mechanism of action  with ED90 value of 28.6 mg/kg. The attachment of the neutral hydrophobic group at position 6 of the benzimidazole moiety enhanced this excellent anti-malarial property (a]benzimidazole (PBI) antimalarial agents having in vitro anti-plasmodial activity with IC50 values of 0.02–0.95 μM against Pf NF54 strain, and 0.02–1.07 μM against multidrug-resistant Pf K1 strain of P. falciparum. Among which, compounds 354–355 exhibited most prominent in vivo efficacy in mouse P. berghei model due to the presence of chlorine group at C-7, C-8 and C-9 position of benzimidazole moiety. In another study, compounds 356–358 displayed excellent activity with IC50 values of 0.69, 1.60 and 1.61 μM, respectively against chloroquine-sensitive 3D7 strain compared to standard chloroquine (IC50 = 1.53 µM)  . Recentllciparum .T. vaginalis, E. histolytica, G. lamblia, L. Mexicana and P. berghei. Compound 361 was found to be the most potent from the series, showing 3- and 9- times more activity than standards metronidazole and pentamidine, respectively. Alp et al.  and 4-phenyl groups (363) at the C-2’ position of amidinobisbenzimidazole moiety contributed for the antiparasitic activity. Among a library of 2-(trifluoromethyl)-1H-benzimidazoles, compounds 364–366 displayed the most prominent in vitro antiparasitic activity against E. histolytica, G. intestinalis, T. vaginalis and T. spiralis . Hernández-Núñez et al. (H-benzimidazol-1-yl)-N-arylacetamide analogues and illustrated 7-fold more potency of compound 370 than the standard benznidazole against G. intestinalis with an IC50 of 3.95 µM, and 4-fold more activity of compounds 370–371 against T. vaginalis in comparison with benznidazole. Flores-Carrillo and co-workers  values of 0.47 ± 0.02, 3.67 ± 0.30, and 0.71 ± 0.22, respectively compared to standard nifurtimox (IC50 = 2.0 ± 0.2). The presence of diethylaminoethyl group substantially augmented the antitrypanosomal property of the compounds (376–378). Unsubstituted or methyl substituted aromatic rings or inclusion of an imidazole ring at C-5 also potentiated the activity  and investigated its pharmacological profile in rat and dog models. Oral administration of 0.1–1 mg/kg azilsartan medoxomil in spontaneously hypertensive rats (SHRs) and renal hypertensive dogs demonstrated better effect in reduction of blood pressure at all doses compared to standard drug olmesartan medoxomil. Abou-Seri et al. (H-benzimidazol-2-yl)-3-pyridinecarbonitrile derivatives. All compounds in the series displayed significant vasodilation properties. Compounds 382–385 showed most prominent activity  compared to standard prazosin hydrochloride (IC50 = 0.487 mM).Sharma et al.  synthesio et al.  reportedi et al.  synthesiex vivo vasorelaxant property in rat aorta rings pre-contracted with phenylephrine. Compounds 386–390 exhibited prominent vasorelaxant activity with EC50 value less than 30 μM. Among a new set of 5-nitro benzimidazole derivatives, compound 391 emerged as the most active agent against AT1 with IC50 value of 1.03 ± 0.26 nM (391) helped the derivative to interact and bind tightly with lipophilic pocket of the receptor. Several benzimidazole derivatives containing indazole moiety were synthesized by Lamotte et al. .Datani et al.  synthesi 0.26 nM . The pree et al.  among wh393–394 were reported to possess comparable activity to positive control losartan  antagonists by high-throughput screening, compound 396 showed similar efficacy as standard drug spironolactone at a dose of 100 mg/kg (p.o.) in rat natriuresis model (Two series of nitric oxide (NO) releasing benzimidazole derivatives were synthesized by coupling benzimidazole biphenyl skeleton with nitro ester and furoxan NO-donor moieties, where compounds losartan . Hao et losartan  designedis model .397 exhibited long-lasting efficiency in decreasing blood pressure, with a maximal lowered response of 35.82 ± 6.20 mmHg at 5 mg/kg and 55.98 ± 4.74 mmHg at 10 mg/kg. The compound also showed potent affinity towards AT1 receptor compared to standard telmisartan with IC50 value of 1.13 ± 1.68 nM. In a separate study, Khan et al. : 0.8 ± 0.1 and 2.3 ± 0.7, respectively) than the both standard losartan and telmisartan  model. A new series of 4-thiazolidinones and 1,3,4-oxadiazoles bearing 2-mercapto benzimidazole moiety were prepared by Shingalapur et al. (403–406 produced notable results in OGTT model. All the potent derivatives contained hydroxyl group which might have contributed for their antidiabetic property. Some 2-benzoyl benzimidazole derivatives were assessed for antidiabetic and lipid-lowering effects (407 appeared to be an effective peroxisome proliferator-activated receptor (PPARγ) partial agonist. Shaikh et al. (408–413) via Passerini reaction and demonstrated their antidiabetic potential using rat OGTT model