Development of White Cabbage, Coffee, and Red Onion Extracts as Natural Phosphodiesterase-4B (PDE4B) Inhibitors for Cognitive Dysfunction: In Vitro and In Silico Studies

Human cognition fundamentally depends on memory. Alzheimer's disease exhibits a strong correlation with a decline in this factor. Phosphodiesterase-4 B (PDE4B) plays a crucial role in neurodegenerative disorders, and its inhibition is one of the promising approaches for memory enhancement. This study aimed to identify secondary metabolites in white cabbage, coffee, and red onion extracts and identify their molecular interaction with PDE4B by in silico and in vitro experiments. Crushed white cabbage and red onion were macerated separately with ethanol to yield respective extracts, and ground coffee was boiled with water to produce aqueous extract. Thin layer chromatography (TLC)–densitometry was used to examine the phytochemicals present in white cabbage, coffee, and red onion extracts. Molecular docking studies were performed to know the interaction of test compounds with PDE4B. TLC-densitometry analysis showed that chlorogenic acid and quercetin were detected as major compounds in coffee and red onion extracts, respectively. In silico studies revealed that alpha-tocopherol (binding free energy (∆Gbind) = −38.00 kcal/mol) has the strongest interaction with PDE4B whereas chlorogenic acid (∆Gbind = −21.50 kcal/mol) and quercetin (∆Gbind = −17.25 kcal/mol) exhibited moderate interaction. In vitro assay showed that the combination extracts (cabbage, coffee, and red onion) had a stronger activity (half-maximal inhibitory concentration (IC50) = 0.12 ± 0.03 µM) than combination standards (sinigrin, chlorogenic acid, and quercetin) (IC50 = 0.17 ± 0.03 µM) and rolipram (IC50 = 0.15 ± 0.008 µM). Thus, the combination extracts are a promising cognitive enhancer by blocking PDE4B activity.


