EJBR2020v10i4art314 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2020; 10(4): 314-325 DOI: http://dx.doi.org/10.5281/zenodo.4009588 Validation of Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease Mohammad Azizur Rahman1,2*, Shahdat Hossain1, Noorlidah Abdullah2, Norhaniza Aminudin2,3 1 Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh 2 Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 3 University of Malaya Centre for Proteomics Research, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia * Corresponding author: E-mail: azizbmb@juniv.edu Received: 08 June 2020; Revised submission: 10 August 2020; Accepted: 27 August 2020 http://www.journals.tmkarpinski.com/index.php/ejbr Copyright: © The Author(s) 2020. Licensee Joanna Bródka, Poland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: Ganoderma lucidum has been hailed as medicinal mushroom. Its effect on memory and learning related behavioral performance along with related protein markers has been evaluated using Alzheimer’s disease (AD) and hypercholesterolemic model rats in the present study. AD model rats were prepared infusing amyloid beta peptide into the right ventricles of the rats. Hypercholesterolemia was evoked feeding 1% cholesterol and 1% cholic acid with basal diet of the rats for 8 weeks. Hot water extract of G. lucidum was ingested orally (200 mg/kg bw) to the HC and AD model rats. Memory and learning related behavioral tests were performed using Barnes maze while protein markers (BDNF, SNAP2, PSD-95, VAchT) were detected using ELISA. Observed findings suggest hypocholesterolemic, lipid profile improving and enhanced cognitive performance of the G. lucidum fed rats. Memory and learning related protein markers also substantiate this fruition. Thus, therapeutic potentiality of Ganoderma lucidum in AD amelioration seems promising. Keywords: Alzheimer’s disease; Ganoderma lucidum; Hypercholesterolemia; Memory and learning; Protein markers of memory and learning. 1. INTRODUCTION Alzheimer’s disease (AD), a neuro-degenerative disorder affects memory and learning abilities and induces behavioral disorder especially in the elderly people [1]. Neurons and neurotransmitters associated with memory, learning and behavioral normalcy become disrupted in the AD patients [1]. Deposition of amyloid beta (Aβ) plaques and/or neurofibrillary tangles (NFTs) in the AD neurons impairs their usual activities [2]. Consequently, AD patients become solely dependent on their family members and care givers and add extra burden as well as negative impact on national and global economy [3,4]. Unfortunately, there is hardly any treatment for more than 40 million AD patients suffering globally [5]. Current COVID-19 epidemic has lessened the attention toward AD medication harshly though the aged people are vulnerable to both AD and COVID-19 [6]. The available therapeutic strategies are aimed at symptom – modifying targets Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 315 European Journal of Biological Research 2020; 10(4): 314-325 rather than preventing the neurons from damages [7]. For example, the drugs (donepezil, galantamin, memantine, rivastigmine) approved by the United States Food and Drug Administration (USFDA) can improve AD symptoms only through modulating brain neurotransmitter release [7]. Though AD symptoms appear in late 60s or later, initiation and progression of AD pathogenesis occurs at or during early 40s [8]. Thus, if protective measures could be taken at or before mid-ages of life, AD progression could be withheld, albeit, reduced [8]. The failure to achieve the ultimate goal in slowing AD progression might remain hidden into multiple causative factors encompassing oxidative stress (OS), hypercholesterolemia, hypertension, genetics, epigenetics as well as adaptive response to some stressors [1,5]. Thus, effective management of the co-existing factors of AD has been highly regarded in the most recent recommendation from the Alzheimer’s association [1,5]. Thus, time is up for considering AD ameliorating therapeutic approaches that would be easy to reach to the common mass of both the developing and developed nations. Hypercholesterolemia refers to the elevated level of cholesterol in the blood [9]. Hypercholesterolemia stems from the increased synthesis and/or decreased removal of endogenous cholesterol and also from the excessive supply of