A dose Dependent hepatoprotective and nephroprotective activity of eucalyptus oil on Streptozotocin induced diabetic mice model
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Eucalyptus globulus are a prime source of global eucalyptus oil production. This oil has therapeutic, flavoring, antimicrobial, and biopesticide properties. This oil has got popularity because they are reported as pharmacologically active.
A study has been conducted with the aim to find dose dependant effect of eucalyptus oil on diabetic mice induced with Streptozotocin (STZ) and analyze the hepatoprotective and nephroprotective nature through biochemical and histological study.
STZ at low dose was used to damage pancreatic β cells and induce type II diabetes on mice model. Oil was used in different percentages namely, 0.5%, 1%, 1.5%, 2% for treating hyperglycemia. Then after a treatment of 28 days, the total body weight, and biochemical parameters including blood glucose, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), Bilirubin, cholesterol, urea, creatinine were checked thoroughly. A complete histological study of different organs like liver, kidney, and pancreas was done.
From the explained experiment the result was quite interesting which showed among all the concentration of essential oil, 1.5% showed maximum effect on hyperglycemia with a rapid approach to normalize high blood glucose level and also other biochemical parameters. The tissue was got rejuvenated and behaved as normal cells after being treated with 1.5% oil.
So the main purpose of the work is to search for an alternative substituent of antidiabetic drug which can work as potentially as conventional drug for diabetes but without having side effects. And Eucalyptus oil proved to be safe and steady source for hyperglycemic patients at a particular dose. So it can be used for further diabetic research.
KeywordsDiabetes Streptozotocin Essential oil Liver Biochemical assay, drug dose dependant
Diabetes mellitus is a metabolic disorder, symptomised by hyperglycemia. This disease is the result of a deficiency in insulin secretion or insulin resistivity of the body .. They disturb the whole endocrine and metabolic balance of the body. According to American Diabetic Association patients having Type II diabetes are also susceptible to cardiovascular disease followed by renal failure, atherosclerosis, gastrointestinal disturbance, eye, skin, nerve complication.
STZ is a naturally occurring nitrosourea product. It is obtained from Streptomyces achromogenes. Lower dose (may be single dose) can cause severe toxicity to β cells in the pancreas which cause necrosis and then hyperglycemia occurs .
Mice model is mostly used in those experiments where lower dose of STZ generally used to induce type II diabetes and experimental drugs are applied .. In the present scenario different conventional marketised antidiabetic drugs like sulfonylurea, glucoside inhibitors, metformin, GLP-I agonist, troglitazones which are hypoglycemic in nature having immense side effects and not suitable for pregnants . Some of the common side effects of these drugs are lower blood sugar level, weight gain, stomach upset, diarrhea, liver diseases, anemia etc. .
Thus, from this point of view scientific interest is now booming throughout the world in the study of medicinal plants, as plants are safe to live cells, having prolonged effect as well as due to lesser side-effects in clinical practice and relatively low costs in their treatment .
Many species of the genus Eucalyptus belonging to Myrtaceae family are used for medicinal purpose. The oil from eucalyptus was isolated from leaves of Eucalyptus globules. Essential oil contains oxygenated monoterpenes namely 1, 8 –eucalyptol, α-terpineol; terpinen-4-ol.These essential oil is also used as traditional medicine as antidiabetic drug ..In 1980 World health organization (WHO) expert committee recommended about Diabetes mellitus that customary plant treatment for Diabetes mellitus justify further evaluation.
But it is a burning question for every drug that after getting treated, how much it is altering the blood parameters and microscopic anatomy of cell and tissue? So for that, a thorough analysis should be done to check the negative effect of drug on biochemicals and cell’s microscopic anatomy.
In the present experiment, Streptozotocin induced diabetic mice were treated with eucalyptus oil at a certain dose was used to treat them without any side effects on the model.
Different biochemical parameters and cellular anatomy was thoroughly studied and can be ensure that eucalyptus oil has a tremendous effect on hyperglycemia, having a hepatoprotective as well as nephroprotective property when treated at a certain dose.
Materials and methods
Binomial name of the plant is Eucalyptus globulus Labill. These plants are commonly known as Tasmanian bluegum or southern bluegum. The plant name has been checked with http://www.theplantlist.org. The accepted name record derives from WCSP(World checklist of selected plant families) data supplied on 2012–03-23. The original publication details are Voy.Rech.Perouse 1:153 1800.
