Therapeutic potential of bitter taste receptor agonists on amyloid β-induced rat model of Alzheimer’s disease

ABSTRACT This study was conducted to investigate the effect of bitter taste receptor (T2R) agonists (caffeine and extracts of myrrh and Boswellia serrata) on amyloid β (Aβ)-induced Alzheimer’s disease (AD) in rats. Rats intracerebroventricularly (ICV) injected with a single dose of Aβ (3 μg/μL/rat) had significantly: 1) lower brain weight, 2) higher short-term memory impairment, 3) lower serum dopamine level, 4) higher AChE activity, 5) higher brain lipid peroxide malonaldehyde, 6) lower brain antioxidant activities of catalase, superoxide dismutase and glutathione peroxidase, 7) higher brain phosphor tau protein level, 8) higher brain levels of apoptotic markers (Bax and caspase 3), 9) lower brain level of antiapoptotic marker Bcl2, 10) downregulated brain expression of the bitter receptor (T2R4) gene, 11) upregulated brain expression of tumor necrosis factor α (TNFα) and nuclear factor κB (NF-κB) genes and 12) marked neuronal degeneration associated with severe vacuolation of neutrophils. All deteriorated effects of Aβ were restored following treatment with caffeine (20 mg/kg), myrrh aqueous extract (10 mg/kg) and Boswellia serrata aqueous extract (400 mg/kg) with best effect for myrrh aqueous extract. These data conclude that bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) had therapeutic potential on amyloid β-induced rat model of Alzheimer’s disease.               


Introduction
The prevalence of neurodegenerative diseases, such as Alzheimer's disease (AD), represents one of the major healthcare problems affecting millions of people worldwide and constitutes an economic burden.A high prevalence of AD was reported in Egypt, especially among communities of farmers and those based in rural areas.Various factors are implicated in this high prevalence, including environmental and genetic factors [1].Several clinical features may precede the incidence of motor symptoms of some neurodegenerative diseases over many years.For example, smell disorders, abnormal sleep and gastrointestinal dysfunction are common in neurodegenerative diseases.Furthermore, clinical and experimental data provide a potential link between neural/behavioral disorders and various inflammatory conditions [2].This necessitates the search for newer tools for screening somatic, autonomic or non-autonomic receptors and/or drugs expressing or activating them.
The resin of Boswellia species and Myrrh, an aromatic oleo gum resin obtained from the stem of Commiphora molmol (family: Burseraceae), is known for their bitter taste.They were mentioned in numerous ancient texts as a traditional natural medicine for many diseases.The gum resin of plants Boswellia species from the Burseraceae family has long been used for the prevention of amnesia and enhancement of memory power.Ethanolic extract of gum resin of Boswellia serrata has been recognized to inhibit the synthesis of pro-inflammatory enzyme, 5-lipoxygenase (5-LOX) [3].Moreover, methanol and hot water extracts of B. serrata showed good antioxidant potential at low concentrations that counteracted the neurotoxic effect of AlCl 3 in the AlCl3-induced model of AD [4].Boswellia serrata extracts consist of different terpenoid compounds with a high abundance of beta-boswellic acid (BBA) [5].BBA improved hippocampal neurite outgrowth and branching in the hippocampal cells, suggesting a possible remedial role for BBA as a useful reagent against neurodegenerative disorders and memory loss [6].Previous studies demonstrated that the extracts of myrrh had antiinflammatory and analgesic effects [7].Aqueous extracts of myrrh resin ameliorated scopolamineinduced memory impairment in mice [8].Sesquiterpenes isolated from the resins of myrrh showed neuroprotective effects against chemically induced neuronal cell death in SH-SY5Y cells [9].It is well known that disturbances of intracellular Ca 2+ homeostasis are an early event in the pathogenesis of AD.Caffeine improved intracellular Ca 2+ levels when given to PS1KI and 3×Tg-AD mice models of AD [10].Caffeine also reverses cognitive impairment and decreases brain amyloid-beta (Aβ) levels in aged AD mice [11].
Alteration in gene expression of taste receptors (T2Rs) is observed in AD.This reinforces the notion that T2Rs in the CNS play novel functions unrelated to taste perception [12].T2Rs are a family of G-protein coupled receptors that respond to a broad range of structurally diverse natural and synthetic bitter compounds including denatonium benzoate, quinine, caffeine, colchicine, yohimbine, saccharin and flufenamic acid.T2Rs are expressed in the brains of mice and rats [13].T2Rs gene expression has also been identified in the human brain [14].However, the exact function in the brain is largely unknown.The study of bitter receptors has significant potential for the identification of their modulatory roles in one of the most common neurodegenerative diseases, AD.Studying mechanisms of activation of these receptors, and more importantly, recognition of functional ligands and specific agonists and/or antagonists will open new therapeutic avenues in the future.There is a shortage in knowledge regarding the effect of caffeine, myrrh, Boswellia serrata extracts on bitter receptors and their neuroprotective effect on AD.
This study aimed to evaluate the possible therapeutic effect of some bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) on Aβ-induced rat model of AD through the evaluation of biochemical and histological changes associated with treatments.