dietary cholesterol or cholesterol precursors [10]. Hypercholesterolemia plays pivotal role in the progression of AD [9-11]. Increased brain level of cholesterol has been found during AD progression [9-11]. The role of cholesterol in AD pathogenesis becomes clearer from the opposite relationship between plasma HDL-cholesterol level and dementia [9-12]. HDL is the main lipoprotein in the human brain and can slow down the in vitro aggregation of Aβ and subsequent toxicity [9-12]. Some studies have indicated 2-3 times greater risk of late-age dementia and AD for those having mid-life hypercholesterolemia [9-12]. Thus, hypercholesterolemia stands as an early risk factor for AD [13-14]. Similarly, hypercholesterolemia is among the major modifiable risk factors of CVD [13-14]. Cholesterol lowering drugs, statins or 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to lower the risk of death or cardiovascular events in patients with or without CVD as well as protective against the risk of developing AD [15-17]. Statins have been found effective in reducing the risk of AD from 60% even up to 74% [15-17]. Statins can directly withstand the production of Aβ and also facilitate their removal from the brain [15-17]. The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice had suggested life-style and dietary modification as the first line and pharmacological intervention through utilization of lipid lowering drugs as the second line of treatment strategy against hypercholesterolemia [18]. Life style modification involves loss of weight, consumption of diet containing less cholesterol and higher saturated fatty acid, giving up of cigarette smoking and alcohol drinking, intake of unsaturated fatty acids such as DHA and regular physical exercise [18]. However, combination of both life style modification and pharmacological treatment sound better in real life [18]. Both edible and medicinal mushrooms lower blood total cholesterol (TC), very low density lipoproteins (VLDL) and low density lipoproteins (LDL) in rats [19-28]. Investigations involving the potentiality of mushrooms as the inhibitor of cholesterol biosynthetic key enzyme 3-hydroxy 3-methyl glutaryl co-enzyme A reductase (HMGCo-AR) have yielded promising outcome [28]. Ganoderma lucidum has been hailed for its immense medicinal values [19, 21-23]. Among them, its antioxidative, anticancer, antibiotic, antiviral, hepatoprotective and neuroprotective effects are most notable [19, 21-23]. Medicinal feat of G. lucidum could be attributed to its content of polysaccharides, polyphenols and triterpenes [23-25]. Despite vast expanse of research concerning G. lucidum, there is hardly any study addressing combined hypocholesterolemic and AD ameliorating effect of this mushroom, though both of these pathophysiologies Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 316 European Journal of Biological Research 2020; 10(4): 314-325 have some common links. Thus, the present study has been designed to elucidate the effect of G. lucidum on experimentally induced hypercholestorlemic and AD model animals. 2. MATERIALS AND METHODS 2.1. Preparation of hot water extract of G. lucidum Purchased from a local farm in Selangor, Malaysia, the fruiting bodies of G. lucidum were identified and authenticated by experts via DNA sequencing by Mushroom Research Centre, University of Malaya herbarium. A voucher specimen was deposited in the University of Malaya herbarium (KLU-M1233). Powdered fruiting bodies of G. lucidum were boiled in distilled water at the ratio of 1: 20 (w/v) for 45 minutes. Cooling was followed by removal of the boiled mushrooms using Whatman No. 1 filter paper. Then, the hot water extracts (HWE) of G. lucidum were obtained using freeze-dryer (Labconco). 2.2. Preparation of AD and HC model animals 2.2.1. Animals, maintenance and dosage of treatment Forty eight wistar male rats (weight range 120 ± 5 gm) were divided into six groups: control (C), G. lucidum HWE fed control (CE), hypercholesterolemic (H), G. lucidum HWE fed hypercholesterolemic (HE), Alzheimer’s disease (A) and G. lucidum HWE fed Alzheimer’s disease (AE) each group containing 8 rats. The extract fed groups (CE, HE and AE) received 200 mg/kbw G. lucidum HWE. Animals had been housed in a 12 hr day night cycle at 25±2°C temperature. All the experimental protocols had been approved by the ethical permission committee, University of Malaya Institutional Animal Care and Use Committee (UMIACUC) [Ethics reference no. ISB/25/04/2013/NA (R)]. 