Abstract: Background and Objective: Stress is described as a physically or mentally change that affects the organism's homeostatic and balance and is considered to cause 75% of all diseases. The E. globulus trees leaf oil polyherbal formulation was evaluated for its antistress activity in the present experiment. Material and Methods: The study employed Wister rats weighing 150-200 g for toxicity testing and Albino mice weighing 15-25 g for the investigation. Acute toxicity studies were carried out for pure essential oil of Eucalyptus globulus following (OECD guideline 423, 2001). The rats were split into 2 groups, each with 3 male rats (n = 3). The experimental group received EOE in an oral dose of 2000 mg kg–1 body weight. Adult male albino mice approximately 20-25 g. were selected for anti-stress testing and split into 5 groups, each with 5 animals. Three activities were selected. FST, TST and anoxia stress tolerance test model, animals were treated with three doses of EOE (100, 200, 400 mg kg–1, p.o). Results: Findings of acute toxicity studies suggested no major toxicity, results for assessment of antistress influences showed that administration of eucalyptus leaf oil at mentioned doses decreases immobility times when compared with control. In the same way, animals treat with diazepam (1 mg kg–1) as predictable, show a major decrease in the immobility time. Conclusion: The current study investigation discovered that eucalyptus oil show antistress activity by preventing stress-induced by FST, TST and anoxia stress tolerance test model.
INTRODUCTION
Stress mostly is the feedback of mind and corpse beside transform into the homeostasis and by acute condition, the homeostatic method of the creature develops into debit and the endurance of the creature is susceptible and triggers a large series of corpse changes called universal version condition. Different medicinal plants Bacopa monnieri, Centella asiatica, Emblica officinalis, Hypericum perforatum, Ocimum sanctum, P. Longman, Withania somnifera are claimed to having anti-stress activity including Immunomodulatory, adaptogenic, anabolic effects1. Stress is distinct as a substantial and emotional alteration to interrupt the homeostasis and the stability of the creature2. Stress causes due to corporeal stressors or similar to deafening sound, large crowd and disorderly environment.
Stress is a non-definite reaction of the corpse identified to change physiological homeostasis of the creature ensuing in a variety of neuronal, endocrine and instinctive dysfunction3. Stress causes a variety of syndrome behavioural confusion-apprehension, despair and other illnesses hypertension, peptic ulcer, diabetes, immunosuppressant, reproductive dysfunction4. Stress show symptom similar to high blood force, upper body ache, pain, distressed abdomen, asthma, arthritis, diarrhoea, constipation. Every occasion worrying state occurs it’s activated the hypothalamic-pituitary axis and middle monoaminergic system. The hypothalamus-pituitary axis enlarges the free corticotrophin release hormones adrenocorticoids and glucocorticoids secretions5. A broad range of drugs is used to care for a variety of types of stress, worry and despair except for nothing of the end of the accessible drug. Various artificial complex similar to benzodiazepines, CNS stimulants such as amphetamines, caffeine, anabolic steroid illustrates the unpleasant cause as a contrast to the polyherbal formulation6. Artificial drugs are superior in speed. India is a mounting countryside so the public cannot exertion the superior rate drug.
The development of such category of disorders (stress, anxiety) so it is essential to discover absent a fresh particle in the marketplace to treat stress7. Stress occurs changes to the creature corpse mass similar to adrenal gland, liver, spleen, testis. stressful occasion occurs to reduce the height of glutathione stage in the brain, peptic ulcer, activate the melon aldehyde level in the brain8. Stress can be induced by various methods in investigational animals like the forced swim endurance model, immobilization stress model, chronic unpredictable stress model. These models are well-recognized in biomedical investigation9. In present age stress and stress linked confusion are an implication reason of illness and causative maybe 75% of all disease10. A herbal formulation Eucalyptus oil is used in the antistress activity. Eucalyptus oil proves the antistress activity. Eucalyptus oil is used for respiratory disorders similar to asthma, allergy. The mucolytic (dipping the thickness of mucus) and bronchodilator (declining conflict in the inhalation airways) properties of Eucalyptus oil help liquefy and relieve nasal and bronchial blockage11.