compared to the standard drug glibenclamide.Several 2-(pyridine-2-yl)-1a et al.  among whr et al.  among wh effects . Compounh et al.  reported414 displayed potent in vivo antidiabetic potential in a 4-week study with diet-induced obesity (DIO) mouse model.Diacylglycerol O-acyltransferase-1 (DGAT-1) is an enzyme which catalyzes the formation of triglycerides from diacylglycerol and Acyl-CoA and thereby plays an important role in intestinal fat absorption. DGAT-1 has emerged as an important therapeutic target for the management of different metabolic disorders, such as obesity and diabetes . Kwak et3). Compounds 415 and 416, upon evaluation of yeast and rat intestinal α-glucosidases inhibitory effect produced most notable results. The IC50 values for compound 416 against yeast and rat intestinal α-glucosidases were reported as 9.1 and 36.7 μM, respectively and thus it appeared to be the most potent benzimidazole among the series. Özil and co-workers .The α-glucosidase inhibitors are an important class of antidiabetic agents because of their property to reduce the postprandial glucose level in type-2 diabetic patients . Mobinik-workers  prepared421 exhibited significant in vitro antidiabetic property  and appeared as a promising antidiabetic agent from the series.Some novel 1,3,4-thiadiazole substituted 2-methyl benzimidazole derivatives were synthesized by conventional methods among which compound property . A serieproperty  and evalh et al.  synthesi(425–430) and reported excellent antidiabetic activity by applying α-glucosidase inhibitory method with a range of IC50 = 0.66 ± 0.05 to 3.79 ± 0.46 μg/ml compared to standard IC50 value of acarbose (1460.28 ± 244.365). Besides, two potent AMP-activated protein kinase (AMPK) activators (431–432) with multi-target antidiabetic property were reported against LPS-activated murine peritoneal macrophages (433) and 1,3-thiazolobenzimidazolone derivative (434), exerted dual effects against dipeptidyl peptidase-IV (DPP-IV) and xanthine oxidase (XO) enzymes with IC50 values less than 200 μM (d]isothiazol-2(3H)-yl) acetate, and reported that compounds 435–436 exhibited potential antidiabetic property by inhibiting Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) 1 and -3.Recently, Bharadwaj et al.  have devrophages . In anotn 200 μM . Finallyn 200 μM  preparedDifferent substituted benzimidazole derivatives have been explored for several years for their anticoagulant activity and potential use in clinical practice. Benzimidazole derivatives acting as anticoagulants are shown in N-alpha-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide), a benzamidine-based powerful inhibitor of thrombin, trypsin and other serine proteases. With the addition of ethyl ester and hexyloxycarbonyl carbamide hydrophobic side chains, improved pharmacokinetic profile was obtained leading to the invention of orally absorbed prodrug, 437 (Dabigatran etexilate). The prodrug 437 reached clinical trials and its active form 438 (Dabigatran) was discovered with excellent thrombin inhibitory potency and tolerability.Thrombin (fIIa) is a multifunctional serine protease responsible for the proteolytic cleavage of fibrinogen. Inhibition of thrombin is a crucial mechanism for inhibition of coagulation. Benzimidazole moiety serves as a suitable template for placing a wide range of substituents required for interaction with thrombin . Hauel e439 with IC50 value of 3.11 ± 0.21 nM appeared to be a potent thrombin inhibitor exhibiting better activity than the standard argatroban (IC50 9.88 ± 2.26 nM). A series of 1,2,5-trisubstituted benzimidazole fluorinated derivatives (440–442) were also evaluated for in vitro inhibitory activity against thrombin , thus showing their potential as thrombin inhibitors. A library of benzimidazole derived 1,3,4-oxadiazole derivatives (443–445) were assessed for ex vivo anticoagulant activity by determining the effect of compounds in increasing prothrombin time (PT) and activated partial thromboplastin time (aPTT)  compared to standard drug acenocoumarol (48 ± 0.5 s). The compounds, however, caused a slight increase in aPTT in comparison with the reference drug, unfractionated heparin (500 IU/kg).Recently, Ren et al.  designedthrombin . Compoune (aPTT) . Compoun446 was found to be the most active compound from the series, indicated by fIXa binding affinity (Ki) value of 0.016 µM.Factor IXa (fIXa), an important coagulation factor, is a useful target for developing potent and selective antithrombotic agents. A research involving pharmacophore modelling of a new series of benzimidazole analogues presented the chemical features necessary for designing fIXa inhibitors and showed that benzimidazole derivatives have the potential to be developed into effective antithrombotic agents . Compoun(447–456) with high docking