Lupeol Isolated from Betula alnoides Ameliorates Amyloid Beta Induced Neuronal Damage via Targeting Various Pathological Events and Alteration in Neurotransmitter Levels in Rat’s Brain

Lupeol, a natural active constituent of Betula alnoides (BA), is well known for its anti-inflammatory, anti-oxidant and neuroprotective activities. In present study the therapeutic potential of lupeol was investigated against amyloid beta (Aβ(1-42)) induced cognitive deficit, neurochemical and biochemical abnormalities in rats. Lupeol was isolated from the BA and its structure was confirmed through nuclear magnetic resonance spectra. Aβ(1-42) (4 μg/4 μL) intracerebroventrically (icv) was administered to rats for the induction of Alzheimer's disease (AD). Lupeol treatment (25 mg/kg/day, 50 mg/kg/day and 100 mg/kg/day per orally) was started one week after Aβ(1-42) infusion up to the 21 st days. Morris water maze from day 16 to 21 and object recognition tasks on day 14 and 15 were performed for memory assessment. On 22nd day, animals were sacrificed and hippocampi were isolated for analysis of biochemical (acetylcholinesterase, lipid hydroperoxide, glutathione and nitrite) and neuro-inflammatory (tumor necrosis factor -α, interleukin (IL)-1β, and IL-6) parameters. In the present study Aβ(1-42) infusion was significantly impaired behavioral memory, increased oxidative stress, decreased antioxidant enzyme and increased pro-inflammatory markers. Treatment of lupeol significantly restored Aβ(1-42) induced behavioral and biochemical abnormalities in rats brain. The findings of the present study suggest that lupeol act through multiple mechanisms and would be used to curb cognitive decline associated with neurodegenerative disorders of AD.


Introduction
Alzheimer's disease (AD) is the most common form of dementia characterized by extracellular deposits of amyloid beta (Aβ  ), intracellular neuro-fibrillary tau tangle, oxidative stress and decline in cognitive functions [1][2][3]. Although the exact pathogenic mechanisms remain unclear, transcriptional dysregulation and impaired cyclic nucleotide signaling have been reported in experimental animals as well as in AD patients [4][5][6]. Recent neuropathological studies have also established a link between morphological and functional changes occurring in the monoaminergic ascending system, particularly in nor epinephrine (NE) and 5-hydroxytriptamine (5-HT), and the pathophysiology of AD. Moreover clinical researchers found that AD patients have complex neurochemical disturbances including the catecholaminergic, cholinergic and glutaminergic neuronal systems [7-10].
Lupeol, a biologically active dietary triterpenoid is found in many medicinal plants and different fruits such as olives, mangoes, and strawberries [11,12]. A variety of medicinal plants such as Betula alnoides, Vernonanthura ferruginea and Zanthoxylum rhoifolium have also been reported to contain lupeol as an active constituent [13][14][15]. Lupeol has been reported to have various pharmacological activities including acetylcholinesterase (AChE) ARCHIVOS DE MEDICINA ISSN 1698-9465

JOURNAL OF NEUROLOGY AND NEUROSCIENCE ISSN 2171-6625
inhibition, anti-inflammatory and anti-oxidant actions [16][17][18]. Further, it has been reported to be effective in various pathologies and recently its neuroprotective effect has been studied [19,20]. Indeed Brimson et al. [21] described a neuroprotective effect of lupeol against glutamate-induced neurotoxicity in HT22 mouse hippocampal cells.
Intra-cerebroventricular (ICV) administration of Aβ  is a well-known model to produce behavioral, biochemical and neuropathological changes similar to that seen in clinical AD and considered as an appropriate animal model of AD [22]. Thus, the present work was carried out to investigate the neuroprotective potential of lupeol against ICV-Aβ  induced cognitive impairment, neurotransmitters deficits, neuroinflammation and oxidative-nitrosative stress in rats.

Drugs and chemicals
The   was purchased from Sigma-Aldrich, USA.
All other chemicals used in the study were of analytical grade. Solutions of the drugs and chemicals were always prepared afresh before use.

Extraction and fractionation of BA (Stem bark)
50 gm of dried and powdered bark of BA was soxhlated with ethanol (60°C to 80°C) yielding 14.6 g of dry ethanolic extract. Lupeol, as a major constituent was separated out using a column chromatography technique [23]. Different spectroscopic methods were used to elucidate the structure of lupeol using Fourier transform infrared spectroscopy (FTIR) -Nicolet S10 (Thermospecific) in CCl 4 at I.S.F. Analytical Lab, Moga. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE-400 Japan (100 MHz and 400 MHz) in chloroform with tetramethylsilane as internal standard at the SAIF, Punjab University, Chandigarh, India.