Pure extracted oil was purchased from Auroshikha,Pondicherry and were used without further purification. To know the chemical constituents of the oil, they were chemically characterized by gas-chromatography. The condition for gas chromatography was mentioned as follows: Gas chromatography: The essential oils were analyzed using a Perkin-Elmer gas chromatograph model 8700, which is equipped with flame ionization detector (FID) and HP-5MS capillary column (30 m × 0.25 mm). Injector and detector temperatures were adjusted at 220–290 °C. Column oven temperature was set from 80 to 220 °C at the rate of 4 °C per minute. Initial and final temperatures were 3 and 10 min, respectively. Helium gas was used as carrier with flow of 1.5 mL per minute. A sample of 1.0 μL was injected, using slit mode (split ratio, 1:100). All quantification was done by a built-in data-handling program provided by the manufacturer of the gas chromatograph (Perkin-Elmer, Norwalk, CT, USA). The composition was reported as a relative percentage of the total peak area .
Adult albino mice of average weight between 29 and 30 g of both sexes were obtained and were acclimatized in the laboratory for 2 weeks under proper aeration and at a temperature of 22°celsius and normal atmospheric pressure and also under proper supervision. They had access to food and pure water ad libitum and the stress level was 0 at the time of experiment.
Diabetes was induced by single intraperitoneal injection of Streptozotocin (STZ) (Sigma Aldrich 75%, USA) (60 mg/kg body weight in 0.1 mol/L citrate buffer, pH 4.5) into 16–18 h fasted mice. 5% glucose solution was supplied to STZ-induced mice for the next 24 h to avoid hypoglycemic mortality. After 96 h of STZ injection, blood sample (10 μL) from mice which were induced with Streptozotocin was collected by tail snip method and glucose was monitored by Accu-Chek (Roche Accu-Chek Advantage® whole-blood glucose monitor).
Fasted mice with glucose concentration greater than 200 mg/dl (11.1 mmol/L) were considered as hyperglycemic .Therefore initial checking of all biochemical parameters without induction of hyperglycemia was consider as day 0.After 96 h (4thday) only blood glucose was checked to confirm the induction of diabetes. Then at 7th, 14th, 21th, 28th day whole biochemical parameters were observed.
Mice grouping and treatment
There were total 56 mice which were grouped into 7 sets containing 8 mice in each group.
Group I-Control (Normal diet). Group II to VII (STZ) and were diabetic. Group III (Glibenclamide [5 mg/kg] . Group IV to VII (0.5%,1%,1.5%,2% Eucalyptus oil [EO]) respectively. EO was prepared by taking different percentage (0.5,1, 1.5, 2) of oil with respect to water as solvent and after immediate sonication which was done by ultrasonic sonicator stabilized the sample and immediately they were injected to the body .
Lethal dose toxicity test
EO were administered to mice at a dose of 0.5, 1, 1.5, 2, 2.5 (ml/kg body weight) and observe the behavioral change and mortality rate for a whole day.
A 28 days treatment was done and body weight and biochemical assays were analyzed. At the end of the treatment, mice from each group’s blood samples were collected from the retro orbital plexus then were sacrificed by anaesthetized. Blood serum was collected by repeated centrifugation at 3000×g for 10 min to assay blood parameters (Bilirubin, Cholesterol, SGPT, SGOT, ALP, GGT, Urea, Creatinine) For hepatoprotective enzymes like SGPT and SGOT tests modified UV (IFCC) kinetic assay was followed where absorbance was recorded at340nm.ALP was checked by p NPP –AMP (IFCC), kinetic assay. Absorbance was taken at 405 nm.GGT test was also done following the SZASZ method. ENZOPAK GGT is formulated on Szasz method recommended by Scandinavian Society for Clinical Chemistry and Physiology.This method is very particular in kinetic, specific and sensitive. Serum absorbance for GGT was taken at 405 nm.Bilirubin and cholesterol test followed Jendrassik and Grof method and CHOD-PAP method. Optical absorbance for Bilirubin was at 546 nm and 505 nm for cholesterol respectively. For nephroprotective enzymes like urea and creatinine Urease, Berthelot, end point assay and modified Jaffe’s reaction initial rate assay was followed. And optical absorption was 578 nm for urea and 505 nm for creatinine. Liver, kidney and pancreas were excised. For histology, excised liver, kidney and pancreas were washed in phosphate buffer and then transfer to 10% formalin fixative solution for 48 h. All three types of tissues were processed for paraffin embedding and were sectioned of 5 μm in microtome. After staining with haematoxylin and eosin, they were examined under microscope (Axio Scope A1-Zesis) at 100X magnification.