Introduction
Cognitive dysfunction is a serious health issue worldwide; this condition is primarily caused by Alzheimer's disease (AD), which is characterized by the aggregation of amyloidβ plaques and neurofbrillary tangles of hyperphosphorylated tau proteins [1].In 2020, dementia was estimated to afect 55 million individuals globally [2].Te declining concentrations of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and brain-derived neurotrophic factor (BDNF) in the hippocampus have been observed to be attributed to AD development [1].Te exploration of nootropics or cognitive enhancers, known as "smart drugs," is catching intrigue on a daily basis.Healthy participants (students and workers) generally take smart drugs to enhance their memory, attention, learning, executive functions, and vigilance.Te benefts and potential risks are associated with the use of the most common synthetic nootropics in healthy adults, including piracetam, benzodiazepine inverse agonists, methylphenidate, donepezil, amphetamine-type stimulants, and unifram analogues [3][4][5].Nootropics that target phosphodiesterases (PDEs) in the brain aid in the normal regulation of neuronal cells [6][7][8].To date, the world is paying more attention to cognitive enhancement using natural sources, but the research on the efects of white cabbage, cofee, and red onion as smart drugs is still unavailable [9,10].
Phosphodiesterase-4 (PDE4), the largest family of PDE, is the most complicated and maybe the most broadly expressed PDE in the body [11], and it particularly hydrolyzes cAMP into its inactive form, 5-AMP [12].Phosphodiesterase-4B (PDE4B) is strongly expressed in the cornu ammonis 2 and cornu ammonis 3 areas of the hippocampus, the cerebellar granular layer, piriform, and parietal cortex [13].Te continuous rise in the production of PDE4 has been observed in the brains of AD patients, specifcally those with hippocampal dysfunction and cognitive impairment [14][15][16].PDE4 inhibitors (frst generation), such as rolipram ameliorate cognition, mitigate despair-like behaviors, anxiety, and promote neuroplasticity in the hippocampus subsequent to transient global cerebral ischemia [17], and they also exhibited serious side efects (nausea and emesis) at therapeutic doses.In similar manner, second-generation PDE4 inhibitors, including rofumilast and cilomilast, augment memory and cognition, increase neurogenesis, and promote neuroplasticity of the hippocampus [18].In clinical trials, the maximal dose remained limited owing to adverse efects [19].For the resolution of this problem, an alternative PDE4B inhibitor, specifcally one that originates from natural sources (plants), is required.According to the previous studies, dichloromethane Uvaria alba (U. alba) subextract was used to target PDE4 B2B and acetylcholinesterase (AChE) in in vitro assay due to its neuroprotective properties, especially in AD for the discovery of drugs [20].Te favonol-enriched n-butanol fraction of U. alba attenuated lipopolysaccharide-induced infammatory responses through the inhibition of proinfammatory NF-κB pathway and the increased NRF 2 levels [21] that are benefcial in neuroprotection.Moreover, in in vitro assay, sappanone A (obtained from heartwood of Caesalpinia sappan L.) efectively suppressed PDE4B1 enzyme activity and decreased TNF-α production in RAW264.7 macrophages leading to dual antioxidant and anti-infammatory activities [22].Vascular dementia, which makes up around 20% of dementia cases, is the second most prevalent type of senile dementia after AD.For vascular dementia, no medications are currently licensed.A wide range of risk factors, such as cerebral hemorrhage, cerebral infarct, arterial hypotension, and cerebrovascular disorders, have been linked to vascular dementia, even if the exact cause of the condition is still unknown.Moreover, changes in cyclic nucleotide levels, neuroinfammation, and excitotoxicity could all be part of the underlying mechanism.In a recent study, twenty-fve α-mangostin derivatives from Garcinia mangostana, administered at a dose of 10 mg/kg, showed impressive therapeutic efects in a vascular dementia model and spared beagle dogs from vomiting, suggesting that these derivatives have potential as a novel antivascular dementia drug [23].Terefore, white cabbage, red onion, and cofee extracts were considered for this study to fnd a natural cognitive enhancer.
White cabbage (Brassica oleracea), family Brassicaceae, is consumed worldwide both as a vegetable and in traditional medicine [24].Tis vegetable has been investigated for antipsychotic [25], anticholinesterase [26], antiinfammatory [27,28], and antioxidant activities [29].White cabbage contains abundant amounts of alkenyl glucosinolate, also known as sinigrin.Previously, sinigrin has been shown to increase the levels of cyclic nucleotides (cAMP) by blocking PDE4 in the lungs [30].On the fip side, cofee (Cofea robusta), family Rubiaceae, is consumed as a beverage, and because of its chlorogenic acid presence, efervescent granules are used as an antidiabetic agent [31,32] as well as often preferred because of its alertness enhancing, drowsiness, and fatigue-removing properties [33].Cofee has a wide range of pharmacological applications [31,32], including anti-Parkinson, anti-Alzheimer, and memory enhancement, and reduces the incidence of dementia [33,34], antiamyloidogenic [35], anti-infammatory [36], antioxidant [37,38], and antiglycation activities [38].Its phytochemical components, such as cafeine and chlorogenic acid, are linked to these efects [33].C. robusta is also a rich source of hydroxycinnamic acid derivatives, such as chlorogenic acid [39].Chlorogenic acid and its products can cross the blood-brain barrier (BBB) and ofer neuroprotection.In addition, chlorogenic acid has been studied for its potential to boost memory in those who have memory defciencies caused by sleep deprivation by inhibiting PDE4 activity [40].On the other hand, red onion (Allium caepa), family Amaryllidaceae, is an extensively consumed vegetable [41]. A. cepa has shown some reported biological activities, namely, antioxidant [42], anti-infammatory [43], neuroprotective properties [44,45], and its favonoids help to reduce the risk of dementia [46]. A. cepa is a complete source of benefcial bioactive compounds, especially favonoids [47]; among them, quercetin is a major phytochemical [48] that can penetrate BBB, inhibit PDE4, and promote the 2 Advances in Pharmacological and Pharmaceutical Sciences activation of kinases (such as protein kinase A).Tese kinases phosphorylate the transcriptional factor cAMPresponsive element-binding protein (CREB).Te CREB induces the expression of genes for neuronal plasticity, ofers neuroprotection, and improves memory function [46].Diferent components [49] or combinations of herbs function synergistically and are, therefore, an essential part of their therapeutic efcacy; many of the phytomedicines on the drug market are complete extracts of plants [50,51].Te three aforementioned were selected for this study because their major compounds (chlorogenic acid, quercetin, and sinigrin) have been previously shown to interact with PDE4.Furthermore, because these plants are safe and widely accessible, further research into their efects, either alone or in combination, can be done in vitro and in silico to increase PDE4B inhibition.
Tere is currently no evidence, either in vitro or in silico, that white cabbage, cofee, and red onion extracts as nootropics alone or in combination inhibit PDE4B and identify the chief bioactive chemicals present in cofee (in aqueous), red onion, and white cabbage (in ethanol) extracts by thin layer chromatography (TLC)-densitometry has been conducted.Tis investigation aimed to identify the major bioactive compounds in white cabbage, cofee, and red onion and to evaluate their activity against PDE4B via in silico and in vitro studies as well as computational pharmacokinetics and toxicities prediction.

