Neuroprotective effects of a combination of Boswellia papyrifera and Syzygium aromaticum on AlCl3 induced Alzheimer's disease in male albino rat

Abstract Alzheimer's disease (AD) is the most common neurodegenerative disease characterized by hippocampal, and cortical neuron deterioration, oxidative stress, and severe cognitive dysfunction. Aluminum is a neurotoxin inducer for cognitive impairments associated with AD. The treatment approaches for AD are unsatisfactory. Boswellia papyrifera and Syzygium aromaticum are known for their pharmacological assets, including antioxidant activity. Therefore, the current study explored the possible mitigating effects of a combination of Boswellia papyrifera and Syzygium aromaticum against aluminum chloride (AlCl3) induced AD. The AD model was established using AlCl3 (100 mg/kg), and the rats were orally administrated with Boswellia papyrifera or Syzygium aromaticum or a combination of them daily for 8 weeks. The Y-maze test was used to test cognition in the rats, while acetylcholinesterase (AChE) and oxidative stress markers were estimated in homogenates of the cerebral cortex and hippocampus. Also, the histopathological examination of the cortex and hippocampus were investigated. The results revealed that administration of either B. papyrifera or S. aromaticum extracts significantly improved the cognitive functions of AD rats, enhanced AChE levels, increased oxidative enzymes levels, including SOD and GSH, and reduced MDA levels in homogenates of the cerebral cortex and hippocampus and confirmed by improvement in histological examination. However, using a combination therapy gave better results compared to a single treatment. In conclusion, the present study provided primary evidence for using a combination of B. papyrifera and S. aromaticum to treat cognitive dysfunction associated with AlCl3 Induced AD by improving the AChE levels and modulating oxidative stress in the brain.

There is a growing body of literature that recognizes B. papyrifera and S. aromaticum for their neuroprotective activity.Oja and his colleagues demonstrate the role of S. aromaticum in mitigating iron-mediated oxidative brain injury in rats (Ojo et al., 2022).Also, a combination of exercise and S. aromaticum reverse the memory deficits, apoptosis, and mitochondrial dysfunction of the hippocampus in AD (Panahzadeh et al., 2022).Additionally, Genus Boswellia acts as a good candidate for neurodegenerative disorders, including AD (Rajabian et al., 2020).Also, many studies reported the beneficial effect of B. papyrifera on learning and memory in rodents (Farshchi et al., 2010;Mahmoudi et al., 2011).However, the neuroprotectant activity of a combination of the two extracts against AD has remained unclear.
Therefore, the present work aims to investigate the neuroprotective roles of a combination of B. papyrifera and S. aromaticum extracts against AlCl 3-induced AD in male rats.This was achieved by measuring the cognitive impairment using the Y-maze test, AChE levels, and oxidative stress biomarkers, as well as investigating the histological picture of the cortex and hippocampus.

Plants and extraction
B. papyrifera dried parts of tree and S. aromaticum dried flowers were purchased from AL-Haraz store in Egypt.The plants were kindly identified by Prof. Dr. A. A. Fayed, Professor of plant taxonomy, Faculty of Science, Assiut University, Egypt.A voucher sample was kept in the Faculty of Science Herbarium, Assiut University, Assiut, Egypt.500 g of both plants were washed by distill water, dried then blended by mortar.The powder of each plant was soaked in 5L of 80% methanol for 3 days in flasks that shake for 170 RPM.The extracts were filtered using 0.45µm filter paper.The methanol of each filtrate was removed using a rotary evaporator at 40 °C then frozen in the refrigerator for further experiments (Melesie Taye et al., 2020).

