Part2: Genistein: A Potential Natural Lead Molecule For New Drug Design And Development For Treating Memory Impairment

For more details, contact tina.xiang@wecistanche.com

Click the link to get part 1: https://www.xjcistanche.com/news/part1-genistein-a-potential-natural-lead-mol-55084007.html

4. Overview of the Mechanisms of Action of Genistein against MI

A number of in vivo evidence-based studies have reported that MI can be caused by a plethora of factors including aging [26], unhealthy lifestyles(i.e., chronic sleep de-privation)[20], neurotoxicants(i.e., lead and STZ) [13,24], neurodegenerative disorders (i.e., AD caused by LPS and β-amyloid)[23,27] and certain non-contagious disease(i.e., diabetes)[21]. The effectiveness of genistein in improving cognitive performances, especially in memory domains, including spatial, recognition, retention, and reference memories, has been reported in several of these studies(Table 1). Overall, genistein confers protection against MI by reducing oxidative stress[19-25,27], attenuating neuroinflammation [19,20,23], enhancing cholinergic neurotransmission [22,27, degrading pathological proteins [13], preventing apoptosis [21], and increasing the expression neuroprotective genes (CREB, CBP, BDNF, IGF-1, and ERK)[19,22].

The overproduction of reactive species especially reactive oxygen species (ROS) and reactive nitrogen species (RNS) coupled with the failure of the antioxidant system in the body can cause a cascade of cellular destruction, including neuronal damage [37]. When ROS and RNSlevels exceed the scavenging capacity of antioxidants in the body, oxidative stress occurs, which is harmful to cellular functions, and especially cognitive performance. The relationship between the onset of neurodegenerative disorders (NDD)(i.e., AD, PD, and HD) is closely linked with oxidative stress and its effect on the formation of neuronal plaques, neurofibrillary tangles (NFIs) as well as the formation of β-amyloid following the overproduction of ROS closely associated with the NDD [38]. In fact, the brain is said to be the most vulnerable to oxidative stress because of its particularly high oxygen consumption, high ROS production by the mitochondria, and low antioxidant capacity [20]. Genistein, an isoflavone mainly found in soybean, has been confirmed to have the ability to alleviate the deleterious effects of oxidative stress on neuronal injuries, such as preventing neuronal death [21], increasing the production of hippocampal glutathione(GSH) and superoxide dismutase (SOD) [20], and lowering lipid peroxidation, ROS, and nitric oxide production [19].

Click here to learn more about products

The activation of the neuro-immune cells (microglia and astrocytes) into pro-inflammatory states (also known as neuro-inflammation) is closely linked with the pathophysiology of NDD including AD [39]. Shahmohammadi et al. [23] and Lu et al.[20] respectively reported that both lipopolysaccharides(LPS) injection and sleep deprivation (SD) can lead to an increased level of pro-inflammatory cytokines and mediators, such as tumor necrosis factor α(TNF-x), nuclear factor activated B-cell (NF-kB), toll-like receptor 4 (TLR4), and interleukin 6(-6). The over-expression of TLR4 will activate neuro-inflammation signaling and increase the TLR4 level, which can worsen the memory processes in the hippocampus. Activation of NF-kB also mediates the production of other pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2), which can induce neuroinflammatory reactions [40]. In β-amyloid MI models, there is a decline in the activity of choline acetyltransferase (ChAT) in the cerebral cortex and hippocampus, along with the decreased production of acetylcholine (Ach) and dopamine [27. The dopaminergic-cholinergic system is closely linked with the mechanism of NDD and a decline in dopaminergic action which will lead to striatal cholinergic interneuron (SCI)over-activation. This phenomenon, in turn, contributes to a reduction in the release of dopamine. Additionally, both the degeneration of cholinergic and dopaminergic neurons are closely involved in the development of NDD[41]. In scopolamine MI models, genistein treatment significantly reduced acetylcholinesterase(AChE), increased ChAT activities, and increased the expression of Ach in the hippocampus compared with scopolamine models untreated with genistein, thus indicating the potential of genistein in increasing AChE activity and lowering Ach levels [22].

In many NDD, the pathological process includes the formation of pathological protein, such as β-amyloid 1-42 and tau protein, while recent studies suggest the utilization of autophagy to degrade these pathological proteins in order to effectively treat NDD [12]. In another study, a high dose of genistein (150 mg/kg/day)was administered in the STZ-induced AD animal model to stimulate autophagy of pathological proteins associated with AD. Genistein treatment significantly alleviated the memory and behavioral disturbances in the AD animal model, along with reducing the levels of pathological proteins in the hippocampus and cerebral cortex. Degradation of pathological proteins through autophagy by the action of genistein is confirmed as shown by the elimination of the primary cause of the disease, which is the accumulation and aggregation of pathological protein [13].