The word stress is distinct by Hans Selye as the amount of all the unfocused change caused by purpose or harm and a condition of disposed of homeostasis. Stress is primarily a mental and physical reaction to a change in homeostatic12. Creative stress is referred to as Eustress, whilst harmful stress is referred to as Distress. Stress is a distracted response of the corpse which has been shown to affect the creature's homeostasis of the body, leading to a variety of neural, endocrine and instinctual dysfunctions13. Stress is defined as the sum of all the body's reactions to a motivation that affects the body's normal physiological state and results in a state of threatened homeostasis and it has been distinguished as the body's inattentive response to any command imposed on it14. Stress, in its most basic sense, is what one feels when one's capacity to cope with life's challenges exceeds one's ability to manage one's load15. Seyle describes stress as "a non-precise reply of the biological system to any demand". Seyle's definition covers all 3 phylogenetic domains (bacteria to human). He emphasized the fact that stress is not alike to emotional or nervous exhaustion since stress can also result in unconscious states in humans and in animals, as well as it can be seen in plants and microbes where the nervous system does not exist even16. Stress has become an unavoidable part of human life in today's environment. Stress must be maintained under control and normal performance must not be jeopardised as a result of extreme stress. Stress is defined as any condition that causes a disturbance in the body's equilibrium. The stress level is extremely high, the human homeostatic mechanism is deficient and the creature's survival is in jeopardy7.
Stress may be a corporal and compound motivation that stimulate understanding and parasympathetic reaction inside a creature. These responses facilitate a creature to struggle for its survival and rise above with well again outcome. Both corporeal and expressive stressors are set into movement centre and undersized respond designed to pot homeostasis17.
Stress occurs in 2 forms eustress and distress. Eustress is good stress. It is essential for a living being to realize duty without causing physiological and psychological hurt. Distress is a bad form of stress that causes harm. It might be due to a variety of physiological environmental and physiological factors. A centrally neural way which judge motivation improved attention and consideration span, suitable anger jointly with a simultaneous embarrassment of vegetative occupation such as feed, sexual behaviour, growth and reproduction are amenities18. Peripheral alter prepare an animal to answer to a stressor and include amplifying heart rate blood pressure, nutrients to organs and, respiration which need extra energy to occupation19.
Hence by going through extensive literature on stress and its mechanisms and by reviewing the above and much more literature about eucalyptus leaf oil present research was designed to study acute toxic effects and antistress potential of eucalyptus leaf oil in rodents.
MATERIALS AND METHODS
Study area: This research was carried out at the Department of Pharmacy, Kota College of Pharmacy, Kota, Rajasthan, India from March-June, 2019.
Collection of essential oils: The pure essential leaf oil of Eucalyptus globulus was purchased from the market. The pure essential oil was volatile in nature, greenish in colour with an aromatic odour. The oil was completely soluble in distilled water.
Animals: The study employed 25 Wistar rats weighing 150-200 g for acute toxicity and Albino mice weighing 15-25 g. They were housed in a cage (22.5-37.5 cm) in a room with standard laboratory conditions, such as a temperature of 23°C, relative humidity of 55.5% and 12:12 hrs of light and dark cycle. The animals were fed a normal pellet diet and had unlimited access to water. The animals were brought to the lab at least 1 hr before the start of the experiment. The tests took place during the day (08:00-16:00 hrs).
Ethical committee: The ethics committee at the institution permitted the study's procedure. The Institutional Animal Ethics Committee (IAEC) authorised the experimental protocol (IAEC/KCP/2019/04) and CPCSEA and ethical guidelines were strictly followed throughout the procedure.