GC-Ms of B. papyrifera and S. aromaticum extracts
B. papyrifera and S. aromaticum methanol extracts were screened using a direct capillary column TG-%MS (30m*0.25mm*0.25mfilm thickness) and a trace GC1310-Isq mass spectrometer (Thermo Scientific, Austin, TX, USA).The column oven's temperature was initially kept at 60 °C, then increased by 5 °C/min to 230 °C and held for 3 min.The final temperature of 290 °C was increased by 30 °C/min and then maintained for three minutes.The injector and MS transfer line were kept at temperatures of 240 and 250 °C, respectively, and helium was used as the carrier gas

Introduction
Alzheimer's disease (AD) is a neurological condition that progresses over time and places a high financial and psychological cost on society (Singh et al., 2016(Singh et al., , 2018)).Over 45 million individuals are estimated to be affected by AD globally, and by 2050, this figure is projected to quadruple every 20 years (Dos Santos et al., 2018;Scheltens et al., 2016).Cognitive impairment, particularly short-term memories, is the earliest presenting symptom of AD, but long-term memories are well-preserved.Executive decision-making and the capacity to do regular work drastically decline as the disease progresses and cognitive impairment becomes apparent.The reduction of cholinergic synapses in the hippocampus and neocortex is a recurrent observation in AD, emphasizing the importance of using an effective approach to control the acetylcholinesterase (AChE) function to overcome this problem (Heo et al., 2004;Loizzo et al., 2008).AD patients have lower levels of the neurotransmitter AChE, which was initially discovered as a synthetic substance in 1867 and is used to transfer nerve signals from one nerve cell to another or through other muscle fibers (Houghton et al., 2006).The U.S. Food and Drug Administration has given the green light to a few AChE inhibitors, including tacrine and rivastigmine, for relieving AD symptoms (J.K. Kim et al., 2009).
Aluminum (Al) is a lethal neurotoxin, and its deposition in the brain contributes to the emergence of neurodegenerative diseases, including AD (Campbell, 2002;Zatta et al., 2003;Kawahara, 2005).Epidemiological research revealed a connection between chronic Al exposure and neurological damage as well as cognitive impairment Long-term dialysis patients who received Al-containing dialysates acquired dialysis dementia (Gupta et al., 2019).A potential source of cognitive damage has been identified by miners' exposure to aluminum powder (Rifat et al., 1990).Animals treated by Al showed AD-like symptoms (Platt et al., 2001;Praticò, 2002).Prolonged exposure to Al disrupts the hippocampal synaptic plasticity due to its deposition in all parts of the rat brain, including the hippocampus, which is the site of learning and memory (Niu et al., 2007).
Currently, the treatment approaches for AD are unsatisfactory.They can only temporarily improve cognitive skills or alleviate symptoms while having numerous adverse effects.In order to treat not just the symptoms of AD but also to cure its pathology with lesser side effects, a class of drugs must be created that can target a wider range of targets (Cummings et al., 2019;Hukins et al., 2019;Yiannopoulou and Papageorgiou, 2020).Numerous active substances were extracted parts mainly from medicinal plants in Europe and Asia and showed promising pharmacological activity against AD (Uddin et al., 2019).Boswellia papyrifera is a floral plant species and frankincense that is indigenous to Ethiopia, and others in Africa.It is also described as Sudanese frankincense.Due to the tree's significant resin, Ethiopia cultivates it (Schmiech et al., 2021).Clove, also known as Syzygium aromaticum, is a dried flower bud from the Myrtaceae family that may be located all over the globe.The marketable component of the clove tree is made up of Boswellia papyrifera and Syzygium aromaticum on AlCl 3 induced Alzheimer's disease with a constant flow rate of 1 ml/min.A diluted sample of 1 µl was automatically fed into the GC's split mode using the Autosampler AS1300.Full scan mass spectra were collected in the range of m/z 40 to 1000 at an ionization voltage of 70 eV.The ion source's temperature was set at 200 °C.The compounds were recognized by comparing the retention timings and mass spectra of the compounds to those in the WILEY 09 and National Institute of Standards and Technology (NIST 11) databases (Deyab et al., 2021).Metabolite identity was reported only when the matching value of the mass spectra comparison was more than 70%.