Preparation of extracts
The resin of Boswellia serrata and myrrh was purchased from local markets in Egypt.These resins were authenticated by Dr. AbdAllah El-Gazar at Faculty of Pharmacy, Kafrelsheikh University, Egypt.The two resins (500 g for each) were extracted with hot water (10 L) for 3 h, and filtrate was evaporated under reduced pressure to give extracts of 180 and 200 g for Boswellia serrata and myrrh, respectively.Aqueous extractions were prepared in phosphate-buffered saline (PBS) for rats treatment.

Experimental animals
This study was conducted on 56 adult male Wistar rats (250-280 g).Animals were maintained under standard laboratory conditions and 12:12 light/dark cycles with free access to food and water ad-libitum and left to acclimatize to the housing conditions for one week.Experiments were conducted after approval of the local Ethics Committee of Kafrelsheikh University with a license number of KFS-VET /1024/2019.

Induction of a rat model of AD
The animals were anesthetized with 5% chloral hydrate and fixed to a stereotaxic apparatus.A single intracerebroventricularly (ICV) injection of 3 μg/μL Aβ (Sigma) dissolved in 0.1M PBS.The site of injection was 2 mm from either side of the midline on a line drawn through the anterior base of the ears [15].
All treatments in GII-IV and GVI-VIII were given orally by gastric gavage for 4 weeks with doses as previously described [4,8,11].At the end of the experiment (after 4 weeks), blood samples were collected from the medial canthus of the eye using a capillary tube and were centrifuged (at 3000×g for 15 min) to get serum for biochemical assays.Rats were decapitated [16], brains excised and weighed, midbrains and striata of one hemisphere dissected out, stored at − 80°C and used for biochemical and molecular analyses.The other hemisphere was embedded in paraffin and sliced for histopathology.

Y-maze test
The Y-maze test was used to evaluate the effect of various treatments on the short-term memory function of rats.The Y-maze is a black, polyvinyl plastic maze consisting of three identical arms (40 cm × 3 cm × 12 cm).In brief, 24 hr before euthanization each rat was placed in the center of the Y-maze 30 min later and was allowed to explore freely through the maze during an 8 -min session.The sequence and total number of arms entered were recorded as described before [17].An entry is counted to have occurred when all four limbs are within the arm.The spontaneous alternation score (%) for each rat is defined as the ratio of the actual number of alternations to the possible alternation number (total number of entries − 2) multiplied by 100.The total number of entries into arms was assessed as a parameter representing locomotor activity [8].

Assessment of dopamine and acetylcholinesterase levels in serum
Biuret Protein Content Assay Kit (Cat.MBS779655) was used to measure the protein concentration.Serum levels of Dopamine (Cat.#SG-20553) and AChE (Cat.#SG -20,512) were determined using rat ELISA kits (Sino gene clon Co., Ltd, Hangzhou, China) following the manufacturer's protocol and as previously described [18].The inter-assay and intra-assay were 10% and 8%, respectively.

Oxidant and antioxidant status in the brain
Homogenates of brain tissue were made in the same way as previously described [19].The brain levels of lipid peroxidation marker malondialdehyde (MDA) and the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) antioxidant enzymes were determined colorimetrically by suitable kits (Biodiagnostics, Egypt) and as previously detailed [20,21].