Preparation of Extracts.
White cabbage (1 kg) and red onion bulb (1 kg) were rinsed with water and chopped separately.Te chopped materials were later placed in an electrical blender one by one and processed to obtain a fner form for extraction.Roasted cofee (1 kg) was also blended with an electrical blender.Te blended samples (white cabbage and red onion) were macerated with pure ethanol 100% (percent) (5 L) for each sample and placed overnight for at least 24 hours.Te cofee was macerated by the infusion method, where ground cofee was boiled in distilled water for 15 minutes.Whatman No. 1 flter paper was used to flter all extracts, and the solvents were evaporated using a rotary evaporator at 50-70 °C, and the extracts were stored in a desiccator at room temperature until dryness.

TLC and TLC-Densitometry
Analysis.TLC analysis was performed on an aluminum plate precoated with silica-gel F254 as the stationary phase and the mixtures of formic acid, ethyl acetate, and aquabidest (1 : 8 : 1.5; 25) and n-hexane, ethyl acetate, and formic acid (6 : 4 : 0.5; 19) as a mobile phase for cofee and red onion, respectively.To visualize the compounds, the TLC spots were observed under a ultraviolet (UV) light at 254 and 366 nm wavelength, as well as with FeCl 3 and citroborate for chlorogenic acid and quercetin, respectively.TLC-densitometry analysis was used to identify chlorogenic acid (1 mg/mL) and quercetin (1 mg/mL) in cofee (10 mg/mL) and red onion (10 mg/mL) extracts solution, respectively.Te maximum wavelengths of 330 nm and 380 nm were set for the TLC-densitometry analysis of chlorogenic acid and quercetin, respectively.

In Silico Molecular Docking Studies.
To determine the binding interaction of the test ligand with PDE4B (4KP6), molecular docking was carried out using the Molecular Operating Environment (MOE) software (MOE 2022.10), and the majority of the default tools (pocket atom, placement method (Triangle Matchter, London dG), refnement method (Induced ft and ASE), and energy minimization as 0.1 kcal/mol) were followed.Protein Data Bank (RCSB PDB, https://www.rcsb.org/)was used to acquire protein crystals of the PDE4B_HUMAN enzyme (UniProt ID: Q07343).For the desired expression, only 4KP6 was selected for docking, and the two-dimensional structures of previously investigated compounds were attained using SMILES ID in PubChem (https://www.pubchem.ncbi.nlm.nih.gov/).

Preparation of Test Ligands.
All test ligands' 3D structures (of total � 61 bioactive compounds from white cabbage, cofee, and red onion; Table 1) were created by MOE by minimizing energy following minimizing energy by MOPAC in MOE (0.1 kcal/mol) and saving them in a (.mdb) fle, and their 2D structures were retrieved from their smiles ID in PubChem.

Preparation of Protein-Ligand Complex.
A total of forty-two protein (PDE4B) crystal structures (including 4KP6) were retrieved from the PDB.Using quickprep in MOE software with the default parameters, the 3D structure of PDE4B (4KP6) was prepared by eliminating metal atoms and minimizing energy by MOPAC in MOE (0.1 kcal/mol).Te crystal protein 4KP6 complex was then saved as an MOE fle for further docking validation.

