Get Permission Pandian and Desai: Effect of eugenolon white blood cells and corticosterone in sub-acute restraint stress-induced wistar albino rats


Introduction

Blood is the medium through which immune cells travel to maintain their normal surveillance and to rapidly reach the site where immune activation is required.1 Hence the quantity of leukocytes in blood represents the distribution pattern of leukocytes in the body and the activation of the immune system.2 Stress is defined as an event of stimulus - mediated reaction in the brain that initiates physiological fight-or-flight system in the body. Stress, that ends within minutes to hours, is called acute stress and it causes the maximum distribution of immune cells in the body.2,3 The hormones such as norepinephrine (NE), epinephrine (EPI), and corticosterone (CORT ) play major role in acute stress response.4,5 NE and EPI, released immediately during stress, mobilize immune cells into blood flow whereas CORT decrease sthe quantity of immune cells in the blood and other tissues.2

Eugenol (2-methoxy-4-(Prop-2-en-1-yl) phenol is a volatile phenolic compound of essential oil obtained from clove (Egeniacaryophyllus). Eugenol is used in preparation of skincare products, cosmetics, flavoring agent dental and pharmaceutical products due to its antiseptic and antispasmodic properties.4 In traditional medicine, eugenol has been used in the treatment of flatulence, cholic, chronic diarrhea, and other gastrointestinal disorders.5 The pharmacological activities of eugenol include anti-oxidant,5 antibacterial,6,7 anti -inflammatory,8 and antipyretic effects.9

Eugenol has been reported to improve motor coordination and decrease plasma corticosterone level in immobilized stress-induced Wistar rats.10 Moreover, it was reported that in vitro eugenol decreases the migration of leukocytes and aids in inflammatory process.11 The effect of eugenol (150 mg/kg) on red blood cells (RBC) in restraint stress-induced rats was studied in our previous study which showed raised RBC count, packed cell volume, and hemoglobin level.12 However, no data was available on the effect of eugenol on the distribution of stress- induced WBCs and corticosterone in rats. Hence, the present study was undertaken to evaluate the se effects.

Materials and Methods

Chemicals

Analytic grade eugenol (C10 H12 O2), a clear to pale yellow oily liquid extracted from certain essential oils especially from clove oil and cinnamon, was purchased from Sigma Chemical Industry.

Animals

Female Wistar albino rats weighing between 150 – 220g were part of in this study, and were housed according to the standard conditions. The study was carried out according to guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India. The study protocol was approved by the Institute’s Animal Ethics Committee (IAEC no. 01 / 17 /2015).

Experimental procedure

Animals were divided into five groups with six animals in each group—Group I (normal control) received standard diet, Group II animals were administrated with vehicle used to emulsify eugenol that is polyglycerol (PG) Intraperitoneal for 15 days and Group III animals were administrated with Eugenol (treated alone - TA) 150 mg /kg/i.p body weight for 15 days. Group IV was subjected to immobilization stress alone (SA) for 15 days (6 hr/day). Group V animals were subjected to stress and were immediately treated with Eugenol 150 mg /kg/i.p body weight for15 days. The dose was selected based on toxicity study.

Immobilization stress induction procedure10

Rats were subjected to restraint stress in a wire mesh restrainer for 6 hours per day for 15 days. The wire mesh restrainer (length : 8 cm, breadth: 4 cm and height: 4 cm) had a wooden base and stainless-steel wire mesh restrainer hinged to the base. A padlock and latch helped to secure the rat in the restrainer.

Blood sample collection13

Blood samples were collected at the end of the study; blood was collected from ventral/ dorsal artery or lateral tail vein by nicking vessel and cannulation was done to minimize contamination of the samples. Precautions were taken to avoid the hemostasis.

Determination of hematological indices

WBC count was performed by Dacie and Lewis method.14 Turk’s fluid was used for TLC (1:20) and cell count was done by using Neubauer counting chamber under light microscope. DLC was performed using method of Mathers et. al.15

Assay of corticosterone

The assay was carried out with slight modification of Singh and Verman16 method and is based on the oxidation of corticosteroids with ferric iron (III) in an acidic medium and subsequent complex with ferrous iron (II) and potassium hexacyanoferrate. Plasma samples (0.5 μl) was mixed with appropriate volumes of working solutions of CORT were transferred into a series of 10 ml volumetric flasks. Sulphuric acid (4N, 2ml) and ferric chloride (0.5%, 2ml) were added to each followed by potassium hexacyanoferrate (III) solution (0.5%, 0.5 ml). The mixture was heated in a water - bath and maintained at 70±2ºC for 30 minutes with occasional shaking and diluted to the 5ml mark with distilled water. The absorbance was measured at 780 nm against the reagent blank.

Statistical analysis

Data were analyzed by ANOVA and Tukey’s multiple comparison tests using SPSS 20 software. P<0.05 was considered statistically significant.