Animals and experimental design
Forty male albino Wister rats (150-200 g body weight), were obtained from a breeding unit at the Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt.Rats were randomly divided into five groups after ten days of acclimatization (eight rats each).Group (1) act as negative control and was gavaged daily for 8 weeks with a saline solution using oral gavage.Group (2): act as positive control and was orally administrated with 100 mg/kg AlCl 3 for eight weeks (Singh et al., 2018).Group (3): act as B. papyrifera treated group and was administrated with AlCl 3 (100 mg/kg) and B. papyrifera methanolic extract (200 mg/kg) once daily for eight weeks using oral gavage (Khajehdehi et al., 2022).Group (4): act as S. aromaticum treated group and was administrated with AlCl 3 (100 mg/kg) and S. aromaticum methanolic extract (200 mg/kg) once daily for eight weeks using oral gavage (Agboola et al., 2022).Group (5): act as B. papyrifera +S.aromaticum treated group and was administrated with AlCl 3 (100 mg/kg) and B. papyrifera + S. aromaticum methanolic extracts (200 mg/kg) once daily for eight weeks using oral gavage.The experimental design was in strict accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Veterinary Institutional Animal Care and Use Committee (VET-IACUC).

Behavioral assessment (Y-maze memory test)
After the last doses of different drugs, rats were submitted to the behavioral analysis room, acclimatized for three h, and tested using the Y-maze test.The device was made of a white wooden maze with three arms that were each 16 cm broad, 50 cm long, and 32 cm high.Each rat from a different group was positioned in one arm and allowed to go vigorously through the maze for five minutes.Four paws had to be inside the arm for entry to be considered legal.The measuring parameters were the number of arm entries and the spontaneous alternation percentage (SAP).SAP is the ability of rats to alternate between different three arms, it was calculated using the Equation 1 below (Kitanaka et al., 2015;Khalil et al., 2020): [( number of variations) / ( total number of arms entries-2)] 100 × (1)

Euthanasia and sampling
24 h after the Y-maze test, rats were sacrificed by cervical dislocation following AVMA Guidelines.Brains were excised gently and washed with cold saline, then divided into two halves.One-half was preserved in 10% neutral buffered formalin for histopathological investigation.The other half was preserved in a deep freezer at -80 o c for subsequent biochemical analysis.

Biochemical assessment
Brain samples were rinsed with physiological buffer saline (100 mM Na2HPO4/NaH2PO4, 0.16 mg/ml heparin, pH 7.4) to get rid of RBCs and clot residues.One gram of tissue samples was homogenized in 5 ml of cold phosphatebuffered saline (50 mM potassium phosphate, 1 mM of ethylenediaminetetraacetic acid [EDTA], pH 7.5) using a sonic homogenizer.All homogenates were centrifuged at 14,000 ×g for 15 min at 4 °C.The supernatant was used to measure AChE content, lipid peroxidation marker (MDA), antioxidant enzymatic activities of SOD, and the levels of GSH.These parameters were measured according to manufacturer protocols (Oxis Research, Portland, USA) (Chang et al., 2013;W. Kim et al., 2014).

Histopathological examinations
The formalinized brains were washed in tap water followed by dehydration using serial dilutions of alcohol.Specimens were then cleared in xylene and embedded in paraffin at 56 °C in a hot air oven for 24 h.Paraffin bees' wax tissue blocks were sectioned at 4 µm thickness using a slide microtome.The obtained tissue sections were collected on glass slides, deparaffinized, and stained with hematoxylin and eosin (H&E).Three brain regions were evaluated in these glass slides, including the prefrontal cortex and hippocampus (CA1 and CA3) areas, and photographed using a light microscope attached to a camera (Olympus BX-53 Olympus Corporation, Tokyo, Japan) (Sevastre-Berghian et al., 2017).

Statistical analysis
one-way analysis of variance followed by post hoc test Bonferroni test was done to analyze the difference between groups using SPSS 24 software (Chicago: SPSS Inc.IBM Corp.).where (P≤0.05considered as significant).Histograms were plotted using GraphPad Prism Version 9 (GraphPad Software Inc., La Jolla, CA, USA).Data were expressed as mean ± standard error (SE).

Behavioral assessment (Y-maze memory test)
The Y-maze test is frequently used to assess the health of rats' hippocampus (Postu et al., 2019).AD rats displayed a substantial reduction in the number of arm entries and SAP% compared to the control group (Figure 1).There was no statistically significant difference between the treated groups and the control group in the number of arm entries.However, administration of B. papyrifera and S. aromaticum alone or in combination considerably increases the SAP% compared to the AD group.When compared to the B. papyrifera and S. aromaticum treated groups, the combination group showed a rise in the number of arm entries and SAP%, although There was no statistically significant difference.

