IntroductionThe purpose of this study was to evaluate robotic-assisted cortical bone trajectory (CBT) screw placement. Early, middle, and late phases of robotic-assisted CBT screw placement were compared for accuracy and screw insertion time by comparing time and accuracy in every phase. MethodsA retrospective review was conducted on the initial 40 patients who underwent spinal fusion using CBT screws in one institution from September 2021 to September 2022 utilizing a spine surgery robot system . The inclusion criterion was one- or two-level posterior lumbar interbody fusion (PLIF). Exclusion criteria were 1) patients who underwent posterior-lateral fusion in other segments, 2) patients who underwent additional decompression in other segments, 3) patients who underwent reoperation, and 4) patients with spondylolysis. The deviation of the CBT screw was evaluated on computed tomography (CT) one week after surgery using the Gertzbein-Robbins grade system. The rate of Grade A was considered the perfect accuracy rate, and the rate of penetration of 2 mm or more  was calculated as the deviation rate. To assess the learning curve, patients were divided into three groups. The first 10 cases were in the early phase group, the subsequent 10 cases were in the middle phase group, and the last 10 cases were in the late phase group. We compared the perfect accuracy rate, deviation rate, operative time, operative time per segment, intraoperative blood loss, registration time, and screw insertion time among the three groups.ResultsThirty patients met the criteria. Overall, the perfect accuracy (Grade A) rate of the screw was 95.3% and the deviation rate was 1.4%. The perfect accuracy rate was 90.4% in the early phase, 95.5% in the middle phase, and 100% in the late phase. The deviation rate was 3.8% in the early phase, 0% in the middle phase, and 0% in the late phase, and there was no statistically significant difference between the three groups. Among the three groups, the operative time, the operative time per segment, the intraoperative blood loss, and the registration time were not significantly different. There was no significant difference in the screw insertion time among the three groups, but it decreased with experience . The screw insertion time of the late phase tended to be shorter than that of the early phase (p=0.052).ConclusionsThe deviation rate of robotic-assisted CBT screw placement with one- or two-level PLIF was 1.4%, which was highly accurate. The deviation rate was 3.8% in the early phase, 0% in the middle phase, and 0% in the late phase. Although the deviation rate was low even in the early period, the screw insertion time in the early 10 cases tended to be longer than that in the late 10 cases. After passing the experience of 10 cases, this study concluded that robotic-assisted CBT screw placement was proficient. Cortical bone trajectory (CBT) is a novel technique for placing pedicle screws (PSs). The CBT screw passes through the medial to lateral and caudal to cranial passages through the pedicle . AdvantaThe CBT screw is inserted toward the superior endplate with 25° to 30° cranial trajectory and 10° lateral trajectory. -7 and thThe purpose of this study was to clarify the accuracy and screw insertion time of robotic-assisted CBT screw placement. Early, middle, and late phases of robotic-assisted CBT screw placement were compared for accuracy and screw insertion time to better understand the learning curve following the introduction of robotics.SubjectsThis study was approved by the institutional review board of St. Marianna University School of Medicine. A retrospective review was conducted on the initial 40 patients who underwent spinal fusion using CBT screws in one institution  from September 2021 to September 2022 utilizing a spine surgery robot system . The inclusion criterion was one- or two-level posterior lumbar interbody fusion (PLIF). Exclusion criteria were 1) patients who underwent posterior-lateral fusion in other segments, 2) patients who underwent additional decompression in other segments, 3) patients who underwent reoperation, and 4) patients with spondylolysis. We indicated PLIF for lumbar spinal stenosis with instability. The spondylolysis was not indicated because of a separation of pars at the insertion point of the CBT screw.Surgical workflowPreoperative computed tomography (CT) images were used to plan screw placements using a dedicated application. Surgery was performed in the prone position, and a fusion area was approached through a midline skin incision. The robot arm unit was attached to the bone mount with a Schanz screw  on the posterior iliac crest, and a robot reference frame was employed. Preoperative CT image data and intraoperative C-arm images were matched by CT-to-fluoro registration. Drilling, tapping, and screw placement were performed without Kirschner wires under the robot arm guidance Figure . Bone, lEvaluationThe deviation of the CBT screw was evaluated on CT one week after surgery using the Gertzbein-Robbins grade system: Grade A: no breach of the cortical layer of the pedicle; Grade B: breaches less than 2 mm; Grade C: breaches of 2 mm or more but less than 4 mm; Grade D: breaches of 4 mm or more but less than 6 mm; Grade E: breaches of 6 mm or more . The preoperative diagnoses were lumbar spinal stenosis in 24 patients and lumbar disc herniation in 6 patients. One-level PLIF was performed in 17 patients, and 13 patients underwent two-level PLIF. The mean operative time was 198.7 ± 46.9 min (one-level: 184.1 ± 49.7 min and two-level: 217.9 ± 36.5 min). The intraoperative blood loss was 138.8 ± 120.3 ml (one-level: 93.5 ± 70.2 ml and two-level: 198.0 ± 147.3 ml). The mean screw insertion time was 134.4 ± 49.1 sec. There were no patients who had a pedicle crack during the screw insertion or who required re-insertion of screws after the first placement. The screw insertion time declined as the number of surgical cases increased  and the operative time per segment  were not significantly different  among the three groups  among the three groups. There was no significant difference in the screw insertion time among the three groups, but it decreased with experience   was set at 0.8, and the effect size was set at 0.4. The total sample size required for the analysis of variance in the three groups was 66. We did not perform an assessment of bone mineral density. We could not compare with conventional CBT screw placement with C-arm images because we have no data of conventional CBT screw placement including the insertion time or fluoroscopic time. The screw insertion was performed by seven surgeons. There may have been differences in screw insertion time and accuracy among surgeons. In the future, we plan to look into how different surgeons' techniques affect how quickly and accurately screws are inserted. We did not evaluate the screw length to confirm whether an ideal CBT trajectory was obtained. The screw length is associated with biomechanics stability and may be relevant to the clinical outcome. We did not evaluate the relationship between screw accuracy and clinical outcome. In the future, we plan to investigate the clinical outcome of PLIF with robotic-assisted CBT screw placement. The deviation rate of robotic-assisted CBT screw placement with one- or two-level PLIF was 1.4%, which was highly accurate. The deviation rate was 3.8% in the early phase, 0% in the middle phase, and 0% in the late phase. Although the deviation rate was low even in the early period, the screw insertion time in the early 10 cases tended to be longer than that in the late 10 cases. After passing the experience of 10 cases, this study concluded that robotic-assisted CBT screw placement was proficient.


A) receptor agonist muscimol (MU). Inhibition of the network bursting activity in response to RF exposure became apparent at an SAR level of 28.6 W/kg and co-occurred with an elevation of the culture medium temperature of ~1°C. Exposure to RF fields preferentially inhibits bursting over spiking activity and exerts fewer constraints on neural network bursting synchrony, differentiating it from a pharmacological inhibition with MU. Network rebound excitation, a phenomenon relying on the intrinsic properties of cortical neurons, was observed following the removal of tonic hyperpolarization after washout of MU but not in response to cessation of RF exposure. This implies that hyperpolarization is not the main driving force mediating the inhibitory effects of RF fields. At the level of single neurons, network inhibition induced by MU and RF fields occurred with reduced action potential (AP) half-width. As changes in AP waveform strongly influence efficacy of synaptic transmission, the narrowing effect on AP seen under RF exposure might contribute to reducing network bursting activity. By pointing only to a partial overlap between the inhibitory hallmarks of these two forms of inhibition, our data suggest that the inhibitory mechanisms of the action of RF fields differ from the ones mediated by the activation of GABAA receptors.Previous studies have shown that spontaneously active cultured networks of cortical neuron grown planar microelectrode arrays are sensitive to radiofrequency (RF) fields and exhibit an inhibitory response more pronounced as the exposure time and power increase. To better understand the mechanism behind the observed effects, we aimed at identifying similarities and differences between the inhibitory effect of RF fields  to the γ-aminobutyric acid type A (GABA Radiofrequencies are electromagnetic waves ranging from 300 kHz to 300 GHz widely used in modern telecommunication technology. The rapid and continuous increase of environmental man-made RF electromagnetic fields (EMF) has raised concerns about their potential risks on human health. In particular, a large body of research has investigated the possible effects of exposure to RF fields used by mobile phones (300–3000 MHz) on the human central nervous system (CNS) (for reviews see –3). AlthEarly research on various neural preparations reported electrophysiological change in response to RF fields –26. SincA receptor agonist, muscimol (MU). The GABAA receptor is the major inhibitory neurotransmitter receptor responsible for fast inhibition in the mammalian brain [In the present study, we have aimed to contribute to the understanding of the mechanisms of action behind the inhibitory effects of RF fields on cultured cortical neural