Validation of Docking Protocols.
To validate the pose or placement algorithm of the docked ligand, 4KP6 was redocked (by applying Induced ft, London dG, and ASE protocols) with its native ligand.Redocking and scoring function validation were used to validate the docking protocol.Te fexible docking on the protein's pocket atoms was done using the induced ft method.Te placement method was chosen as Triangle Matcher and London dG, and the scoring function was chosen as ASE.Before molecular docking of the test ligands, pose validation and scoring function of known ligands were analyzed.To evaluate pose validation, the binding afnities for ligand-enzyme complexes were calculated as kcal/mol, and the root mean square deviation (RMSD) was computed.Te RMSD value was regarded to be within the threshold limit, <2 Å (angstrom).Te association between docking score and IC 50 values of recognized ligands was calculated in order to examine the scoring function validation.

Docking of Test Ligands with Protein.
All the prepared 61 test ligands (excluding their six repetitions of the same compounds in the table) from white cabbage, cofee, and red onion (Table 1) were docked with the prepared PDE4B (4KP6) using the Induced ft, London dG, and ASE protocols, with 10 poses being set for each component.

Profles of ADME. SwissADME software (Molecular
Modeling Group, Swiss Institute of Bioinformatics, 2019) was used to computationally predict the ADME parameters of sixty-one secondary metabolites from white cabbage, red onion, and cofee extracts.Pharmacokinetic profles were assessed using Lipinski's "rule of fve," which examines a drug's biochemical characteristics that may afect how well it absorbs and permeates through cell membranes.A compound must meet at least three of Lipinski's criteria (molecular weight <500 Da, estimated lipophilicity (log P) <5, number of hydrogen-bond donors <5, and number of hydrogen-bond acceptors <10) in order to show drug similarity.Additionally, for in silico toxicity prediction, which considers the possible mutagenicity, tumorigenicity, irritating efects, and reproductive toxicity of the secondary metabolites from white cabbage, red onion, and cofee extracts, the OSIRIS property explorer program (Tomas Sander, Idorsia Pharmaceuticals Ltd., 2017) was utilized [20].

Statistical Analysis.
Te data are presented as the mean ± standard errors of means (SEM).To generate sigmoidal % activity versus concentration graphs, IC 50 was calculated using nonlinear regression curve ftting.One-way ANOVA was applied for analysis followed by Tukey's multiple comparison test, and a p value of 0.05 was established as the statistically signifcant value.GraphPad Prism version 5.0 was used to analyze the data.

Identifcation of Chief Bioactive Compounds by TLCdensitometry Analysis.
We identifed chlorogenic acid in cofee.On a silica-gel F254 plate, the compound was detected at UV 254 and 366 nm (Figures 1(a)(i) and 1(a)(ii), respectively).TLC-densitometry analysis was performed to confrm this fnding (Figure 1(b)).Quercetin was detected at UV 254 and 366 nm in red onion (Figures 1(c)(iii) and 1(c)(iv), respectively).Tis result was confrmed by TLCdensitometry analysis (Figure 1(c)).In the current docking research, it was discovered that the triazine ring of native ligand had a π-hydrogen bonding with Ile410 and its nitrogen on amine group on the same ring with Asn395 while its triazole ring had π-hydrogen interaction with Phe414 and π-π binding with Phe446; besides this, nitrogen in its triazole ring represented attachment with Met431 (Figures 2(a Quercetin (PubChem CID: 5280343), the bioactive component in red onion, and reference drug rolipram (PubChem CID: 5092) have also been studied to fnd out how well they interact with PDE4B (PDB ID: 4KP6).Figure 3 shows the 3D interaction of rolipram (binding energy −22.801 kcal/mol) with amino acids of PDE4B (PDB ID: 4KP6) in Figure 3 In order to evaluate chlorogenic acid (PubChem CID: 1794427) and alpha-tocopherol's (PubChem CID: 1742129) afnity for the PDE4B (PDB ID: 4KP6), chlorogenic acid, a cofee bioactive component, has also been studied.In Figure 4, Figure 4(a) depicts the 3D interaction of chlorogenic acid (binding energy −21.501 kcal/mol) with amino In the table (Table 2), the compounds' names, their structures (along with rolipram, as a reference), free bind energies (∆G bind [kcal/mol {kilocalorie/molar}]), and pIC 50 values have been enlisted.