Drug and chemicals: All of the chemicals and reagents utilised were of the highest quality. The various reagents and solvents also taken from the Department of Pharmacy, Kota College of Pharmacy, Kota, such as normal saline (0.9 g of NaCl in 100 mL distilled water), formalin saline (10% v/v -10 mL of formalin in 90 mL distilled water, picric acid (for animals marking), Ethylene Diamine Tetraacetic Acid (EDTA) (2% use in vial because of anticoagulant property). The eucalyptus oil emulsion in water with a variety of concentrations including 4, 6, 8, 12, 18 and 30% was prepared by using tween-80 and span-80 used as the emulsifier. The standard and test drug used for this study are as follow:
| Standard drug | : Diazepam (1 mg kg–1) is the standard medication |
| Test drug | : 100, 200 and 400 mg kg–1 eucalyptus oil |
Acute toxicity study: Pure essential oil of Eucalyptus globulus was subjected to acute toxicity tests after (OECD guideline 423, 2001). Animals were separated into 2 groups, each with 3 male rats (n = 3) and fasted overnight. The control group received normal saline, while the test group received 2000 mg kg–1 body weight of emulsion oil in water orally. The animals were examined individually after dosing at least once during the first 4 days and every day afterwards for a total of 14 days. Changes in skin, fur, eyes, mucous membranes, respiratory, autonomic, central nervous system and behaviour patterns were all seen. EOE was administered orally to the sample population at a dose of 2000 mg kg–1 body weight. The animals were examined individually after dosing at least once during the first 4 days and every day afterwards for a total of 14 days. There were anomalies in the epidermis, fur, eyes, mucous membranes, respiratory, autonomic, central nervous system and behaviour patterns20.
Forced swim test: The Forced Swim Test, developed by Porsolt, is the most extensively used behavioural tool for proving antistress behaviour in mice. The procedure was similar to what has been described previously. Mice were made to swim in an open glass container (25×15×25 cm) filled with clean water to a height of 15 cm and kept at a temperature of 26 degrees Fahrenheit. Animals could not hold themselves up at this altitude of water by manipulating the container's base or surface fortifications with their hind paw or tail. Since water has been used to indicate changing activities, the water in the container was distorted after each animal was subjected to FST. During the first 2 min of the trial, every animal displays a strong faction. The period of stillness was carefully recorded for the entirety of the totality 6 min test stage, which was then 4 min21,22.
Tail suspension test: The tail suspension model is a behavioural model for the study of antistress activities in rodents. The animals were allowed to adapt to the laboratory at least before 1-2 hrs. Each animal was individually balanced to the bench's edge, 50 cm above the ground, using adhesive tape placed around 1 cm from the tip of the tail. All animals below must be acoustically and visually separated from other animals for the duration of the experiment. The total time spent immobile was meticulously documented at 6 min. Because it did not display any corpse movement, hung unresponsively and was still, the animal was judged lifeless. The experiment was carried out in a dimly lit room with each mouse being used only once. The animals' immobility in reaction to a medical treatment that the animals in the study were familiar with was recorded by the observer23,24.
Anoxia stress tolerance test: Five groups of Albino mice including both genders were formed, each with 5 animals. Normal saline was given to the 1st group, while the 2nd, 3rd and 4th were given uncontaminated essential eucalyptus oil with dosages of 100, 200 and 400 mg kg–1, p.o. and stress to the 2nd, 3rd and 4th groups. Group 5 mice were agitated and administered diazepam (1 mg kg–1). For a total of 21 days, the drug action was handed out daily. At the end of the week, i.e., the 1st, 2nd and 3rd weeks after pharmaceutical treatment, the animal was demonstrating anoxia tolerance time. A hermetic container with a 1 L air capacity was utilised to persuade to anoxia stress. All of the animals were maintained in the hermetic container and the time it took for the first seizure signal to appear was timed. The animals were then removed from the vessel and if required, resuscitated10.
RESULTS
Acute toxicity study: As per OECD guideline 423 for acute oral toxicity at a dose of 2000 mg kg–1, the rats in the eucalyptus oil group did not show any signs of toxicity at this dose level over the 14 days observation period. The surviving rat showed no undesirable clinical symptoms at the dosing level evaluated. There is no change in the behaviour of the animals' stool, urine or eye colour. All of the treated animals appeared normal and displayed no aberrant behaviour. They all had a regular breathing pattern. Sedation, convulsions and lacrimation symptoms were completely absent. The treated rat group showed no signs of illness or mortality. Based on these findings, 2 dose levels of eucalyptus oil were chosen for anti-stress activity: 200 and 400 mg kg–1 (Fig. 1).