networks by performing a direct comparison with the inhibitory effects of the GABAan brain , 41. Sigan brain  wherein Primary cultures of neocortical neurons were prepared from embryos of gestating Sprague-Dawley rats . Experiments involved six gestating rats. All procedures were carried out in compliance with the European Community Council Directive for the Care and Use of laboratory animals (2010/63/EU) and protocols were approved by the Bordeaux Ethics Committee for Animal Experimentation (CEEA—050).5 cells. Cells were left to sediment and adhere on the MEA chip for up to 2 h and the MEA chambers were then filled with 1 mL of NBM. MEAs were kept in individual petri dishes at 37°C in a humidified incubator with 5% CO2 until mature neural network development. Culture mediums were half-exchanged every 48 h until taking recordings.Preparation of primary neural cultures was carried out using the methods described in , 28. In In the present study, a modified version  of a 60-2. Preamplification gain was 1200 and signals were acquired and digitized at 10 kHz/channel with an MCS-dedicated data acquisition board . Signals were recorded and visualized with the MC Rack (MCS GmbH) software. After 30 min of baseline recording, neural cultures were exposed for 15 min either to a sham treatment (SH), a pure continuous carrier radiofrequency (RF) at 1800 MHz, or to the GABAA receptor agonist muscimol (MU), . After treatment, post-treatment activity was continuously monitored for 45 min. Data from cultures aged between 17 and 27 days in vitro (DIV) were included in the present study .The experimental setup for simultaneous electrophysiological recordings and exposure to RF fields or pharmacological agents comprised an MEA coupled to an open transverse electromagnetic cell (TEM) , 42, 43 R) of a metric (M) during the exposure phase (MExposure) relative to the baseline reference phase (MBaseline).Processing and analysis of multi-channel data were performed with the software package SPYCODE  developeNorm. RMSE) as follow:The level of synchronicity for descriptors of bursting activity across MEA channels was evaluated with the coefficient of variation (CV) defined as the ratio (expressed in %) of the average channel standard deviation to the metric mean value . The lower the CV, the higher synchronization across MEA channels , 48. Int‘Y’ is the averaged normalized value of MBR or MSR over all MEA channels (K) and ‘y’ is the averaged normalized value of MBR or MSR at the level of the individual channel (k). For example, a Norm. RMSE value equal to 0.5 indicates that the mean inter-channel variation to the mean is of 50%. Computation methods for the metrics described above are reported in Where APIB) were roughly twice as numerous (~1.9) as sorted APs outside bursts (APOB) and that this proportion decreased to ~1.3 and ~1.1 respectively for the AUX and MIN AP clusters  software over a period of 30 min including 15 min of baseline (pre-exposure phase immediately prior to treatment) and 15 min when neural cultures are continuously exposed to the treatment. To ensure reliable sorting between the two recording phases, pre-exposure and exposure phases were merged into a single file with the MC_dataTool (MCS GmbH) software. Detection threshold was set at five times the standard deviation of the channel noise level and waveform sample-wide containing single event was set at 4 ms . Note that this method of detection differs from the one used in SPYCODE. AP sorting was performed using the T-Dist E-M method  and analyses were executed in batch mode. This method enabled detecting on average 67, 991 ± 10,655 (Mean ± SEM) APs per MEA and to sort on average 40,135 ± 6,114 APs per MEA . On averclusters . As ~89%p-value < 0.05 was considered statistically significant. Effect size , when reported, was calculated with the “rcompanion” [Statistical analysis was performed using the R software  and the mpanion”  R packagmpanion”  and ‘ggpmpanion”  R packagmpanion”  was usedmpanion” . PairwisA receptor agonist MU under similar levels of inhibition, the relation between MBR and MU concentration was first evaluated (50-MBR) was estimated to be ~0.25 μM, a value in agreement with other studies on basic receptor and neural culture pharmacology [A photograph of the setup illustrating the different parts is shown in valuated . MU exermacology , 56–58.The inhibitory effects of RF fields and MU were then compared with respect to an SH group after data normalization see . Definitp < 0.001; MU: ~57% reduction, SH/MU, p < 0.001) and MSR . Inhibitory effects of MU on bursting and spiking activities were on average stronger than for RF exposure .Exposure to RF fields (SAR of 28.6 W/kg) or MU (0.25 μM) both reduced MBR ; SH-MSR = 0.37 (0.30); p = 0.0327). RF- and MU-induced inhibition were both associated with a comparable level of spatial variation of bursting activity across the MEA channels (RF = 0.20 (0.22); MU = 0.28 (0.12); p = 0.3059). The degree of spatial variability in MBR was not different from the intrinsic spatial variability observed in response to SH exposure (p = 0.3024). In the same way as for the data for MBR, the data for MSR indicated that RF- and MU-induced inhibition caused spiking activity to vary equivalently in space (RF = 0.54 (0.16); MU = 0.65 (0.21); p = 0.1848) but spatial fluctuations of MSR were higher than for the intrinsic variation observed with SH exposure ; although as in SH exposure, spatial variations of MBR were lower than for MSR (p < 0.001). Collectively these data indicate that RF-induced inhibition occurred within the MEA space as diffusely as the pharmacological inhibition induced by MU.Inhibition of neural network activity was evaluated in the spatial domain by quantifying the inter-channel variability of MBR and MSR variations across all channels of the MEA layout by computing the normalized root mean square error . MU, but not RF exposure, increased IBSR  and spiking rate (SR) over time . In respSimilarities and differences in the temporal domain between the two treatments are once again exemplified in The inhibitory effects of RF fields and MU were next analyzed and compared at the level of single-unit activity by evaluating changes in AP waveforms . Definitp = 0.3511—p = 0.2859—p < 0.001; SH/MU, p = 0.0018; RF/MU, p = 0.6065—p < 0.001; SH/MU, p = 0.0038; RF/MU, p = 0.3547—p = 0.0374; SH/MU, p = 0.0224; RF/MU, p = 0.5659—rising = 0.475, ε²falling = 0.194; MU: ε²rising = 0.384, ε²falling = 0.180) suggesting that narrowing of the AP half-width in response to RF and MU exposure occurred primarily through a mechanism that increases the depolarization slope.The average effects on AP waveform in response to RF fields and MU are shown from two representative single units and their respective phase plots in In the present study, exposure to RF fields were performed at an SAR level of 28.6 W/kg, a value ~1.4 times lower than levels used in , 28. IndA receptor agonist MU. Our results showed that in contrast to MU, RF exposure preferentially inhibits bursting over spiking activity. Although spiking activity was reduced by RF exposure, inhibition was more variable and weaker than for bursting activity. Other studies with cultured networks of cortical neurons also reported that MU equivalently inhibits spiking and bursting activity [A receptors from synaptically released GABA, whereas the second depends on sustained activation of peri- and extrasynaptic GABAA receptors by ambient GABA. In our experiments, continuous application of MU in the culture medium activates both synaptic and peri-extrasynaptic GABAA receptors, which ultimately leads to a tonic neural hyperpolarization. Neuronal excitability is in essence equivalently reduced throughout the network subcomponents and an equivalent reduction in activity patterns based on regular spiking, intrinsically bursting neurons as well on network collective bursting behavior is observed. As RF exposure differentially impacted spiking and bursting activity, one may argue that cell hyperpolarization is not the main force driving the inhibitory effects of RF on neural networks. Studies on the effect of RF exposure on the membrane potential of excitable cells (cardiomyocytes and neurons) has led to conflicting results, with some showing no effect [We have previously reported that exposure to RF fields decreases the bursting activity of cultured networks of cortical neurons  and thatactivity , 57. GABactivity . The firo effect , 63, 64,o effect , and somo effect . DetaileIh), subthreshold membrane oscillations and T-type calcium current, above which high frequency action potentials fire for a brief period [At the cellular level, cortical neurons can generate bursts based on intrinsic properties such as hyperpolarization-activated current  From left to right: Mean AP detection for unsorted and sorted AP fraction ; Relative fraction of sorted APs attributed to Major (MAJ), Auxiliary (AUX) and Minors (MIN) clusters; Mean AP count for sorted AP occurring either inside (AP IB) or outside (AP OB) bursts period. Data collected over 15 min during the pre-exposure phase from 15 cultures of the RF group used here as representative. (B) Example of sorted AP waveforms after principal component analysis and hierarchical classification, overlay of 125 waveforms per cluster with averaged waveform highlighted, data from one channel of the same culture. Scale: (y): 40 μV; (x): 500 μs. (C) Illustration of the metrics used to quantify changes in AP waveforms. FWHM: full width at half maximum. As recorded extracellularly the AP waveform is inverted. Scale: (y): 10 μV; (x): 500 μs.(TIF)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(1) Indicates significant difference between SH-RF and SH-MU pairs (p < 0.05) and *(2) indicates significant difference between MU-SH and MU-RF pairs (p < 0.05), ns indicates no significant differences between groups. Pairwise comparison done with Kruskal-Wallis test followed by Conover’s all-pairs posthoc test. SH, n = 12; RF, n = 15; MU, n = 8.Data expressed as Median (IQR). *(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Tablep < 0.05) between MU-SH and MU-RF pairs. Pairwise comparison done with Kruskal-Wallis test followed by Conover’s all-pairs posthoc test. SH, n = 12; RF, n = 15; MU, n = 8.Data expressed as Median (IQR). * Indicates significant difference ((TIF)Click here for additional data file.