score. The study uncovered that all the compounds showed more than 50% in vitro thrombin inhibitory property at 1 mg/ml concentration, where the IC50 values of compounds 447, 450 and 456 were 1.92, 2.17 and 1.54 nM, respectively, comparable to the IC50 value of positive control dabigatran (1.20 nM). The derivatives 425 and 428, previously reported in this review for antidiabetic property, exerted anticoagulant activity by augmenting the clotting duration. However, only compound 425 exhibited excellent inhibition (93.4%) of epinephrine-induced platelet aggregation  model. The synthesized compounds displayed anticonvulsant property at a dose of 20 mg/kg (i.p.) compared to standard phenytoin and compounds t of all . A serieactivity . CompounH-benzo[d]imidazol-2-yl)methyl}-3-arylthioureas which have been mentioned earlier for their analgesic potential. The research group in a previous study reported about the anticonvulsant and cytotoxic effects of the same series of compounds. Compounds 463–465 were found to possess potent anticonvulsant property in comparison with standard drug phenytoin (466 displayed the most promising result in inhibiting convulsion induced in mice by both methods (N-phenyl-1H-benzo[d]imidazole-1-carbothioamides (467–471) from the reaction of 2-mercaptobenzimidazole with hydrazine hydrate, substituted acetophenones and phenylisothiocyanate. Compounds 467–471 were found to be active compounds in MES and scPTZ models, and devoid of neurotoxicity -1henytoin . A serie methods . Bhrigu toxicity .472 at a dose of 50 mg/kg (i.p.) appeared to be the most potent among the derivatives (H-benzimidazol-1-yl]-N0-[(Z)-phenylmethylidene] acetohydrazide (473–474) were evaluated employing MES and scPTZ methods. Compounds 473–474 were found to be effective anticonvulsant agents  compared to control which was attributed to its good binding property with the GABAA receptor (476–480 exhibited notable anticonvulsant potency (around 70–80%) in comparison to standard drug phenytoin.A series of benzimidazole substituted semicarbazones were synthesized and tested for anticonvulsant activity using MES model. Compound ivatives . In anott agents . A new sreceptor . Recentlreceptor  synthesiNeurological disorders comprise the disorders of central and peripheral nervous system. Alzheimer disease (AD), dementia, stroke, Parkinson’s disease, multiple sclerosis, brain tumor etc. are the most common diseases affecting a large number of people. AD is a chronic neurodegenerative disorder and the most common form of dementia manifested by loss of memory, language, cognitive functions, behavior and emotion. The factors which mostly contribute in the development of AD include the levels of acetylcholine (ACh) and deposits of neurotoxic amyloid-β-peptide (Aβ) . A numbe481 (IC50 = 1.5 ± 0.043 µM) and 482 (IC50 = 2.4 ± 0.049 µM) were found to possess better activity than the standard EDTA (IC50 = 274 ± 0.007 µM). The presence of 2,3-dihydroxyphenyl (481) and 4-hydroxyphenyl (482) moiety might have contributed for their notable property (483–484) as potent and selective inhibitors of Phosphodiesterase 10A (PDE10A). Compound 484 showed 55% receptor occupancy (RO) of PDE10A at 30 mg/kg p.o. in rat brain. Further research led to the identification of compound 484, with improvements in in vivo efficacy (57% RO in rats at 10 mg/kg p.o.), rat clearance and oral bioavailability  profile resulting in notable in vivo efficacy as NPY Y5 receptor antagonist.Tamura and co-workers synthesized a series of benzimidazole derivatives for neuropeptide Y (NPY) receptor antagonistic activity which is an important pharmacological target for the treatment of different neurodegenerative disorders. Compound ng agent . In anotng agent , compounH-benzimidazole derivatives as inhibitors of c-Jun N-terminal kinases (JNK3), a group of degenerative signal transducers and potential target of neurodegenerative diseases, e.g. Alzheimer’s and Parkinson’s diseases. Majority of the compounds exhibited high affinity (Kd = 10 µM–46 nM) to JNK3 when investigated through SPR, JNK3 kinase assay and cell-viability of human neuroblastoma cells. The most potent compound 488 showed notable cell protective effect (IC50 = 1.09 µM) against toxicity induced by anisomycin  is a mitochondrial metalloprotease capable of degrading amyloid-β peptide and increasing its proteolysis in human neuronal cells. Identification of potential agonists of hPreP is of great importance for Alzheimer’s drug design. Compounds  disease .491 (IC50 = 6.4 µM) was found to be around 20 times more potent than DFB (IC50 = 136 µM) (Among the therapeutic agents used for the treatment of traumatic brain injury (TBI), positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGluR5) are important. He et al.  designed 136 µM) .492–494) under microwave