INTRODUCTION

Aluminium (Al), the third most common element approximately 8% of total mineral components in the earth’s crust found combination with oxygen, silicon,

ARTICLE INFORMATION:

*Corresponding Author: drshariqali@yahoo.com Received 5th August, 2015

Accepted after revision 28th November, 2015 BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 NAAS Journal Score : 3.48

©A Society of Science and Nature Publication, 2015. All rights reserved.

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"uorine and other elements in the soil, rocks, clays and gems has a signi!cant toxic potential for humans (Ver- straeten et al., 2008). Aluminium enters the human body via food, air, water and drugs, and is present in many manufactured foods such as processed cheese, baking

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Laxman Kumar and Sharique A. Ali

powders, cake mixes, frozen dough, pancake mixes (Lev- esque et al., 2000) and pharmaceutical products, espe- cially antacids (Wang, et al., 2000). Chronic exposure to aluminium is involved in neuro-degenerative disorders, such as Alzheimer’s disease (Flora et al., 2005) dialysis, Parkinson’s dementia (Hirsch et al., 1991) and hepato- toxicity (Yumoto et al., 1993, Muhammad et al, 2014 Crane et al 2014).

It has the potential to cause neurological disor- ders in human and animals, it’s accumulation in the brain has been linked to various neurodegenerative diseases (Yokel, 2002; Zatta et al., 2003). Aluminium ions alter the structure of cellular membranes, inhibit many enzymes like alkaline phosphatase and acetylo- cholinesterase, and adenyl cyclise (Zatta et al., 2002; Platt et al., 2001). Aluminium forms complex with ATP (Al-ATP) as it has strong af!nity for phosphate ion, which is 107 times stronger than Mg 2+ ion. Therefore, it can be hypothesized that the synthesis of GSH hin- dered due to the less availability of ATP by altering the synthesis of g-Glutamylcysteine (g-GluCys) synthetase and glutathione synthetase, enzymes involved in GSH synthesis. Thus decreased activity of glutathione syn- thetase leads to reduced GSH level (Nehru et al., 2005).

Salt et al., (1998) reported that plant extracts detoxify various kinds of environmental pollutant. Plants have been used to treat various diseases and have been an exemplary source of medicine over the years (Ates et al., 2003). Aloe vera is one such ancient plant whose medici- nal properties have been known since centuries and has wide range of therapeutic applications such as wound healing effect, reduction of blood sugar in diabetes, for soothing burns, for easing intestinal, for curing ulcers and for reducing arthritic swellings (Shelton 2003; Davis et al., 1994). A. vera gel contains anthroquinones (aloin, aloe-emodin) which may have a variety of properties of anti oxidant agent, including the protective role for heavy metal toxicity (Flora et al., 2005; Yadav et al., 2009; Zubaydi et al., 2009). The goal of this study was to investigate the protective role of Aloe vera on alumin- ium induced changes in brain enzymes of albino rats.

MATERIALS AND METHODS

Healthy adult albino rats (Rattus norvegicus) weighing 175 ± 5 gm were used for the experiments, procured from Mhow, Bhopal (MP) India, and maintained in our laboratory. The rats were acclimatized in laboratory con- ditions for two weeks and were maintained at 28 ± 20C room temperature and relative humidity (60 ± 10%) with a 12 hours light-dark cycle in the animal house of bio- technology laboratory, Sai!a Science College, Bhopal. Food and water were provided ad libitum throughout the

experiment to avoid effects of starvation. No mortality was observed during the acclimatization period and dur- ing whole experimentation period up to 60 days.

Aloe vera plant leaves were used for the present study. Leaves of A. vera were collected in and around the Bho- pal. Preparation of A. vera (leaf gel) extracts was done according to the method of Arunkumar et al., (2009) with slight modi!cations. Skin of the leaves were pealed and the gel inside was used for extraction. 100 gms of the gel was added to 250 mL of ethanol and extracted using the Soxlet assembly. Later on, the solvent of the extracted material was removed at low temperature in a rotary vacuum evaporator and the resulting dried extract was lyophilized in a freeze dryer.

All the experimental animals were divided into three groups as group I, II and III.

Group I: - This group of 10 (5+5) animals was fed with normal diet and water ad libitum, as control group. Group II: - This group of 10 (5+5) animals was fed with normal diet and aluminium in a dose of 98 mg/kg of body weight orally for 30 and 60 days. Group III: - This group of 10 (5+5) animals was fed with normal diet and received aloin (100mg/kg body weight) and aluminium sulphate (98 mg/kg body weight) for 30 and 60 days. Animals were sacri!ced by cervical dislocation on 30th and 60th days respectively. Liver and kidney were iso- lated and kept in ice cold conditions for experiment.