Diabetes-induced cerebral ischemia-reperfusion (IR) can trigger apoptosis, leading to neuronal death. Rajput et al. [21] investigated the effect of genistein in preventing neural cells apoptosis in an STZ-induced diabetic animal MI model. Genistein helps in obstructing neuronal apoptosis through some mechanisms, including(1)reducing the production and aggregation of β-amyloid,(2) reducing the phosphorylation of tau protein, and (3) eliminating ROS and free radicals, all of which are closely linked with neuronal apoptosis [42]. The transcription of factors, such as brain-derived neurotrophic factor (BDNF), cAMP-response element-binding protein (CREB), CREB-binding protein (CBP), insulin-like growth factor-1 (IGF-1), and extracellular signal-regulated kinase (ERK), are closely related to cognitive performance, especially with memory.

BDNF is a growth factor closely linked with the development and proliferation of neural cells and disturbance in BDNF levels would result in memory dysfunction. Memory disorder is also correlated with the perturbation of CREB levels. The main function of IGF-1 is to ameliorate the effect of growth hormone(GH) in humans. Its other effect concerns the proliferation of neurons in the brain, where the under-expression of IGF-1 is closely linked with memory impairment. Additionally, ERK plays an important role in synaptic plasticity and the development of memory domains [19,22].

The reported studies used a minimum of 0.5 mg/kg and a maximum of 150 mg/kg dose of genistein with treatment duration ranging from 4 to 90 days. Based on our review of the literature, we noticed that the obtained results were not compared to standard drugs. However, on the other hand, preclinical investigations strongly suggested that genistein is effective in enhancing the cognitive function of MI animal models, even at a low dose level thus indicating its potential efficacy for treating MI.

5. Pharmacokinetics and Bioavailability of Genistein

Due to its low molecular weight and lipophilic property, genistein is rapidly absorbed into the body. Yang et al. [43]reported a high absorption(77%)of genistein in rat/mouse while the lowest absorption reported was 28%where genistein showed different absorption depending on the region of the intestinal tract; duodenum(44%), colon (35%), ileum (18%), and jejunum(16%). Interestingly, there are gender differences in its absorption, as female rats had a relatively complete absorption (~100%) of genistein as compared to male rats (56%). Nevertheless, it is unclear why the discrepancies occur.

Jaiswal et al. [44] stated that orally, only 20-40%of genistein is absorbed which eventually contributes to the low bioavailability, with time to the maximal absorption (Tmax)at 5-6h and a half-life of 8h. Kwon et al.[45] reported that genistein, which is the glycoside form of genistein that is in a free form, has a greater bioavailability following oral administration. The occurrence is most likely due to the ability of genistein to be absorbed in both forms (genistein and genistein) in the intestinal tract where the former can passively be transported across the enterocytes membrane and directly enter the circulation with the help of sodium-dependent glucose transporter (SGLT1). Genistein is rapidly distributed throughout the body and is abundant mostly in the gastrointestinal tract （18.5 μg/g） and liver（0.98 μg/g）.The half-maximal effective concentration （EC50）of genistein exceeded in other tissues suggesting a better competition with estradiol to activate the estrogen receptors (ERa and ERβ).

Genistein, one of the dietary isoflavones, is metabolized in the intestine, which includes reactions such as oxidation, reduction, and conjugation. In fact, the metabolism of genistein is closely associated with the metabolism of endogenous estrogen, in which phase 2(conjugation) reactions are predominantly higher as compared with phase 1 (oxidation and reduction) metabolic reactions. The two major metabolic pathways for genistein are glucuronidation and sulfation, with the enzymes UDP glucuronosyltransferase(UDT) and sulfotransferase (SULT) mediating the conjugation reactions. Yang et al. [43]reported a high metabolic rate of genistein in the intestine following oral, intravenous, and intraperitoneal administration. Due to the high activity of UDT and SULT in the intestine, most of the dietary genistein are metabolized in the enterocytes before being transported to the liver where the remaining aglycone genistein is metabolized. It was also reported that UDT and SULT are also present in high concentrations in other tissues (kidney, heart, lung) indicating the possibility of genistein being metabolized in these sites. Nevertheless, the main sites for genistein excretion are biliary and renal excretions. In the urine, genistein is mainly excreted in its conjugated forms,i.e., monoglucuronide(53-76%), diglucuronide (12-16%), and sulfoglucuronide (2-15%). Overall, genistein is mainly excreted in the glucuronide

form compared to its aglycone, as evidenced by the high concentration of glucuronide genistein in the bile [44].