Results

Table 1
Groups WBC (×103 /mm3) Platelets (×106 /mm3)
Group I (Control) 9.51 ± 1.33 267.70 ± 3.66
Group II (PG) 11.43 ± 1.30 262.53 ± 4.06
Group III (TA150mg) 12.28 ± 1.31 180.15 ± 1.74
Group IV (SA) 13.38 ± 1.071 54.25 ± 3.35
Group V (T/S 150mg) 12.97 ± 0.81 238.80 ± 1.63

Effect of eugenol on WBC and platelet count

[i] WBC-White blood cells; PG-polyglycerol, TA - treated alone, SA-stress alone, T/S-stress+treatment,

Effect of eugenol on leukocyte count

As per the leukocyte count of different groups of animals (Table 1), a significant increase in TLC was observed in SA and T/ S in contrast to the control group(p<0.0001). However, no significant difference was observed between leukocyte count of T / S and SA groups (Table 2).

Effect of eugenol on Platelets

The platelets count of different groups of animals (table 1) indicated significantly decreased platelet count in SA (p<0.0001) as compared to other groups. Whereas, results of T/ S indicated significantly (p<0.0001) increased platelet count when compared with TA and SA (Table 2).

Table 2
Groups WBC Platelets
Difference P value Difference P value
PG-Control 1.916 0.066 -5.170 0.051
SA-Control 3.863 0.000*** -213.446 0.000***
TA-Control 2.771 0.003* -87.553 0.000***
T/S-Control 3.458 0.000*** -28.900 0.000***
SA-PG 1.946 0.060 -208.276 0.000***
TA-PG 0.855 0.722 -82.383 0.000***
T/S-PG 1.541 0.193 -23.730 0.000***
TA-SA -1.091 0.513 125.893 0.000***
T/S-SA -0.405 0.975 184.546 0.000***
T/S-TA 0.686 0.851 58.653 0.000***

Comparison between groups

[i] WBC-White blood cells; PG-polyglycerol, TA - treated alone, SA-stress alone, T/S - stress+treatment, ‘ *** ’ P<0.000 1, ‘ ** ’ P<0.01, ‘ * ’ P<0.05

Effect of eugenol on differentiation leucocyte count

Table 3
DLC (×103 /mcL) Group I (Control) Group II (PG) Group III (TA 150mg) Group IV (SA) Group V (T/S 150mg)
Neutrophils 28.21 ± 3.96 31.52 ± 3.27 30.79 ± 2.58 25.54± 2.31 31.07 ± 2.96
Lymphocytes 66.39 ± 4.12 70.68 ± 5.09 57.85 ± 3.72 67.79± 6.70 67.73 ± 4.03
Basophils 0.70 ± 0.35 0.36 ± 0.11 0.47 ± 0.27 0.1± 1.52e-17 0.46 ± 0.44
Monocytes 2.66 ± 1.16 2.97 ± 1.91 4.24 ± 1.48 1.77± 0.42 2.58 ± 1.20
Eosinophils 2.13 ± 0.59 2.77 ± 1.61 2.13 ± 0.62 0.52± 0.27 1.13 ± 0.55

Effect of eugenol on differential leucocyte count

[i] DLC-differential leukocyte count; PG-polyglycerol, TA - treated alone, SA-stress alone, T/S - stress+treatment.

The differential leukocyte count of all groups, as represented in table 3, indicates significantly decreased counts of neutrophils, basophils, eosinophil, and monocytein SA group when compared with control, PG, TA and T / S (p<0.05). Similarly, significant decreased lymphocytes count was observed in TA when compared with control, SA (p<0.05) and PG (p<0.001). However, significant increased monocytes count was observed in TA when compared with control and SA (Table 4).

Table 4
Groups Neutrophils Lymphocytes Basophils Monocytes Eosinophils
Dif. P Dif. P Dif. P Dif. P Dif. P
PG-Control 3.31 0.36 4.29 0.55 -0.34 0.27 0.30 0.99 0.64 0.70
SA-Control -2.66 0.57 1.39 0.98 -0.60 0.01* -0.89 0.77 -1.61 0.02*
TA-Control 2.58 0.59 -8.54 0.03* -0.22 0.65 1.57 0.27 -0.00 1.00
T/S-Control 2.86 0.50 1.34 0.98 -0.24 0.60 -0.08 0.99 -1.00 0.29
SA-PG -5.97 0.01* -2.89 0.83 -0.26 0.53 -1.20 0.53 -2.25 0.001**
TA-PG -0.72 0.99 -12.83 0.000*** 0.11 0.95 1.26 0.48 -0.64 0.70
T/S-PG -0.44 0.99 -2.95 0.82 0.10 0.97 -0.38 0.98 -1.64 0.02*
TA-SA 5.24 0.04* -9.93 0.01* 0.37 0.19 2.46 0.02* 1.61 0.02*
T/S-SA 5.53 0.03* -0.05 1.00 0.36 0.22 0.81 0.82 0.61 0.73
T/S-TA 0.28 0.99 9.88 0.01* -0.01 0.99 1.65 0.23 -0.99 0.29

