Neuroprotective effect of Conessinin on Elevated oxidative stress induced Alzheimers’disease in rats

Nagaraju Bandaru¹*, Chandrasekhar Komavari², Uma Sankar Gorla¹, GSN Koteswarao¹, Umasankar Kulandaivelu¹, A. Ankarao¹

¹Department of Pharmacology, College of Pharmacy, Koneru Lakshmaiah Education Foundation,

Vaddeswaram, Guntur, Andhra Pradesh, India - 522502

²Department of Pharmacology, Shri Vishnu College of Pharmacy, Bhimavaram, Andhra Pradesh - 534202

*Corresponding Author E-mail: bnagaraju@kluniversity.in

ABSTRACT:

Background: Alzheimer disease (AD) is a progressive dementia affecting a large proportion of the aging population. There is evidence that brain tissue in patients with AD is exposed to oxidative stress during the course of the disease. Conessine is a natural steroidal glycoside, which has been reported to exert various biological activities such as antioxidant and anti-inflammatory effect. Aim: The present study aimed to investigate the effects of Conessine on neurobehavioral activity and superoxide dismutase (SOD), glutathione reductase (GRx) and catalase (CAT) enzymes activity, malondialdehyde (MDA) levels in hippocampal area of rats in an experimental model of AD. Methods: The AD was induced in animals by intracerebroventricular injection of STZ (icv-STZ) unilaterally. Animals were treated with the Conessine (20 mg/kg body weight), then after three successive weeks, recognition memory was examined (passive avoidance test and novel object recognition test) and antioxidant parameters were evaluated. Results: In our study behavioural testes showed improvement on memory retrieval and recognition memory consolidation. Furthermore the Conessine increased the activity of antioxidant enzymes SOD, glutathione GRx and CAT levels and decreased MDA in the hippocampal area. Conclusion: These results suggested that Conessine may inhibit STZ-induced oxidative stress, and that it may possess therapeutic potential for the treatment of AD.

KEYWORDS: Alzheimer’s, Oxidative stress, Memory, anti oxidant, Conessine.

1. INTRODUCTION:

Alzheimer’s disease (AD) is a neurodegenerative disorder that it is the most common cause of dementia in the old age who are slowly deprived in memory and the ability to carry out the simplest tasks¹. Those with AD begin to lose their cognitive abilities, including behavioural disorders and loss of functional autonomy². Both genetic and environmental factors are regarded as a risk factor of AD.

Free radicals, increased oxidative stress and mitochondrial dysfunction, which finally cause the neuronal/synaptic dysfunction and neurodegenerative^3,4. The hippocampal damage is considered as a major contributor to the development of cognitive dysfunction in AD, including learning and memory impairments. (The brain’s neurons are sensitive to oxidative damages because of their higher metabolic activity and loss of antioxidant capacity. Oxidative stress occurs due to misbalance in oxidant A.D. [5,6] and antioxidant factors and has been known to play a critical role in the neurodegenerative diseases such as Parkinson’s disease and AD⁷. Although reactive oxygen species (ROS) play a critical role in the several cellular and signalling pathways at enzyme's activity, over production of ROS causes damage to biomolecules such as lipid and protein⁸. However, the genetic and biochemical information demonstrating that oxidative stress is increase in AD and treatment with antioxidants has been useful in animal models. Recently, a developing investigation has focused on the potential of natural antioxidants to aid in the protection of cognitive function during aging, while reducing risk for AD and other dementing disorders⁹. The capacity of steroidal alkaloids to act as an antioxidant is dependent upon their molecular structure, the position of hydroxyl groups, and other substitutions in the chemical structure of these polyphenols. Conessine carries its antioxidant properties by direct radical scavenging and enhancement of antioxidant defence of the cell and the ability to cross the blood-brain barrier has proven to be a natural treatment for various disorders of the central nervous system¹⁰.

2. MATERIALS AND METHODS:

All procedures in this study were in agreement with the Guide of Care and were approved by the ethics committee on animal experimentation of the Shri Vishnu College of Pharmacy. 30 male Wistar rats weighting 220 ± 20g at the time were used. The animals were housed seven cages, in a colony room with a 12h light/dark cycle (8:00–20:00 lights on) at 22±2°C. They had free access to food and tap water except during the time of experiments. All animals were allowed to adapt to the laboratory conditions for at least one week.