In Vitro PDE4B1
Assay.An in vitro assay was performed to confrm the activities of the tested extracts, combination extract (white cabbage, red onion, and cofee), standards, and combination standard (sinigrin, quercetin, and chlorogenic acid) (Figure 6; Table 4).All samples were assayed at a concentration of 100 µg/ml with serial dilutions, and it was found that the sinigrin standard showed a very weak PDE4B1 inhibitory activity (Figure 6(a); Table 4); alternatively, white cabbage extract (Figure 6(b); Table 4) presented higher PDE4B1 inhibition than sinigrin standard.Similarly, the quercetin standard (Figure 6(c); Table 4) displayed PDE4B1 inhibitory activity which was greater than white cabbage extract but less than red onion extract (Figure 6(d); Table 4).Moreover, red onion extract showed this efect signifcantly more than the quercetin standard, and it is probably owing to the rest compounds such as polyphenols and organosulfates present in the extract.Likewise, chlorogenic acid (Figure 6(e); Table 4) exhibited PDE4B1 inhibition more than red onion while less than cofee extract (Figure 6(f ); Table 4).Te cofee extract showed a PDE4B1 inhibitory efect signifcantly greater than chlorogenic acid, and this maybe due to additional compounds such as cafeine present in the extract.Lastly, it was observed that combination extract (Figure 6(h); Table 4) represented signifcantly excellent PDE4B1 inhibitory activity with IC 50 at a concentration of 0.12 ± 0.03 µM among all under test standard and reference samples, even over combination standard (Figure 6(g); Table 4); however, our results were in accordance with the reference (rolipram).In the present study, rolipram (Figure 6(i); Table 4) was employed as a reference drug against all the test extracts and their standards and exhibited a PDE4B1 inhibitory activity slightly less than combination extracts, and contrarily, near to combination standard.IC 50 values (PDE4B1 inhibitory activities) of test compounds, extracts, and rolipram are summarized in Table 4 while Figure 6 represents nonlinear regression curves between PDE4 B1% activity and concentration (Log (µM)) of nine tested samples.

Discussion
Plants are the hidden source of mysterious therapeutic components [209].Over the last several decades, the therapeutic value of natural products has long gone unreported     Advances in Pharmacological and Pharmaceutical Sciences        Advances in Pharmacological and Pharmaceutical Sciences

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Advances in Pharmacological and Pharmaceutical Sciences in health services [210].Alternatively, numerous available data indicate that people used to consume herbs to treat a variety of ailments [211].Contrary to popular belief, plants play a crucial role in the provenance of current allopathic medications [212].Plant products have been examined based on folklore uses to enhance the quality of life and defne the scientifc horizon [213].Cabbage, cofee, and red onion are rich sources of glucosinolates, phenolic acids, and favonoids, respectively.Tese phytochemicals participate in controlling multiple diseases, such as neurodegenerative diseases, especially memory dysfunction by targeting PDE4 [30,39,40,46,47].Te chemical profle of cofee and red onion extracts was analyzed using a TLC-densitometry.Cofee and red onion extracts were dissolved in methanol as stated in the method section and analyzed on a precoated silica-gel TLC plate for the identifcation of chlorogenic acid and quercetin as major compounds.Te current study detected chlorogenic acid and quercetin in cofee and red onion, respectively, and evaluated their in silico and in vitro activities against PDE4B1 as well as pharmacokinetic and toxicity studies.In our previous study, Ahmad et al. 2023 followed the same procedure that identifed sinigrin (with an Rf 0.77 at maximum wavelength 399 nm) in white cabbage by using TLC-densitometry analysis [214]; hence, we excluded sinigrin in our current analysis.Molecular docking was carried out to gain insight into the binding afnity and potential interaction of PDE4B (PDB ID: 4KP6) with the bioactive compounds of white cabbage, cofee, and red onion.Te protein targets were validated (redocked) prior to conducting the docking research, and RMSD values were used as a parameter.RMSD is a distinctive characteristic that demonstrates the capacity of both native ligand complexes and proteins to duplicate themselves in the development of an appropriate confguration.Te preferred RMSD value is 1 Å, though 2 Å is also appropriate [215].Sixty-three compounds were tested against PDB4B (PDB ID: 4KP6) by performing their molecular docking with MOE.Te authors found that 28 compounds interacted with PDE4B (PDB ID: 4KP6) by binding majorly with amino acid residues (Phe 446 and Ile 410 ); however alpha-tocopherol, which is one of the previously reported bioactive components of white cabbage, Advances in Pharmacological and Pharmaceutical Sciences       [216,217].