2.2.2. Induction of hypercholesterolemia Hypercholesterolemia to the H rats was evoked by adding 1% cholesterol and 1% cholic acid (for intestinal better absorption of cholesterol) with the basal diet of the rats [23]. 2.2.3. Preparation of AD model rats Alzheimer’s disease model rats (A) were prepared by infusing Aβ1-42 (ab120959, Abcam, USA) to the cerebral ventricles following the method of Abdullah et al. [29]. 2.3. AD studies 2.3.1. Barnes maze study Memory and learning related behavioral performance of the rats was evaluated using Barnes maze. A Barnes maze made of stainless steel and having a diameter of 150 cm was used (Fig. 1). It contained 20 holes, each having a diameter of 10.5 cm. One of the holes contained the dark escape box. The maze top has been placed on a metal stand and elevated 100 cm above the floor. Intra- and inter-maze cues of different types such as colored paper-shapes (round, square and triangle) had been placed as the landmarks for cognition and spatial memory. The surface of the maze had been brightly illuminated using flush (120 W) light based on the principle that the rodents tend to hide in the dark corner as compared to the illuminated open center of a circular surface. In Barnes maze, spatial learning and memory is measured based on the rodents’ ability to learn and remember the location of the hidden escape box using the visual cues. After experimentation with each rat, the target hole and the whole maze was cleaned with 70% ethanol. All the sessions were recorded Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 317 European Journal of Biological Research 2020; 10(4): 314-325 using video camera (HDR CX130E, Sony, Japan) and Arcsoft showbiz software and the video files were tracked with the tracking software Kinovea. 2.4. Sacrifice of the animals Twenty four hours following the last treatment and test, the rats were kept fasting overnight. Then, the rats were anesthesized with intra-peritoneal injection of sodium pentobarbital (35 mg/kbw) and sacrificed. 2.5. Statistical analyses Conducting all the experiments in triplicate, data have been presented as mean±SEM. Using SPSS version 16, ANOVA was performed following least significance difference at 95% level. 3. RESULTS AND DISCUSSION 3.1. Hypocholesterolemic effect of G. lucidum HWE 3.1.1. Effect of G. lucidum HWE on body weight change Gradual increase in body weight (ranging from 140 ± 5 g up to 280 ±2 g) of the rats of all the groups was noticed throughout the experimental period (Fig. 1). However, the hypercholesterolemic rats (H) gained the maximum weight and their rate of becoming weighty surpassed that of the others. Weight gain tendency of the normo-cholesterolemic rats was moderate and feeding of G. lucidum HWE resulted in decreased weight gain as time passed by. Similarly, G. lucidum HWE fed AD rats experienced relatively lower body weight growth than their non-fed counterparts. These findings indicate towards body weight lowering potencies of the G. lucidum HWE up on the experimental animals. Figure 1. Effect of G. lucidum HWE upon body weight change of the rats. Data are expressed as mean±SEM of triplicate measurements. Data were analyzed with one-way ANOVA and post-hoc Tukey’s HSD test (P≤0.05). Where, C = Control, CE = G. lucidum HWE fed control, H = hypercholesterolemic, HE = G. lucidum HWE fed hypercholesterolemic, A = AD model rats and AE = G. lucidum HWE fed AD rats, respectively (n=8 for every group). Current findings are in agreement with several others who reported a gradual increased body weight in the cholesterol fed rats [30]. As the present experimental animals were almost matured from their beginning of the experiment, decreased body weight gain of the G. lucidum HWE fed rats might occur due to decreased Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 318 European Journal of Biological Research 2020; 10(4): 314-325 fat (triacylglycerol) deposition, decreased cholesterol biosynthesis or increased lipolysis and/or the combined effect of all. 3.1.2. Effect on plasma TG level Feeding of hypercholesterolemic diet to the rats resulted in their increased plasma TG levels (Table 1). The hypercholesterolemic (H) rats had 1.88 times higher plasma TG level compared to the controlled (C) rats indicating the increased