2.1.Experimental design:

In the experimental research, male adult rats were randomly divided into 4 groups (n = 6). The following groups of animals were used: control group, vehicle group (received distilled water by gavage), donepezil (2mg/kg) group, streptozotocin (STZ) group (received 3 mg/kg icv-STZ), Conessine treated groups (received 20 mg/kg at three weeks by gavage). Rats were anesthetized intraperitoneally with ketamine hydrochloride (100mg/kg) and xylazine (5mg/kg) and fixed in a stereotaxic frame. The stainless steel guide cannula (21-gauge) was implanted unilaterally in the: lateral ventricle (AP: +0.8 mm; L: +1.4 mm; D: +3.6 mm). It was then fixed to the skull with acrylic dental cement. Thereafter, all the animals in the experimental groups were lesion by 3mg/kg icv-STZ unilaterally.

2.2 Behavioural assessment:

2.2.1. Passive avoidance test:

The dark box and lighted box with the same measures (20×20×20cm) are the main compartments of the passive avoidance apparatus. The boxes are separated by a guillotine door (8×8cm). The lighted box was illuminated with a lamp (60 W, positioned above the apparatus). The floor of the dark compartment was made of stainless steel (0.5cm diameter) separated by a distance of 1 cm. Intermittent electric shocks (50 Hz, 5 s), 1.5 mA intensity were delivered to the grid floor of the dark compartment by an isolated stimulator. Training was terminated when the rat remained in the illuminated part for 120 consecutive seconds. On the retention test that given 24 h after the acquisition trial, the rat was again placed into the illuminated part and the step-through latency and the time spent in the dark part were recorded as a measure of retention performance. If animal remained in a light compartment and did not cross within 300 s to the dark compartment, (where the foot shock had been given) the session was ended and score of 300 was assigned¹¹.

2.2.2.Morris water maze:

Morris water maze employed in the present study was a model to evaluate spatial learning and memory. Escape from water itself acts as motivation and eliminates the use of other motivational stimuli such as food and water deprivation. Water provides uniform environment and eliminates interference due to olfactory clues.¹² Animals were trained to swim to a platform in a circular pool (180cm diameter*60cm) located in a sound attenuated dark test room. The pool was filled with water (28°C) to a depth of 40cm. A movable circular platform, 9cm in diameter and mounted on a column, was placed in the pool 2cm below the water level for escape latency time (ELT), while during time spent in the target quadrant (TSTQ) the platform was removed. Four equally spaced locations around the edge of the pool (N, S, E, and W) were used to divide the pool into 4 quadrants and one of them is used as start point, which was same during all trials. The pool was filled with opaque water to prevent visibility of the platform in the pool. The escape platform was placed in the middle of one of the random quadrants of the pool and kept in the same position throughout the experiments. Animals received a training session consisting of day 7 to 10 and ELT was recorded. ELT defined as the time taken by the animal to locate the hidden platform. ELT was noted as an index of learning.

2.2.3.Biochemical parameters assay:

Two days after completion of behavioural studies that all animals groups were anesthetized with ketamine and xylazine and sacrificed 2 h after the final dose of Conessine treatment and then brain were collected. Rat’s brains were cut into coronal slices of hippocampus 2 mm thickness using a rat brain matrix (Ted Pella, Redding, CA, USA). Next, the hippocampal slices were fixed in −80°C refrigerator. The hippocampal samples were homogenized in phosphate saline buffer and then centrifuged. The supernatant was separated and used for assays of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRX) activities, malondialdehyde (MDA) and protein level content.

2.2.4.Determination of SOD activity:

The assay of SOD activity was examined according the method of Genet with some modification¹³. Briefly, 50mM sodium phosphate buffer was mixed with EDTA (0.0018mM), pyrogallol (0.003mM) and 20μl enzymatic extract. The decrease in absorbance was then followed at 420nm for 180 s at 25°C against a blank containing all the ingredients without the homogenate tissue. One unit of enzyme is defined as the amount of enzyme that causes half maximal inhibition of pyrogallol autoxidation.

