Advances in Pharmaceutical Research


Research Article [Artilce ID-9636944]

Neuroprotective Activity of Santalum album Seeds in Sepsis Encephalopathy

Ojha Asmita1, Saraf Shailendra2, Jangra Meenu2
1Om Bioscience and Pharma College, Panchayanpur, Roorki, Uttarakhand, India
2 Faculty of Pharmacy, Babu Banarasi Das Northern India Institute of Technology, Lucknow, UP
 

Received: 11 Jan, 2019; Revised: 28 Feb, 2019; Accepted: 15 Mar, 2019; Published: 02 Apr, 2019

DOI - 10.36218/APR/9636944
DOI URL- http://doi.org/10.36218/APR/9636944

Copyright 2019 This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Correnspondence should be addressed to Ojha Asmita: kasmitbhardwaj@gmail.com


Abstract

Septic encephalopathy is a systemic inflammatory disease and is characterized by change in neurotransmitter release, dysregulated production of cytokines, organ dysfunction and long-term cognitive and emotional impairments. In the present study, it was investigated whether administration of ethanolic extract and chloroform fraction of Santalum album can affect the behavioural, oxidative stress parameters and brain oedema in sepsis-surviving mice. Septic encephalopathy was induced in albino mice of either sex (20-30 gm body weight) by cecal ligation and puncture technique. Ethanolic extract of Santalum album was prepared by microwave assisted extraction and various fractions were prepared. Behavioural parameters like neurological examination and exploratory behaviour were studied after 3 hours and 24 hours of sepsis induction. Santalum album extract showed significant (p<0.001) improvement in depression, memory, neurological severity score and exploratory behaviour in surviving animals. Further glutathione level and catalase level was found to be elevated with ethanolic extract and chloroform fraction at 200mg/kg body weight whereas level of malondialdehyde (MDA) was found to decrease in chloroform fraction, at a dose of 200 mg/kg body weight. Brain oedema was found to be increased approximately 20% in diseased group, which was reduced after treatment. Thus, based on the results, it may be concluded that Santalum album shows neuroprotective effect against septic encephalopathy via reduction of oxidative stress and improvement in learning and memory, neurological severity score and exploratory behaviour.


INTRODUCTION

Sepsis is an infectious disease. Its sequelae are among the most frequent causes of mortality and morbidity in intensive care units (ICU). During sepsis development, several neurological abnormalities may be observed, such as disorientation, confusion, agitation, lethargy, and coma. Recent treatments for sepsis have focused on decreasing mortality through aggressive resuscitation.

Interventions such as early quantitative resuscitation have been adopted and have reportedly resulted in significant mortality reduction [1]. Several conditions commonly associated with sepsis (hepatic or renal dysfunction, electrolyte abnormalities, acid-base disturbances, glycemia disturbances, hypotension, hypoxemia, sedation, body temperature disturbances, and neuroendocrine dysfunction) make SAE evaluation difficult [2, 3].

Survivors from the intensive care unit may present long-term cognitive impairments, including alterations in memory and anxiety, which are associated with a decrease in quality of life [4, 5]. Network of inflammatory mediators is generated, including TNF-a, IL-1, and other cytokines and chemokines that activate leukocytes, promote leukocyte–vascular endothelium adhesion, and induce endothelial damage [6]. In India, people have used sandal stem (Santalum spp.) medicinally to remedy many ailments [7]. S. album is mainly used to treat skin itching, inflammation, facial acne and headaches. The essentials oil of S. album has shown the antioxidant, hypotensive and antimicrobial activity [8]. Among others, immunomodulatory [9], anti-inflammatory, and antirheumatic activities have been described.

MATERIALS AND METHODS

Experimental animals

Ten weeks old Male Swiss mice weighing 20 to 25g were used. The animals were kept at a constant temperature (25°C) with the access to pellet diet and water in a room with a 12 h light/dark cycle. The protocol was approved in accordance with the ethical guidelines of the Institutional.