atherogenic propensity of the H rats. Feeding of G. lucidum HWE lowered plasma TG level in both the control, hypercholesterolemic and AD rats. Plasma TG lowering effect of the G. lucidum HWE was in the order of controlled (20%) > hypercholesterolemic (16%) > AD (11%) rats (Table 1). TG lowering effect of G. lucidum HWE was statistically significant in each group compared to their respective controls (Table 1). Increased TG levels in the hypercholesterolemic rats might be due to the decreased clearance of TG owing to the lowered lipoprotein lipase (LPL) activity or due to increased deposition of LDL-C [31]. TG lowering effect of the HWE G. lucidum might be mediated through increased inhibition of the HMGR by the phenolics content of the HWE of G. lucidum [23, 28]. Table 1. Lipid profile of the experimental animals. Plasma parameter (mg/dL) C CE H HE A AE TG 120.73 ± 0.63a 100.93 ± 0.85b 197.0 ± 0.880c 160.33 ± 0.90d 145.93 ± 0.70e 135.33 ± 0.52f TC 94.6 ± 0.74a 85.9 ± 0.91b 139.2 ± 0.84c 110.4 ± 1.0d 118.67 ± 0.85e 103.87 ± 0.56f HDL-C 28.06 ± 0.42a 34.47 ± 0.55b 27.94 ± 0.58c,e 34.33 ± 0.40d,f 27.73 ± 0.40c,e 33.4 ± 1.35d,f LDL-C 37.97 ± 0.58a 25.68 ± 0.10b 68.86 ± 0.90c 44.60 ± 1.34d,f 57.75 ± 0.91e 40.8 ± 0.57d,f VLDL-C 25.54 ± 0.13a 21.78 ± 0.17b 39.40 ± 0.18c 32.47 ± 0.18d 29.19 ± 0.14e 25.67 ± 0.10f Data are expressed as mean±SEM of triplicate measurements each (n=8). Mean values containing different lower case superscripts are statistically significant at P≤0.05 level with one-way ANOVA and post-hoc Tukey’s HSD test of every triplicate data. Where, TG = triacylglycerol; TC = total cholesterol; HDL-C = high density lipoprotein-cholesterol; LDL-C = low density lipoprotein cholesterol; VLDL-C = very low density lipoprotein cholesterol; C = Control, CE = G. lucidum HWE fed control, H = hypercholesterolemic, HE = G. lucidum HWE fed hypercholesterolemic, A = AD model rats and AE = G. lucidum HWE fed AD rats (n=8, in every group), respectively. 3.1.3. Effect on plasma TC level Feeding of hypercholesterolemic diet to the rats resulted in 1.62 times increased plasma TC levels (Table 1). Later on, treating the rats with G. lucidum HWE lowered plasma TC level significantly (P≤0.05) in all the rat groups. TC lowering effect of G. lucidum might be mediated by competitive inhibition of HMG Co-A reductase, the rate limiting step of cholesterol biosynthesis, or through inhibition of 14α demethylase by the ganoderic acids or by impaired intestinal absorption by β-D glucan present in the G. lucidum HWE [24]. 3.1.4. Effect on plasma HDL-C level In the present study, the hypercholesterolemic and AD rats had lowered plasma HDL levels compared to the normo-cholesterolemic controls (Table 1). Feeding of G. lucidum HWE increased plasma HDL-C levels significantly (P≤0.05) in all the rat groups. However, the rate of increasing was highest in the Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 319 European Journal of Biological Research 2020; 10(4): 314-325 normocholesterolemics and the increasing trend was C (27.5%) > H (22.80%) > A (20.20%) rats (Table 1). Lowered level of HDL-C in the hypercholesterolemic rats of the present study might be due to the accelerated clearance of apo A1 from the plasma following hypercholesterolemia in the H and A rats [32]. On the other hand, significantly increased (P≤0.05) plasma HDL-C level in the G. lucidum HWE fed rats indicates increased clearance of TC from the peripheral tissue to the liver for excretion that points towards CVD ameliorating effect of G. lucidum HWE. 3.1.5. Effect on plasma LDL-C and VLDL-C level Plasma LDL-C level increased 1.92 times in the hypercholesterolemic and 1.42 times in the AD rats, compared to the respective controls (Table 1). Similarly, plasma VLDL-C level increased by 1.76 times in the H and by 1.65 times in the A rats. Increased levels of LDL-C and VLDL-C in the hypercholesterolemic rats might arise from their intake of hypercholesterolemic diet that might have downregulated the hepatic LDL receptors of the H rats [32]. Ganoderma lucidum HWE supplementation caused significant lowering effect upon plasma level of both LDL-C and VLDL-C in all the rat groups (Table 1). Ganoderma lucidum HWE caused significantly lowering effect upon the plasma LDL-C level of both H and AD rats as compared to the controlled. Ganoderma lucidum HWE mediated decreased cholesterol absorption and biosynthesis might cause decreased availability of hepatic cholesterol for lipo-protein biosynthesis in the extract fed rats (CE, HE, AE). As a consequence, decreased VLDL secretion in the plasma along with decreased conversion of VLDL into LDL may end into lowered plasma LDL level [32]. Mechanistically, G. lucidum HWE induced increased LDL receptor in the rat hepatocytes may contribute to the LDL lowering effect that resulted in lowered secretion of LDL in the rat plasma [32]. Increased clearance of LDL from the blood of the mushroom-fed rats may also have been involved [32]. 