Brain tissue was homogenized with 5 volume of ice cold 5mM Tris HCL ph 7.4 containing 30 mM sucrose. The homogenate was centrifuged at 1500 xg for 10 minutes to remove nuclei and cell debris. The resultant super- natant was used for further analysis. The experimen- tal chemicals were obtained from Sigma Chemical Co. USA of analytical grade. The following Brain enzymes i.e. Acetylcholinesterase, Sodium Potassium ATPase and Reduced glutathione from rat brain of aluminium sul- phate per se exposed and Aloe vera plus aluminium sul- phate exposed and non exposed (Control) was assayed as per the following standard procedures.

Acetylcholinesterase activity of control as well as experimental rats were assayed as per the method of Ellman et al., (1961).Sodium Potassium ATPase Sodium Potassium ATPase (Na+K+ATPase activity was assayed as per the method of Svoboda et al., (1984).Glutath- ione (GSH) Reduced glutathione was determined by the method of Moron et al., (1979)

RESULTS AND DISCUSSION

In the present investigation, analyses of brain enzyme were done in albino rats subjected to different durations of aluminium sulphate administration. The values of brain enzymes (i.e. acetyl cholinesterase, Sodium Potas-

sium ATPase and Glutathione) of rats exposed to alu- minium sulphate per se and in combination with aloin for a period of 30 days and 60 days with well matched controls are reported here (table 1 to 6 and !gure 1 to 6).

The effects of aluminium sulphate and in combina- tion with standard aloin on acetyl cholinesterase (AChE) activity during the 30 days and 60 days experimental period were showed in table 1,2 and !gure 1,2. There was no much "uctuation in the AChE activity in the brain tissue of control animals throughout the experi- mental period. The data indicated that aluminium sul- phate caused signi!cant decrease in acetyl cholinesterase activity as compared to control animals after 30 days of exposure period. The value decreased from the control value of 41.80 ± 1.985μmol/min/mg protein to16.38 ± 0.4954μmol/min/mg protein (Table 1; Figure 1).

While, in combination of aluminium sulphate with standard aloin did not show as much signi!cant reduc- tion in acetyl cholinesterase activity where the value was found to be 27.02 ± 1.0740 μmol/min/mg protein as compared to the aluminium sulphate per se value of

16.38± 0.4954 μmol/min/mg protein and control value of 41.80 ± 1.985 μmol/min/mg protein (Table 1; Figure 1). When the period of exposure was further increased

Table 1: Showing values of acetylcholinesterase of brain of rats exposed to aluminium sulphate (98 mg /kg of body weight) per se and in combination with aloin for a period of 30 days with well matched controls.

FIGURE 1: Showing values of acetylcholinesterase of brain of rats exposed to aluminium sulphate (98 mg /kg of body weight) per se and in combination with aloin for a period of 30 days with well matched controls.

Laxman Kumar and Sharique A. Ali

Table 2: Showing values of acetylcholinesterase of brain of rats exposed to aluminium sulphate (98 mg /kg of body weight) per se and in combination with aloin for a period of 60 days with well matched controls.

FIGURE 2: Showing values of acetylcholinesterase of brain of rats exposed to aluminium sulphate (98 mg /kg of body weight) per se and in combination with aloin for a period of 60 days with well matched controls.

up to 60 days the activity of acetyl cholinesterase was decreased drastically from the control value 40.00 ± 0.7071 μmol/min/mg protein to 14.01 ± 0.8765 μmol/ min/mg protein which is more prominent in compared to the 30 days treated group. But when aloin was given to rats after aluminium sulphate intoxication for 60 days the value was elevated up to 34.64 ± 1.9380 μmol/min/ mg protein in comparison to the per se value of 14.01 ± 0.8765 μmol/min/mg protein (Table 2; Figure 2).

The effects of aluminium sulphate and in combination with standard aloin on enzyme Na+/K+ ATPase activity during the 30 days and 60 days experimental period are shown in table 3, 4 and !gure 3,4. The data obtained from the experiments clearly indicated that after alu- minium sulphate intoxication Na+/K+ ATPase activity was decreased in 30 days exposed animals where the value was found to be 43.52 ± 1.066 nm/mg/protein which is quite less than the control value of 63.71±.6665 nm/mg/protein (Table 3; Figure 3).

On the other hand after co treatment of aloin and aluminium sulphate, decline in Na+/K+ ATPase activity was further continued where the value was found to be 36.71±1.099 nm/mg/protein which is also less than the control value as well as aluminium sulphate per se treated value (Table 3, Figure 3).