Preparation of extract

Plants were collected from local market, Lucknow, and authenticated by Dr. Suneeta, NISCAIR, New Delhi. 100gm of Santalum album was extracted by microwave assisted extraction in three cycles for 20min.

METHODOLOGY

Microwave Facilitated Extraction

Microwave assisted extraction technique is less tedious and reduces the time [10]. The powdered stem was extracted in a Microwave synthesizer, at a power of 350 W for 20 minutes, using ethanol as the solvent. The solvent: sample loading ratio was 10:1 and pre-leaching time was 10 minutes. The crude extract obtained was collected and concentrated in a vacuum rotary evaporator.

Surgical procedure and CLP model

Sepsis was induced by CLP [11]. Briefly, they were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), given intraperitoneally. Under aseptic conditions, midline laparotomy was performed to expose the cecum and adjoining intestine. The cecum was tightly ligated, below the ileocecal valve, and was perforated with needle. The cecum was then squeezed gently to extrude a small amount of feces through the perforation site. The cecum was then returned to the peritoneal cavity, and the laparotomy was closed with. Animals were resuscitated with regular saline (50 mL/kg) subcutaneously (s.c.) immediately. All animals were returned to their cages with free access to food and water. In the sham-operated group, the mice were submitted to all surgical procedures, but the cecum was neither ligated nor perforated.

Forced swim test

Mice were individually forced to swim in open glass chamber (25 ×15 × 25cm) containing fresh water to a height of 15 cm and maintained at 26±1°C.

At this height of water, animals were not able to support themselves by touching the bottom or the side walls of the chamber with their hind‐paws or tail. Each animal showed vigorous movement during initial 2 min period of the test. The duration of immobility was manually recorded during the next 3 min of the total 5 min testing period. Mice were immobile when they ceased struggling and remained floating motionless in water, making only those movements necessary to keep their head above water [12].

Assessment of learning and memory

Morris water maze was employed to evaluate learning and memory. It consists of a circular water tank (diameter 150 cm and height 45 cm), filled with water maintained at 25°C. The water is made opaque with a white colour. The tank is divided into four equal quadrants with the help of two threads, fixed at right angle to each other on the rim of the pool. A platform (10 cm2) of 29 cm height is in one any quadrant. The position of platform and clues were kept consistent throughout the training session. Each mouse was allowed to explore the pool for 90 s. The mean time spent by the mouse in target quadrant for searching the hidden platform was noted as an index of retrieval [13].

Open field test

Mice were allowed to explore the open field apparatus for 5-minutes while taking scores of their behaviours. After the 5-minutes test, the mice were scooped up from the open field and returned to their home cages. The behaviours scored included: Number of line crossing; frequency with which the mice crossed one of the grid lines with all four paws, Centre square entries; frequency with which the mice crossed one of the red lines with all four paws into the central square, Duration of stay in the central square [14].

Behavioural score

The neurological examination scores were assigned according to Bederson’s modified neurological examination test. Briefly, the consciousness, performance in a smooth climbing platform, walking and postural reflexes were assessed. For walking and posture, mice were allowed to move about freely on the floor and were observed [15, 16].

Estimation of oxidative stress

Measurement of Catalase activity

Catalase activity was determined using Aebi’s method. Fifty microliters of the lysate were added to a cuvette containing 2 ml of phosphate buffer (pH 7.0) and 1 ml of 30 mM H2O2. Catalase activity was measured at 240 nm for 1 min using spectrophotometer.

The molar extinction coefficient of H2O2, 43.6M cm-1 was used to determine the catalase activity. One unit of activity is equal to 1 mmol of H2O2 degraded per minute [17].

Measurement of Glutathione level

Glutathione (GSH) levels in tissue homogenized were determined according to the method of Ellman. In which 0.5 ml of 4% sulphosalcylic acid was added to equal volume of tissue homogenate for precipitation of protein and then it was centrifuged. After centrifugation 0.5 ml of clear supernatant was mixed with 4.5 ml (DTNB) reagent [0.m1M DTNB in 0.1 m phosphate buffer pH8. The light absorbance of the mixture was measured spectrophotometrically at 412 nm after 2 minutes [18].