6. Challenges and Opportunities to Improve the Drug Delivery of Genistein for MI

The data acquired from DruLiTo software [46] indicate that genistein is a good potential drug-like molecule and can be a good therapeutic agent for a variety of disorders including neurodegenerative disorders (Table 2). Nevertheless, despite its promising therapeutic activity against MI as confirmed in several in vivo animal models studies, confirming its potential against Ml in human clinical trials remains a challenging obstacle. Several difficulties hindering its clinical effectiveness include its poor water solubility, insufficient targeting of pathological protein, rapid metabolism, and excretion, as well as low bioavailability following oral administration. Additionally, genistein can cross the blood-brain barrier(BBB) rapidly easily, making the brain another of its site of action (Figure 5)[44]. Although genistein is widely found in leguminous plants mainly in soybean, which is commonly consumed, phase I clinical trials can be commenced without any problems as soybean is deemed to be safe for human consumption and has been used in many traditional cuisines, especially in the Asian regions [8].

Low serum level of genistein following oral administration is a disadvantage, restricting its transport to the site of action and for the exertion of pharmacological actions. The use of nanoformulation to deliver genistein into the body may be an effective method to enhance its water solubility, permeability, bioavailability, as well as its overall therapeutic response. In addition to nanoformulation, other genistein formulation systems can also be considered to enhance its delivery into the human body, including administering as tablets (facilitates the immediate release of genistein), solid lipid microparticles (to enhance bioavailability), microparticles (to enhance water solubility and bioavailability), solid lipid nanoparticles (to enhance bioavailability), hydrogel matrix(to improve water solubility), micelles(to improve bioavailability after oral administration), nanostructured lipid carriers(to enhance oral bioavailability), and liposomes(to enhance permeability and retention) [44].

In addition, drug metabolism and pharmacokinetics (DMPK) research is crucial in order to comprehend both the efficacy and safety of genistein against NDD and its indirect protective effect against MI. Genistein is structurally modifiable to enhance its DMPK properties, along with amplifying its overall therapeutic properties including improving its aqueous solubility, increasing permeability and retention as well as ameliorating toxicity and adverse reactions. Finally, the mutual cooperation and understanding between experts in medicinal and organic chemistries are essential in modifying genistein for therapeutic and commercial use through drug research and development. The findings can be used to develop a potential natural lead compound for drug design and development in treating MI.

7. Possible Structural Modifications and Derivatives of Genistein

Chemists have been prompted to develop many derivatives of genistein (1) because of the recognized biological benefits. A series of naturally occurring genistein (1) and biochanin A(2) molecules, as well as their analogs (4-6), were synthesized and evaluated for antioxidant activity[471. The 7-O-carboxymethyl-genistein(8) was synthesized from genistein(1) or from sophoricoside (7) where the carboxylate group increased the water solubility of genistein(1) by more than a thousand-fold that are reported as valuable drug-like candidates in pharmacological actions [48].

The cytotoxic actions of the synthesized genistein amino acid derivatives (9a-9d)were in fact reported to be more effective than genistein (1)[49]. The 6-carboxymethyl genistein(10) has been identified as a new synthetic estrogen receptor modulator and an excellent substrate for building conjugates with anthracyclines [50,51]. Additionally, the antiosteoporotic and anti-proliferative properties of the derivatives (1la-e) have been described by a group of researchers [52,53]. Further, to improve solubility, a genistein-piperazine complex(12)was developed, in which the hydroxyl group at C-7 of genistein (1) was hydrogen-bonded to the nitrogen of piperazine [54].