Real-time PCR
Real-time polymerase chain reaction (qPCR) was utilized to analyze the expression of proinflammatory genes (TNFa and NfKb) and the bitter taste receptor T2R4 in the brain.Total RNA was extracted from brain tissue using Gene JET RNA Purification Kit (Thermo Scientific, # K0731, USA) and its purity and concentration were assessed before the addition of reverse transcriptase (Thermo Scientific, #EP0451) to get cDNA.The qPCR mixture contained cDNA, 2XMaster Mix (QuantiTect SYBR Green) and primers (Table 1).The Step One Plus thermal cycler (Applied Biosystem, USA) was used, with the following thermal cycle settings: one cycle of initial denaturation (at 94°C for 4 min), followed by 40 cycles of denaturation (at 94°C for 40 s) and annealing extension (at 60°C for 40 s).Using the Livak approach and as previously published [24][25][26], we compared the relative expression [fold change mean±standard error of mean (SEM)] of all candidate genes to that of β actin (an internal control).

Histological examination
Following their overnight fixation in 10% formalin, brain specimens from cerebral cortex including the hippocampus were histologically processed to obtain paraffin sections (5 µm).All tissue slides were stained with hematoxylin and eosin and examined by a light microscope [27].

Statistical analysis
Data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Tukey test as a post hoc test.All statistical analyses were performed using the GraphPad Prism software.Probability values of ≤ 0.05 were considered statistically significant.

Effect of various treatments on brain weight of Aβ-induced AD rats
The absolute and relative weight of brains in the four control groups (G1-GIV) was insignificantly (p > 0.05) different (Table 2).However, Aβ-induced AD rats (GV) exhibited significantly (p ≤0.05) lower absolute and relative brain weight when compared to the control groups.This reduced absolute and relative brain weights were restored following treatment with caffeine (GVI), myrrh aqueous extract (GVII) and Boswellia serrata aqueous extract (GVIII).

Effect of various treatments on Aβ-induced memory impairment
The Y-maze test was used to evaluate the effect of various treatments on the Aβinduced short-term memory impairment in rats.The results of this behavioral test (Figure 1) showed no significant (p > 0.05) changes in spontaneous alternation % among the control groups (GI-GIV).In contrast, rats injected with Aβ (GV) showed significantly (p ≤0.05) reduced spontaneous alternation % (suggesting impaired short memory) compared to control groups.This decreased spontaneous alternation was significantly (p ≤0.05) restored following treatment with caffeine (GVI), myrrh aqueous extract (GVII) and Boswellia serrata aqueous extract (GVIII), implying enhanced memory (Figure 1).Rats treated with myrrh aqueous extract (GVII) exhibited the best improvement followed by Boswellia serrata aqueous extract (GVIII).

Effect of various treatments on dopamine and AChE levels
Figure 2 shows no statistical (p > 0.05) changes in the serum levels of dopamine and AChE among the control groups (GI-GIV).However, GV (Aβ group) had significantly (p ≤0.05) decreased dopamine and significantly (p ≤0.05) increased AChE serum levels relative to the control groups.On the other hand, rats in GVI (caffeine group), GVII (myrrh aqueous extract group) and GVIII (Boswellia serrata aqueous extract group) had significantly (p ≤0.05) higher dopamine and significantly lower AChE levels, with better improvement in GVII followed by GVIII, as compared to GV (Figure 2).

Effect of various treatments on brain levels of phosphor tau protein and apoptotic markers
GV (Aβ group) exhibited significantly (p ≤0.05) higher concentrations of phosphor tau protein and the apoptotic markers (Bax and caspase 3) and a significantly (p ≤0.05) lower concentration of the antiapoptotic marker Bcl2 in the brain than in the control groups (GI-GIV, Figure 3).However, treated groups GVI (caffeine group), GVII (myrrh aqueous extract group) and GVIII (Boswellia serrata aqueous extract group) had significantly (p ≤0.05) lower concentrations of phosphor tau protein, Bax and caspase 3 and a significantly (p ≤0.05) higher concentration of the Bcl2 in the brain, with best effects in GVII followed by GVIII, than in GV (Figure 3).There was no significant (p > 0.05) difference among the control groups (GI-GIV).

Effect of various treatments on brain oxidative and antioxidant status
Aβ-induced AD rats (GV) had significantly (p ≤0.05) higher levels of lipid peroxidation marker MDA and lower activities the antioxidant enzymes (CAT, GPx and SOD) than all control groups GI-GIV (Table 3).Treatment with caffeine (GVI), myrrh aqueous extract (GVII) and Boswellia serrata aqueous extract (GVIII) restored these markers to levels near that of the control groups with best improvement (lowest MDA and highest GSH and SOD) observed in myrrh aqueous extract-treated group (GVII), followed by Boswellia serrata aqueous extract-treated group (GVIII) and finally caffeine-treated group (GVI).