Alcohol use disorder (AUD) is characterized by enhanced cue-reactivity and the opposing control processes being insufficient. The ability to inhibit reactions to alcohol-related cues, alcohol-specific inhibition, is thus crucial to AUD; and trainings strengthening this ability might increase treatment outcome. The present study investigated whether neurophysiological correlates of alcohol-specific inhibition (I) vary with craving, (II) predict drinking outcome in AUD and (III) are modulated by alcohol-specific inhibition training. A total of 45 recently abstinent patients with AUD and 25 controls participated in this study. All participants underwent functional magnetic resonance imaging (fMRI) during a Go-NoGo task with alcohol-related as well as neutral conditions. Patients with AUD additionally participated in a double-blind RCT, where they were randomized to either an alcohol-specific inhibition training or an active control condition (non-specific inhibition training). After the training, patients participated in a second fMRI measurement where the Go-NoGo task was repeated. Percentage of days abstinent was assessed as drinking outcome 3 months after discharge from residential treatment. Whole brain analyses indicated that in the right inferior frontal gyrus (rIFG), activation related to alcohol-specific inhibition varied with craving and predicted drinking outcome at 3-months follow-up. This neurophysiological correlate of alcohol-specific inhibition was however not modulated by the training version. Our results suggest that enhanced rIFG activation during alcohol-specific  inhibition (I) is needed to inhibit responses when craving is high and (II) fosters sustained abstinence in patients with AUD. As alcohol-specific rIFG activation was not affected by the training, future research might investigate whether potential training effects on neurophysiology are better detectable with other methodological approaches. Alcohol use disorder (AUD) is a leading cause for societal and individual burden of disease , 2 and tOn a behavioral level, patients with AUD displayed inhibitory deficits –10 when Dysregulations in the inhibitory control network in AUD and other addictive disorders have been repeatedly observed , 17, 18 Following the logic that response inhibition is especially crucial in the context of alcohol-related cues, which may trigger strong appetitive processes , 39–41, Taken together, neuroscientific and experimental research in AUD suggests that inhibition is crucial, and probably also particularly difficult, when it must be exerted in an alcohol-related context which provokes craving.via the improvement of alcohol-specific inhibitory capacities. Such a working mechanism would potentially also induce changes in the neurophysiological correlates of alcohol-specific inhibition.Following a translational approach, such research led to the development of an alcohol-specific inhibition training (Alc-IT), which was designed to improve patients' inhibitory control over their responses to alcohol-related stimuli , 46. AlcThe presented study thus aims to (I) replicate earlier reports indicating that neuronal activation related to alcohol-specific inhibition increases with craving; (II) test whether neuronal activation related to alcohol-specific inhibition is related to drinking outcomes and (III) investigate whether the neurophysiological signature of alcohol-specific inhibition can be altered through alcohol-specific inhibition training.In the context of a randomized-controlled, double-blind, clinical trial investigating the effects of alcohol-specific inhibition training on drinking outcomes , the pret−value ≤ 63] as well as non-problematic drinking behavior , right-s, AUDIT  < 8; Alcs, AUDIT  < 2] wers, AUDIT  < 8 per n et al. . Three pAt baseline, a trained study member verified AUD diagnosis with the Diagnostic Expert System for Psychiatric Disorders . A ques(3−monthfollowup) – PDA(Baseline)] was used in those fMRI analyses, which investigated the relation between the neurophysiology of alcohol-specific inhibition and drinking outcomes.Assessment of alcohol consumption was assessed at baseline (assessing drinking in the 90 days prior to detoxification entry) and 3-month follow-up (assessing drinking in the 90 days following treatment discharge) with the TLFB interview . From TLALC, 416 GoNEU, 64 NoGoALC, and 64 NoGoNEU trials, leading to a Go/NoGo ratio of 6.5. The trials were presented in a pseudorandomized order with a mean of 7.5 Go-trials  between two NoGo-trials. The task was subdivided into two blocks with a break of 1 min and 1 s. Photographs were displayed for 900 ms with a 100 ms inter-stimulus interval blank screen. The GNG-task was created and response data was logged with the E-Prime 2.0 software .The task used in this study to assess BOLD-responses of alcohol-specific and neutral inhibition was equal to the task in Batschelet et al. . BuildinFunctional and anatomical MRI data acquisition was conducted at the University Hospital of Bern, using a Siemens Magnetom Prisma scanner with 3 Tesla magnetic field strength and a head coil with 64 channels. For functional image acquisition during the above described GNG-task, a multi-band echo planar imaging (EPI) sequence was run . For subsequent image distortion correction, a b0 protocol was run to acquire 4 field map images (2 phase/amplitude each) with the same image geometry as in the EPI-sequence, with TR/TE1/TE2 = 591/4.92/7.38 ms. Anatomical images were obtained using an MP2RAGE sequence . Note that, while the combination of a fast event-related task design with BOLD-fMRI is not optimal, such a combination has—despite the drawbacks and a reduced signal-to-noise-ratio—yielded important insights into the neural basis of inhibition in prior studies , 63.3 FWHM.Task-fMRI images were preprocessed using the routines implemented in SPM12. Initially, the origins of all functional and anatomical images were reoriented to the anterior commissure. The field map images were used to construct the voxel displacement map for unwarping, which was applied after realignment that involves participant's motion correction. Subsequently, slice time correction was run as well as coregistration of the functional images to the anatomical image. A brain tissue segmentation was performed to obtain forward deformation fields for the image normalization procedure, which transformed the images to the MNI standard space. Finally, all functional images were smoothed using a 3D-Gaussian Kernel with 6 mmALC_correct, NoGoNEU_correct, GoALC_correct, GoNEU_correct, NoGoALC_error, NoGoNEU_error, GoALC_error, and GoNEU_error). The six individual motion parameters derived from realignment were entered as regressors of no interest to the design matrix. Finally, the GLM was estimated. For calculation of contrast images, only parameter estimates of regressors corresponding to event types with correct participant responses were included, since this study focused exclusively on successful inhibition. Thus, the following four contrasts were computed: (NoGoALC + NoGoNEU) > (GoALC + GoNEU), NoGoALC > GoALC, NoGoNEU > GoNEU, (NoGoALC > GoALC) > (NoGoNEU > GoNEU).In order to extract functional activation images for each GNG stimulus condition, contrast images at the subject level were generated . For this purpose, the preprocessed images were entered to a general linear model (GLM). In detail, a design matrix was constructed with the predefined GNG task events consisting of stimulus type , response type , and participant's response accuracy . We used the canonical hemodynamic response function (HRF) as a basis function that was convoluted with the onsets of the resulting eight event types . To this end, a whole-brain linear regression was performed with NoGoALC > GoALC) > (NoGoNEU > GoNEU), which isolates alcohol-specific inhibitory activation, was used for exploring whether the neurophysiological signature of alcohol-specific inhibition is predictive for drinking outcomes. More detailed, the individual planned contrast whole brain images (from the pre-training fMRI session) of N = 35 patients with AUD were used in a whole brain linear regression with the drinking outcome (indicated by PDA Δ) as the covariate of interest. This analysis thus used the fMRI data from the pre-training session to predict drinking outcome at 3-months follow-up. For this analysis, only gray matter voxels were used by applying a binary mask derived from the mean over all individuals' MP2RAGE gray matter segmented image. Brain regions yielded by this analysis as reflecting a neural correlate of alcohol-specific inhibition which is predictive of relapse, will be used as regions of interest (ROIs) in the third analysis (see below).Second, a planned contrast  and the three within-subject factors response type (NoGo/Go) × stimulus type (ALC/NEU) × pre-post (pre-training/post-training). A significant 4-way interaction was needed to confirm a training-effect on alcohol-specific inhibition in the potential ROI that is predictive for drinking outcome.Third, to investigate whether the thus functionally defined, relapse-predicting, alcohol-specific inhibitory activation can be altered through training, the MNI-coordinates of potential effects yielded by the second whole-brain analysis served as regions of interest (ROIs). From these ROIs, beta-values were extracted for each subject, for which a complete dataset was obtained. The analytic sample of this third analysis consisted of t-tests run separately for the AUD and HC groups with the aim of contributing to the research on general inhibition in AUD. As general inhibition was not the main focus of this study, these results can be found in the For completeness, a basic analysis used the (NoGoALC + NoGoNEU) > (GoALC + GoNEU) contrast (pre-training images only) in one-sample ALC > GoALC and OCDSimp, linear regression with NoGoNEU > GoNEU and OCDSimp, linear regression with planned contrast and PDA Δ, and one-sample t-tests with NoGo > Go), SPM12 routines were applied with a family-wise error (FWE) correction at critical p-value threshold of 0.05, with a cluster size of 0. The repeated-measures ANOVA involving beta-values of the ROI was computed with SPSS. Anatomical brain and activation illustrations were created using the MRIcroGL software (https://www.nitrc.org/projects/mricrogl/). Finally, statistics necessary to produce illustrations  was used to assess group differences of demographics and clinical scores. All variables except the BDI II score were non-normally distributed. Therefore, to