Accepted Manuscript

Sub-chronic oral toxicity of Cuminum cyminum L.’s essential oil in female Wistar rats

Mohsen Taghizadeh, Seyed Naser Ostad, Zatollah Asemi, Mohaddese Mahboubi,

Sara Hejazi, Reza Sharafati-Chaleshtori, Aliakbar Rashidi, Hosein Akbari, Nasrin
Sharifi

PII: S0273-2300(17)30176-9
DOI: 10.1016/j.yrtph.2017.06.007
Reference: YRTPH 3854

To appear in: Regulatory Toxicology and Pharmacology

Received Date: 9 August 2016

Revised Date: 14 June 2017
Accepted Date: 16 June 2017

Please cite this article as: Taghizadeh, M., Ostad, S.N., Asemi, Z., Mahboubi, M., Hejazi, S., Sharafati-
Chaleshtori, R., Rashidi, A., Akbari, H., Sharifi, N., Sub-chronic oral toxicity of Cuminum cyminum L.’s
essential oil in female Wistar rats, Regulatory Toxicology and Pharmacology (2017), doi: 10.1016/
j.yrtph.2017.06.007.

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Title Page

Sub-chronic oral toxicity of Cuminum cyminum L.’s essential oil in female

Wistar rats

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Mohsen Taghizadeh a, Seyed Naser Ostad b, Zatollah Asemi a, Mohaddese Mahboubi c, Sara

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Hejazi c, Reza Sharafati-Chaleshtori a, Aliakbar Rashidi a, Hosein Akbari d, Nasrin Sharifi a

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Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of

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Medical Sciences, Kashan, I.R. Iran
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Department of Pharmacology and Toxicology, Tehran University of Medical sciences, Tehran, I.R.
Iran
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Department of Microbiology, Medicinal Plant, Research Center of Barij Essence, Kashan, I.R. Iran

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Department of Biostatistics, Kashan University of Medical Sciences, Kashan, I.R. Iran
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Correspondence to:
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Nasrin Sharifi, PhD

Research Center for Biochemistry and Nutrition in
Metabolic Diseases, Kashan University of
Medical Sciences, Kashan, I.R. Iran
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Tel: +98 3155540021

Fax: +98 3155620608
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Email: sharifi.nsr@gmail.com

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Author Contribution

Ostad, SN. and Taghizadeh, M. designed and supervised the research. Sharifi, N. involved in

study design and wrote the manuscript. Asemi, Z. and Akbari, H. contributed to statistical

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analysis of the data. Mahboubi, M. and Hejazi, S. assessed the haematological and

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biochemical laboratory tests. Sharafati-Chaleshtori, R. and Rashidi, A. evaluated the

histopathological samples.

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Sub-chronic oral toxicity of Cuminum cyminum L.’s essential oil in 2

female Wistar rats 3

ABSTRACT 4

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The current study was performed to evaluate the toxicity of Cuminum cyminum L. (C. 5

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cyminum)’s essential oil after 23 days and 45 days of repeated oral administration in 6

female Wistar rats. A total of 80 healthy female Wistar rats were randomly selected 7

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and divided into 4 groups. The rats were gavaged with C. cyminum’s essential oil at 8

dose levels of 0, 250, 500 and 1000 mg/kg/day. Clinical signs, body weight, 9

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hematology, serum biochemistry and organ histopathology were assessed once after 10
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23 days and again after 45 days passed from the start of the intervention. Oral 11
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administration of C. cyminum’s essential oil had no observed adverse effects on 12

clinical signs, mortality, body weight, hematology, biochemistry and organ histology 13
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(liver, kidneys, spleen and lungs) in a sample of healthy female Wistar rats after 23 14
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days and 45 days from the start of the study. However, an increase in serum levels of 15

alanine transaminase (ALT) was found only at dose level of 1000 mg/kg/d C. 16
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cyminum’s essential oil, after the 23-days interval. We conservatively defined the 17
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non-observed adverse effect level (NOAEL) for C. cyminum’s essential oil as 500 18
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mg/kg/d in female Wistar rats. The present study results should be treated with 19

cautious in terms of the other organs' toxicity. 20

Key words: C. cyminum’s essential oil, sub chronic toxicity, Wistar rats 21

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1. INTRODUCTION 1

Cuminum cyminum L. (C. cyminum) is one of the commercial spice plants belonging 2

to the Apiaceae family and is widely used to enhance food flavor and aroma (Pandey 3

et al., 2015). The plant is mostly cultivated in India, Iran and Mediterranean region. 4