Effect of various treatments on inflammation and bitter taste receptor
Changes in the expression of bitter receptor (T2R4) gene and inflammation-related TNFα and NF-κB genes were assessed in the brain using qPCR, and the results showed a significant downregulated expression of T2R4 and a significant (p ≤0.05) upregulated expression of TNFα and NF-κB in rats injected with Aβ (GV) relative to the control groups (GI-GIV, Figure 4).In contrast, treated groups (GVI -GVIII) exhibited significantly (p ≤0.05) upregulated T2R4 and significantly (p ≤0.05) downregulated TNFα and NF-κB in the brain, with best improvement in myrrh aqueous extract group (GVII), compared to GV.None of the treated group returns the altered expression to level similar to the control groups, and no significant (p > 0.05) difference among the four control groups (GI-GIV) was noticed (Figure 4).

Effect of various treatments on Aβ-induced brain tissue histopathology
Figure 5 shows the results of histopathology examination to brain sections from all groups.Brain of normal control animals (GI -GIV) showed normal neurons (arrows) and fibers.However, brain of control positive animal injected with Aβ (GV) showed marked neuronal degeneration associated with severe vacuolation of neuropil.On the other hand, brain of diseased animal treated caffeine (GVI) showed a mild neuronal degeneration with limited neuronal vacuolation.Moreover, brain of diseased animal treated with myrrh showed mild degree of nuclear pyknosis and neuronal vacuolation.Additionally, brain of diseased animal treated with Boswellia serrata aqueous extract (GVIII) showed pericellular vacuolation.

