Neuroprotective role of Allium cepa and Allium sativum on Hippocampus, striatum and Cerebral cortex in Wistar rats

 

Shyamala Nayak¹, Nayanatara Arun Kumar²*, Anupama Hegde¹, Rekha D Kini², Vandana Blossom³, Roopesh Poojary²

¹Department of Biochemistry, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal.

²Department of Physiology, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal.

³Department of Anatomy, Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal.

 

ABSTRACT:

Natural products are gaining much importance in light of the serious side effects posed by drugs of chemical origin. High intake of foods rich in antioxidants reduces the risk of neurodegenerative disorders. Traditionally, Allium sativum L.(garlic) and Allium cepa. L (onion) has received considerable attention for their therapeutic benefits around the globe. The present study assesses the free radical scavenging role of Allium sativum and Allium cepa in cerebral cortex, striatum, and hippocampus. Adult wistar rats of either sex were grouped as control group (Group I) treated with normal saline and the two experimental group were treated with the aqueous bulbous extracts of dehydrated Allium sativum (Group II) and Allium cepa (Group III) was considered as treated groups. Homogenates of hippocampus, striatum and cerebral cortex were analyzed for biochemical and neuronal analysis. LD₅₀ value of these extracts in rats was found at a dose of 500 mg/kg BW. A Significant decline (P<0.05) in the MDA level was observed in the hippocampus, striatum and cerebral cortex in group II when compared to group III. Total antioxidant level, GSH, SOD level was significantly high (P<0.001) in the treated groups. Neuronal increase was significant in Group II (P < 0.01) when compared to Group III. Allium sativum and Allium cepa was found to have a challenging role in hampering oxidative stress in Hippocampus, striatum and cerebral cortex, the target regions in neurological disorders. However, ample number of studies are required to establish their mechanism of action as a progression to clinical approach.

 

 

 

INTRODUCTION:

The nervous system is one of the major control systems regulating various functions such as motor activity, learning, memory and cognition. Hippocampus, cortex and striatum mediate various functions¹. The maintenance of healthy functional and structural state of the nervous tissues is of utmost importance. Neurodegenerative diseases have become the problem of major health concern².

 

This increased incidence of these neurodegenerative diseases is the leading cause of death. Due to increased life expectations, there is the probability of the increased incidence of these disease^3. Currently there is no potent therapeutic approach available for the treatment of neurodegenerative diseases.

 

The brain is more susceptible to damage from free radicals caused by oxidation processes due to more lipid content and increased consumption of oxygen. Oxidative stress is one mechanism that inextricably linked to neurodegenerative disorders. Reactive oxygen species (ROS) are generated within the biological system^4-6. Increased concentrations of ROS can lead to the disruption of balance between pro-oxidant and antioxidant levels leading to various degenerative disorder⁷. There is a huge clinical advantage in preventing nerve damage and death of the nerve cell. Several researchers have been interested in screening new therapies using antioxidant agents. Research has been diverted towards the use of natural antioxidants in combating oxidative stress. In addition to their antioxidant activity the natural agents acts at the molecular level. The components derived from medicinal plants plays an pivotal role in maintaining the normal brain functions. The sources from plants contains abundant molecules available for improving human health. The medicinal components in vegetables and fruits decreases the risk of neurological disorders^8-9.. Therefore, consuming surplus quantity of vegetables and fruits reduces the risk of diseases caused by dysfunction of neurons.

 

Allium cepa L. (onion) and Allium sativum L. (garlic) belongs to the family Alliaceae are grown worldwide¹⁰. These are used as the main source of food components. Antimicrobial property of the garlic makes it an effective agent against bacterial, viral, fungal and parasitic infection. Anticancer effect of garlic is well documented in the literature^11-13. Human studies using garlic powder demonstrated its role in immunity and phagocytosis. Onion is also one of the multipurpose food plants possessing strong nutritional and health benefits for eras. Literature survey documents the presence of pharmacologically important active constituents in onion and garlic. The various antioxidants and pharmacological properties of the onion and garlic has been documented in various research^14-15. Literature lacks information on the neuroprotective role of the onion and garlic on distinct brain regions. This study was focused to explore the neuroprotective role of Allium sativum and Allium cepa on the selective brain tissues such as cortex, striatum and hippocampus targeting mainly towards therapeutic role against neurodegenerative disorders.