test differences between the two AUD subgroups, non-parametric Kolmogorov-Smirnov tests were run. For differences over all three groups, non-parametric Kruskal-Wallis tests were computed. Behavioral GNG data  were non-normally distributed and therefore analyzed with ANOVA-type non-parametric statistics using the nparLD package in R . This antrations , 4 were p-values can be found in While the three study groups did not differ in age, the HC group showed a higher education level than the AUD group. As expected, the HC group scored lower in BSCL GSI, OCDSimp, and OCDSsum scores than the AUD group. All group means as well as group statistical (1) = 7.25, p = 0.007]. EOCs, the main indicator of inhibitory performance, did however not differ between patients with AUD and HCs. The RTs of EOC were significantly shorter in NEU than ALC , and shorter in HC compared to AUD , although the two-way interaction was not significant . Concerning the number of EOOs the AUD group performed worse than the HC group . Again, the interaction was not significant . RTs in correct Go trials were shorter in HC compared to AUD , with no significant stimulus type × group interaction .Analyzing the GNG behavioral data from the pre-training measurement, both AUD and HC made more EOCs with NEU trials than ALC trials as confirmed by a significant main effect of stimulus type . The two-way  and three-way interaction (stimulus type × pre/post-training × training type) were also significant . Follow-up analyses of these interactions showed that only in the NEU condition, but not in the ALC condition, the two-way interaction (pre/post-training × training type) was significant . Subsequent simple effects analyses indicated that EOCs in the NEU condition only decreased in the control training , and not the Alc-IT training type . When analyzing the two-way interaction of stimulus type and pre/post-training separately for each training group, this interaction was not significant in both groups .The next analysis compared the patients' GNG behavioral data between the two training groups  and both timepoints (pre taining/post training) and stimulus types . This analysis indicated that numbers of EOC were higher in NEU than ALC . In the number of EOO, main effects of pre/post-training , and stimulus type  were found. In particular, the number of EOO was higher pre- compared to post-training, and higher for ALC compared to NEU. Finally, correct response RTs in the Go condition were shorter post than pre training RTs . Means and standard deviations of the analyzed GNG behavioral variables can be found in Such effects were not found in the RTs of EOC , showing higher activation in NoGo compared to Go trials. Moreover, a significant three-way interaction of response type × pre- post-training × training type  indicated that Alc-IT has an effect on inhibition activation in general, but not specifically on alcohol-related inhibition. This interaction can be seen in F = 1.6, p = 0.213, η2 = 0.053], opposing to our hypothesis of a beneficial effect of Alc-IT on alcohol-specific inhibition .This analysis aimed at investigating whether Alc-IT had a beneficial effect on the alcohol-related inhibitory activation in the rIFG ROI identified in the previous analysis. Analyzing fMRI data collected during an alcohol-related GNG task, this study set out to investigate whether neurophysiological correlates of alcohol-specific inhibition (I) vary with craving ; (II) are related to drinking outcomes in AUD  and (III) can be altered through alcohol-specific inhibition training .In a small cluster of the right inferior frontal gyrus (rIFG), the neurophysiological correlates of alcohol-related  inhibition increased with craving, indicating that brain activation during alcohol-related inhibition was higher in those subjects, which experienced higher levels of craving. This finding extends earlier reports of craving being linked to enhanced neurophysiological activation in cingulate areas during alcohol-specific inhibition , 42 to tThe rIFG is part of the cognitive control network , 67 and Again in the rIFG, neurophysiological activation during alcohol-specific inhibition was related to a better outcome, as indicated by a higher percentage of days abstinent at 3-month follow-up. Those patients displaying a higher activation difference in the rIFG for alcohol-related  inhibition reported more days abstinent at 3-months follow-up. The outcome was thus better in those patients, who managed to recruit enhanced neuronal resources during inhibition when the inhibitory system had to oppose cue-induced appetitive processes.As summarized above, the rIFG is closely linked to effective inhibition and has been linked to response suppression in the face of craving and cue-induced reactions , 70. OurTaken together, the present results indicate that higher rIFG activation is necessary to inhibit responses to alcohol-related cues when craving is high. In line with this, as most patients with AUD probably experience situations with high craving when returning to everyday life after residential treatment, enhanced IFG activation during alcohol-specific inhibition predicts a better outcome at 3-months follow-up.post-hoc with no significant results). To be complete, one should mention that the neurophysiological correlate of general  inhibition was differentially affected in the group receiving Alc-IT when compared to control training. As there was however no theoretical justification for a ROI-analysis focusing on this three-way interaction in the rIFG, this finding has to be seen as highly exploratory.A training effect on the neurophysiological correlate of alcohol-specific inhibition in the rIFG could not be observed; rIFG activation was not modulated by patients engaging in alcohol-related inhibition training (Alc-IT) vs. an unspecific inhibition training (control). Thus, we cannot conclude that this neurophysiological correlate, which—according to the analyses reported above—varies with craving and predicts drinking outcome, is affected by the alcohol-specific inhibition training. In that respect, our results differ from reports on another cognitive training intervention, approach bias training, where training effects on brain activity could be observed, albeit in ROI analyses focusing on other brain regions and during different tasks , 73. OnepFWE−corr = 0.030; T = 5.95; kE = 3; x = 48 mm; y = 12 mm; z = 8 mm). Besides the fact that the application of the FWE-correction used here is a common way to minimize type-I errors, one should however take into account two important aspects: (I) Alcohol-specific inhibitory activation, as we attempted to isolate it in this study, is reflected in a highly specific effect. Our analyses thus had to concentrate on those inhibitory sub-processes that vary with the context in which inhibition had to be carried out. On the one hand, such a highly specific effect might be represented on smaller sub-regions. On the other hand, such an effect might also be harder detectable in a noisy signal such as BOLD. One might therefore consider the application of higher field strengths , where a higher signal should be expected and where the higher spatial resolution might also allow a better investigation of subregions of IFG. (II) The spatial extend of the rIFG clusters amounted to 32 and 72 mm3, respectively. With a neuronal density of several thousand per mm3, we should not preclude that activation of such a specific cognitive process is possible within these small regions, unless there exists indication that it is impossible. In addition, it appears unlikely that two different whole-brain analyses yielded exclusively two highly proximal clusters in a region central to inhibition  during NoGo trials). This is inconsistent with the overall pattern yielded by meta-analyses summarizing behavioral studies on inhibitory performance , 9 and wA second aspect concerns the fact that no effect of the Alc-IT training intervention on alcohol-related EOCs could be detected. As studies on behavioral effects of cognitive training interventions are often larger, this might either be due to a limited power or to the fact that Alc-IT really did not affect alcohol-related errors of commission.In summary, the present study corroborated the central role of the rIFG in the inhibitory control network in AUD by indicating that rIFG activation during alcohol-related inhibition is related to craving and drinking outcome. Therefore, as has been proposed before in the context of cocaine addiction , future maria.stein@upd.unibe.ch.The datasets presented in this article are not readily available because no consent for this was collected from patients. Processed data and corresponding processing subroutines can be requested from the corresponding author. Requests to access the datasets should be directed to ClinicalTrials.gov (NCT02968537). The patients/participants provided their written informed consent to participate in this study.The studies involving human participants were reviewed and approved by the Local Ethics Committee (KEK-number: 2016-00988) and registered at the Swiss National Clinical Trials Portal (SNCTP000002043) and MG collected the data, conceptualized and performed data and statistical analyses, and wrote the manuscript. LS and FM designed the study and supervised data collection and data curation. RT and HB collected the data. AF conceptualized and performed data and statistical analyses. SS collected the data and conceptualized data analyses. YM supervised data collection and data curation. MS designed the study, supervised data collection and data curation, conceptualized data and statistical analyses, and wrote the manuscript. All authors contributed to discussion and interpretation of the results, revised, and approved the final version of the submitted manuscript.This study was funded by the Swiss Foundation for Alcohol Research (SSA-Nr. 283) and the Swiss National Science Foundation .