Animals
Male wistar rats (250-300 g) were obtained from Central Animal House of I.S.F. College of Pharmacy, Moga, Punjab (India). Animals were kept in polyacrylic cages (4/cage) and maintained under standard husbandry conditions (room temperature 22 ± 1°C and relative humidity of 60%) with a 12 h light/dark cycle (lights on at 8 AM). The protocol was reviewed and approved by the Institutional Animal Ethics Committee and the experiments were carried out according to Indian National Science Academy guidelines.

Experimental procedure
Animals were anesthetized with ketamine (100 mg/kg, i.p) and xylazine (5 mg/kg, i.p) and were fixed on steriotaxic apparatus. Skull was exposed after midline sagittal incision and holes on both sides of the brain were drilled according to Paxinos and Watson [24]. Animals were divided into five groups and each group comprised of 10 animals. The treatment schedule and the interval for estimation of various parameters were described as; Group1: served as double vehicle control, received artificial cerebrospinal fluid (ACSF) in a volume of 4 µl in each ventricle on day 1% and 2% dimethyl sulfoxide per orally (p.o., as a vehicle of lupeol) for 21 days. Group 2: Rats were infused with ICV-Aβ  (4 μg/4 μL) dissolved in ACSF in a volume of 4 µl in each ventricle on day 1. Group 3, 4 and 5 received lupeol at doses of 25 mg/kg, 50 mg/kg, 100 mg/kg p.o. respectively 1 week after the ICV-Aβ  infusion starting from day 7 and continued once daily at 10 am daily for a period of 21 days. The doses of lupeol were selected on the basis of earlier reports in which significant antioxidant and neuroprotective properties were demonstrated [18,25].

Object Recognition test (ORT):
The ORT was performed as described by Giorgetti et al. [26].
Spatial navigation task: Spatial learning and memory of animals in a Morris water maze (MWM) was tested by the method described by Morris [27].
Brain homogenate preparation: Terminally, on day 22, rats were sacrificed and hippocampus from the brain was separated, weighed and then homogenized individually. The various biochemical parameters were determined separately in hippocampal supernatant collected following homogenization. Animals were perfused with phosphate buffer saline before decapitation from the heart to remove blood from the brain tissues completely and its interference with the homogenate readouts. After perfusion, animals were sacrificed by decapitation and brains were removed and rinsed with ice-cold isotonic saline. The rat hippocampal tissues were then homogenized with ice cold 0.1 M phosphate buffer (pH 7.4) in a volume 10 times the weight of the tissue. The homogenate was centrifuged at 10,000 g for 15 min (4°C) and aliquots of supernatant were separated and used for biochemical analysis. Protein was measured in all brain samples by the method of Lowry et al. [28] using bovine serum albumin (1 mg/ml) as a standard.

AChE activity:
The quantitative measurement of AChE activity in brain was performed according to the method described by Ellman et al. [29].

Malondialdehyde (MDA) estimation:
The quantitative measurement of MDA end product of lipid peroxidation-in brain homogenate was performed according to the method of Wills [30].

Nitrite estimation:
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay using Greiss reagent (0.1% N-[1naphthyl] ethylene diaminedihydro chloride, 1% sulfanilamide and 2.5% phosphoric acid) as described by Green et al. [31].

Statistical analysis
The values were expressed as mean ± standard error mean (SEM). P<0.05 was set to be statistically significant. The results were analyzed using Analysis of variance using statistical Graph Pad Prism software. In ORT, time of exploration during T1 and T2 on familiar and novel object was analyzed by paired student t-test.

Isolation of lupeol
The melting point of the compound, lupeol was found to be 213°C to 215°C. The FTIR spectra gave information about the functional groups or chemical entities present in lupeol which was found in accordance with the literature data of lupeol [33]. Further 1H NMR spectrum of the compound revealed the presence of seven tertiary methyl protons, a sextet of one proton at δ 2.17 and presence of olefinic protons at (H-29a and b) which is characteristic of lupeol [34]. On the basis of FTIR and NMR spectra structure of compound was drawn which was in good conformity for the structure of lupeol (Figure 1).

Effect of lupeol on memory performance in MWM task of ICV-Aβ (1-42)-infused rats
In the MWM, animals were trained for 5 days starts on day 17 of Aβ

Effect of lupeol on non-spatial memory performance in ORT in ICV-Aβ (1-42)-infused rats
There was no significant difference observed between the Aβ (1-42) -infused and Lupeol-treated rats for familiar objects ( Figure  3A). When Aβ  infused rats were exposed to familiar and novel objects in ORT, they were unable to discriminate between familiar and novel objects and spend almost equal time to explore the similar and novel objects as compared to control group ( Figure 3B). Whereas, lupeol (25 mg/kg, 50 mg/kg and    100 mg/kg) treatment produced significant improvement in discriminating ability between familiar and novel object in Aβ  infused rats ( Figure 3C).