Determination of lipid peroxidation

To 1ml of brain homogenate which was prepared in potassium chloride (KCl) solution, 0.5ml of thiobarbituric acid (0.8%) and 0.5ml of trichloroacetic acid (30%) was added. It was covered with Aluminium foil and kept on water bath for 30 min at 80°C.

After 30 min tubes were taken out and kept in ice cold water. After 15 min tubes were centrifuged at 3000 rpm for 15 min. Then absorbance was taken using spectrophotometer at 540 nm against blank as 1ml distilled water with 0.5ml TBA and 0.5ml TCA [19].

Nitrate/Nitrite level

The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay with Greiss reagent (0.1% N-(1-napththyl) ethylene diamine dihydrochloride, 1% sulfanilamide and 5% phosphoric acid) equal volume of the supernatant and the griess reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540nm using Spectrophotometer. The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve [20].

Brain oedema

Brain edema was evaluated by the drying-weighing method based on the measurement of the water content of brain [21]. After removal the brain tissue was placed on a filter paper for the removal of the excess water. The brain was put in an incubator with constant temperature and humidity to be dried for 24 h at 100°C; 24 h later the dried brain was reweighed. The percentage of water was calculated according to the following formula:

%H2O = wet weight - dry weight x100

wet weight

In vitro anti-inflammatory activity

Hypotonic solution induced haemolysis or membrane stabilizing activity

The test sample consist of erythrocyte suspension 0.030ml mixed with 5ml hypotonic solution (154mM NaCl in 10mM Sodium Phosphate Buffer at pH 7.4) containing Herbal Preparation 100 and 200 μg/ml. The control sample consisted of 0.030ml RBC suspension mixed with hypotonic buffered solution alone. The standard drug acetylsalicylic was treated similar to test at 100 and 200 μg/ml concentrations. The experiment was carried out in triplicate. The mixtures were incubated at 10 minutes at room temperature, centrifuged for 10 minutes at 3000rpm and absorbance of the supernatant was measured spectrophotometrically at 540 nm [22-23]. The percentage inhibition of haemolysis or membrane stabilization was calculated by following equation.

% Inhibition of haemolysis = A1 –A2x 100

A1

Where: A1= Absorbance control, A2= Absorbance of sample

Effect on Protein Denaturation

Test solution consisted of 1ml of concentrations of Herbal Preparation 100 and 200μg/ml. Similarly, standard acetylsalicylic acid 100 and 200 μg/ml was made. Standard and test sample were mixed with 1ml of egg albumin solution (1mM) and incubated at 27 ±1˚C for 15 minutes. Denaturation was induced by keeping the reaction mixture at 70˚C in a water bath for 10 minutes. After cooling the turbidity was measured spectrophotometrically at 660 nm [23, 24]. Percentage inhibition of denaturation was calculated from control where no drug was added.

% inhibition of protein denaturation can be calculated using formula:

%inhibition = 100 x [ Vt/Vc -1].

Where: Vt = absorbance of test sample, Vc= absorbance of control

 

RESULTS AND DISCUSSION

After 24 hr, ethanolic extract and chloroform fraction of Santalum album at dose 100mg/kg and 200mg/kg body weight showed significant difference in forced swim test when compared to vehicle (p<0.001) (Figure 1).

Figure 1: Effect of Santalum album on depression

Values are expressed as mean±SEM. **p <0.01 versus vehicle treated group, #p< 0.001 versus vehicle treated groups, ap<0.001 versus sham control. SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post-test.

Effect on learning and memory

Learning and memory was assessed by Morris water maze test. Higher dose of Santalum album 200mg/kg BW (SEE200) showed a significant difference in cognitive behaviour after 3 hr and 24 hr (p<0.05 and p<0.001 respectively). SCF100 and SCF200 showed significant difference after 3 hr and 24hr (p<0.001) (Figure 2).