Discussion
Currently, there is a lack of definitive cure for neurodegenerative diseases such as AD.Furthermore, the existing pharmacological treatment has many adverse reactions, specifically on the long run, and is associated with concomitant drug-drug interactions [28].This necessitates the search for alternative treatment options.This study aimed to explore the impact of bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) on Aβ-induced rat model of AD.
The obtained results revealed a significant reduction in the weight of brains in Aβinduced AD rats as compared to the control groups.Similar reduction in brain weight was noticed in rats injected with Aβ by another study [29].This reduction may be attributed to Aβ destructive effect on neuronal tissues [28,30].On the other hand, AD rats treated with bitter taste receptor (T2R) agonists including caffeine, myrrh aqueous extract and Boswellia serrata aqueous extract showed a significantly higher brain weight than Aβ-injected rats.This indicates that these T2R agonists could ameliorate Aβ bad effects on the brain.Alzheimer's patients often experience a decline in memory and other cognitive abilities associated with dopamine reduction alongside a rise in the cholinergic nerve function marker AChE [31,32].Moreover, animal models of AD exhibited higher levels of AChE activity and lower level of dopamine in serum of AD rats than in control rats [33,34].In consistence, we also found significant reduction in short-term memory and cognitive abilities (as revealed by Y-maze test), reduced dopamine level and significant increase in AChE activity in the serum of Aβ-induced AD rats.In support, extract of gum resin of Boswellia serrata was reported to improve  Note: Data were presented as means ± SEM.Small (a-e) letters showed the marked change at P ≤ 0.05.The significance was expressed by dissimilar letters [a (the highest value) and e (the lowest value)] in the same column.
the memory [3].Inhibition of AChE, an enzyme that hydrolyzes acetylcholine and regulates cholinergic function, can relieve AD symptoms [35].The mechanisms by which AChE inhibitors relieve AD involve induction of antioxidant enzymes activity, diminishing of oxidative stress, protection of neurons from Aβ-triggered damage and inhibition of cytokines production from microglia and monocytes [35].Because the majority of AChE inhibitors cause several side effects (hepatotoxicity, nausea and diarrhea [28], searching for alternative AChE inhibitors with less or no side effects becomes an urgent demand.Herein, we provide bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) as new natural AChE inhibitors as these compounds have the ability to not only inhibit AChE activity but also to improve memory impairment in β-induced AD rats.The present study revealed that administration of Aβ significantly increased levels of lipid peroxidation marker MDA and significantly decreased activities of the antioxidant enzymes (CAT, GPx and SOD) as compared to all control groups.Previous studies exhibited that Aβ is a neurotoxic protein which induces the accumulation of intracellular neurofibrillary tangles that is considered as the major feature of AD [36,37].This peptide aggregates to cause oxidative stress which in turn causes neuronal apoptosis in AD [36,38].Oxidative stress has been linked to AD. Herbal supplements have been studied extensively for their potential to reduce the oxidative stress caused by Aβ accumulation in brain cells [39,40].In accordance, we found that treatment with caffeine (GVI), myrrh aqueous extract (GVII) and Boswellia serrata aqueous extract (GVIII) restored oxidative and antioxidant markers to levels comparable with the control groups with best effect for myrrh aqueous extract.This infers that by reducing the amount of intracellular ROS produced, caffeine, myrrh and Boswellia serrata aqueous extracts could protect the brain against the apoptosis caused by Aβ.It is well established that Aβ not only induces oxygen-free radical release but also represses the activities of antioxidant enzymes to achieve its neurotoxic impact [38].Indeed, we found that rats injected with Aβ had elevated levels of the lipid peroxidation marker MDA.Thus, Aβ may harm or kill neuronal cells by inducing membrane lipid peroxidation, which disrupts the membrane ion channel and induces apoptosis [41].
Neuronal malfunction and apoptosis are caused by apoptosis-related pathways that are activated by Aβ deposition in AD [39,42].Previous research have demonstrated that caspase 3 plays a critical role in Aβ-induced apoptosis in AD brains [28,42,43] and natural remedies helped reduce this impact [28,39,40].In agreement, we also found significantly higher concentrations of caspase 3 in the brain of Aβ-injected rats and this effect was ameliorated by bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata).To further understand the potential signaling molecules that regulate Aβ-induced apoptosis and how this might be ameliorated by bitter taste receptor agonists, we looked upstream of caspase 3, the final product of the apoptotic cascade.The apoptotic Bax and the anti-apoptotic Bcl2 lie upstream to caspase 3 in the intrinsic apoptotic pathway [44].We found increased level of Bax and decreased level of Bcl2 in the brain of Aβ injected rats and this deleterious effect was reversed after the treatment with bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata).Other investigations have also found that Aβ may activate similar apoptotic pathway [28,42,43,45].Collectively, our results indicate that agonists of the bitter taste receptor (such as caffeine and myrrh and Boswellia serrata extracts) significantly reduced Aβ -induced apoptosis in the brain by increasing levels of the anti-apoptotic marker Bcl2 and decreasing levels of the pro-apoptotic markers Bax and caspase 3.
Previous research has shown that invitro stimulation with Aβ causes pro-inflammatory cytokine (TNFa, IL6, IL1b and NFkB) activation in AD patients [28,46].Similarly, we also found a significant upregulation of TNFa and NFkB genes in the brain of Aβ -injected rats.Moreover, agonists of the bitter taste receptor (such as caffeine and myrrh and Boswellia serrata extracts) inhibit TNFa, IL1b and NFkB, making it a potent anti-inflammatory with neuroprotective effects [7,9,11,47,48].Consistent with these findings, we also discovered that rats-treated with these bitter taste receptor agonists showed a substantial downregulation for TNFa and NFkB pro-inflammatory cytokines.The primary cellular alterations associated with AD are inflammation and oxidative stress.Therefore, therapies aimed at AD prevention may benefit from including anti-inflammatory and antioxidants.Patients who used NASIDs for 2 years had a lower incidence of developing AD [49].In the present study, memory and cognition were both improved by bitter taste receptor agonists, and they also have a neuroprotective impact, unlike NASIDs, which had no such effect.As a result, these bitter taste receptor agonists might replace NASIDs in the role of antiinflammatory in preventative therapeutics for AD because of its lower risk and lower cost.
Certain foods can modify receptor-mediated signaling cascades in the brain, including G-protein-coupled receptors (GPCRs), ion channels and others.Neurons in the brain may respond to various tastants; for example, both sucrose and quinine, which are sweet and bitter, stimulated neurons in the nucleus accumbens (NAc) [50].However, the quinine evoked a different group of NAc neurons than the sucrose did, suggesting that there may be distinct circuits for processing unpleasant and rewarding stimuli.According to Srinivas et al. [51], quinine can block gap connections between neurons.Additionally, it has been established that bitter taste receptors (T2Rs) are present in brain cells of mice and rats [13] and human [14].In AD, T2Rs were also differentially expressed in the brain tissue [12].To the best of our knowledge, this could the first report to show a significant downregulation of the T2R4 gene in the brain of rats injected with Aβ.On the other hand, administration of T2Rs agonists (caffeine, myrrh and Boswellia serrata aqueous extracts) significantly upregulated T2R4 expression in the brain relative to Aβinjected rats.It has been established that quinine enters the brain through blood brain barrier (BBB) [32].It is worth to know whether the active ingredients of T2Rs agonists (caffeine, myrrh and Boswellia serrata aqueous extracts) can also pass through BBB at physiological amounts capable of activating T2Rs.
At cellular level, brains of rats injected with Aβ showed marked neuronal degeneration associated with severe vacuolation of neuropil.In agreement, other studies showed neuronal degeneration after injection of Aβ [52,53].In contrast, brain of diseased animal treated bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) showed a mild neuronal degeneration with limited neuronal vacuolation and nuclear pyknosis indicating neuronal improvement.
Induction of apoptosis, inflammation and oxidative stress by Aβ were restored by caffeine, myrrh and Boswellia serrata aqueous extracts.These results infer the potent neuroprotective potential of these T2Rs agonists.In support, previous studies similar neuroprotective effects for B. serrata extracts against AlCl 3 -induced AD in rats [4].Beta-boswellic acid and incensole acetate, two major components of B. serrata extracts, can ameliorate the neurotoxic effects of Aβ through inhibition of apoptosis, inflammation and oxidative stress [5,6,28].Moreover, previous studies also reported a neuroprotective effect for myrrh resin and its components on SH-SY5Y neuronal cells [9] and scopolamine-induced memory impairment mice [8].Caffeine can reverse memory loss and decrease Aβ levels in the brains of aged mice with AD [11] through the induction of Ca 2+ inside the neurons thereby restoring the intracellular Ca 2+ homeostasis [10].Among the three T2Rs agonists used in the present study, the myrrh aqueous extract showed the best neuroprotective effect.