 

MATERIAL AND METHODS:

Ethics:

All the procedures for the animal experiments were reviewed and approved by the Institutional Animal Ethical Committee, Kasturba Medical College, Mangalore (F. No. KMC/MNG/IAEC/19-2018; approved date: June 30^th 2018). All the animal studies were conducted and maintained according to the guidelines proposed by the Committee for Control and Supervision of Experimentation on Animals (Reg. No. 213/CPCSEA), Government of India.

 

Experimental Animals:

Adult Wistar rats, weighing 150-200g body weight were selected for all invivo studies. The experimental animals were obtained from Central animal house, Kasturba Medical College, Mangalore (Reg.No.213/CPCSEA). The experimental rats were placed in polypropylene cages with paddy husk beddings. Throughout the research period, animals were maintained at 25 ± 2^°C temperature and 50 ± 5% humidity with 12 h of light/dark cycles. Rats were provided with ad libitum access to laboratory food (commercial mice pellets from VRK nutritional solutions, India) and water.

 

Procurement of Allium sativum and Allium cepa:

Dehydrated powder of Allium sativum and Allium cepa was received as a Gift sample from Mevive International TM Coimbatore, Tamilnadu, India.

 

Toxicity study and Dosage fixation:

An acute toxicity study was carried out as prescribed by the Organization for Economic Cooperation and development guidelines. Prior to experimentation, Wistar rats were fasted overnight (water withheld for 3–4 h) and fixed doses of onion and garlic powder dissolved in distilled water 1mL with the dosage as per 50, 200, 400, 500, 1000 and 2000mg/kg body weight were administered. The extract was tolerated as no death was found up to the maximum dose administered. Rats were observed individually after dosing for the first 30 min periodically, and daily thereafter for 14 days for any toxicity signs such as gross changes in the skin, fur, eyes, mucous membranes, or circulatory, respiratory, autonomic, and central nervous systems, or changes in behavior pattern. On the basis of earlier studies report and our observation the effective dose of 500mg/kg body weight was selected for both the herbal powder further studies. The treatment was given for 30 days.

 

Experimental design:

Eighteen Wistar albino rats were divided into three groups (n=6; n= number of animals in each group) as follows:

 

Group-1(control), administered with distilled water

Group-2 - Allium sativum, 500mg/kg body weight

Group-3 – Allium cepa, 500mg/kg body weight. The treatment was given for 30 days

 

Preparation of sample:

Brain dissection:

At the end of the 30th day the animals were anaesthetized with a combination of ketamine and xylazine (50mg/ml of ketamine and 20 mg/ml of xylazine). Brain was dissected out by making midline incision to view the skull. Then, a small incision from the caudal part of parietal bone and a firm cut in the anterior part of the frontal bone were made to remove the brain more easily. Isolated brains were dissected on ice (using large and small curved serrated forceps) to obtain cerebral cortex, striatum and hippocampus according to the method of Glowinski and Iversen¹⁶. The isolated brain parts homogenates were prepared with ice cold phosphate buffer saline pH 7.4 using a homogenizer (RO-1727A Remi Motors, India). Homogenization of brain tissue was done under prechilled conditions. The homogenate obtained was centrifuged in cold centrifuge. The supernatant obtained was used for various biochemical estimations as follows.

 

Lipid peroxidation assay:

The lipid peroxidation products in the homogenate were measured through the estimation of Thiobarbituric acid reactive substances was done as per the method of Rao et al¹⁷. Tissue homogenate (1mL) was precipitated with 2.5mL of ice cold trichloroacetic acid (TCA). The samples were centrifuged at 3000g for 10 min. From the obtained homogenate, 2ml of the supernatant, 0.67% of thiobarbituric acid (TBA) was added and kept in boiling water bath for 10 min and cooled. The pink chromogen that was developed was read immediately at 532nm. Thiobarbituric acid reactive substances TBARS concentration was calculated using molar extinction co-efficient of chromophore (1.56×105 (mol/L)^-1cm^-1 and the values were expressed in nmloes/L.