Data expressed as Mean ± standard deviation and all analysis was carried out by Microsoft office excel and Origin software (version Pro 8.5; Origin Lab Corporation, Northampton, MA 01060, USA.
Results and discussion
Biochemical aspects of the blood of different group of mice serum depicted through different graphs.
In Fig. 2 c] Normal Bilirubin range was observed in control group but higher in STZ induced group. Bilirubin is the main bile pigment which formed because of breakdown of heme of red blood cells by reticuloendothelial system. Unconjugated bilirubin is carried by Albumin to the liver where it is conjugated with glucuronic acid and becomes water soluble. Total Bilirubin concentration always increases severely in hepatocellular disease. Here also increased Bilirubin was observed as STZ caused hepatocellular damage but after EO treatment again normal range of Bilirubin was seen, especially when treated with 1.5%.EO. [18, 19, 20]. Figure 2 d , showed the distinct variation of total cholesterol among normal, diabetes and diabetes recovered mice. Control group have normal range of cholesterol, followed by increased in cholesterol range in group II. Cholesterol is a waxy substance made by the body and found in some animal-based foods. Blood cholesterol levels describe a group of fats also known as lipoproteins that include HDL and LDL. Cholesterol can be harmful by contributing to narrowed or blocked arteries. Result showed that conventional drug Glibenclamide drags the level of cholesterol into normal range. Like the same EO of 1.5% also depicted the same result. As 261 mg/d L is quite high at 7th day but it reduces drastically to 122 mg/d L on 28th day .
Lethal dose toxicity test
Survival rate was 100% and no reports of death was seen even after 28 days.
But in case of STZ treated tissues (Fig. 7 b), the structural morphology was not normal; size of the islet cell was shrunken with architectural disarray (marked) and number of acinar cells decrease around the islet cell. When the tissue was treated with Glibenclamide then in usual manner the tissue got recovered and normal morphology was gained gradually. When EO was treated, 0.5% didn’t show any significant change but 1% showed proper structure of islet cells (Fig. 7 e) with increased number of acinar cells. But best result was obtained when 1.5% was treated, at that time better restoration of the beta cell in comparison to other dose was observed. Normal size of islet cells with normal number of acinar cells with less necrotic cell and contracted blood vessels were observed (Fig. 7 f). When 2% was applied there was improvement in the number of acinar cells but no improvement was took place for islet of langerhans cells [4, 32]. As the dose of Streptozotocin was 60 mg/kg body weight which has been consider as a moderate dose for the induction of diabetes and it didn’t damaged all of the pancreatic cells which was proved by the Histopathological image of pancrease as shown in Fig. 7 and also proved that 1.5% dose of Eucalyptus oil has the capability to regenerate the damaged pancreatic cells. As this work is a basic preliminary work which emphasized only the dose dependant activity of the eucalyptus oil on diabetes, so insulin release was not considered as a checking parameter.
So the main purpose of the work is to search for an alternative substituent of antidiabetic drug which can work as potentially as conventional drug for diabetes but without having side effects which was evidenced by biochemical and histopathological analysis and also therapeutically competent at a particular dose of 1.5%.So eucalyptus oil proved to be safe and steady source for hyperglycemic patients at a particular dose. Though it is a dose dependant study of the essential oil but further research need to upgrade the information and also the molecular pathway behind the dose dependant activity need to be searched further.
The team would like to acknowledge the department of pharmacy Jadavpur University for their kind help and also acknowledge Dr. Avery Sengupta and Prof.Achntya Saha from University of Calcutta for providing us the instrument facility.
This work has not been funded by any source/organization.
Monalisa Chakraborty: Design of experiment and perform the experiment, Data analysis and interpretation, Writing section.
Biswajoy Bagchi: Design of experiment, Data analysis and interpretation, Sukhen Das, Ruma Basu, Papiya Nandy: Checking writing section, Interpretation of experimental data, significance of result.
The author declares that they have no competing interest.
Regarding the animal ethics, consent was approved by department of pharmaceutical technology Jadavpur University, Kolkata, India.
Serial/Registration number: 1526–1805/GO/Re/S/15/CPCSEA 10/06/2015,Jadavpur University,188,Raja S.C. Mullick Road,Kolkata − 700,032,West Bengal.
Also followed the NIH guidelines for the welfare of laboratory animals and also obeys the guideline for the care and use of laboratory animals. [Eighth Edition Committee for the Update of the Guide for the Care and Use of Laboratory Animals Institute for Laboratory Animal Research Division on Earth and Life Studies (National research council.)]
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