Conclusions
Bitter taste receptor agonists (caffeine and extracts of myrrh and Boswellia serrata) protect rat brain from Aβ -induced oxidative stress, inflammation and apoptosis.The neuroprotective effect of these agonists could be attributed to their antioxidant activity, anti-inflammatory and anti-apoptotic potentials.Although our work contributes to the development of therapeutics for AD by expanding our knowledge of how bitter taste receptor agonists can protect against Aβ -induced neurodegeneration, further investigations are required to give more details regarding the underlying mechanism of bitter taste receptor agonists action to protect neurons from damage by Aβ in AD.

Figure 1 .
Figure 1.Effect of caffeine, myrrh and Boswellia serrata aqueous extracts on Aβ-induced short-term memory impairments as detected by the Y-maze test.The spontaneous alternation score was recorded in all groups.Data are presented as mean ± SEM (n = 7) and columns with different letters [a (the highest value) and c (the lowest value)] are significantly different at p < 0.05.

Figure 2 .
Figure 2. Effect of caffeine, myrrh and Boswellia serrata aqueous extracts on serum levels of dopamine and AChE in Aβ-induced AD rats.Data are presented as mean ± SEM (n = 7) and columns with different letters [a (the highest value) and d (the lowest value)] are significantly different at p < 0.05.

Figure 3 .
Figure 3.Effect of caffeine, myrrh and Boswellia serrata aqueous extracts on brain levels of phosphor tau protein and apoptosis-related markers (Bax, caspase 3 and Bcl2) in Aβ-induced AD rats.Data are presented as mean ± SEM (n = 7) and columns with different letters [a (the highest value) and e (the lowest value)] are significantly different at p < 0.05.

Figure 4 .Figure 5 .
Figure 4. Effect of caffeine, myrrh and Boswellia serrata aqueous extracts on the expression of T2R4, TNFa and NFkB in the brain as detected by qPCR.Data are presented as mean ± SEM (n = 3) and columns with different letters [a (the highest value) and e (the lowest value)] are significantly different at p < 0.05.

Table 2 .
Effect of caffeine and aqueous extracts of myrrh and Boswellia serrata on absolute and relative brain weight of Aβ-induced AD rats.
Note: Data are presented as mean ± SEM (n = 7) and column with different letters [a (the highest value) and b (the lowest value)] is significantly different at p ≤ 0.05.Relative body weight (%) = [absolute brain weight (g)/body weight (g)] x 100.

Table 3 .
Effect of caffeine and aqueous extracts of myrrh and Boswellia serrata on brain MDA, CAT, SOD and GPx in Aβ-induced AD rats.