Organ weight observation: The weight of the animals' essential organs was calculated. The weights of the major organs were affected by the extent of body water. The results showed no significant changes in the weights of the vital organs such as kidneys (0.35±0.09-0.33±0.08 g), liver (02.92±0.05-2.95±0.08 g), heart (0.36±0.05-0.35±0.06 g), lungs (0.96±0.18-0.86±0.12 g), spleen (0.24±0.05-0.26±0.04 g), brain (1.01±0.24-0.86±0.15 g) and testis (0.37±0.006-0.14±0.008 g) after the treatments (Table 1).
Haematological analysis: Haematological values measured showed insignificant changes in the values of lymphocytes which were decreased from 82.30±1.57-72.63±2.49 (109 L–1), Hb levels from 14.39±0.11-14.4±0.28 g dL–1 and WBCs from 12700±47.55-40665.65±14.69.31 (109 L–1).
| Fig. 1: | Effect of eucalyptus oil 2000 mg kg–1 on body mass index Mean±SEM for n = 3 |
| Fig. 2(a-n): | Histopathology of the different control group Normal control (a, c, e, g, i, k, m) and treated group (b, d, f, h, j, l, n) of organs brain, heart, kidney, lungs, spleen, testis and liver at the resolution of 40× |
| Table 1: | Changes in organ weight after 14 days treatment with Eucalyptus globulus leaf oils as compared to control rats (without treatment) | |
| Organ | Control (g) | Treated (g) |
| Liver | 02.92±0.05 | 2.95±0.08 |
| Lung | 0.96±0.18 | 0.86±0.12 |
| Brain | 1.01±0.24 | 0.86±0.15 |
| Spleen | 0.24±0.05 | 0.26±0.04 |
| Heart | 0.36±0.05 | 0.35±0.06 |
| Kidney | 0.35±0.09 | 0.33±0.08 |
| Testis | 0.37±0.006 | 0.14±0.008 |
| Values are reported as Mean±SEM (n = 5) and were analyzed using one-way ANOVA and Dunnett's test | ||
The experimental group's MCV was much higher (99.91±0.56) than the control group (89.11±1.60). Other haematology measures changes were recorded as RBCs from 4.82±0.064-4.22±0.061, (1012 L–1) MCH from 29.8±0.37-32.95±0.174 (pg), MCHC from 32.95±0.235-32±0.424 (fl), Monocytes from 0.02±0.70-1.66±0.15 (109 L–1), granulocytes from 15.64±1.32-22.30±2.35 (109 L–1) and PLT from 4.3±0.12-4.16±0.205 (109 L–1), in the treated animal groups when compared with normal controls (Table 2).
Histopathology: When compared to the control group, the organs of the animals fed Eucalyptus globulus leaf oil at a level of 2000 mg kg–1 showed no colour changes. Histopathological investigation of liver, kidney, lungs, heart, brain and spleen from both the control and treated rats revealed no obvious alterations at the end of the experimentation stage.
| Fig. 3: | Effects of EOE and diazepam also on the period of immobility time were studied in the FST Results are given as Mean±SEM (n = 5). In comparison to the relevant control group and ***p<0.001 was found |
| Fig. 4: | Effects of EOE and diazepam on the duration of immobility time in TST Results are expressed as Mean±SEM (n = 5) and ***p<0.001 as compared to the respective control group |
The microscopic analysis revealed that all of the organs from the oil-treated rat did not show any changes in cell structure and that the oil-treated organs were more or less comparable to the control organs (Fig. 2a-n).
Antistress activity: The following results were obtained in a different model of screening the antistress drug.