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Flavonoids are a group of naturally occurring polyphenolic secondary metabolites which have been reported to demonstrate a wide range of pharmacological properties, most importantly, antidiabetic and anti-inflammatory effects. The relationship between hyperglycaemia and inflammation and vascular complications in diabetes is now well established. Flavonoids possessing antidiabetic properties may alleviate inflammation by reducing hyperglycaemia through different mechanisms of action. It has been suggested that the flavonoids’ biochemical properties are structure-dependent; however, they are yet to be thoroughly grasped. Hence, the main aim of this review is to understand the antidiabetic and anti-inflammatory properties of various structurally diverse flavonoids and to identify key positions responsible for the effects, their correlation, and the effect of different substitutions on both antidiabetic and anti-inflammatory properties. The general requirement of flavonoids for exerting both anti-inflammatory and antidiabetic effects is found to be the presence of a C2–C3 double bond (C-ring) and hydroxyl groups at the C3’, C4’, C5, and C7 positions of both rings A and B of a flavonoid skeleton. Furthermore, it has been demonstrated that substitution at the C3 position of a C-ring decreases the anti-inflammatory action of flavonoids while enhancing their antidiabetic activity. Correlation is discussed at length to support flavonoids possessing essential pharmacophores to demonstrate equipotent effects. The consideration of these structural features may play an important role in synthesizing better flavonoid-based drugs possessing dual antidiabetic and anti-inflammatory effects. A meta-analysis further established the role of flavonoids as antidiabetic and anti-inflammatory agents. Flavonoids are a type of plant secondary metabolite with a polyphenolic structure, and they are one of the most common families of natural products (NPs) ,2. FlavoDiabetes is one of the chronic diseases that can lead to death, and it was the seventh-highest cause of death in the United States in 2017 based on 83,564 death certificates . AccordiThe rising significance of inflammation in T1D and T2D has increased interest in targeting inflammation to enhance disease prevention and treatment. Timely, encouraging preliminary results are shown in the clinical studies employing both T1D and T2D anti-inflammatory therapy, such as monoclonal antibodies and IL-1 antagonists, IKKbeta-NF-kappaB inhibitors , and tumour necrosis factor (TNF) inhibitors, and supported the inflammation role in this context ,15. As pDistinguished scientific databases and search engines, namely, Google Scholar, Springer Link, Science Direct, Scopus, Wiley Online Library, PubMed, and Web of Science were thoroughly considered to find the relevant references and literature to complete this review. Additionally, the refereed non-indexed journals were also taken into consideration to collect all key information and to make certain that no well-documented information was left out. In this regard, a total of 102 articles from 2000 to 2022 focusing on diabetes, inflammation, polyphenolic compounds, flavonoids, the in vitro antidiabetic effects of flavonoids, the in vivo antidiabetic effects of flavonoids, the in vitro anti-inflammatory effects of flavonoids, the in vivo anti-inflammatory effects of flavonoids, the structure–activity relationship study of flavonoids for in vitro antidiabetic effects, the structure–activity relationship study of flavonoids for in vivo antidiabetic effects, the structure–activity relationship study of flavonoids for in vitro anti-inflammatory effects, and the structure–activity relationship study of flavonoids for in vivo anti-inflammatory effects, which were identified as keywords. After thorough study and investigations of all searched articles, 67 research manuscripts were finally selected to gather all the required information.1), rhamnetin (2), genistein (3), eriodyctiol (4), luteolin (5), kaempferol (6), fisetin (7), phloretin (8), catechin (9), epicatechin (10), epigallocatechin (11), and myricetin (12). It was discovered that 1 was the most potent COX-2 transcription suppressor, whereas 9 and 10 had the least inhibitory action. Briefly, 1 belongs to the flavonol class, and 10 belongs to the flavan-3-ol class; however, the presence of C2–C3 bond unsaturation and the oxygenation at C4 in 1 is thought to be responsible for its suppressing capabilities. It was also reported that the C2–C3 double bond of the C-ring, as can be seen in 4 and 5, caused a minute effect on COX-2 transcriptional activity. Next, all tested flavonoids with a 4-oxo group were potent suppressors of COX-2 transcriptional activity, except 12, which concluded that oxygen at C4 is critical for bioactivity. It was also corroborated by the fact that the compounds without oxygen at C4, such as 9, 10, and 11, showed either minimum or no inhibitory effect. Subsequently, it was discovered that the number of hydroxyl groups on the B-ring may be substantial in COX-2 transcriptional activity. It was supported by the findings which stated that compounds containing hydroxyl groups at C3′ and C4′, such as 4, 7, 5, 1, and 2, can significantly suppress COX-2 transcription. Meanwhile, compounds that bear three hydroxyl groups on the B-ring such as 11 and 12 had an absence of inhibitory activity. The free 7-hydroxyl group with low electron density in the A-ring was a significant structural feature for the suppression of COX-2 transcriptional activity. The authors concluded that the presence of a 4-oxo group in the C-ring, a 3′,4′-dihydroxy  structure in the B-ring, and a low electron density in the 7-oxygen group in the A-ring are required for exerting anti-inflammatory activity via the suppression of COX-2 transcriptional activity, as shown in Flavonoids have been reported to exert anti-inflammatory activity through different ways. Mutoh et al. evaluated the inhibitory activity of twelve different flavonoids on the transcription of the cyclooxygenase-2 (COX-2) gene in a human colon cancer cell line, namely DLD-1 cells . The fla1), luteolin (5), catechin (9), and taxifolin (13) [Next, flavonoids have also been reported to exert their anti-inflammatory activity through lipoxygenase (LOX) enzyme inhibition. Redrejo-Rodriguez et al. studied the inhibitory activity of LOX enzymes of four different flavonoids, namely, quercetin (lin (13) . It has 5), 3′,4′-dihydroxyflavone (14d), 3′,4′,7-trihydroxyflavone (16d), 3′,4′,5-trihydroxyflavone (15d), and quercetin (1). As reported by the authors, all the above-mentioned compounds contained catechol moiety in the B-ring, which may be responsible for the inhibitory activity. It was also reported that the number of hydroxyl groups in the A-ring did not appear to be a factor for the inhibition. However, the presence of a hydroxyl group at the C3 of the C-17c ring significantly reduced the LTB4 inhibitory activity, which was proved by compound 1 which exerted 2.5 times less potent inhibition than 5. Next, 5 was shown to be more effective than 1, possibly due to the latter’s high hydroxyl group count, which reduces hydrophobicity and inhibits flavonoid intercalation in the hydrophobic cavity that serves as the enzyme’s active site access channel. After that, the C2–C3 double bond appeared to be important for the inhibition as the tested flavanones had lower potency compared to flavones and flavonols. Cases 1 and 5 were reported to exhibit higher inhibition than non-planar compounds, 13. Therefore, it can be said that planarity may influence flavonoids’ ability to interact with LOX enzymes. Notably, in order to demonstrate LTB4 enzyme inhibition, the structure–activity relationship study signalled the catechol group in the B-ring, the number and position of hydroxyl groups, the double bond between C2 and C3, and planarity appear to influence the inhibitory activity.Ribeiro et al. conducted a systemic investigation on the inhibition of the generation of leukotriene B4 (LTB4) and neutrophils by synthetic and natural flavonoids . Figure 5) greater effectiveness than the quercetin (1). Apart from that, the presence of catechol moiety in the A-ring or B-ring can increase the inhibitory action. Following that, sugar moiety can significantly reduce the inhibition as it can decrease flavonoids’ hydrophobicity, which leads to a decrease in affinity towards LOX’s active site. The absence of the C2–C3 double bond of the C-ring appeared to decrease the inhibitory action as depicted by naringenin (18), hesperidin (19), epicatechin (10), and taxifolin (13). On the other hand, 1 that bears the C2–C3 double bond was reported to be more potent than the abovementioned flavonoids. The authors discussed that the C2–C3 double bond is significant as it completes a conjugated binding system that extends through all three rings and the carbonyl group of the C-ring, thereby stabilising the complexes or radical intermediates generated by flavonoids.Additionally, using linoleic acid as a substrate, Sadik et al. studied the anti-inflammatory effects of 18 flavonoids on rabbit reticulocytes and soybean 15-LOX . Firstly1) and its primary metabolites in suppressing inflammatory eicosanoid production from human leukocytes, demonstrating the role of metabolic transformation on flavonoid bioactivities [1 and its metabolites used in the test. Briefly, 1 potently inhibited LTB4 synthesis in leukocytes, and its activity was found to depend on the specific structural properties, specifically the C2–C3 double bond in the C-ring. It is a structural necessity for 1 to inhibit LTB4 since its absence eliminated the inhibitory activity. Furthermore, conjugation at the 3′-OH of 1 such as 3′-O-methylquercetin (20) and quercetin-3′-O-sulfate (21) decreased LTB4 inhibition by up to half, and glucuronidation at the 3-OH such as quercetin-3-O-glucuronide (22) and 3′-O-methylquercetin-3-O-glucuronide (23) also greatly diminished the LTB4 inhibitory action. Interestingly, the 3′-OH of the B-ring was more important in inhibiting LTB4 than the 3-OH of the C-ring. This was demonstrated when 1 was compared to structural analogues such as luteolin (5) and kaempferol (6) since the structural analogues exhibited a drop-in activity compared to 1 due to the absence of 3-OH in the C-ring (as seen in compound 5) and 3′-OH in the B-ring (as shown in compound 6).Similarly, Loke et al. compared the activity of quercetin , cynaroside (24), and cesioside (25) were isolated from H. corniculata. Then, isoorientin (26) was obtained from G. tenella and G. azurea, as well as stereolensin (27) from P. rotundifolia and P. incarnata. The structures of these flavonoids are shown in 5 had the highest inhibition on both LTB4 and TXB2 synthesis compared to other compounds. Then, it was followed by 24 and 25, with moderate inhibition on both syntheses. Meanwhile, 26 and 27 selectively showed good TXB2 inhibition. From