 

Estimation of total antioxidants (TAO):

The total antioxidants level was estimated according to the method described by Koracevic et al¹⁸. Each sample had its own control in which Fe-EDTA mixture, hydrogen peroxide and sodium benzoate were added after 20% acetic acid. For each series of analysis a negative control was prepared, except that sample homogenate was replaced with 0.1 M Sodium phosphate buffer, pH7.4. Uric acid (1Mm/L) was used as standard. The reaction mixture was incubated at 37ºC for 60 minutes, then 20% Acetic acid and 0.8% TBA were added and incubated for 10 minutes at 100ºC, then cooled in ice bath. The absorbance was measured at 532 nm. The total antioxidants level is expresses as μmol/L.

 

Estimation of Reduced Glutathione:

Tissue GSH concentration was estimated according to the method described by Ellman¹⁹. One milliliter of supernatant was precipitated with 1ml of metaphosphoric acid and cold digested at 4ºC for 1 h. The samples were centrifuged at 1,200g for 15 min at 4ºC. To 1ml of this supernatant, 2.7ml of phosphate buffer and 0.2ml of 5, 5’ dithio-bis-2-nitrobenzoic acid (DTNB) were added. The yellow color developed was read immediately at 412nm using a Systronic-117 UV-Visible spectrophotometer. The values were expressed as mg/gm. tissue.

 

Estimation of Superoxide dismutase activity:

Superoxide dismutase activity was measured according to the method of Marklund and Marklund²⁰. The assay was based on the ability of the enzyme to inhibit auto-oxidation of pyragallol oxygen. Stock solution of pyragallol was made in 10 mMHCl. One unit of the enzyme was defined, as the amount of SOD required for producing a half maximal inhibition of autooxidation. Absorbance was measured for 5 min at 420nm, and expressed as units/min/mg of tissue protein.

 

Neuronal assay of frontal cortex and hippocampus (Neuronal density by Nissl staining):

The Brain tissue was stored in 10% formalin brain for 48 hours for the process of fixation. Once the fixation has been done the paraffin blocks were made. Coronal sections of frontal cortex and hippocampus using rotary microtome of thickness about 6-7μ thickness were made and the sections are stained with crystal violet stain. The Quantitative analysis was performed under light microscopy (20X 300X300 µ areas in the dentate gyrus (DG). 150x150 micron area was selected for counting. Hippocampus area (CA1, CA2, CA3, and CA4) was also selected for quantifications²¹.

 

Quantification of neurons:

Number of neurons in unit volume (cells/mm³) was quantified in dentate gyrus and hippocampal regions. Numbers of cells in ten randomly selected 1000 μ² areas were counted in each section. A total of 12-15 sections were selected from each brain (every 15^th section). Finally number of cells/mm³ was calculated using stereo investigator principle²¹

 

Statistical analysis:

Results are expressed as Mean±SD and comparisons between groups were made by means of an unpaired Student t-test. Differences were considered significant at P< 0.05.

 

RESULTS:

A significant decline (P<0.05) in the MDA level was observed in the hippocampus, striatum and cerebral cortex in group II when compared to group III. Even though onion treated rats also showed a decreased MDA level in distinct brain tissues, the decrease was not statistically significant. Total antioxidant level, GSH, SOD level was significantly high (P<0.001) in the treated groups (Table.1). Comparatively, extracts of garlic showed more potent effects (P<0.001) than onion treated groups.

 

Table1: Superoxide dismutase, reduced glutathione, total antioxidant, and lipid peroxidation level in the distinct brain tissues in garlic and onion treated Wistar rats.

Groups	Parameters	HIPPOCAMPUS	STRATI,	CPRTEX
CONTROL (Group I)	SOD U/mg protein	10.66±0.095	10.94±0.03	11.35±0.179
	TOA µmol/L	0.77±0.02***	0.98±0.07	1.71±0.10
	GSH mg/tissue	189.10±0.47	206.73±8.87	166.8±2.84
	LPO nmols/L	590.5±8.54	326.5±10.48	427.5±6.34
Allium sativum (Group II)	SOD U/mg protein	18.648±0.39	29.59±0.37***	26.641±0.36***
	TOA µmol/L	1.955±0.024	2.78±0.10***	2.34±0.0.***
	GSH mg/tissue	672.70±0.68	693.03±4.34***	682.29±3.41***
	LPO nmols/L	581.16±5.91	289.16±6.43	384±4.28
Allium cepa (Group III)	SOD U/mg protein	16.92±0.19***www	26.011±0.34***www	24.80±0.53***www
	TOA µmol/L	1.39±0.058***www	1.891±0.02***www	2.145±0.02***www
	GSH mg/tissue	543.66±1.45***www	566.41±5.96***www	558985.32***www
	LPO nmols/L	498.14±6.87w	311.8±7.18w	401.2±8.92w