3.2. AD ameliorating effects of G. lucidum HWE 3.2.1. Barnes maze study Compared with the respective controls, the G. lucidum HWE fed rats made less mistake in finding the escape box (Fig. 2). This observation corresponds towards enhanced memory and learning abilities of the G. lucidum HWE fed rats. To the best of our knowledge, ours is the first report concerning Barnes maze study of G. lucidum HWE fed rats and thus comparative discussion could not be furthered. Ganocomponents of different structure and function might have imparted AD ameliorating effect observed in this behavioral study [20-24]. 3.2.2. Effect of G. lucidum HWE on memory and learning related markers 3.2.2.1. BDNF Brain derived neurotrophic factor (BDNF), a neuroprotectin group of growth factor, is involved in neuronal survival and functioning. Its pivotal role in in non-neuronal cells such as in smooth muscle and endothelial cells have also been documented [34]. Though BDNF has been regarded as a pro-atherogenic factor, recent studies have reported its ameliorating effects on CVD [35]. Elevated BDNF levels have been reported to be associated with lowered CVD morbidity and mortality [35]. In the present study, we observed significantly reduced level of BDNF in the AD rats compared to those of the controls (Fig. 3). However, BDNF level increased in the G. lucidum HWE fed rats: in the HC rats, Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 320 European Journal of Biological Research 2020; 10(4): 314-325 the level increased much than those of the AD rats. Thus, increased BDNF level in the G. lucidum HWE fed rats indicate both AD and hypercholesterolemia ameliorating effects. In this regard, our findings coincide with those of Kaess et al. [34]. Observed memory and learning related behavioral attainment of the G. lucidum HWE fed rats are also substantiated by this marker [35-38]. Tri-terpenoids and phenolics present in G. lucidum HWE might have imparted BDNF and memory enhancing as well as hypocholesterolemic effects [39]. Figure 2. Total errors of the rats in finding escape cage. Data are expressed as mean±SEM (n=8; each trial is the average of six sessions). Data were analyzed with one-way ANOVA and post-hoc Tukey’s HSD test (P≤0.05). Here, C = Control, CE = G. lucidum HWE fed control, A = AD model rats, AE = G. lucidum HWE fed AD rats, H=hypercholesterolemic and HE = G. lucidum HWE fed hypercholesterolemic rats, respectively. Figure 3. Effect of G. lucidum HWE on memory and learning related markers. Mean values containing different lower case superscripts are statistically significant at P≤0.05 level with one-way ANOVA and post-hoc Tukey’s HSD test (n=8). Here, BDNF = Brain derived neurotrophic factor, SNAP = Synaptosomal associated protein, PSD95 = Post-synaptic density protein 95 KD, TNFα = Tumor necrosis factor alpha, C = Control, CE = G. lucidum HWE fed control, H = hypercholesterolemic, HE = G. lucidum HWE fed hypercholesterolemic, A = AD model rats and AE = G. lucidum HWE fed AD rats, respectively. Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 321 European Journal of Biological Research 2020; 10(4): 314-325 3.2.2.2. SNAP 25 Synaptosomal-associated protein 25 KD (SNAP 25), a pre-synaptic membrane protein plays important role in maintaining long-term potentiation (LTP) and working memory [40-41]. Reduced level of SNAP25 had been reported in AD brains [40-41]. Similar trend was observed in the hippocampi of the AD rats in the present study (Fig. 3). Interestingly, G. lucidum HWE fed rats showed significantly (P ≤ 0.05) soared level of SNAP 25 in their hippocampi that might impart improved cognitive performance of the G. lucidum HWE fed rats. SNAP25 has also been implicated in elevating serum triacylglycerol level and weight increment [42]. Thus, the increased SNAP25 level in