the bioactivity studies, a structural activity relationship was summarized. For the non-selective inhibition of TXB2 and LTB4 synthesis, the authors discussed that in the A-ring, the presence of hydroxyl group at C7 and meta hydroxyls at C5 and C7 were important. In addition, the presence of ortho hydroxyl groups at C3′ and C4′ on B-ring also plays an important role in the inhibition. It was proved that the absence of these three characteristics will decrease the inhibition as demonstrated in 24 and 25. For selective TXB2 synthesis inhibition, the structures of 26 and 27 were studied. It was reported that the direct attachment of the sugar moieties to OH- or carbon at C6 of the A-ring while retaining flavone basic hydroxyl groups will improve the inhibition against TXB2 synthesis.Odontuya et al. studied the SAR for the anti-inflammatory effect of flavonoids that were isolated from several plants . There wdiverse array of flavonoids 38 in number for their anti-inflammation potential through their COX-2 mRNA inhibition [5) and apigenin (17c) showed greater inhibition compared to their isomers. In contrast, sugar substitutions and the -OH group at C3 caused lower COX-2 mRNA inhibition. Similarly, the C2–C3 double bond was also responsible for the decrease in inhibition.Li et al. evaluated a hibition . Then, aMurraya paniculata (L.) Jack (Rutaceae) [30 as it had the weakest activity among the three compounds.Wu et al. isolated three flavonoids from a famous traditional Chinese medicinal plant, utaceae) . Then, tVitex grandifolia which is traditionally used by the Yoruba community in southwest Nigeria to treat various disorders [26), orientin (31), and isovitexin (32) . The authors discussed the structure–activity relationship of these three flavonoids. Firstly, the C2–C3 double bond might influence the anti-inflammatory activity of flavonoids. Next, the -OH groups at C3′ and C4′, as shown in 26 and 31, can increase the anti-inflammatory activity. In addition, the presence of sugar moiety in the A-ring compared to the B- and C-rings will lead to better anti-inflammatory activity.Bello et al. examined three flavonoids isolated from isorders . The flaxin (32) . The ant17c), luteolin (5), and quercetin (1) portrayed complete inhibition; meanwhile, diosmetin (17f) and chrysin (17a) showed weak inhibition. On the other hand, hesperidin (19), kaempferol (6), genistein (3), and daidzein (33) were reported to be completely inactive against iNOS. For COX-2 inhibition, only 17c and 17f exerted inhibition. The authors discussed the structural requirements needed for both iNOS and COX-2 inhibition. It was highlighted that the presence of hydroxyl groups at the C2′ and C4′ of the B-ring may influence both activities. In contrast, methoxy and hydroxyl groups at C3 can reduce the iNOS inhibition. Secondly, for the inhibition of cytokine production such as TNF-α, IFN-γ, and IL-2, it was reported that all tested flavonoids showed good and effective inhibition. The authors summarized five structural requirements for the inhibition of cytokine production. The C2–C3 double bond, C4′ hydroxyl group, C3′ hydroxyl group, the absence of the C3 hydroxyl group, and the C5 hydroxyl group favoured the cytokine production inhibition. Moreover, the introduction of sugar moiety at C3 needs to be avoided as it causes a complete reduction in inhibition. Thirdly, similarly, all flavonoids showed excellent antiproliferative activity on both unstimulated splenocytes and concanavalin A-treated cells. It was also mentioned that about an average of 40% of splenocytes were reduced after the addition of the selected flavonoids. The C2–C3 double bond and the B-ring hydroxyl groups, particularly the C5 hydroxyl group, were reported to be important to these antiproliferative activities. Lastly, the reduction in splenocytes’ viability was also assessed. It was discussed that the 1, 17c, 5, and 3 displayed stronger activity compared to other flavonoids. The authors mentioned that the C3′ and C4′ hydroxyl groups can increase the activity of the reduction in splenocytes’ viability.López-Posadas et al. studied 14 flavonoids’ anti-inflammatory effects on rat splenocytes and carried out their structure–activity relationship . Five di6), quercetin (1), apigenin (17c), chrysin (17a), diosmetin (17f), luteolin (5), daidzein (33), genistein (3), and hesperidin (19) on the macrophages derived from the bone marrow of mice [17f and 5) and flavonols (6 and 1) exhibited the significant inhibition of M-CSF–macrophage proliferation. Meanwhile, isoflavones and flavanones displayed weak antiproliferative activity. Therefore, it can be concluded that the iso-position of the B-ring and the absence of the C2–C3 double bond can lead to lower antiproliferative activity. Secondly, the effect of flavonoids on the inhibition of TNF-α was conducted. It was reported that 5, 1, and 3 exerted good TNF-α inhibition. The authors discussed that the C3′ and C4′ hydroxyl groups as in luteolin and 1 favoured the inhibition. In addition, the one hydroxyl group as in 3 can also lead to better TNF-α inhibition. The author stressed that the absence of hydroxyl groups in the B-ring was detrimental and led to the negative results of TNF-α inhibition. Thirdly, iNOS inhibition showed that 1, 17c, 5, and 17f were able to inhibit iNOS and nitric oxide (NO) at low concentrations. The authors mentioned that the B-ring position, the C2–C3 double bond, and the hydroxyl group will influence the inhibition. Lastly, the authors suggested that 5 and 1 are the best naturally occurring flavonoids as anti-inflammatory agents due to the abovementioned characteristics.Comalada et al. evaluated the effect of naturally occurring flavonoids viz. kaempferol ( of mice . The ant2) production in rat peritoneal macrophages between flavonoids subclasses [2 production. In contrast, flavan-3-ol displayed weak inhibition. The C2–C3 double bond and 4-oxo of C-ring were discussed to be the important characteristics of a good inhibitor. In addition, the authors stressed that the absence of the C2–C3 double bond will lead to a loss of inhibition activities. When the tested flavonoids were compared, it was reported that the hydroxyl groups at the C5 and C7 positions led to a lower IC50 value, indicating better inhibition compared to the compounds without OH groups at C5 and C7. Similarly, in isoflavones alone, the hydroxyl group at C5 was reported to show better activity compared to compounds without the -OH residue at C5. Meanwhile, in flavones and flavonols, the absence of hydroxyl groups in the B-ring showed better inhibition compared to compounds that had OH groups such as at C3′ and C4′.Takano-Ishikawa et al. compared the structure–activity relationship of inhibition on lipopolysaccharide-induced prostaglandin E2 (PGEbclasses . There w17c) and luteolin (5) showed good inhibition with IC50 values of 23 and 27 μM, respectively. Meanwhile, naringenin (18) and apiin (34) showed the weakest inhibition with IC50 values up to 100 μM. The authors proposed that the C2–C3 double bond and the 5,7-dihydroxy groups at the A-ring favoured the inhibition of NO production. In addition, methoxy groups at C8 of the A-ring and the 4′ or 3′,4′-vicinal substitutions at the B-ring may also lead to better inhibition. In contrast, the 3-OH at the C-ring showed weaker activity which can be seen in flavonol derivatives such as flavonol, galangin (35), and quercetin (1). As mentioned before, 18, a flavanone derivative, showed the weakest activity. Hence, it can be concluded that the C2–C3 double bond and the planar ring are important structural requirements to exhibit good inhibition of NO production.Kim et al. evaluated the effect of naturally occurring flavonoids on NO production in the macrophage cell line . Among t36a–y, and their chemical structures are demonstrated in 36g which contained hydroxyl groups at C3′ and C4′ of the B-ring showed maximum inhibitory action compared to other synthesized compounds. Therefore, the authors concluded that the catechol group may be responsible for NO inhibition. The statement was also supported by the fact that 36e with a hydroxyl group at C4′ exerted better NO inhibition than 36f with methoxy groups at C3′ and C4′. Halogenation at C4′ as shown in 36i, 36j, and 36k either slightly lowered or showed no increase in the NO inhibitory action. Similarly, the authors reported that trimethylsilyl (TMS), alkyl, or aryl substituents at the C4′ position did not increase the inhibition action. Pivaloyloxy group at C7 of the A-ring and the hydroxyl groups at C5 and C7 as shown in 36v–y exhibited moderate inhibitory action but still showed lower activity than 36g, which possesses hydroxyl groups at the C3′ and C4′ positions. An et al. synthesized several flavone derivatives and evaluated their anti-inflammatory action via NO inhibition . As depiArtocarpus heterophyllus and tested their NO inhibitory activities [39 and 40 exhibited significant inhibitory action compared to other isolated compounds. On the other hand, 38 and 41 were among the compounds that showed the weakest activities. Therefore, the authors concluded that the presence of hydroxyl groups at C5 and C7 of the A-ring, as well as at C4′ of the B-ring, were assumed to influence the flavonoids’ NO inhibitory activities.Liu et al. isolated several flavonoids from tivities . The che17c) and luteolin (5) exerted the most significant inhibition compared to other flavonoids. Both compounds contained hydroxyl groups at the C5 and C7 positions of the A-ring, the C2–C3 double bond, and oxygenation at C4 of C-ring. Therefore, it can be said that these structural features are important in the inhibitory activity. It was also discovered that chalcones with a broken C-ring, such as phloretin (8), exhibited equal efficacy to 17c, indicating that the co-planar structure was not required or essential for inhibition. Pelargonidin (42), on the other hand, demonstrated poorer inhibition than 17c and 8 in the absence of a C2–C3 double bond but with a closed C-ring, indicating that the C2–C3 double bond is a significant structural characteristic. Flavans, (+)-catechin (9), and (−)-epicatechin (10) with no C2–C3 double bond also had lower activity. After that, daidzein (33) and genistein (3) with the absence of hydroxyl groups at the C5 and C7 positions, respectively, had weak inhibition of LPS-induced IL-8 release. Therefore, the findings support the statement that the hydroxyl groups at the C5 and C7 