Laxman Kumar and Sharique A. Ali

Table 3: Showing the effect of Aluminium sulphate (98 mg /kg of body weight) on the brain enzyme, Na+/K+ ATP ase activity (nm/mg/protein) of albino rats along with treatment of Aloin (100 mg /kg of body weight) plus aluminium (98 mg /kg of body weight) for 30days of exposure along with well matched control animals.

FIGURE 3: Showing the effect of Aluminium sulphate

(98 mg /kg of body weight ) on the brain enzyme, Na+/ K+ ATP ase activity (nm/mg/protein ) of albino rats along with treatment of Aloin (100 mg /kg of body weight) plus aluminium (98 mg /kg of body weight) for 30days of exposure along with well matched control animals.

Table 4: Showing the effect of Aluminium sulphate (98 mg /kg of body weight ) on the brain enzyme, Na+/K+ ATP ase activity (nm/ mg/protein ) of albino rats along with treatment of Aloin (100 mg /kg of body weight) plus aluminium (98 mg /kg of body weight) for 60days of exposure along with well matched control animals.

FIGURE 4: Showing the effect of Aluminium sulphate

(98 mg /kg of body weight) on the brain enzyme, Na+/ K+ ATP ase activity (nm/mg/protein) of albino rats along with treatment of Aloin (100 mg /kg of body weight) plus aluminium (98 mg /kg of body weight) for 30 days of exposure along with well matched control animals.

When the period of exposure was further increased up to 60 days the activity of Na+/K+ ATPase was drastically reduced from the control value 63.71±.6665 nm/mg/ protein to 34.23±1.982 nm/mg/protein which is more prominent in compared to the 30 days treated group. But when aloin was given to rats after aluminium sul- phate intoxication for 60 days the value was elevated up to 42.71±1.443 nm/mg/protein in comparison to the per se value of 34.23±1 nm/mg/protein (Table 4, Figure 4).

Reduced glutathione (GSH) contents in brain tissue homogenates were estimated using a colorimetric tech- nique. As indicated by the data showed in table and !g- ure 5, 6. Aluminium sulphate alone caused signi!cant reduction in reduced glutathione level in both 30 and 60 days treated animals. In 30 days intoxicated animals the level of glutathione was reduced from the control value of 7.040 ± 0.1691 m mole g-l to 4.060 ± 0.1806 m mole g-l (Table 5; !gure 5).

When the duration of exposure was increased up to

60 days the reduction of glutathione level was reduced more drastically in comparison to the 30 days treated animals where the level of glutathione was found to be 2.780 ± 0.2627 mmole g-l which is quite low in com- parison to the control value of 7.160 ± 0.1077 mmole g-l (Table 5; !gure 5).

Table 5: Showing glutathione of rat brain tissue (m mole g-l) in aluminium sulphate per se and Aluminium sulphate (98 mg /kg of body weight) with aloin exposed rats along with a period of 30 days of exposure with control values.

FIGURE 5: Showing glutathione of rat brain tissue (m mole g-l) in aluminium sulphate (98 mg /kg of body weight) per se and aluminium sulphate with aloin exposed rats along with a period of 30 days of exposure with control values.

Table 6: Showing glutathione of rat brain tissue (m mole g-l) in Aluminium per se and aluminium sulphate (98 mg /kg of body weight) with aloin exposed rats along with a period of 60 days of exposure with control values.

FIGURE 6: Showing glutathione of rat brain tissue (m mole g-l) in aluminium sulphate (98 mg /kg of body weight) per se and aluminium sulphate with aloin exposed rats along with a period of 60 days of exposure with control values.

In an another set of experiment when rats were co treated with aloin and aluminium sulphate, aloin almost nullify the toxic effect of aluminium sulphate as indi- cated by the glutathione level which is now found to be near about control range. In this set of experiment the level of reduced glutathione was found to be 3.526 ±

Laxman Kumar and Sharique A. Ali

0.1911 mmole g-l in 30 days exposed animals and 5.296

±0.2180 mmole g-l in 60 days exposed animals (Table and !gure 6, 6).

Acetyl cholinesterase is found at mainly neuromus- cular junctions and cholinergic brain synapses, where its activity serves to terminate synaptic transmission. Ace- tylcholine is a neurotransmitter which enables chemi- cal communication to occur between a nerve cell and a target cell. This target cell may be another nerve cell, muscle !ber or gland (Richetti et al 2011). The results of the present study thus have clearly indicated that alu- minium sulphate signi!cantly altered the level of acetyl cholinesterase activity in rat brain where the level was found to be highly decreased in both the treated groups. In contrast to this elevated level of acetyl cholinesterase was noticed in aloin and aluminium sulphate co treated groups, indicating the protective role of aloin against aluminium sulphate toxicity.