2.2.5. Determination of CAT activity:

Catalase activity was assayed following the method of. Briefly, the reaction mixture consisted of 50mM sodium phosphate buffer pH 7.0, 10mM hydrogen peroxide and 20μl of the enzymatic extract. The absorbance of the supernatant was then measured spectrophotometrically at 240nm for 5min at 25°C against a blank containing all the reagents except the homogenate tissue. The enzyme activity is expressed as μmol of H2O2consumed/min/mg protein.

2.2.6. Determination of glutathione reductase (GRX) activity:

Glutathione reductase activity was assayed following the method of Pinto¹⁴. Briefly, the reaction mixture contained 0.1mM phosphate buffer (pH 7.0), 125mM NADPH and 20μL of the enzyme solution in a final volume of 1ml at 30°C. The absorbance was measured at 340nm. One unit of enzyme is defined as 1μmol of NADPH oxidized/min/mg protein.

2.2.7 Estimation of lipid peroxidation:

Lipid peroxidation was measured by the method of Esterbauer and Cheeseman¹⁵. In brief, the samples containing 1mg protein was mixed with 0.5ml of a solution of tricolor acetic acid (20%) and 1ml of a solution of thiobarbituric acid (0.67%) and incubates for 1 h–100°C. After cooling, the precipitate was removed by centrifugation. The absorbance of reaction mixtures was measured at 535 nm using a blank containing all the reagents except the tissues homogenates.

3.StatisticalAnalysis:

Results were analyzed by one-way analysis of variance, followed by Dunnett's test using Graph pad Prism software and expressed as mean±standard error of mean (n = 6).

P<0.001 was considered statistically significant.

4. RESULTS AND DISCUSSION:

In this research study, we have demonstrated that Conessine administered at doses of 20mg/kg for 3 weeks significantly improved learning and recognition memory consolidation and increased hippocampal antioxidant indexes in the Alzheimer’s model.(Graph1) The present study showed that STZ increase escape latency time in Morris water maze. (Figure 1, graph 2) Conessine one reversed recognition memory in this test by decrease escape latency time. Another study showed that flavonoids such as hesperidin and glycosides enhanced learning and memory through various mechanisms such as elevating brain-derived neurotropic factor (BDNF) levels^16,17.

Oxidative stress caused by an imbalance between oxidant and antioxidant systems is aggravated during aging and AD. An accumulation of ROS, which is particularly characteristic of oxidative stress, is mainly produced by mitochondria and causes damage to lipids, cellular proteins, nucleic acids and glucose. The consequences of such damage are seen as lipid peroxidation, protein oxidation, DNA oxidation, and glycoxidation¹⁸. Our data also concluded that during Alzheimer model, glutathione level decreased and lipid peroxidation increased and Conessine treatment reversed them in the hippocampal area. Similar studies have shown that treatment with other exogenous antioxidant such as turmeric, saffron and vitamin E as a potent antioxidants reduces neurogenic damage and treat patients with AD^19,20. Glutathione is the most current antioxidant in the brain and plays a role in the detoxification of ROS. Levels of glutathione decrease with age and in AD. Decreased intracellular glutathione leads to the release of pro-inflammatory factors TNF-α, IL-6 and nitrite ions, and the activation of P38 MAPK, JNK and NF-κB in microglia and astrocytes^21,22. We found that Conessine significantly increased activity of CAT, SOD, GRX and decrease MDA levels in hippocampal area.(Graph 3)

Our research indicated that implications of Conessine significantly increases on cognitive function, enzymatic activity and GSH level.

Finally in our study, Conessine may have a potential neuroprotective activity and promises a therapeutic value in neuropathological conditions including AD.

5. CONCLUSION:

The most important protective mechanism offered by Conessine is through its ability to decrease hippocampal MDA and to increase levels of GSH and antioxidant enzymatic activity. We have demonstrated that Conessine significantly reduced oxidative stress and increased antioxidant enzymes activities in rat model of AD. AD patients might benefit from using Conessine to increase their anti-oxidation capacity.

6. ACKNOWLEDGMENT:

Authors are thankful to management of K L College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, Andhra Pradesh and Shri Vishnu College of Pharmacy, Bhimavaram.

7. CONFLICT OF INTEREST:

No conflict of interest.

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Received on 27.08.2019 Modified on 18.10.2019

Research J. Pharm. and Tech 2020; 13(6): 2703-2707.

DOI: 10.5958/0974-360X.2020.00481.3