Biochemical assessment
Cortical AChE levels were markedly elevated (p< 0.05) in AD rats compared to the control group.However, administration of B. papyrifera and S. aromaticum individually or in combination dramatically decreased (p< 0.05) these levels compared AD group.However,  AChE levels were slightly decreased in hippocampal samples of the AD group compared to the control.While administration of these plants singly or in combination enhances AChE levels reaching normal levels as shown in Figure 2. Additionally, MDA levels were substantially elevated in cortical and hippocampal samples of AD rats (p< 0.05) and slightly reduced upon using B. papyrifera and S. aromaticum individually or in combination where treatment using S. aromaticum showed the lowest levels in the treatments as represented in Figure 3.Moreover, cortical and hippocampal SOD levels were dramatically decreased in the AD rats compared to the control group.However, the administration of B. papyrifera and S. aromaticum individually or in combination significantly increased these levels compared to AD rats (Figure 4).The cortical and hippocampal GSH levels dramatically reduced in AD rats compared to the control group.However, administration of B. papyrifera and S. aromaticum individually or in combination slightly elevated the GSH levels compared to AD rats (Figure 5).

Histopathological examinations
The impact of B. papyrifera and S. aromaticum was confirmed by testing histopathological sections from both the cerebral cortex as well as hippocampus of the brains of different tested groups of animals.Rats in the control group displayed a normally distributed structure of neurons, neuroglia, and neuropil in H&E-stained cerebral cortex slices (Figure 6a).Contrarily, AD rat's cerebral cortex exhibited pyknotic pyramidal cells with neurofibrillary tangles, perineuronal gaps, and neuropil vacuolation (Figure 6b).While, examination of B. papyrifera rat brain sections revealed triangular-shaped pyramidal cells with nearly normal structure, vesicular stained nuclei, and some pyknotic neurons with neurofibrillary tangles could be seen (Figure 6c).Also, S. aromaticum rat brain sections showed deteriorated and pyknotic neuronal cells with neuropil   Boswellia papyrifera and Syzygium aromaticum on AlCl 3 induced Alzheimer's disease vacuolation in their cerebral cortex (Figure 6d).Conversely, the cerebral cortex of rats treated with a combination of B. papyrifera and S. aromaticum displayed nearly normal triangular-shaped neurons with big vesicular nuclei and few neurons that looked pyknotic with pericellular space (Figure 6e).
Concerning the hippocampus, Rats in the control group displayed a normal three-layer structure.The neuropil's molecular layer was made up of neurons and neuroglia, the pyramidal layer was made up of triangular-shaped neurons with massive, vesicular nuclei, and the polymorphic layer was made up of neurons and neuroglia (Figure 7a).However, the hippocampus of AD rats showed numerous structural alterations and hippocampal three layers; molecular, pyramidal, and polymorphic layers respectively.The molecular layer had neuropil vacuolation.The pyramidal layer showed neuronal cells with neurofibrillary tangles and some neurons appeared pyknotic with pericellular space.Also, neuropil vacuolation was observed (Figure 7b).Hippocampus of B. papyrifera treated rats revealed pyramidal neurons with nearly normal triangular shapes but few neurons are still pyknotic with pericellular space.There was neuroglia pericellular space in both molecular and polymorphic layers.Perivascular space was also noticed (Figure 7c).While rat's hippocampus of S. aromaticum treated rats revealed degenerated pyramidal neurons with pericellular space, pyknotic neuroglia with pericellular space, and few pyramidal neurons appeared normal (Figure 7d).The hippocampus of rat's brains treated by a combination of B. papyrifera and S. aromaticum showed nearly normal-shaped pyramidal neurons with diminished neuropil vacuolation.Some neuroglia appeared pyknotic with pericellular space (Figure 7e)