irradiation and assessed their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. Compounds 492–494 displayed more than 25-fold more selectivity towards BuChE than AChE ((495–496) showed selective inhibition on BChE and appeared to be the most potent BuChE inhibitors  (497 was found to be the most potent AChE inhibitor (IC50 = 0.93 ± 0.04 µM) with marked selectivity ratio (13.68)  enzyme at a concentration of 0.5 and 1 mM, respectively and exerted prominent IOP lowering effect (51.56%) compared to the standard fasudil  . Besides (13.68) . Similarntration . The nit fasudil .500–501 were reported as promising AChE and BuChE inhibitors , highly neuroprotective against hydrogen peroxide mediated toxicity, metal chelators, and free radical scavengers in the treatment of Alzheimer’s disease (502–503 demonstrated duel effects as anti-Alzheimer agents by beta amyloid cleavage enzyme-1 (BACE1) inhibition and neuroprotection  and 65.7% hBACE1 inhibition. Besides, the compound (505) increased glutathione (GSH) level, reduced ROS production, and subsequently opened a door for further development to be established as a promising treatment option against Alzheimer’s disease  and twofold more activity than another standard acetohydroxamic acid (IC50 = 42.00 ± 1.26 µM). Several 1-alkylbenzimidazoles and 1,3-dialkyl benzimidazolium salts were evaluated for their tyrosinase inhibitory activity among which compound 508 emerged as the most potent derivative (IC50 = 0.31 mM)  .509 appeared to be the most potent with a binding affinity value (KiCB1) of 0.53 nM . More recently, they synthesized a new series of benzimidazole derivatives which displayed more selectivity towards human Cannabinoid 2 (CB2) receptor than that towards CB1 receptor (511 demonstrated the best binding affinity (Ki = 0.08 µM), high selectivity index (KiCB1/KiCB2 >125) and low toxicity.A series of 2-pyridylbenzimidazole derivatives were designed and synthesized as Cannabinoid 1 (CB1) receptor agonists amongst which compound  0.53 nM . The sam 0.53 nM . Compounreceptor . Compoun512 displayed prominent MCH receptor 1(MCH-R1) binding affinity (IC50 = 1 nM), as well as low human ether-a-go-go-related gene (hERG) binding affinity thereby ensuring low risks of cardiovascular diseases, metabolic stability and preferable pharmacokinetic profile , an attractive target for developing anti-obesity agents. Compound  profile . Igawa et MCH-R1 .in vivo corticotropin releasing factor type 1 (CRF1) receptor antagonistic property. Compound 514 with an electron withdrawing cyano group at the 4-position of benzimidazole nucleus emerged as the most promising among the series. In continuation with their research to develop CRF1 receptor antagonists, compound 515 was reported as a potent CRF1 binding inhibitor (IC50 = 4.1 nM) and in vitro CRF1 antagonist (IC50 = 44 nM) (516 displayed very prominent CRF1 receptor binding activity (IC50 = 58 nM) and oral bioavailability (F = 68% in rat model) indicating its potential in developing clinically effective CRF1 receptor antagonist in the future (Mochizuki and co-workers  assessed= 44 nM) . More ree future .517 (Ki = 13.8 nM) exhibited highest affinity towards human GnRH receptor and emerged as the most prominent GnRH receptor antagonist ((518–521) as intraocular pressure (IOP) reducer in the treatment of dexamethasone-induced ocular hypertension. Among these, compound 521 exerted the best anti-glaucoma action by the maximum IOP reduction of 22.32% from baseline following single drop administration (0.1%)  receptor antagonists. The derivative tagonist . Furthern (0.1%) .522 at a dose of 2.34 mg/kg showed prominent anxiolytic, antidepressant and anticonvulsant activities in both rat and mice models. Vasil’ev et al.  and collagen (1 µg/ml)-induced human platelet aggregation in an in vitro model. Its inhibitory effect might be attributed to the presence of 1-imidazolyl moiety at one end carrying a long chain of three sugar moieties. A series of benzimidazole derivatives bearing a sterically hindered phenolic group in their structures were evaluated for in vitro antiplatelet activity using the Adenosine diphosphate (ADP)-induced platelet aggregation model of rabbit’s plasma  in Balb-c mice. The compounds displayed PPARγ agonist activity, indicating their potential in the management of morphine-induced paradoxical pain. Akhtar et al. .Moreover, Raka et al.  reportedHeterocyclic compounds possess versatile applications, such as antibacterial, antiviral, antitubercular, anticancer, anti-inflammatory, analgesics, herbicidal, fungicidal, insecticidal, antidiabetic, antihypertensive, and so on . Some ofAmong the heterocyclic compounds, benzimidazole derivatives have been playing the most pulsing and eye-catching pioneer role in synthetic pharmaceutical and agrochemical sectors for the last