Comparison between groups

[i] Dif.-difference, P-pvalue, PG-polyglycerol, TA - treated alone, SA-stressalone, T/S-stress+ treatment, ‘ *** ’P<0.001, ‘ ** ’P<0.01, ‘ * ’P<0.05

Effect of eugenol on CORT

The plasma CORT levels in different groups were—control: 1.85±0.761, PG: 1.81±0.767, TA: 1.67±0.668, T/S: 2.15±0.86, and SA : 2.87±1.15. However, no significant differences were observed in mean plasma CORT concentration of groups when compared with each other.

Discussion

Stress can be psychological or physical and causes the release of norepinephrine and epinephrine which in turn elevates the release of blood lymphocytes, monocytes, and neutrophils whereas, CORT decreases immune cells in blood and tissues.2 The aggregation of leukocytes in the blood causes immune -enhancement by availing maximum leukocytes at the site of activation.12,17

The acute stress in human causes increase in immune cell distribution in blood as compared to the resting- state; whereas rodents demonstratedecreased immune cells in blood. This is maybe due to the initial aggregation of immune cells and reflect trafficking of immune cells.2 The study of Rosenberger et al. described that response to stress causes transfer of leukocytes inside the blood within a minute, decreases monocytes, and lymphocyte number, and increases the number of neutrophils.18 In the present study leukocytes concentration decreases in stress- induced rats which may again be due to initial aggregation and reflect trafficking of immune cells. Whereas, eugenol treated group did not show any significant alteration in leukocyte counts. In contrast, a short- term decrease in blood leukocyte numbers represents trafficking of cells out of the blood to the target organs2 Similarly, in the present study, monocytes and neutrophils were decreased in the stress-induced animals.

The study of Malyszko et al.19 and Takeda et al.20 explained acute water immersion restraint and acute cold - restraint stress which further causes reduced collagen-induced aggregation in whole blood and ADP-induced aggregation in platelet-rich plasma, respectively. Similarly, in the present study, decreased platelet count in animals by acute restraint stress were observed. However, increased platelets count was observed in eugenol and stress-treated animal groups. Similar results were reported by Hata et al.21

The study performed by Pitman et al.22 reported increased basal CORT level inrestrain stress-induced rats on day 2 and 3. Although fromday 4-6, the levels were not significantly increased due to habituation to the stress. Similarly, the study of Sadler et al.23 suggest no significant increase in the CORT level in the mice on 14 day restraint due to habituation to stressor.  Hypothalamic-pituitary-adrenal axis is less responsive to the repeated restrain stress after 8 or 14 days of restraint.24,25 In this study, no significant increase in CORT levels were observed among the groups of animals, which may be due to habituation to the stressor. In contrast with results of the present study, the study of Pandian et al. reported significant increase in plasma CORT level in restraint stress- induced for 15 days for 6 hours among the groups when compared with control group. Whereas, stress and eugenol-treated group shows significantly decreased CORT level when compared with stress -alone group.

In this study the administration of eugenol (150 mg/kg) in T/S and TA for 15 days increased the TLC compared with control group rats whereas, the effect of eugenol on DLC did not show significant difference in neutrophil and lymphocytes count in T/ S groups when compared with other groups and no significant differences were observed between basophils, monocytes eosinophils of T/S when compared with SA group.

The present study showed the effect of eugenol on WBC and CORT in sub-acute restraint stress-induced rats. The blood samples were collected at the end of the study which may be resulted in fluctuated results. Hence, further studies are required to determine the effect of eugenol on WBC and CORT at regular time intervals and effect of eugenol on other stress hormones.

Conclusion

Stress-induced distribution of WBCs was not significantly attenuated by eugenol as was observed in stress and eugenol-treated rats and no changes were observed in CORT in all groups of animals. Therefore, additional studies are required to know the action of eugenol on chemical mediators released by stress which is responsible for WBCs and platelets distribution in blood.

Source of funding

None.

Conflict of interest

None.