Effect of lupeol on AChE activity in Aβ (1-42)-infused rats
ICV infusion of Aβ  produced significant increase in AChE activity as compared with vehicle treated groups. Lupeol (25 mg/kg, 50 mg/kg and 100 mg/kg) attenuated Aβ  -induced elevation in AChE activity (Figure 4) and restored the values as that of vehicle control rats.

Effect of lupeol treatment on brain oxidativenitrosative stress (MDA, GSH and nitrite) levels in ICV-Aβ (1-42) injected rats
The level of MDA and nitrite were increased and GSH was decreased significantly in Aβ  infused groups as compared of control group. Lupeol (25 mg/kg, 50 mg/kg and 100 mg/kg) treatment significantly restored the MDA, nitrite and GSH levels as compared with those of Aβ (1-42) -infused rats ( Table 1).

Effect of lupeol treatment on brain proinflammatory cytokines levels in ICV-Aβ (1-42)-infused rats
The levels of TNF-α, IL-6 and IL-1β were increased significantly in hippocampus following Aβ  infusion as compared with control group. Lupeol (25 mg/kg, 50 mg/kg and 100 mg/kg) treatment significantly restored the hippocampal pro-inflammatory cytokines level as compared with Aβ  infused rats (Table 2).

Discussion
The present study demonstrates the neuroprotective potential of lupeol against Aβ  induced behavioral, biochemical and neurochemical abnormalities in rats. ICV infusion of Aβ  in rats is known to produce cognitive deficit and other neuropathological changes similar to those seen in AD and thus considered as a suitable model system of AD [23,35]. In the present study, ICV-Aβ  infusion produced cognitive impairment, oxidative stress, neuroinflammation and hippocampal neurotransmitter deficit demonstrating similar changes following Aβ  infusion in rats [35]. Aβ  is an oligomer, reported to cause destruction in the selective brain regions such as hippocampus, cortex and perirhinal cortex that affects spatial and non-spatial memory respectively [36][37][38]. MWM and ORT were used to assess spatial and non-spatial memory respectively. In the present study, Aβ  infused rat showed poor learning and consolidation of memory in MWM task. In addition, these animals were unable to discriminate between novel and familial objects indicating Effect of lupeol in ORT in Aβ  infused rats. The values are expressed as mean ± SEM (n=10). (A) The performance of rats in (T1) sample phase trial with two similar objects (F01 and F02). No significant difference was observed between the untreated and Lupeol-treated rats.

Figure 3A
Effect of lupeol in ORT (Familiar and novel objects) in Aβ  infused rats. The values are expressed as mean ± SEM (n=10). The performance of rats in the (T2) test phase trial, wherein rats exposed with two dissimilar objects, one already exposed F01 (considered as familiar object). * P<0.001 vs. exploration time of familiar objects.
The values were expressed as mean ± SEM The results were analyzed using two-way ANOVA followed by Bonferonni test for multiple comparisons and one way ANOVA followed by Tukey's test, for multiple groups using statistical Graph Pad Prism software (version 5.0, La Jolla, CA, USA). @ P<0.001 vs. Vehicle; * P<0.001 vs. Aβ  .   .
The values were expressed as mean ± SEM The results were analyzed using two-way ANOVA followed by Bonferonni test for multiple comparisons and one way ANOVA followed by Tukey's test, for multiple groups using statistical Graph Pad Prism software (version 5.0, La Jolla, CA, USA). @ P<0.001 vs. Vehicle; * P<0.001 vs. Aβ  . which stay in good agreement with its previous findings [51]. However, lupeol treatment significantly attenuated Aβ  induced oxidative-nitritive stress and decreased the levels of pro-inflammatory cytokines, confirming its antioxidant and antiinflammatory properties [52]. Earlier reports have also suggested that oral administration of lupeol decreases the secretion of pro-inflammatory cytokines such as TNF-α, and IL-1β and inhibit the over activation of microglia and astrocytes [52]. It has been reported that lupeol act through various mechanism and downregulates various apoptotic molecules like Bax, cytochrome C and caspases that leads to neuronal death [52]. Moreover Lupeol has also been reported to exhibit neuroprotective action against the glutamate induced exitotoxicity in mouse hippocampal cells [22]. Thus, the observed anti-inflammatory antioxidant effects of lupeol help to decrease the neuronal oxidative stress and may offer novel strategies in the treatment of age-related AD.

Conclusion
In the current study, lupeol halt Aβ  induced behavioral, biochemical and neurochemical abnormalities in rats. Lupeol was able to improve behavioral changes, restore neurotransmitters levels, decreased oxidative-nitrosative stress and reduced inflammatory processes. Together these findings suggest multiple therapeutic approach of lupeol towards Aβ  induced dementia and suggest lupeol as a potential therapeutic molecule to treat AD like symptoms.