Figure 2: Effect of Santalum album on learning and memory

Values are expressed as mean±SEM. **p <0.01 versus vehicle treated group, #p< 0.001 versus vehicle treated groups, ap<0.001 versus sham control. SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post-test.

Effect on exploratory behaviour

Exploratory behaviour was assessed by open field. After 24 hr when vehicle treated animal was placed in open field it showed anxiety like behaviour. Ethanolic extract of Santalum album at 100mg/kg and 200mg/kg BW (SEE100 and SEE200) showed significant difference after 24 hr compared to vehicle treated group (p < 0.001). Similarly, SCF100 and SCF200 showed a significant difference in open field after 24hr (p<0.001) (Figure 3).

Figure 3: Effect of Santalum album on exploratory behaviour

Values are expressed as mean±SEM. #p< 0.001 versus vehicle treated groups, ap<0.001 with sham control.SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, two-way ANOVA followed by Bonferroni post-test.

Effect on neurological severity

Assessment of neurological severity included the consciousness, performance in a smooth climbing platform, walking and postural reflexes. In neurological behaviour there was no difference in control and treatment group after 3hr when compared to vehicle but after 24hr the neurological severity score was found to be significantly higher in diseased group (p <0.001). Ethanolic extract of Santalum album at lower and higher dose 100mg/kg BW and 200mg/kg BW showed significant difference after 24 hr (p <0.001). Similarly chloroform fraction of Santalum album at lower dose and higher dose showed significant difference after 24hr (p <0.001) (Figure 4).

Figure 4: Effect of Santalum album on neurological severity

Values are expressed as mean±SEM. #p< 0.001 versus vehicle treated groups, ap<0.001 versus sham control. SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100 Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, two-way ANOVA followed by Bonferroni post-test.

Glutathione estimation in brain homogenate

Level of antioxidant was found to be reduced in diseased group when compared to control group. Santalum album ethanolic extract and chloroform fraction at dose 100mg/kg BW showed a increase in glutathione level but it was not improved significantly. At higher dose 200mg/kg BW extract and fraction caused a significant increase in glutathione level (p<0.05) (Figure 5).

Figure 5: Effect of Santalum album on Glutathione level in brain homogenate

Values are expressed as mean±SEM. *p <0.05 versus vehicle treated groups, ap<0.001 versus sham control. SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post test

Effect on Catalase in brain homogenate

Level of catalase was found to be very low in vehicle group when compared to control group. Lower dose of Santalum album of ethanolic extract and chloroform fraction was not found to be effective against oxidative stress. Treatment with ethanolic extract at dose 200mg/kg BW showed marked increase in catalase activity (p<0.01) when compared to vehicle and treatment with chloroform fraction at 200mg/kg BW showed an increase in catalase activity (p<0.001) it was found to be most active in comparison to all groups (Figure 6).

Effect on Nitrite/Nitrate level

Nitrite (NO2–) and nitrate (NO3–) are known inert oxidative end products of endogenous nitric oxide. Level of Nitrate/Nitrite was increased in diseased group p<0.001, when compared to sham control. At lower dose of ethanolic extract and chloroform fraction of Santalum album it has been found that level of Nitrite/Nitrate was not significantly decreased, but it was significantly reduced in ethanolic extract treated group at 200mg/kg body weight p<0.05 and chloroform fraction group at 200mg/kg body weight p<0.05 when compared to vehicle treated group (Figure 7).

Figure 6: Effect of Santalum album on Catalase activity

Values are expressed as mean±SEM. **p <0.01 versus vehicle treated group, #p< 0.001 versus vehicle treated groups, ap<0.001 versus sham control. SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post-test.

Figure 7: Effect of Santalum album on Nitrite/Nitrate level

Values are expressed as mean±SEM. *p <0.05 versus vehicle treated group, ap<0.001 versus sham control.SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post test

 

Effect on Malondialdehyde level

Malondialdehyde (MDA) is an aldehyde considered to be the terminal compound and the most important marker for monitoring lipid peroxidation. Level of malondialdehyde was found to be increased in vehicle group when compared to control. Ethanolic extract of Santalum album at both doses decreased level of MDA p<0.05 and chloroform fraction was found to be more active at both doses (p<0.01) (Figure 8).