couple of decades . As havi1) proton pump inhibitors (PPIs) exemplified by omeprazole, esomeprazole, lansoprazole, pantoprazole, etc., 2) anthelmintic drugs demonstrated by mebendazole, albendazole, oxibendazole, etc., 3) non-sedative H1-receptor blockers or antihistamine presented by astemizole, norastemizole, emedastine, mizolastine, etc., 4) angiotensin II receptor blocker illustrated by telmisartan, candesartan, azilsartan, and 5) calcium sensitized cardiac agents instanced by isomazole  have become resistant due to long-term exposure against several cancers, such as hepatocellular carcinoma . It is aIt is crystal clear that an exciting number of benzimidazole pharmacophore derivatives have become potential drug candidates to treat several disease conditions in ongoing clinical trials. However, this promising moiety is copious to be further investigated for designing a number of different target molecules, which might reveal some encouraging outcomes in medicinal research. It can easily be assumed at the current stage that several new strategies might be designed and developed for multi-target agents with the highest affinity, specificity, and enhanced bioactivities. In the future, the benzimidazole scaffolds might be fused with another heterocyclic ring system to be explored several novel chemically stable and potent pharmacological agents against several rare or critical diseases or with better pharmacokinetic and pharmacodynamic profiles. This might be an outrageous and worldwide revolutionary achievement in the next century. Toxicity, drug resistance, and poor bioavailability are the significant concerns during drug development or the underlying reasons behind the compounds be untouched in the human trial. Structural modifications of the existing synthesized molecules might be an excellent technique to be brought about improved or novel activities with a better safety profile. Besides, the prodrug strategy can also be applied to ameliorate the bioavailability of the present derivatives. Furthermore, combinations of the benzimidazole derivatives with other existing drugs might be explored for finding synergistic effects or exciting pharmacological activities against multidrug-resistant microorganisms.Moreover, several benzimidazole-containing clinically used medications, e.g., omeprazole, lansoprazole, pantoprazole, and rabeprazole, are actually in the form of prodrugs for which the onset of action is slower. Thus, there is an increased demand for producing the active form of drugs directly from benzimidazoles, and for this, researchers are paying attention to novel benzimidazole-derived therapeutics with lesser toxicity and better pharmacodynamic profile. In this case, PPIs containing benzimidazole ring might be modified, or the benzimidazole pharmacophore might be replaced by imidazole ring with the improved biological profile. The non-approved benzimidazole scaffolds for cancer therapy having eminent pharmacokinetics and safety profile might be repurposed to treat several life-threatening cancers with a lack of safe and effective treatment. Some anthelmintic drugs containing benzimidazole nuclei, such as flubendazole, albendazole, and mebendazole, could be considered to apply against various types of cancer in future investigations. Besides, stereochemistry could be regarded as to trace and explore more potent isomers of the existing approved benzimidazole derivatives because one enantiomer may exert distinct biochemical property than the other enantiomer.Benzimidazole, an essential nitrogen-containing heterocyclic moiety, can be found in various therapeutically used compounds playing a vital role in treating many diseases. Many efforts have been given so far on the development of target-based benzimidazole derivatives, and the interest to produce new therapeutically active agents to treat different diseased conditions has grown noticeably during the last few years. However, this field has several challenges, especially to bring the numerous synthesized compounds into the clinical trial, which showed valuable pharmacological properties in different studies, and later to ensure their availability in the market and clinical practice. The present review has focused on the current status of benzimidazole moiety, emphasizing the SAR of different benzimidazole-based compounds explored by scientists around the world. So far this is the most inclusive and informative account about biological and therapeutic potential of benzimidazole derivatives. With a compilation of information from more than 250 latest pieces of literature, we aimed to aid the researchers, medicinal chemists, and drug designers with valuable and comprehensive knowledge and provide them the rationale to develop target-oriented and clinically useful benzimidazole-based molecules.