References

1 

T A Springer Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigmCell199476301314

2 

F S Dhabhar W B Malarkey E Neri B S Mcewen Stress-induced redistribution of immune cells-From barracks to boulevards to battlefields: A tale of three hormones-Curt Richter Award WinnerPsycho Neuroendocrinol201237913451368

3 

F S Dhabhar B S Mcewen Acute stress enhances while chronic stress suppresses immune function in vivo: a potential role for leukocyte traffickingBrain Behav Immun199711286306

4 

E Basch A Gasparyan N Giese S Hashmi M Miranda D Sollars Natural standard monograph (www.naturalstandard.com) copyright© 2008J Diet Suppl20085117146

5 

G P Kamatou I Vermaak A M Viljoen Eugenol; from the remote Maluku Islands to the international market place: a review of a remarkable and versatile moleculeMolecules20121769536981

6 

A M Leite E O Lima E L Souza Mffm Diniz V N Trajano I A Medeiros Inhibitory effect of β-pinene, α-pinene and eugenol on the growth of potential infectious endocarditis causing gram-positive bacteriaBraz J Pharmacol Sci200743121126

7 

S M Ali A A Khan I Ahmed M Musaddiq K S Ahmed H Polasa Antimicrobial activities of eugenol and cinnamaldehyde against the human gastric pathogen Helicobacter pyloriAnn Clin Microbiol Antimicrob2005420

8 

H H Leem E O Kim M J Seo S W Choi Antioxidant and anti-inflammatory activities of eugenol and its derivatives from clove (Eugenia caryophyllata Thunb.) Korean J Food Sci20114013611370

9 

J Feng J M Lipton Eugenol: antipyretic activity in rabbitsNeuropharmacol19872617751778

10 

M Pandian Selvan R Rajan Effect of 4-Allyl-2-Methoxyphenol (Eugenol) on Motor Co-Ordination in Subacute Restraint Stress Induced Wistar Albino RatsJ Appl Pharm Science2016611120125

11 

C F Estevo-Silva R Kummer F C Fachini-Queiroz R Grespan G A De Melo S Baroni Anethole and eugenol reduce in vitro and in vivo leukocyte migration induced by fMLP, LTB 4, and carrageenanJ Nat Med2014683567575

12 

M Pandian R D Padmaja R Ravindran Effect of 4-Allyl-2-Methoxyphenol (Eugenol) On Red Blood Cells In Subacute Restraint Stress Induced Wistar Albino RatsJ Dent Med Sci201812178185

13 

S D Christensen L F Mikkelsen J J Fels T B Bodvarsdottir A K Hansen Quality of plasma sampled by different methods for multiple blood sampling in miceLab Anim2009436571

14 

J V Dacie S M Lewis Practical Haematology. 11th edLongman Group Ltd. Hong Kong20011117

15 

R A Mathers G O Evans J Bleby Platelet measurements in rat, dog, and mouse blood samples using the Sysmex XT2000iVComp Clin Pathol201322815821

16 

D K Singh R Verma Spectrophotometric determination of corticosteroids and its application in pharmaceutical formulationIran J Pharmacol Ther2008716161

17 

Dhabhar FS. Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathologyNeuroimmunomodulation2009165300317

18 

P H Rosenberger J R Ickovics E Epel E Nadler P Jokl J P Fulkerson Surgical stress-induced immune cell redistribution profiles predict short-term and long-term postsurgical recovery: A prospective studyJ Bone Joint Surg200991122783

19 

J Malyszko T Urano Y Takada A Takada Time-dependent changes in platelet aggregation, fibrinolytic activity, and peripheral serotonergic measures in rats subjected to water immersion restraint stressPathophysiol Haemost Thromb1994244236242

20 

H Takeda M Asaka K Matsuno T Ohtaki T Miyazaki Stress-induced gastric mucosal lesion and platelet aggregation in ratsJ Clin Gastroenterol199214145148

21 

T Hata A Kawabata T Kita E Itoh Y Nishimura Changes in platelet count and related parameters in SART-stressed mice and the action of administered NeurotropinJpn J Pharmacol1988474349356

22 

D L Pitman J E Ottenweller B H Natelson Plasma CORT levels during repeated presentation of two intensities of restraint stress: chronic stress and habituationPhysiol Behav19884314755

23 

A M Sadler S J Bailey Repeated daily restraint stress induces adaptive behavioural changes in both adult and juvenile micePhysiol Behav2016167313323

24 

N Grissom V Iyer C Vining S Bhatnagar The physical context of previous stress exposure modifies hypothalamic-pituitary-adrenal responses to a subsequent homotypic stressHormones Behav20075195103

25 

V Viau P E Sawchenko Hypophysiotropic neurons of the paraventricular nucleus respond in spatially, temporally, and phenotypically differentiated manners to acute vs. repeated restraint stressJ Comp Neurol2002445293307



jats-html.xsl


This is an Open Access (OA) journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

  • Article highlights
  • Article tables
  • Article images

View Article

PDF File   Full Text Article


Copyright permission

Get article permission for commercial use

Downlaod

PDF File   XML File  


Digital Object Identifier (DOI)

Article DOI

https://doi.org/10.18231/j.ijcap.2019.094


Article Metrics






Article Access statistics

Viewed: 1793

PDF Downloaded: 660