the HC rats might have been due to elevated expression of SNAP25 in those rats. 3.2.2.3. PSD 95 Post-synaptic density and maturation of the excitatory synapses are maintained by the post-synaptic density protein 95 KD (PSD95) [43]. Inverse relationship between Aβ and PSD95 level has been reported in the AD hippocampi [44]. In line with this, lowered level of PSD95 has been observed in the AD rats hippocampi, compared with those of the normal (Figure 3). There was an increment of PSD95 level in the G. lucidum HWE treated rats’ hippocampi that correspond to their improved memory and learning abilities (Fig. 3). Besides, level of PSD95 had been reported to be increased in hypercholesterolemic rats [45]. Elevated level of PSD95 in the HC rats of the present study coincide with the reported findings [45]. 3.2.2.4. TNFα Tumor necrosis factor alpha (TNFα), a neurotoxin, acts as pro-AD signaling agent, promotes atherogenesis and disrupts lipid metabolism [46-48]. Increased level of TNFα in the AD and HC rats correspond towards disrupted cognitive performance and dyslipidemia of the respective rat groups (Fig. 3). These AD hallmarks have been ameliorated with G. lucidum HWE treatment that might be attributed towards its content of phenolics, triterpenoids and polysaccharides [19-23]. 3.2.2.5. VAchT Cholinergic neurotransmission is regulated by the vesicular acetylcholine transporter (VAchT) and its reduced level impairs cognitive performance, another hallmark of AD [49]. AD and HC rats of the present study showed lower level of VAchT while those of the G. lucidum HWE treated showed enhanced level (Fig. 3). Thus, AD and HC rats had suffered from disrupted cholinergic neuronal activities while G. lucidum HWE treatment could ameliorate this disruption [50-51]. 4. CONCLUSIONS Infusion of soluble Aβ1-42 to the rat cerebral ventricles affected AD model rats’ memory and learning related behavioral tasks indicating the effectiveness of the current model of AD studies. Hypercholesterolemic model rats also showed poor performance in behavioral tests. Feeding of G. lucidum HWE to the AD and hypercholesterolemic rats improved their memory and learning abilities. Memory-related protein marker tests also indicate hypercholesterolemia and AD ameliorating effect of G. lucidum. Thus, G. lucidum could be regarded as an AD and hypercholesterolemia ameliorating agent. However, further study is needed for formulating therapeutic dosage. Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 322 European Journal of Biological Research 2020; 10(4): 314-325 Authors' Contributions: NAb, NAm and SH planned and supervised the research, edited the manuscript. MAR conducted the research, statistical analyses and prepared the manuscript. The final manuscript has been read and approved by all authors. Conflict of Interest: The author has no conflict of interest to declare. Acknowledgment: The authors gratefully thank University of Malaya and the Ministry of Higher Education, Malaysia for the HIR-MOHE Research Grant F000002-21001 funding and Mohammad Azizur Rahman is grateful to Jahangirnagar University and the University Grants Commission of Bangladesh. REFERENCES 1. https://www.alz.co.uk/research/WorldAlzheimerReport2018.pdf?2 2. Hardy JA, Higgins GA. Alzheimer's Disease: The amyloid cascade hypothesis. Sci. 1992; 256: 184. 3. Michael SM, Jeffrey LC, Tara F, Jeffrey G. The spectrum of behavioral changes in Alzheimer's disease. Neurol. 1996; 46: 130-135. 4. Reisberg B, Borenstein J, Salob SP, Ferris SH. Behavioral symptoms in Alzheimer's disease: phenomenology and treatment. J Clin Psychiat. 1987; 48: 9-15. 5. Alzheimer’s Association. Alzheimer’s Association Report. Alzheimer’s disease facts and figures. Alzh Dement. 2018; 14: 367-429. 6. Sheraton M, Deo N, Kashyap R. A review of neurological complications of COVID-19. Cureus. 2020; 12(5): e8192. 7. Mielke MM, Jeannie-Marie L, Chris DC, Robert CG, Maria CN, Kathleen AWB, et al. Effects of FDA approved medications for Alzheimer’s disease on clinical progression. Alzh Dement. 2012; 8: 180-187. 8. Hamel E, Jessika R, Brice O, Xin-Kang T. Neurovascular and cognitive failure in Alzheimer’s disease: benefits of cardiovascular therapy. Cell Mol Neurobiol. 2016; 36: 219-232. 9. Reitz C. Dyslipidemia and the risk of Alzheimer’s disease. Curr Atheroscler Rep. 2013; 15: 307-313. 