positions of the A-ring are important for inhibitory action. Next, the authors mentioned that the hydroxyl group at the C3 position of the C-ring, as can be seen in naringenin (18), can improve inhibitory action. Zhang et al. investigated the inhibition of IL-8 released by lipopolysaccharide-stimulated bronchial epithelial cells by 17 flavonoids . It was 17c), luteolin (5), quercetin (1), and myricetin (12) inhibited LPS-induced TNF-α that had been produced from a macrophage. Therefore, the authors hypothesized that the presence of hydroxyl groups at the C5 and C7 positions of the A-ring, as well as at the C4′ position of the B-ring, seemed to be important features. Moreover, galangin (35) and chrysin (17a) exhibited weaker and an absence of inhibitory activities, respectively, when compared. Therefore, it can be suggested that an absence of the hydroxyl group at C4′ of the B-ring can reverse or reduce the bioactivity. Following that, for in vivo assay, the tested flavonoids were orally supplied to mice. It was reported that 5 and 17c decreased serum TNF-α production after oral administration. The presence of hydroxyl groups at the C4′ (B-ring), C5, and C7 (A-ring) positions seemed to be important for oral inhibition, as both 5 and 17c contained these features. Furthermore, as 5 exhibits stronger inhibition, the hydroxyl group at the C3′ position of the B-ring may be responsible for the greater inhibition observed with 5. Ueda et al. evaluated the inhibition of flavonoids against TNF-α production and discussed the related structural features that are responsible for inhibition . For an 17e with dihydroxy groups at C2′ and C3′ exerted the maximum NO inhibition when compared to other synthesized compounds. Briefly, 43a, 43b, and 43g that contained one hydroxyl group either at C2′ or C3′ also exhibited significant inhibition, albeit weaker than 43e. Therefore, it was proved that hydroxyl groups at C2′ and C3′ are important structural requirements for NO inhibition. The fact was further corroborated when the methylation of C2′-OH, C3′-OH, or both C2′ and C3′-OH, as can be seen in 43j, 43k, and 43n, respectively, caused inactivity. It was also reported that substitution at the C4′ position, as seen in 43c, 43f, and 43h, can lead to weaker inhibitory action. Jiang et al. synthesized 19 flavanonols and evaluated their NO inhibitory action in RAW macrophage 264.7 cells, as well as discussed the structural activity relationship . The synMorus alba L. (Moraceae), commonly known as mulberry. The isolated prenylated flavonoids, namely, kuwanon C (43s), kuwanon T (43t), sanggenon H (43u), morusin (43v), morusinol (43w), cyclomorusin (43x), kuwanon S (43y), kuwanon E (43z), and kuwanon U (43za) (p < 0.01), morusinol (p < 0.01), and cyclomorusin (p < 0.05). It was revealed that flavonoids with an isoprenyl group at C-3 (including 43s) were weak COX-2 inhibitors with less activity than the reference inhibitor, i.e., indomethacin. The results also showed that, in comparison to 43s, modifications of the isoprenyl moieties at C-3 and C-8 in 43w did not significantly influence the activity [Culenova et al. isolated several prenylated flavonoids from the root bark of U 43za)  were evaza  were M. alba L.) leaf flavonoids. Initially, different ethanol concentration -based extracts were prepared to obtain flavonoid-rich extracts. These extracts inhibited the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells. All extracts increased the antioxidative capacity by decreasing the reactive oxygen species (ROS) production and the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and improving the metal ion chelating activity and reducing power. The results revealed that the extract prepared using 30% ethanol exhibited the best anti-inflammatory and antioxidative activities. The LC-MS analysis revealed the presence of 24 different flavonoids in the resultant extracts. Finally, a nontargeted metabolomic analysis confirmed that quercetin (1), kaempferol (6), and their derivatives in 30% ethanol extract were more abundant than the other two extracts and may be the main flavonoids involved in anti-inflammatory and antioxidative effects. Furthermore, 30% ethanol extract of mulberry leaf was also evaluated for the pharmacological activities in dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice. The same extract alleviated the clinical symptoms, reduced the secretion of inflammatory cytokines, and inhibited the activation of the inflammatory pathway in DSS-induced colitis mice [Lin et al. further evaluated the anti-inflammatory and antioxidative activities of mulberry  are indispensable for the anti-inflammatory function of flavones, and -OH at the C-5 and C-4′ positions were found to enhance, while -OH at the C-6, C-7, C-8, and C-3′ positions was found to diminish their activity. Moreover, the C2–C3 single bond and -OH at the C-3 and B-ring positions weakened flavone aglycones’ activity. It was also discovered that most of the flavone aglycones showed activity through the NF-κB, MAPK, and JNK-STAT pathways, and their possible cell binding targets were found to be kinase, aryl hydrocarbon receptor (AhR), G-protein coupled receptors, and estrogen receptors .44), which bears hydroxyl groups at six different positions to be the most efficient of all the flavonoid compounds examined. The positions include C3 of the C-ring, C3′ and C4′ of the B-ring as well as C5, C6, and C7 of the A-ring. After that, it was summarized that the presence of hydroxyl groups at C3′ and C4′ in the B-ring and the C2–C3 double bond were found to be critical variables for inhibition, as is illustrated in Flavonoids have been reported to demonstrate antidiabetic effects through various molecular mechanisms of action. Kato et al. investigated the structural requirements of flavonoids with glycogen phosphorylase inhibitory activity. Glycogen phosphorylase is one of the enzymes that catalyse the breakdown of glycogen into glucose in the liver, and its inhibition has been shown to modulate the glucose level associated with T2D . The res17a) that is bearing C5-OH. It was hypothesized that the hydroxyl group at C5 may not be essential for inhibition against aldose reductase. Then, it was reported that diosmetin 7-O-β-D-glucopyranoside (45) had weaker inhibition compared to diosmetin (17f). Therefore, it can be assumed that 7-O-glucosyl moiety can lead to a reduction in inhibitory action. The presence of the hydroxyl group at C3 was shown to reduce inhibition, which is supported by the fact that 3-O-methyl or 3-O-monosaccharide derivatives are better inhibitors than the comparable free flavonols at the C3 position. Next, apigenin (17c) inhibited more effectively than kaempferol (6), implying that the presence of the 3-OH group is not essential for the higher inhibition. Lastly, the authors mentioned that flavonoids with catechol moiety at the B-ring (hydroxyl groups at C3′ and C4′) demonstrated better inhibition than flavonoids with pyrogallol moiety .Matsuda et al. studied the aldose reductase inhibitory action of flavonoids and the structure–activity relationship . Aldose Matsuda et al.  further Next, Matsuda et al. investigated the effect of 44 flavonoids on the adipogenesis of 3T3-L1 adipocyte cells . The str46) , human recombinant aldose reductase (HRAR), and advanced glycation end products (AGE). For RLAR inhibition, 46 showed a significant IC50 value (0.30 μM) when compared to the control, epalrestat (0.07 μM). For HRAR inhibition, 46 also showed a remarkable IC50 value (0.17 μM) compared to the control, epalrestat (IC50 0.15 μM). Meanwhile, for AGE inhibition, 46 portrayed a stronger inhibition with a lower IC50 value (17.89 μg/mL) when compared to the control, aminoguanine (IC50 81.05 μg/mL). The authors discussed that there are three important structural characteristics that contributed to the remarkable RLAR, HRAR, and AGE inhibitions. Firstly, flavonols with a prenyl group at the C8 position and C3′,4′-dihydroxyl groups lead to more potent inhibition. Next, the presence of the methoxy group at C-5 also caused stronger inhibition. Lastly, the essential structural characteristic that contributed to the strong inhibition is the presence of the hydroxyl group at the C-4′ position.Jung et al. investigated a prenylated flavonol called sophoflavescenol (46)  for its Potentilla anserina L. [47 and 50–54 belong to the biflavonoid category and portrayed remarkable IC50 values, which ranged from 2.57 to 8.96 μM. Next, the substitution of the gallolyl moiety instead of the glucose moiety at C-2″ can significantly improve the inhibition activity. It can be seen in compound 49 with the gallolyl moiety at C-2″ (IC50 = 1.05 μM) compared to compounds 48 with the glucose moiety and 1 (82.47 and 75.80 μM). The presence of a hydroxyl group in the B-ring was discovered to be essential, as evidenced by compound 55′s weak inhibition (IC50 = 155.57 μM).Yang et al. reported the identification of 30 different phenolic compounds from the rhizomes of erina L. . All com56a–e and glucuronirated flavonols 56f–i from Tamarix gallica on α-glucosidase inhibitory activity [56a and 56e, it was suggested that the hydroxyl group at C3′ with glucuronic acid and methyl ester was responsible for α-glucosidase inhibition.Hmidene et al. studied the effect of five simple flavonols activity . The acaSedum dendroideum leaf extract [57) but not in 58 and 59, and the results showed 57 had higher hypoglycaemic activity compared to 58 and 50. Next, to test the importance of the rhamnosyl unit at C7, 60 (with rhamnosyl unit at C7) and 61 (without rhamnosyl unit at C7) were tested. The results supported that a rhamnosyl unit at C7 is important as 60 exhibited hypoglycaemic activity after 120 min, but 61 showed activity after 60 min and lost activity at 120 min.Da Silva et al. investigated the in vivo antidiabetic activity of kaempferol derivatives that were isolated from  extract . There w50 values of 7.6 ± 0.4 μM and 15 ± 3 μM. Based on Proença et al. evaluated the series of 40 flavonoids for an in vitro α-glucosidase inhibition. The compounds were grouped into five groups . After t62  with an IC50 value of 44 ± 3 μM. Then, based on this most-active compound, an activity pattern was created by the authors. Based on Similarly, Proença et al. studied the same series of 40 flavonoids. However, the bioactivity tested was slightly different, in which inhibition against pancreatic α-amylase was evaluated . Like α-50 value. The values