Advances in Pharmacological and Pharmaceutical Sciences
∆G bind is a crucial parameter because it shows how strongly compounds attach to the receptor.Even, the stability and strength of the interaction between an enzyme and its ligand are also indicated by a meager ∆G bind .Te pharmacological impacts are infuenced by ∆G bind [215].Te compounds' ∆G bind was identical to one another, with the standard compound having the lowest free-bond energy.As a result, white cabbage, cofee, and red onion can be able to target PDE4B because their compounds (sinigrin, chlorogenic acid, and quercetin, respectively) interacted with PDE4B during docking, and they may also be an excellent source of a natural cognitive enhancer that can help treat memory impairment.Terefore, it can be suggested that white cabbage, cofee, and red onion extracts should be researched more thoroughly in in vivo investigations.Drug development has advanced to a new stage that uses computer-based drug design in predicting the ADME characteristics of the medications [218].A free online tool called SwissADME was used to assess the pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small compounds.Most of the compounds among sixty-one compounds from white cabbage, red onion, and cofee extracts exhibited to have no predictive toxicities during computational analysis.However, one by third part of the analyzed compounds possess predictive harmful efects which were predicted by SwissADME and OSIRIS.In a previous study, though there was a low GI absorption, quinadoline pharmacophore had an adequate ADME profle with logP and solubility in acceptable limits [219].Te fndings from brain and intestinal estimated permeation predictive model (BOILED-Egg) approach for the GI tract and brain permeation for the prediction of the top-ranked secondary metabolites were corroborated with the recent earlier study [20].Te results of this study indicate that additional research on the bioactive compounds of white cabbage, red onion, and cofee extracts against their respective target proteins is necessary to fnd new therapeutic leads against memory function and AD, as the top-ranked ligands have good pharmacokinetic features.
Te idea of virtual screening of secondary metabolites from white cabbage, cofee, and red onion extracts was sparked by the high global prevalence of memory impairments and the absence of efective and safe treatments.
According to the previous studies, the computational identifcation of kobophenol A as a promising lead drug against SARS-CoV-2 infection was confrmed through experimental validation, demonstrating its ability to prevent the binding of S1-RBD from SARS-CoV-2 to the host ACE2 receptor.Te data obtained indicated that kobophenol A might be explored further as a pharmacological treatment for SARS-CoV-2 infection that is safe, efcacious, and nontoxic [220].According to the in silico ADMET prediction, quinadoline B confers high drug-likeness, poor blood-brain barrier penetrability, and high gastrointestinal absorption, in accordance with Lipinski's rule of fve.Te complexes generated between the top-ranked ligands and their respective protein targets were dynamically stable and had high total free energy of binding, as demonstrated by MD modeling and subsequent total free energy calculation.Consequently, these substances may serve as models or models for the creation of multitargeting ligands directed against SARS-CoV-2 [221].In another study, MD simulations were performed on the compounds that rated highest in order to assess the stability of the systems over a predetermined period of time and to thoroughly examine the binding mechanism of the most promising derivatives.Ultimately, fve compounds with a better afnity for SARS-CoV-2 RdRp were found using the in silico searching procedure, piqueing researchers' interest in pursuing additional research on these compounds as potential antiviral medicines.Notably, the suggested computational methodology ofers a convenient computational procedure for hitto-lead optimization, with implications for anti-SARS-CoV-2 drug development and generally in the drug optimization process [219].One more study revealed that the complexes formed between the protein targets of the top-ranked ligands were found to be thermodynamically stable by MD modeling.Tese results, along with the advantageous pharmacokinetic characteristics of the top-ranked ligands, call for additional research on U. alba's secondary metabolites in relation to their individual target proteins in order to identify novel therapeutic approaches against AD and cancer [20].However, the current work on cabbage, cofee, and red onion extracts could serve as a foundation for the future search and creation of unique cognitive-enhancing drugs.
PDE4B is widely expressed in the diferent brain regions.PDE4B1, a long isoform of PDE4B [222], is mainly present in hippocampal CA2 and CA3 regions being more specifc than the PDE4B gene knockouts, thereby considering it a possible therapeutic target in cognition by promoting neuroplasticity [222,223] while PDE4B2B, a short isoform, usually expressed in cortex and hippocampus [224] and imparts crucial role in neuroinfammation [225,226].Its structural superposition showed a little conformational change, suggesting that the inhibitors might not be PDE4 subfamily-specifc [227].To determine the IC 50 values of the tested compounds (sinigrin, quercetin, and chlorogenic acid and their combination), white cabbage, cofee, and red onion and their combination extracts against PDE4B1, an in vitro assay was performed.Rolipram was used as a reference drug for PDE4B1 inhibitory activity.Tis study revealed diferent IC 50 values between sinigrin and Advances in Pharmacological and Pharmaceutical Sciences white cabbage extract against PDE4B1 that may be due to the extract containing other phytoconstituents.In one study, sinigrin enhanced intracellular cAMP levels by PDE4 inhibition at its highest concentration 10 −5 mol/L [30].However, literature is still lacking studies on sinigrin and white cabbage extract in connection with PDE4B1.Likewise, quercetin and red onion extract were assayed against PDE4B1 in order to determine IC 50 values.Our fndings indicated that red onion extract had more PDE4B1 inhibitory activity as compared to quercetin, which is a naturally occurring PDE4-selective inhibitor present in vegetables, tea, and fruits [228].Although quercetin inhibitory activity for PDE4D is available currently, a study gap for quercetin and red onion extract on PDE4B1 is still present.However, only quercetin 3-O-α-L-arabinopyranosyl-(1⟶2)-O-α-L-rhamnopyranoside, a derivative of quercetin, was found to show inhibitory activity against PDE4B with an inhibition 100% in an in vitro assay [229].Similarly, chlorogenic acid and cofee extract were assayed against PDE4B1 in order to examine their PDE4B1 inhibitory activities.Te results of the current study indicated that cofee extract had more PDE4B1 inhibitory activity as compared to chlorogenic acid.Tough chlorogenic acid was evaluated against PDE4 only in previous studies, the data are not available on chlorogenic acid and cofee extract against PDE4B1.Te authors employed rolipram as a reference drug against tested compounds (sinigrin, quercetin, and chlorogenic acid and their combination), combination (white cabbage, cofee, and red onion and their combination) extracts for PDE4B1 inhibitory activity.Our investigation exhibited that rolipram inhibited PDE4B1 more than all tested samples except combination extracts.In a previous study, Xia et al. determined the PDE4B1 inhibitory activity of rolipram in an in vitro fuorescence polarization assay where it showed IC 50 at a concentration of 0.13 ± 0.02 µM [216].Additionally, rolipram was also analyzed against PDE4B1 in an in vitro enzymatic assay and showed IC 50 at a concentration of 0.15 ± 0.02 µM by Tang et al. [230].Our results for all test compounds, extracts, and reference rolipram were consistent with the earlier studies against PDE4 B. As per the previous report, a single-dose triple combination has more efcacy, safety, and fewer side efects than high-dose single therapy and/or twice-a-day double therapy [231].Terefore, based on our in vitro experiment, a single-dose triple combination of extracts treatment can be safer and more efective than mono and double extracts in treating memory function.However, a further, in vivo approach is guaranteed to strengthen this evidence.
To explain how cabbage, cofee, and red onion extracts work, the author made a proposed mechanism of action targeting PDE4B that can help in further in vivo studies and drug development as well (Figure 7).According to the current fndings of the molecular analysis, cabbage, cofee, and red onion extracts work to prevent memory loss by acting as a PDE4B inhibitor.Te previous study embarks that inhibition of PDE4B enzyme triggers the activation of cAMP/PKA/CREB/BDNF signaling pathway and subsequently raises the expression of BDNF protein in the hippocampus, thereby improving memory function [232].According to the previously reported study fndings [30,40,46], chlorogenic acid, quercetin, and sinigrin from cofee, red onion, and white cabbage extracts, respectively, have both in vitro and in vivo PDE4 inhibitory actions.Efective methods for raising intracellular cAMP levels include inhibition of this enzyme which is benefcial for improving memory function [30,39,46,233].Te cabbage, cofee, and red onion extracts activate the adenylate cyclase pathway, as cAMP/PKA/CREB/BDNF pathway, thereby increasing the level of BDNF, a protein found in synapses, BDNF controls the expansion, upkeep, and maturation of brain neurons, expression in the hippocampus and preventing the memory loss.Te brain's temporal lobe, cerebral cortex, and hippocampal regions are where this protein is primarily expressed.BDNF is crucial for preserving the form and functionality of neurons.A defciency of neurotrophic protein can raise the risk of neuronal loss, which can cause damage and death to brain neurons.Tis loss of neurons impairs brain function physiologically and results in cognitive issues, including memory loss [1].However, the compounds present in white cabbage, cofee, and red onion extracts have the ability to block PDE4B and reverse memory loss.It is unknown if PDE4B negatively infuences the pathway involved in CREB-mediated neuronal survival.Te discovery of PDE4B-selective inhibitors has opened up new avenues for research into the dual function of PDE4B as a pro-survival and anti-infammatory target for a variety of neurological conditions and traumas especially in cognition.