10. Sparks DL, Stephen W, Scheff JC, Hunsaker III, Huiachen L, Teresa L, David RG. Induction of Alzheimer-like β-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol. Exp Neurol. 1994; 126: 88-94. 11. Refolo LM, Pappolla MA, Malester B, LaFrancois J, Bryant-Thomas, Wang R, et al. Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model. Neurobiol Dis. 2000; 7: 690-691. 12. Refolo LM, Pappolla MA, LaFrancois, Malester J, Brian S, Stephen DJ, et al. A cholesterol-lowering drug reduces β-amyloid pathology in a transgenic mouse model of Alzheimer's disease. Neurobiol Dis. 2001; 8: 890-899. 13. Anstey KJ, Lipnicki DM, Low LF. Cholesterol as a risk factor for dementia and cognitive decline: a systematic review of prospective studies with meta-analysis. Am J Ger Psychiat. 2001; 16: 343-354. 14. Mathew A, Yoshida YM, Kumar T, Sakthi D. Alzheimer's disease: Cholesterol a menace? Brain Res Bull. 2011; 86: 1-12. 15. DeKosky ST. Statin therapy in the treatment of Alzheimer disease: what is the rationale? Am J Med. 2005; 118(12): 48-53. Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 323 European Journal of Biological Research 2020; 10(4): 314-325 16. Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G. Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol. 2000; 57(10): 1439-1443. 17. Fassbender K, Simons M, Bergmann C, Stroick M, Lütjohann D, Keller P, Hartmann T. Simvastatin strongly reduces levels of Alzheimer's disease β-amyloid peptides Aβ42 and Aβ40 in vitro and in vivo. Proc Nat Acad Sci. 2001; 98(10): 5856-5861. 18. Perk J, De Backer G, Gohlke H, Graham I, Riner Z, Cifkova, R. European Association for Cardiovascular Prevention & Rehabilitation (EACPR); ESC Committee for Practice Guidelines (CPG). European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice. Eur Heart J. 2012; 33(13): 1635-1701. 19. Rahman MA, Abdullah N, Aminudin N. Evaluation of the antioxidative and hypo-cholesterolemic effects of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (Agaricomycetes), in ameliorating cardiovascular disease. Int J Med Mushroom. 2018; 20(10): 961-969. 20. Rahman MA, Abdullah N, Aminudin N. Corroborative assessment of mushroom as the graceful ageing and lifespan promoting agent. Biointerf Res Appl Chem. 2017; 7(3): ID 2048. 21. Rahman MA, Abdullah N, Aminudin N. Ganoderma lucidum (P.) Karst modulates memory and learning related behaviour in Alzheimer's diseased rats. Behav Brain Res. 2017; 3(2): 45-55. 22. Rahman MA, Abdullah N, Aminudin,N. Ganoderma lucidum (P.) Karst ameliorates Alzheimer's diseased rat brain proteomics. J Proteom Res. 2017; 4(2): 152-161. 23. Rahman MA, Abdullah N, Aminudin N. Anti-oxidative and hypocholesterolemic potentiality of the solvent partitioned fractions of Ganoderma lucidum (Curtis) P. Karst (Lingzhi mushroom). Int J Med Mushroom. 2017. 24. Rahman MA, Abdullah N. Aminudin N. Interpretation of mushroom as a common therapeutic agent for Alzheimer’s disease and cardiovascular diseases. Crit Rev Biotechnol. 2015; 36(6): 1131-1142. 25. Yahaya NFM, Rahman MA, Abdullah N. Therapeutic potential of mushrooms in preventing and ameliorating hypertension. Trends Food Sci Technol. 2014; 39(2): 104-115. 26. Rahman MA, Abdullah N, Aminudin N. Lentinula edodes (shiitake mushroom): an assessment of in vitro anti-atherosclerotic bio-functionality. Saudi J Biol Sci. 2018; 25(8): 1515-1523. 27. Rahman MA, Abdullah N, Aminudin N. Antioxidative effects and inhibition of human low density lipoprotein oxidation in vitro of polyphenolic compounds in Flammulina velutipes (Golden Needle Mushroom). Oxid Med Cell Long. 2015: ID 403023. 28. Rahman MA, Abdullah N, Aminudin N. Inhibitory effect on in vitro LDL oxidation and HMG Co-a reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) persoon (Lion’s Mane Mushroom). BioMed Res Int. 2014: ID 828149. 29. Abdullah MA, Hashimoto M, Katakura M. Neuroprotective effect of madecassoside evaluated using amyloid Β1-42-mediated in vitro and in vivo Alzheimer’s disease models. Int J Ind Med Plant. 2014; 47: 1669-1682. 30. Otunola GA, Oloyede OB, Oladiji AT, Afolayan AA. Effects of diet-induced hypercholesterolemia on the lipid profile and some enzyme activities in female wistar rats. Afr J Biochem Res. 2010; 4(6): 149- 154. Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 324 European Journal of Biological Research 2020; 10(4): 314-325 31. Nofer JR, Kehrel B, Fobker M, Levkau B, Assmann G, von Eckardstein A. HDL and arteriosclerosis: beyond reverse cholesterol transport. Atheroscler. 