reported were myricetin (12) (IC50 = 11.63 ± 0.36 μM) > apigenin-7-O-glucoside (63) (IC50 = 22.80 ± 0.24 μM) > fisetin (7) (IC50 = 46.39 ± 0.34 μM). Then, by using the 3D-quantitative structure-activity relationship model, structural characteristics that were needed for good inhibition were summarized. There are four important characteristics that can be seen in meta-position. Next, minor and electron-donating groups, as well as hydrogen bond donor groups, were favoured at C3 of the C-ring. After that, at C7 of the A-ring, bulky and hydrogen acceptor groups were favoured. Then, for insulin sensitization activity, all compounds were tested by using molecular docking and in vitro evaluation with insulin-resistant HepG2 cells. The results showed five flavonoids that exerted good insulin sensitization activity which were baicalein (64), isorhamnetin-3-O-rutinoside (65), 63, kaempferol-7-O-β-glucoside (66), and cyanidin-3-O-glucoside (67). There was no structural analysis conducted on these five flavonoids for insulin sensitization. However, from both studies, the authors concluded that compound 63 can be used in diabetes management in the future as it exerted excellent activity in both α-glucosidase inhibition and insulin sensitization.Next, Jia et al. studied several dietary flavonoids for their α-glucosidase inhibitory and insulin-sensitizing potentials . For α-gBoesenbergia rotunda, which is known as fingerroot. Based on 68), pinostrobin (69), and alpinetin (70). Meanwhile, chalcones are cardamomin (71) and boesenbergin B (72), whereas dihydrochalcones are panduratin A (73) and isopanduratin (74). For the evaluation of AGE inhibition, two methods were conducted, namely AGE inhibition assay and methylglyoxal (MG) trapping activity. MG is a known precursor of glycation. It was reported that most compounds showed greater AGE inhibition than the control, aminoguanidine. Then, for MG trapping activity, all compounds showed comparable activities compared to aminoguanidine. Briefly, 68 was the most active compound for MG trapping activity, with an EC50 value of 63.22 ± 10.12 µM. By using the structure of 68 and other compounds, the SAR of flavonoids on the MG trapping activity was summarized. Firstly, hydroxy groups can improve the inhibition, while methoxy and geranyl groups can reduce the inhibition. Next, the presence of a methoxy group at the C7 position of dihydrochalcone can improve the activity compared to methoxy groups at the C5 position. After that, for α-glucosidase inhibition, only bioactivity studies were conducted, while no SAR was conducted. It was reported that 68 also demonstrated an inhibitory effect against α-glucosidase.Potipiranun et al. isolated several flavonoids to test their activities on antidiabetic complication (AGE inhibition) and α-glucosidase inhibition . Three fXiao et al. reviewed an α-glucosidase inhibitory effect of dietary flavonoids and summarized important structural characteristics that were responsible for the inhibition . The sumMahapatra et al. reviewed all SAR studies that were related to the antidiabetic activities of chalcones from 1977 to 2014 . Then, t75c–d and 76b  exerted higher activity compared to the control, bavachinin (EC50 = 18.74 μM). Then, the authors reported that removing the C7-methoxy group which can be seen in 75a or removing the C6-isopentenyl chain and then replacing it with a geranyl chain (as can be seen in 75e) can reduce the PPAR-γ activation. In contrast, the replacement of isopentenyl with isopentyl at C6 of the A-ring (75d) can improve the activity. The presence of an electron-donating group (75b) or electron-withdrawing group (75c) at C3′ was found to increase the PPAR-γ activation. Moreover, it was found that the activity was reduced by oxidising the C-ring of flavanone 75b to flavone 75a. Interestingly, oxidising the C-ring of flavanone 75d to produce flavone 76b boosted PPAR-γ activation.Du et al. synthesized several flavonoid derivatives to be tested as PPAR-γ agonists . The syn77a with trihydroxyl groups was the most potent inhibitor, with an IC50 value of 45 μM. Then, it was found that compounds 77b–j in which there was an absence of any hydroxyl group at C5, C6, and C7 showed either weaker inhibition or inactivity. Most importantly, 77g without C6-OH showed no inhibition, which led the authors to conclude that C6-OH is essential for inhibitory action. Next, 77t was also shown to exert weaker inhibition when the hydroxyl group was added to position C8, despite having three hydroxyl groups pattern at C5, C6, and C7. Following that, the inclusion of an electron-withdrawing or electron-donating group at C8, as can be seen in 77n–q and 77s–u, resulted in either inactivity or became weak in terms of inhibition, despite the fact that 77r lost a little amount of activity. Compound 77v that contained bulky piperidino-methyl group at C8 was also found inactive. Hence, the researchers speculated that 77r’s less bulky fluorine at C8 was responsible for its moderate activity compared to others. It can be concluded that C6-OH and substitution at C8 can influence the inhibition.Gao et al. synthesized several flavone derivatives and investigated the effect of the A-ring hydroxyl groups on α-glucosidase inhibition . The flaTetracera indica (Houtt. ex Christm. and Panz.) Merr. (Dilleniaceae) and Tetracera scandens (Linn.) Merr. (Dilleniaceae) leaf extracts and then evaluated their antidiabetic effects via α-glucosidase and dipeptidyl peptidase IV (DPP-4) inhibition assays [1) possessing a catechol moiety showed the highest inhibition compared to other isolated compounds. Isoscutellarein (78) and kaempferol (6) with C4-OH showed weaker inhibition compared to 1. Therefore, the catechol group, in which the hydroxyl groups at C3′ and C4′ of the B-ring, were thought to be crucial in α-glucosidase inhibition. Next, for DPP-4 inhibitory action, 1, 78, hypoletin (79), and 6 showed remarkable inhibition. The presence of hydroxyl groups can be considered to affect the inhibitory effect of DPP-4. The absence of a C2–C3 double bond and a 4-oxo group can further reduce the inhibition of α-glucosidase and DPP-4, according to the results of the study.Sarian et al. isolated several flavonoids from the n assays ,65,66,67In p = 0.0301). According to the Q-test, the true outcomes appear to be heterogeneous (Q (16) = 168.4474, p < 0.0001, I² = 90.5015%).p < 0.0001). According to the Q-test, the true outcomes appear to be heterogeneous (Q (29) = 155.3766, p < 0.0001, I2 = 81.3357%).In contrast, the inhibition of the generation of leukotriene B4 (LTB4) by human neutrophils, nuclear factor kappa B (NF-κB) inhibition, the inhibition of the generation of leukotriene B4 (LTB4) by human neutrophils, pancreatic α-amylase inhibitory activity, rat lens aldose reductase (RLAR) inhibitory activity, human recombinant aldose reductase (HRAR) inhibitory activity, the advanced glycation end products (AGE) inhibitory activity assay, and the DPP-4 enzyme inhibitory assay were performed as only one study; therefore, a heterogenic analysis was not possible.As earlier described, inflammation is strongly associated with diabetic pathogenesis in both T1D and T2D. As a result, an antidiabetic drug possessing anti-inflammatory capabilities has the great potential to be a promising treatment for both T1D and T2D ailments. Flavonoids have been identified as one of several therapeutic therapies for diabetes mellitus due to their remarkable dual antidiabetic and anti-inflammatory capabilities. 2, and IL-8. Moreover, the C2–C3 double bond and C=O at C4 in the C-ring are also found to be important for the anti-inflammatory action of flavonoids. The inclusion of a sugar moiety and hydroxyl group, particularly at the C3 position of the flavonoids skeleton, on the other hand, can reduce anti-inflammatory activity. According to the findings in this review , hydroxySimilarly, according to Flavonoids are one of the major secondary metabolites of plants that have been reported to demonstrate various pharmacological effects including antidiabetic and anti-inflammatory activities. Since flavonoids comprise the same core scaffold, the functional variation is largely associated with the presence of different substituent groups in the different positions of the flavonoid’s skeleton. Flavonoid–protein  interactions are important phenomena dictating the flavonoids’ beneficial distinct pharmacological properties. The relationship between the chemical constitution fragment and pharmacological effects has revealed that the presence of appropriate bio-isosteres as side chains can significantly affect the biological activity of flavonoids on the same target receptor. Hence, the structure–activity relationship analysis of flavonoids is important in understanding the dual mechanistic view of flavonoids in demonstrating the anti-inflammatory and antidiabetic effects. It can be concluded that for a flavonoid molecule to exert anti-inflammatory and antidiabetic activities together, the presence of a C2–C3 double bond (C-ring) and hydroxyl groups at positions C3′, C4′ (B-ring), C5, and C7 (A-ring) are essential structural requirements. Substitution at the C3 location, on the other hand, has been shown to lower flavonoids’ anti-inflammatory activity while increasing their antidiabetic activity. Therefore, the modification of these positions with the introduction of appropriate bio-isosteres will certainly play an important role in synthesizing more potent flavonoid-based drugs possessing essential pharmacophores to display equipotent antidiabetic and anti-inflammatory effects. Different in vitro assays using a variety of biological test systems should further be chalked out to confirm these flavonoids as safe antidiabetic and anti-inflammatory agents and help us understand various mechanisms that would equally alleviate hyperglycaemia and inflammation. Moreover, the knowledge of structure−activity relationships in these assays may further prove to be helpful in evaluating the potential of the in vivo antidiabetic and anti-inflammatory activities of flavonoids. A meta-analysis of the context of the antidiabetic and anti-inflammatory effects of flavonoids further established the role of these polyphenolic compounds as antidiabetic and anti-inflammatory agents. This review provides a theoretical foundation for the development of high-bioactive and low-toxicity flavonoid-based active pharmaceutical ingredients to tackle diabetes and inflammation concurrently.