Several plant extracts do have enzyme protective effects and can reverse the alterations caused by the metals (Muhammad et al., 2014). The neuroprotective ef!cacy of Cucumis melo seed oil in protecting the cad- mium induced changes in acetyl cholinesterase (AChE) activity was investigated in the rat brain. The results showed that rats intoxicated with cadmium for 4 weeks signi!cantly reduced the AChE levels in brain, also observed that administration of oil for 4 weeks in cad- mium intoxicated rats signi!cantly elevated the activity of AChE ( Somade et al., 2014).

Sodium, potassium-adenosine 5’-triphosphatase is an ion-transporting enzyme. Na+/K+-ATPase is a transmem- brane protein found in higher eukaryotes that transports Na+ and K+ across the plasma membrane to maintain ionic gradients. These gradients, in turn, facilitate other secondary active transport systems such as Na+/amino acid and Na+/glucose cotransport in animals, Na+/K+- ATPase is very important for the proper functioning of cells and tissues and in the induction of cytotoxicity, especially in nerve cells (Crane et al., 2014; Wright et al., 1989). Above results of present study clearly demon- strated that aluminium sulphate signi!cantly inhibited Na+, K+-ATPase activity where the level of this enzyme was dropped down in quit signi!cant way in both 30 as well as 60 days treated group. It is also interesting to note that aloin counter balanced the toxic effect of aluminium sulphate which is indicated by the above data where level of Na+, K+-ATPase enzyme was found to be near as the control rats in both 30 and 60 days treated groups. Alcoholic seed extract of Celastrus pan- iculatus could potentially prevent aluminium induced neurotoxicity in the cerebral cortex, hippocampus and cerebellum of the rat brain. It was found that aluminium administration signi!cantly decreased the level of GSH and the activities of Na+/K+ ATPase, Ca2+ ATPase and

Laxman Kumar and Sharique A. Ali

Mg2+ ATPase as compared with control rats (Thangara- jan Sumathi et al., 2013).

Glutathione (GSH) plays an essential role in the intra- cellular antioxidant defense against oxidant radicals, especially the OH radical. The GSH level in the brain provides indirect information on oxidative stress of the brain. It is the major free radical scavenger in the brain. Diminished GSH levels elevate cellular vulnerability towards oxidative stress; characterized by accumulating reactive oxygen species ( Dringen et al., 2000; Pravat et al., 2012). In interpretation of the above data, it has been found that there is a clear cut inhibition of GSH val- ues in all the rats exposed to aluminium sulphate in all durations of treatments, whether of short or long term exposures. In the present study, aluminium has strongly altered the normal values of GSH in brain tissues of all exposed rats, however interestingly it has been observed that co treatment with aloin markedly, counter acted the toxic effects of aluminium sulphate, by a synergistic action of ain at the cellular levels.

Ogungbe and Lawal, (2008) reported marked protec- tive effects of ethanolic extracts of garlic, Allium sativum and ascorbic acid on cadmium-induced oxidative stress in rats, which had induced reduced glutathione levels. It was observed that there was signi!cant increase in GSH con- tent in the liver and kidney of ethanolic extract garlic pre- treated rats as compared to controls. Similarly, supporting the data of our present work, Sharmila et al., (2009) have shown that there is a signi!cant therapeutic ef!cacy of oral administration of Ocimum sanctum (200 mg/kg, once daily), post arsenic exposure (100 ppm in drinking water) in rats. They observed that animals exposed to arsenic showed a signi!cant inhibition of GSH activity in blood, where as administration of O. sanctum post arsenic expo- sure, exhibited signi!cant recovery and restored blood GSH levels, as seen in the present !ndings.

The results of the present study clearly indicated that aluminium sulphate has signi!cantly altered the nor- mal levels of acetyl cholinesterase, Sodium Potassium ATPase and glutathione of rat brain enzymes, where the levels were found to be highly decreased in both the treated group. But in contrast to this elevated level of acetyl cholinesterase, sodium potassium ATPase and glutathione were noticed in aloin and aluminium sul- phate co treated groups, indicating protective role of aloin against aluminium sulphate toxicity.

ACKNOWLEDGEMENT

Authors are grateful to Principal Sai!a College of Sci- ence Bhopal and Secretary, Sai!a Education Society Bhopal, for providing necessary facilities and encour- agement.

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