Forced swim test: When compared to control (150.17±4.9), animals treated with Eucalyptus globulus leaf oil (100, 200 and 400 mg kg–1) showed a significant decrease in their immobile periods (82.83±2.37, 60.67±1.98 and 47.67±2.46 correspondingly<0.001). Similarly, mice given diazepam (1 mg kg–1) demonstrated a significant reduction in immobility time (38.52.92, p<0.001), as expected (Fig. 3).
Tail suspension test: In this test animals treated with Eucalyptus globulus leaf oil (100, 200 and 400 mg kg–1) showed a decrease in their immobility times which was significant (135.33±3.19, 113.17±2.81 and 96.17±2.45, respectively, p<0.001) when compared with control (160.17±3.62). Similarly, animals treated with diazepam (1 mg kg–1), as expected, showed a significant decrease in the immobility time (73.33±2.11, p<0.001) (Fig. 4).
| Fig. 5: | Effects of Eucalyptus globulus on anoxia stress tolerance time immobility (min) Values are reported as Mean±SEM (n = 5) and were analysed using one-way ANOVA and dunnett's test. *Statistical significance vs. control (p<0.05) |
| Table 2: | Effect of Eucalyptus globulus leaf oil 2000 mg kg–1 on haematological parameters | |
| Blood parameters | Control group | Oil treated group |
| Hemoglobin (g dL–1) | 14.39±0.11 | 14.4±0.28 |
| Total leucocytes count (109 L–1) | 12700±47.55 | 40665.65±14.69.31 |
| Differential leukocytes count | ||
| Granulocytes (109 L–1) | 15.64±1.32 | 22.30±2.35 |
| Lymphocytes (109 L–1) | 82.30±1.57 | 72.63±2.49 |
| Monocytes (109 L–1) | 0.02±0.70 | 1.66±0.15 |
| RBC count (1012 L–1) | 4.82±0.064 | 4.22±0.061 |
| Platelet count (109 L–1) | 4.3±0.12 | 4.16±0.205 |
| MCV (fl) | 89.11±1.60 | 99.91±0.56 |
| MCH (pg) | 29.8±0.37 | 32.95±0.174 |
| MCHC (g L–1) | 33.50±0.235 | 32±0.424 |
| PCV (L L–1) | 43±0.46 | 44±0.608 |
|
Values are reported as Mean±SEM (n = 5) and were analyzed using one-way ANOVA and dunnett's test, TLC: Total leukocyte count, DLC: Differential leukocyte count, RBC: Red blood cells, MCV: Mean corpuscular volume, PCV: Packed cell volume and MCHC: Mean corpuscular haemoglobin concentration |
||
Anoxia stress tolerance test: Mean±SEM was used to express the outcomes of the Anoxia stress tolerance test. Anoxia stress tolerance time was significantly (p<0.05) improved in the EOE (400 mg kg–1) and Diazepam (1 mg kg–1) treated groups on the 7th, 14th and 21st days. After the 2nd and 3rd weeks of EOE (200 mg kg–1) treatment, there was an increase in anoxia tolerance time but the 7th day result was not statistically significant (Fig. 5).
However, at the end of the 1st, 2nd and 3rd weeks of treatment, the effect of EOE (400 mg kg–1) on anoxia stress tolerance time in mice was not statistically significant.
DISCUSSION
Experimental findings suggest anti-stress influences of Eucalyptus globulus leaf oil. Results of FST concludes that leaf oil significantly decreased the immobile periods in the treatment group as compared to control animals. Outcomes of TST and AST also suggested the anti-stress activity of the leaf oil, in TST, administration of leaf oil at different concentrations caused a dose-dependent decrease in immobility times as compared to control animals, similarly, the experimental facts of AST summarized that treated animals exhibited fewer immobility durations as compared to controls. The most significant results were exhibited by the rats treated at 200 mg kg–1 dose. Stress is described as a physical or psychological change that affects the organism's equilibrium and balance25. Physical stressors such as loud noise, large crowds and congested surroundings generate stress26. Stress is a non-specific bodily reaction that disrupts the organism's physiological balance, resulting in a variety of neuronal, endocrine and basic dysfunctions27. In the modern era, stress and stress-related disorders are a significant cause of disease and contribute perhaps 75% of all illnesses28. A polyherbal formulation of eucalyptus oil is used in the antistress activity. Eucalyptus oil shows the antistress activity. Eucalyptus oil is used for respiratory disorders like asthma, allergy29. The mucolytic (reducing the viscosity of mucus) and bronchodilator (decreasing resistance in the breathing airways) properties of Eucalyptus oil help liquefy and relieve nasal and bronchial congestion29. Eucalyptus oil is proving to be used as an antistress but there is no scientific data available.