Discussion
Herbal medicines have traditionally been used to treat AD-related illnesses and enhance cognitive performance.Numerous possible uses for plant antioxidant properties in human healthcare exist (Shudo et al., 2009;Chen et al., 2022).In our study, the ameliorative effects of a combination of B. papyrifera and S. aromaticum were investigated against AD rats.The chemical profile of these plants was identified using GC-MS analysis.It could be noticed that B. papyrifera has eight major compounds.Investigators have reported more than 300 bioactive molecules from alkaloids, flavonoids, and other organic compounds derived from different parts of Broussonetia genus (Chen et al., 2022).Shudo et al. reported the role of flavanol derivatives in the suppression of cholinesterase enzymes linked to AD (Shudo et al., 2009).Moreover, Ryu et al. explained the role of retinoids in the maintenance of neural cells and their promising roles in neurodegenerative disorders (Ryu et al., 2012).Furthermore, analysis of S. aromaticum methanol extract revealed the presence of eight major compounds.On the other hand, eugenol and caryophyllene were detected as major compounds of S. aromaticum extract with antioxidant potential (Teles et al., 2021).AD was induced in our study using AlCl 3. Al for both people and animals is primarily sourced from manufactured meals and drinking water.It can enter the body through a variety of medical uses, such as dental resin composites or vaccination adjuvants, skin contact, and the breathing  of dust (Ausiello et al., 2013;Newairy et al., 2009).In our investigation, the spatial working memory of the AD group was severely diminished as reflected by the decrease in the number of arm entries and the decrease in the SAP%.The SAP depends on the natural tendency of the rats to alternate between different three arms.However, treatment with B. papyrifera and S. aromaticum and/or a combined group showed a substantial improvement in  Boswellia papyrifera and Syzygium aromaticum on AlCl 3 induced Alzheimer's disease spatial working memory as visualized by an increment in the SAP%.These results were in agreement with other previous studies that reported the roles of natural products such as Pinus halepensis and coconut oil in neuroprotection due to their antioxidant capabilities (Postu et al., 2019;Khalil et al., 2020).
The role of AlCl 3 could be explained through an allosteric association between Al and the peripheral anionic position of the enzyme molecule, exposure to AlCl 3 boosted cholinesterase activity (Abd-Elhady et al., 2013).Although the pathways causing neuronal loss are not fully understood, the idea of apoptosis has been put forth (Sargholi Nootarki et al., 2015).However, the administration of B. papyrifera and S. aromaticum singly or in combination enhances AChE activity compared to the AD group.The bioactive components in X. parviflora extract may be able to prevent the death of neurons by acting as antioxidants and anti-apoptotic agents (Shaba, 2017).
Oxidative stress is one of the main hypotheses that was responsible for declining brain performance in AD rats (Yuan et al., 2012).Chronic Al intake alters SOD, CAT, and GSH levels, and their activities drastically dropped with rising MDA levels (Breijyeh and Karaman, 2020).However, administration of B. papyrifera and S. aromaticum singly or in combination reduces MDA levels and enhances SOD and GSH levels compared to the AD group.S. aromaticum was discovered to be capable of regulating scavenging reactive oxygen species (ROS), while also raising the fraction of anti-oxidant mechanisms, as demonstrated by a previous study (Shekhar et al., 2018).In accordance with Rajabian et al., reported the molecules derived from Boswellia species to modulate mechanisms regulating neurodegenerative disorders (Rajabian et al., 2020).Furthermore, Miran et al., reported that B. sacra is an essential origin of terpenoids with biomedical applications for many diseases (Miran et al., 2022).Besides, Amir Rawa et al. identified several molecular mechanisms used by Syzygium species in neuroprotection, including suppression of pro-inflammatory mediators, prevention of microglial invasion, and modulation of ß-cell insulin release (Amir Rawa et al., 2022).Also, some studies proposed that the bioactive molecules in herbal extracts protect sensitive neurons, increase memory and blood flow, stimulate neural performance, and stimulate neurogenesis by decreasing ROS accumulation (Botton et al., 2010;Spencer, 2010;Yadang et al., 2020).For instance, Botton and his colleagues investigate the memory enhancer role of caffeine against scopolamine in adult mice (Botton et al., 2010).Also, Spencer demonstrates the mechanisms of flavonoids in the brain (Spencer, 2010).While Yadang and his colleagues found a beneficial role for Carissa edulis (Forssk.)against AD induced by scopolamine in mice (Yadang et al., 2020).
These changes were associated with neurofibrillary tangles, perineuronal gaps, and neuropil vacuolation in both the cortex and the hippocampus of AD rats.Alzheimer's patients' brains comprise amyloid "plaques" and neurofibrillary tau protein "tangles," as well as significant loss of neurons in different areas of the brain (Walton, 2007).Neurons and their connections in brain regions, including entorhinal cortex and hippocampus are frequently destroyed by AD.Whereas the hippocampus rapidly loses structure, which is related to the functional separation from other brain regions (Carmona and Pereira, 2013;Rao et al., 2022).However, the administration of B. papyrifera and S. aromaticum singly or in combination was able to reverse these changes and maintain the brain architecture.These findings were in agreement with previous studies (Khalil et al., 2020;Postu et al., 2019), who reported the roles of natural products such as Pinus halepensis and coconut oil in maintaining the brain architecture due to their antioxidant capabilities (Postu et al., 2019;Khalil et al., 2020).