To boost genistein's biological activity by enhancing its cellular absorption rate and extending its stability as well as blood circulation, Meng et al. [55]identified a number of genistein fatty acid esters (13a-13c). On the other hand, Matsumoto et al. [56] developed genistein derivatives (14a-14c) as nitric oxide donors, which have the potential to inhibit tyrosine kinase activity while also increasing nitric oxide levels. Kohen et al. [50] reported genistein derivatives (15a and 15b) with much more enhanced antiproliferative activity than genistein (1). Additionally, 7-O-modified derivatives of genistein (16a-16c, 17a, and 17b)were reported by Zhang et al. [57]

In another study, Li et al. [58] synthesized and investigated deoxybenzoin genistein derivatives for their antibacterial activity, and reported that the dimeric deoxybenzoin (18a-18e) derivatives are generally more active than genistein (1). Besides, Rusin et al. [59]and Rusin et al. [60] discovered a new genistein glycoside (19). The glycoside form of genistein (20a-20d) and O-tetradecanoyl-genistein(21) can reduce the clinical manifestations of experimental autoimmune encephalomyelitis, a murine autoimmune disease used to study multiple sclerosis [61]. Furthermore, anti-Alzheimer's activity has been studied for compounds 22a-22c and 23a-23h. Similarly, cholinesterase inhibition, metal-chelating activity, and human hepatoma cell viability were investigated for a series of new genistein-polyamine conjugates (24a-24h) [62].

It remains unclear whether the structural modifications of genistein can improve its physicochemical and biological properties, including its pharmacological actions against MI. Using various chemical processes, many analogs of genistein have also been produced (Figure 6). The synthesis of more potent genistein-derived compounds in silico may pave the way for new drug discovery and development. Nonetheless, more in vitro and in vivo research is needed to demonstrate the safety and efficacy of all semisynthetic genistein derivatives. In the future, more research into the structure-activity relationship (SAR) of genistein will be required to obtain various other unique compounds that can be developed from it. In addition to conducting the further study in order to better understand the therapeutic abilities of genistein against MI, additional experimentation to support brain targeting drug-delivery of genistein is required (Figure 7). Additionally, the implementation of randomized control trials can further strengthen the claims in this review.

8. Conclusions

Overall, genistein is a promising natural lead for improving cognitive performance in all three types of Mlmodels in animals (pharmacological, toxicological, and genetic models. Genistein attenuates neuronal damage via several mechanisms as described in this review and has demonstrated good potential to be investigated as a novel therapeutic agent in the treatment of Ml. Conclusively, through this review, it has been implicated that genistein in Ml animal models acts mainly through the antioxidant defense mechanism, conferring protection against inflammation, enhancing neurotransmission, inducing autophagy, and the prevention of apoptosis, and increasing the expression of neuroprotective genes. An in silico method to select more potent genistein-derived molecules could pave the way for new drug development and discovery. Nonetheless, more in-vitro and in-vivo research is needed to show that all semisynthetic genistein derivatives are safe and effective. More research into the structure-activity relationship (SAR) of genistein will be required in the future to produce a variety of additional unique molecules from it. Altogether, genistein is a potential natural lead for the design and development of a novel neuroprotective drug, in our perspective and based on scientific evidence.

Author Contributions: Writing—original draft, S.F, M.A.A.Y., M.S., and N.K.F; conceptualization, M.A.A.Y., and M.S.; supervision, S.E, M.S.and N.K.F; resources, S.E., M.A.A.Y., M.S., S.H.G. N.N.I.M.R., P.T.L., S.R., V.S., A.K.A., S.J., YS.W., D.U.M., K.V.S.and N.K.F.; data curation, S.F., M.A.A.Y., M.S., S.H.G.N.N.IM.R., PTL., S.R., VS., A.K.A., S.J., YS.W., D.U.MKVS.andNKE: writing—review and editing, S.F., M.A.A.Y., M.S., S.H.G., N.N.I.M.R., PTL., SR.V.S., A.K.A.S.J. Y.S.W., D.U.M., K.VS.and N.K.F.All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

References

1. Brewer, J.B.; Gabrieli, J.D.; Preston, A.R.; Vaidya, C.J.; Rosen, A.C. Chapter 5–Memory. In Textbook of Clinical Neurology; Goetz, C.G., Ed.; Elsevier: Amsterdam, The Netherlands, 2007; pp. 71–86.

2. Caselli, R.J.; Boeve, B.F. Chapter 33–The degenerative Dementia. In Textbook of Clinical Neurology; Goetz, C.G., Ed.; Elsevier: Amsterdam, The Netherlands, 2007; pp. 687–717.

3. Jankovic, J. Chapter 34–Movement disorders. In Textbook of Clinical Neurology; Goetz, C.G., Ed.; Elsevier: Amsterdam, The Netherlands, 2007; pp. 722–750.

4. Kinsella, L.J.; Riley, D.E. Chapter 40–Nutritional deficiencies and syndromes associated with alcoholism. In Textbook of Clinical Neurology; Goetz, C.G., Ed.; Elsevier: Amsterdam, The Netherlands, 2007; pp. 882–902.