2002; 161(1): 1-16. 32. Baba S, Natsume M, Yasuda A. Plasma LDL and HDL cholesterol and oxidized LDL concentrations are altered in normo- and hypercholesterolemic humans after intake of different levels of cocoa powder. J Nutr. 2007; 137(6): 1436-1441. 33. Kaess BM, Preis SR, Lieb W, Beiser AS, Yang Q, Chen TC, et al. Circulating brain-derived neurotrophic factor concentrations and the risk of cardiovascular disease in the community. J Am Heart Assoc. 2015; 4(3): e001544. 34. Bahls M, Könemann S, Markus MRP, Wenzel K, Friedrich N, Nauck M, et al. Brain-derived neurotrophic factor is related with adverse cardiac remodeling and high NTproBNP. Sci Rep. 2019; 9(1): 15421. 35. Tyler WJ, Alonso M, Bramham CR, Pozzo-Miller LD. From acquisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learn Mem. 2002; 9(5): 224-237. 36. Siuda J, Patalong-Ogiewa M, Żmuda W, Targosz-Gajniak M, Niewiadomska E, Matuszek I, et al. Cognitive impairment and BDNF serum levels. Neurol Neurochir Pol. 2017; 51(1): 24-32. 37. Balducci C, Beeg M, Stravalaci M, Bastone A, Sclip A, Biasini E, et al. Synthetic amyloid-β oligomers impair long-term memory independently of cellular prion protein. PNAS. 2010; 107(5): 2295-2300. 38. Mizuno M, Yamada K, Olariu A, Nawa H, Nabeshima T. Involvement of brain-derived neurotrophic factor in spatial memory formation and maintenance in a radial arm maze test in rats. J Neurosci. 2000; 20(18): 7116-7121. 39. Zhang XQ, Ip FC, Zhang DM, Chen LX, Zhang W, Li YL, et al. Triterpenoids with neurotrophic activity from Ganoderma lucidum. Nat Prod Res. 2011; 25(17): 1607-1613. 40. Gosso MF, de Geus EJC, van Belzen MJ, Polderman TJC, Heutink P, Boomsma DI, Posthuma D. The SNAP-25 gene is associated with cognitive ability: evidence from a family-based study in two independent Dutch cohorts. Mol Psychiat. 2006; 11: 878-886. 41. Greber S, Lubec G, Cairns N, Fountoulakis M. Decreased levels of synaptosomal associated protein 25 in the brain of patients with Down syndrome and Alzheimer's disease. Electrophor. 1999; 20: 4-5, 928- 934. 42. Musil R, Spellmann I, Riedel M. SNAP-25 gene polymorphisms and weight gain in schizophrenic patients. J Psychiatr Res. 2008; 42(12): 963-970. 43. Chen KH, Reese EA, Kim HW, Rapoport SI, Rao JS. Disturbed neurotransmitter transporter expression in Alzheimer disease brain. J Alzh Dis. 2011; 26(4): 755-756. 44. Sultana R, Banks WA, Butterfield DA. Decreased levels of PSD95 and two associated proteins and increased levels of BCl2 and caspase 3 in hippocampus from subjects with amnestic mild cognitive impairment: insights into their potential roles for loss of synapses and memory, accumulation of Aβ, and neurodegeneration in a prodromal stage of Alzheimer's disease. J Neurosci Res. 2010; 88(3): 469-477. 45. Ya BL, Liu WY, Ge F, Zhang YX, Zhu BL, Bai B. Dietary cholesterol alters memory and synaptic structural plasticity in young rat brain. Neurol Sci. 2013; 34(8): 1355-1365. 46. Fon Tacer K, Kuzman D, Seliskar M, Pompon D, Rozman D. TNF-alpha interferes with lipid homeostasis and activates acute and proatherogenic processes. Physiol Genomics. 2007; 31(2): 216-227. Rahman et al. Ganoderma lucidum against hypercholesterolemia and Alzheimer’s disease 325 European Journal of Biological Research 2020; 10(4): 314-325 47. Nguyen PA, Won JS, Rahman MK, Bae EJ, Cho MK. Modulation of Sirt1/NF-κB interaction of evogliptin is attributed to inhibition of vascular inflammatory response leading to attenuation of atherosclerotic plaque formation. Biochem Pharmacol. 2019; 168: 452-464. 48. McAlpine FE, Lee JK, Harms AS, et al. Inhibition of soluble TNF signaling in a mouse model of Alzheimer's disease prevents pre-plaque amyloid-associated neuropathology. Neurobiol Dis. 2009; 34(1): 163-177. 49. Rodrigues HA, Fonseca Mde C, Camargo WL, Lima PMA, Martinelli PM, Naves, LAP, Guatimosim C. Reduced expression of the vesicular acetylcholine transporter and neurotransmitter content affects synaptic vesicle de78342istribution and shape in mouse neuromuscular junction. PLoS One. 2013; 8(11): e78342. 50. Ullrich C, Pirchl M, Humpel C. Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits. Mol Cell Neurosci. 2010; 45(4): 408-417. 51. Prado VF, Martins-Silva C, de Castro BM, Lima RF, Barros DM. Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition. Neuron. 2006; 51(5): 601-612.