Forced Swim Test (FST) induced stress animal model extensively studied and served as an established model to examine antistress effects the different pharmacotherapeutic moieties in several research protocols30. FST is based on the principle that the rodents develop immobility after some time if forced to swim in a non-escapable jar filled with water31. Researchers examined and proved that when animals were forced to swim as per standardized conditions of the test during the first 2 min of the test, each animal moves around energetically while in the next 4 min of the 6 min test, the duration of immobility develops32,33. Findings of this study also authenticate the previous works, results depicts that animals administered Eucalyptus globulus leaf oil (100, 200 and 400 mg kg–1) had less immobility time than control animals. The standard medication diazepam reduced immobility time as well.
Tail Suspension Test stress model has been widely used behavioural paradigm for detecting antistress activity in rats25, briefly, the animals were individually suspended from the table's edge, 50 cm above the floor, using adhesive tape put 1 cm from the tip of the tail. During the test, each animal was sonically and visually segregated from the other animals. A total of 6 min of immobility was manually recorded. When an animal did not move its body, hung passively and entirely still, it was deemed immobile25. Scientists had authenticated it in numerous researches33-35. Current findings also support the previous outcomes both treated groups showed a reduction in immobility time. When compared to the control group, this rise was substantial.
Further to support the research outcomes and to make the confirmatory remark on the antistress effect in different experimental works the anoxia stress tolerance test had been widely used previously36. Various scientists studied the antistress effect of the drugs using the AST test previously37, Briefly, To induce anoxia stress, a hermetic vessel with a 1 L air capacity was utilised. Each animal was housed in its hermetic vessel and the time it took to show the 1st signs of seizure was recorded. The animals were then removed from the vessel and resuscitated if necessary. The findings of the anoxia stress tolerance test were calculated as Mean+SEM. Anoxia stress tolerance time was significantly (p 0.05) improved in the EOE (400 mg kg–1) and Diazepam (1 mg kg–1) treated groups on the 7th, 14th and 21st days. After the 2nd and 3rd weeks of EOE (200 mg kg–1) treatment, there was an increase in anoxia tolerance time but the 7th day result was not statistically significant. However, at the end of the 1st, 2nd and 3rd weeks of treatment, the effect of EOE (400 mg kg–1) on anoxia stress tolerance time in mice was not statistically significant.
CONCLUSION
Findings of the experiments conducted conclude that when animals were stressed using the FST, TST and Anoxia stress tolerance test models and were treated with three doses of EOE (100, 200 and 400 mg kg–1, p.o.) the immobility periods were reduced significantly. In the same way, animals treat with diazepam (1 mg kg–1) as predictable, show a major decrease in the immobility time. The current investigational protocol discovered and confirmed that eucalyptus oil possesses antistress activity by preventing stress-induced by FST, TST and Anoxia stress tolerance test model.
SIGNIFICANCE STATEMENT
The present research protocol was intended to study the antistress effect of eucalyptus globra leaf oil. Outcomes of the research attested to the hypothesis and were also in agreement with the earlier findings of the scientists. The Eucalyptus leaf oil showed promising antistress activity in different FST, TST, AST models of stress induction. These results can help future researchers to do some extensive experiments at a molecular level to further establish the drug for clinical research. This will serve humanity in terms of reduction of suffering and economic loss.
ACKNOWLEDGMENT
This work was funded by the Deanship of Scientific Research at Jouf University Saudi Arabia under Grant No (DSR-2021-01-0318).