5. Brown, P. Chapter 43–Transmissible spongiform encephalopathy. In Textbook of Clinical Neurology; Goetz, C.G., Ed.; Elsevier: Amsterdam, The Netherlands, 2007; pp. 953–965.

6. Menze, E.T.; Esmat, A.; Tadros, M.G.; Abdel-Naim, A.B.; Khalifa, A.E. Genistein improves 3-NPA-induced memory impairment in ovariectomized rats: Impact of its antioxidant, anti-inflammatory and acetylcholinesterase modulatory properties. PLoS ONE 2015, 10, e0117223. [CrossRef]

7. Kˇrížová, L.; Dadáková, K.; Kašparovská, J.; Kašparovský, T. Isoflflavones. Molecules 2019, 24, 1076. [CrossRef] [PubMed]

8. Rizzo, G.; Baroni, L. Soy, soy foods and their role in vegetarian diets. Nutrients 2018, 10, 43. [CrossRef] [PubMed]

9. Han, B.-Z.; Rombouts, F.M.; Nout, M.R. A Chinese fermented soybean food. Int. J. Food Microbiol. 2001, 65, 1–10. [CrossRef]

10. Nazari-Khanamiri, F.; Ghasemnejad-Berenji, M. Cellular and molecular mechanisms of genistein in prevention and treatment of diseases: An overview. J. Food Biochem. 2021, 45, e13972. [CrossRef]

11. Qiang, X.; Sang, Z.; Yuan, W.; Li, Y.; Liu, Q.; Bai, P.; Shi, Y.; Ang, W.; Tan, Z.; Deng, Y. Design, synthesis and evaluation of genistein-O-alkylbenzylamines as potential multifunctional agents for the treatment of Alzheimer’s disease. Eur. J. Med. Chem. 2014, 76, 314–331. [CrossRef]

12. Pierzynowska, K.; Gaffke, L.; Cyske, Z.; W˛egrzyn, G. Genistein induces degradation of mutant huntingtin in fibroblasts from Huntington’s disease patients. Metab. Brain Dis. 2019, 34, 715–720. [CrossRef]

13. Pierzynowska, K.; Podlacha, M.; Gaffke, L.; Majkutewicz, I.; Mantej, J.; W˛egrzyn, A.; Osiadły, M.; My´sli ´nska, D.; W˛egrzyn, G. Autophagy-dependent mechanism of genistein-mediated elimination of behavioral and biochemical defects in the rat model of sporadic Alzheimer’s disease. Neuropharmacology 2019, 148, 332–346. [CrossRef]

14. Yu, H.-L.; Li, L.; Zhang, X.-H.; Xiang, L.; Zhang, J.; Feng, J.-F.; Xiao, R. Neuroprotective effects of genistein and folic acid on apoptosis of rat cultured cortical neurons induced by β-amyloid 31–35. Br. J. Nutr. 2009, 102, 655–662. [CrossRef] [PubMed]

15. Zhou, X.; Yuan, L.; Zhao, X.; Hou, C.; Ma, W.; Yu, H.; Xiao, R. Genistein antagonizes inflammatory damage induced by β-amyloid peptide in microglia through TLR4 and NF-κB. Nutrition 2014, 30, 90–95. [CrossRef] 16. Shi, D.-H.; Yan, Z.-Q.; Zhang, L.-N.; Wang, Y.-R.; Jiang, C.-P.; Wu, J.-H. A novel 7-O-modified genistein derivative with acetylcholinesterase inhibitory effect, estrogenic activity, and neuroprotective effect. Arch. Pharmacal Res. 2012, 35, 1645–1654. [CrossRef] [PubMed]

17. dos Santos Petry, F.; Hoppe, J.B.; Klein, C.P.; Dos Santos, B.G.; Hözer, R.M.; Bififi, F.; Matté, C.; Salbego, C.G.; Trindade, V.M.T. Genistein attenuates amyloid-beta-induced cognitive impairment in rats by modulation of hippocampal synaptotoxicity and hyperphosphorylation of Tau. J. Nutr. Biochem. 2021, 87, 108525. [CrossRef] [PubMed]

18. Park, Y.-J.; Ko, J.W.; Jeon, S.; Kwon, Y.H. Protective effect of genistein against neuronal degeneration in ApoEE / mice fed a high-fat diet. Nutrients 2016, 8, 692. [CrossRef]