***P<0.001:versis Grpi[ II; www P<0.001, wP<0.05; Group II versus Group III

 

 

Fig. 1: Neuronal count in rat brain regions in Allium sativum and Allium cepa treated Wistar rats

 

There was no significant change in the neuronal count in the prefrontal cortex and hippocampus in the treated group when compared to the control group. Comparatively, the neuronal increase was significant in Group II (P < 0.01) when compared to Group III. Garlic treated groups showed a potent significant effect when compared to onion treated (Figure.1-4)

 

 

Fig. 2: Neuronal count in the cortex and various hippocampus regions (DG, CA1, CA2, CA3, CA4) in Normal control Wistar rats

 

 

Fig. 3: Neuronal count in the cortex and various hippocampus regions (DG, CA1, CA2, CA3, CA4) in Allium sativum treated Wistar rats

 

 

Fig. 4: Neuronal count in the cortex and various hippocampus regions (DG, CA1, CA2, CA3, and CA4) in Allium cepa treated Wistar rats

 

DISCUSSION:

Onion and garlic is one of the traditional herbal medicine used in the prevention of various alignments. Neurological disorders increases the production of reactive oxygen species. Rise in the lipid peroxidation level suggests the increased free radicals. Hence, lipid peroxides serve as a prime marker of oxidative damage in various diseases^22-23. The observed decrease in the level of malondialdehyde in the brain tissues in treated rats indicates the suppressed level of free radical production Increased level of antioxidant enzyme observed in this study after the treatment with garlic and onion are in consistent with the reports by several authors^10-12.

 

Superoxide dismutase (SOD) is needed for the proper functioning of the brain. It is a potent enzyme that regulates the level of superoxide anion radical in the brain. The antioxidant system of SOD protects the brain against toxicity caused by the free radicals. In the present study the increased level of SOD in the treated rats indicated the protective role of garlic and onion against brain damage. Glutathione also protects the cells from oxidative damage thus maintaining homeostatis. The present results supports the antioxidant nature of Allium sativum and Allium cepa.

 

Hippocampus plays an important role concerned with learning, memory and emotional behavior²⁴ and also control the secretion of adrenocorticotrophic hormone. Damage to the neurons of the hippocampus is seen in various neurodegenerative diseases. Bilateral hippocampal has been linked to memory deficits²⁴. Nonsignificant increase in the neurogenesis was observed in the treated group when compared to the control group.. The increased number of immature neurons signifies the reorganization of the hippocampus. The garlic treated group showed increased neurogenesis.

 

 

The intake of certain plant products in vivo and in vitro could be related to the diverse antioxidant nature. The phytochemicals is mainly attributed to the protective role of in medicinal plants. The sulphuric compounds in garlic and onion might be responsible for the protective effects. Further, the immune modulatory present in this extract plays an very important role in the immune resistance combating oxidative stress¹⁰.

 

In conclusion, the neuroprotective role of garlic and onion might be due to the presence of efficient antioxidant enzymatic systems potentiating its role in the specific role in brain tissue. Comparatively, the extracts of Allium sativum showed more potent effects than that of the onion. Further in depth studies Allium sativum and Allium cepa could be explored on hippocampus, striatum, and cerebral cortex aiming as a therapeutic target in some animal neurodegenerative models.

 

ACKNOWLEDGEMENT:

We are very thankful to Mevive International TM Coimbatore, Tamilnadu, India for providing us the dehydrated powder of Allium sativum and Allium cepa as a gift sample and sincere gratitude to Manipal Academy of Higher Education for providing us all the facilities for the smooth conduction of the research.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 26.06.2020           Modified on 16.07.2020

Accepted on 31.07.2020         © RJPT All right reserved