Conclusion
Herbs have a long history of usefulness and safety, which is probably because of their variety of constituent parts and how these parts engage with the body's various physiological goals.The present study highlighted some of the proposed functions of B. papyrifera and S. aromaticum extracts to enhance the status of AD which is a growing disease in many communities.These combinations improve cognitive dysfunction, enhance AChE activity, reduce the cortical and hippocampal MDA levels, and increase the SOD and GSH levels.These changes were associated with restoring the cortical and hippocampal normal structure.The limitation of this study is that we did not use LC-MS analysis to verify the neuroprotective activities of the extracts.Therefore, future research could be done separately on these extracts to include LC-MS analysis and to explore their underlying mechanisms and different molecular pathways.

Figure 2 .
Figure 2. Effects of the B. papyrifera and S. aromaticum and/or combination on the (a) Cortical AChE; and (b) Hippocampal AChE on the brain homogenates of the AlCl 3 induced rats.Values are means ± S.E.M. (n = 8 animals per group).Statistical significance was determined by one-way ANOVA Followed by Bonferroni's post hoc analyses -* compared to the control group; @ compared to AlCl3 group (p < 0.05).

Figure 1 .
Figure 1.Effects of the B. papyrifera and S. aromaticum and/or combination on the spatial working memory of AlCl 3 induced rats using Y-maze test.(a) number of arm entries (b) and SAP%; spontaneous alternation.Values are means ± S.E.M. (n = 8 animals per group).Statistical significance was determined by one-way ANOVA Followed by Bonferroni's post hoc analyses -* compared to the control group; @ compared to AlCl 3 group (p < 0.05).

Figure 3 .
Figure 3. Effects of the B. papyrifera and S. aromaticum and/or combination on the (a) Cortical MDA, (b) and Hippocampal MDA on the brains of the AlCl3 induced rats.Values are means ± S.E.M. (n = 8 animals per group).Statistical significance was determined by one-way ANOVA Followed by Bonferroni's post hoc analyses -* compared to the control group; @ compared to AlCl 3 group (p < 0.05).

Figure 4 .
Figure 4. Effects of the B. papyrifera and S. aromaticum and/or combination on the (a) Cortical SOD; superoxide dismutase (b) and Hippocampal SOD on the brain homogenates of the AlCl 3 induced rats.Values are means ± S.E.M. (n = 8 animals per group).Statistical significance was determined by one-way ANOVA Followed by Bonferroni's post hoc analyses -* compared to the control group; @ compared to AlCl 3 group (p < 0.05).

Figure 5 .
Figure 5. Effects of the B. papyrifera and S. aromaticum and/or combination on the (a) Cortical GSH, (b) and Hippocampal GSH on the brain homogenates of the AlCl 3 induced rats.Values are means ± S.E.M. (n = 8 animals per group).Statistical significance was determined by one-way ANOVA Followed by Bonferroni's post hoc analyses -* compared to the control group; @ compared to AlCl 3 group (p < 0.05).

Table 1 .
Different compounds present in Boswellia papyrifera extract using GC-MS analysis with their retention time (RT), area percentage, and molecular mass.

Table 2 .
Different compounds present in S. aromaticum extract using GC-MS analysis with their retention time (RT), area percentage, and molecular mass.