Conclusion
In summary, quercetin and chlorogenic acid were identifed by TLC-densitometry as the major compounds in red onion and cofee, respectively.Te molecular docking study showed that among 28 bioactive components, alphatocopherol had the best binding interaction with the PDE4B whereas chlorogenic acid and quercetin demonstrated moderate interaction.In this experiment, sinigrin did not show the interaction with PDE4B.Te majority of compounds presented no toxicities.Te combination extracts showed stronger inhibition against PDE4B1 as compared to the combination standard and reference (rolipram).Further research on an in vivo model is needed to confrm this activity.
) and 2(b)).On the other hand, the known ligand (ZINC000043194322) (binding energy −35.123 kcal/mol) was found to interact in 2D with amino acids of PDE4B (PDB ID: 4KP6) in Figure2(c), whereas Figure2(d) represents a known ligand's 2D association with amino acids.Additionally, the known ligand's benzene ring displayed a π-π interaction with Phe446, and the pyridine ring had π-hydrogen bonding with Met347; however, the oxygen on the pyrimidine ring served as a sidechain donor at Met431 (Figures2(c) and 2(d)).
(a) and the 2D interaction in Figure 3(b).Tis study revealed that the nitrogen in the pyrrolidine ring in rolipram contributed sidechain to Asp 395 and the benzene ring interacted with Phe 446 by π-π binding interaction (Figures 3(a) and 3(b)).Alternatively, Figure 3(c) exhibits 3D interaction of quercetin (binding energy −17.252 kcal/mol) with amino acids of PDE4B (PDB ID: 4KP6) while Figure 3(d) represents 2D interaction of known ligand with amino acids.Te quercetin's benzene-1,2-diol ring interacted with Phe 446 through π-π binding and π-hydrogen binding with Ile 410 , while its hydroxyl group served as a donor to Asn 395 in these interactions.Moreover, the hydroxyl group on the benzene-1,2-diol ring of quercetin served as a donor to Tr 414 (Figures 3(c) and 3(d)).