Brain oedema

Brain oedema was evaluated by the drying-weighing method based on the measurement of the water content of brain. Higher dose of ethanolic extract caused reduction in water content. Chloroform fraction also showed a significant decrease in brain oedema at different dose (figure 9).

Figure 8: Effect of Santalum album on Malondialdehyde (MDA) level

Values are expressed as mean±SEM. *p <0.05 versus vehicle treated group,**p<0.01 versus vehicle treated group, ap<0.001 versus sham control.SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kgBW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dennett’s post-test.

Figure 9: Effect of Santalum album on brain oedema

Values are expressed as mean±SEM. *p <0.05 versus vehicle treated group, **p<0.01 versus vehicle treated group, #p<0.001 versus vehicle group ap<0.001 versus sham control.SEE100: Ethanolic extract of Santalum album at 100mg/kgBW, SEE200: Ethanolic extract of Santalum album at 200mg/kg BW, SCF100: Chloroform fraction of Santalum album at 100mg/kg BW, SCF200: Chloroform fraction of Santalum album at 200mg/kg BW, one-way ANOVA followed by Dunnett’s post-test.

Membrane stabilizing activity

Membrane stabilizing activity was measured by induction of inflammation by hypotonic solution in red blood cells. in control group there was no inhibition on haemolysis. Aspirin was taken as standard and it caused concentration dependent inhibition of haemolysis. Ethanolic extract and chloroform fraction of Santalum album at concentration 100 µg/ml caused inhibition on haemolysis but it was not found to be significant, while at 200µg/ml extract and fraction significantly (p<0.001) protected the lysis of erythrocyte membrane induced by hypotonic solution as compared to control group (Figure 10).

Figure 10: Effect of Santalum album on heamolysis

Values are expressed as mean±SEM. **p <0.01 versus control group, #p<0.001 versus control. ASP100 Aspirin at concentration 100 µg/ml, ASP200: Aspirin at 200 µg/ml concentration, SEE100: Ethanolic extract of Santalum album at concentration 100 µg/ml, SEE200: Ethanolic extract of Santalum album at 200 µg/ml, SCF100: Chloroform fraction of Santalum album at 100µg/ml, SCF200: Chloroform fraction of Santalum album at 200 µg/ml,one-way ANOVA followed by Dunnett’s post-test.

Effect on Protein denaturation

Protein denaturation occurred in egg albumin by heat.Aspirin was taken as standard. It caused inhibition of protein denaturation. Ethanolic extract and chloroform fraction at different concentration caused a significant reduction in protein denaturation. As the concentration of extract and fraction was increased there was also increase in anti-inflammatory activity (Figure11).

Figure 11: Effect of Santalum album on protein denaturation

Values are expressed as mean±SEM. *p <0.05 versus control group, #p<0.001 versus control.ASP100 Aspirin at concentration 100 µg/ml, ASP200: Aspirin at 200 µg/ml concentration, SEE100: Ethanolic extract of Santalum album at concentration 100 µg/ml, SEE200: Ethanolic extract of Santalum album at 200 µg/ml, SCF100: Chloroform fraction of Santalum album at 100µg/ml, SCF200: Chloroform fraction of Santalum album at 200 µg/ml, One-way ANOVA followed by Dunnett’s post-test.

CONCLUSION

Sepsis caused behavioural and biochemical changes in the brain. Plant treatment significantly improved survival rate and prognosis of the disease as assessed by biochemical, and neurological parameters. Sepsis caused an increase in brain oedema by increasing water content which was reduced by plant treatment. Although we have observed neuroprotection at the selected dose, still there is a need for further studies to clarify the therapeutic window for neuroprotection against oxidative injury induced by sepsis. These findings, taken all together, may suggests that Santalum album may have beneficial effects in septic encephalopathy by improving biochemical, behavioural changes.


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