Table 1 :
List of 67 reported test compounds from white cabbage, cofee, and red onion extracts excluding six repeated components.

Table 4 :
Summary of PDE4B1 inhibitory activities (IC 50 values) of test compounds, extracts, and rolipram.Te values were the average of the three independent experiments, expressed as means standard deviation (SD).IC 50 � median inhibitory concentration and µM � micromolar. * 28 Advances in Pharmacological and Pharmaceutical Sciences exhibited the strongest binding afnity for PDE4B (PDB ID: 4KP6) by interacting with Phe 446, Ile 410 , and Gln 443 residues while chlorogenic acid with Phe 446 , Ile 410 , and His 234 and quercetin with Phe 446, Ile 410 , and Asp 395 interacted moderately.Unfortunately, sinigrin did not represent any interaction with PDE4B (PDB ID: 4KP6).Te present study also exhibits that rolipram showed its afnity by creating an attachment with Phe 446 and Asp 392 residues.In an earlier investigation, test ligands interacted with PDE4 (1XMU) amino acids (Phe 446 , Asn 395 , Gln 443 , and Ile 410 ) [216].Similarly, in another study, rolipram generated a contact with Phe 446 , Asp 392 , and Ile 410 residues of PDE4 [217].It means that interaction with any one of the amino acids, such as Phe 446 , Asp 392 , and Ile 410 , can result in the inhibition of PDE4B activity.Based on these investigations, our docking results are in accordance with Xia et al. reports