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Also it is grown in China, Mexico and Chile. C. cyminum is consumed in two forms: 5

powdered and seeds. C. cyminum’s seeds are added to the foods such as cheese and 6

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bread in Middle East countries (Johri, 2011). There are many known benefits for C. 7

cyminum consumption in different traditional medicines. For example, in Iranian 8

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traditional medicine, C. cyminum is used for its carminative, eupeptic, analgesic, anti- 9

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microbial, anti-bronchitis and galactagogue effects (Johri, 2011). 10
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The major volatile components of C. cyminum’s essential oil include cuminaldehyde, 11

cymene and terpenoids (Sowbhagya, 2013). The beneficial effects of the C. cyminum 12
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that were found in experimental studies have been attributed to these bioactive 13
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compounds. Some antifungal and anti-bacterial effects have been investigated for 14
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cuminaldehyde (De Martino et al., 2009). Recent cellular studies suggest that 15

cuminaldehyde may have protective effects on neurodegenerative disease such as 16

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Parkinson's (Morshedi et al., 2015). The results of in vitro and in vivo studies 17

revealed anti-carcinogenic potential for C. cyminum’s essential oil (Gagandeep et al., 18

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2003). The results of previous studies also found the antioxidant activity for C. 19
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cyminum which was supposed to be related to its flavonoids components (Bettaieb et 20

al., 2010). C. cyminum’s oil also is known to have several other pharmacological 21

properties, such as anti-diabetic, anti-hypertensive and immunomodulative effects 22

based on the evidence derived from in vitro and in vivo studies (Chauhan et al., 2010; 23

Dhandapani et al., 2002; Kumar et al., 2009). These medical properties have attracted 24

the attention towards the use of C. cyminum as herbal remedy for several diseases. 25

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Most of the previous studies examined the effects of C. cyminum in cellular and 1

animal model of diseases and consequently the future clinical trials would be 2

designed to confirm the beneficial effects of C. cyminum on disease treatment in 3

human subjects. However, when a plant extract is used in pharmacologic doses, it 4

may have some adverse effects on human body. As a result, it is necessary to assess 5

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toxicity and safety of an herbal medication before its administration to human 6

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subjects. To the best of our knowledge, the non-observed adverse effect level 7

(NOAEL) for C. cyminum’s essential oil has not yet been investigated in a systematic 8

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way. Therefore, to provide data for establishing safe dosage and treatment duration 9

for clinical application, the current study was performed to evaluate the toxicity of C. 10

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cyminum’s essential oil after 23 days and 45 days of repeated oral administration in 11
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female Wistar rats. 12
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2. MATERIALS & METHODS 13

2.1. Preparation of C. cyminum’s essential oil 14

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Dried C. cyminum, obtained from Kashan, Iran, was identified as Cuminum cyminum 15

L. by Dr. Valiollah Mozafarian from Tarbiat Modares University, Tehran, Iran. The 16
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voucher specimen (BE.2128) was deposited in the herbarium of the Department of 17

Phytochemistry, Barij Essence Pharmaceutical Company, Kashan, Iran. Once approved by 18

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botanical expert, approximately 2 kilograms of C. cyminum’s seeds were used to 19

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extract about 70 grams of essential oil. The essential oil of C. cyminum was extracted 20

by hydro-distillation method using a Clevenger-type apparatus (Clevenger, 1928). 21

Then, one random sample of the obtained essential oil was analyzed by gas 22

chromatography (GC-3800, Varian, USA) to determine its ingredients. 23

2.2. Experimental animals 24

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The study was conducted in accordance with the Guide for the Care and Use of 1

Laboratory Animals, National Research Council , 2011. eighth edition. The animals 2

were randomly selected from the healthy female Wistar rats at seven weeks of age 3

which were purchased from the Pasture Institute of Iran. Wistar rats were housed at 4

least for 7 days prior to the start of study to adapt to their environment. The rats were 5

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kept in the temperature of 23±2.2°C and 12-h-light/12-h-dark cycle. The animals were 6

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housed in groups of four in stainless steel cages. All of them received appropriate care 7

according to the guide for the use of laboratory animals. The study protocol and 8

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procedures were approved by the Animals Ethics Committee of Tehran University of 9

Medical Sciences. Furthermore, the study was conducted based on the Good 10

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Laboratory Practice (GLP) protocol developed by Iranian Food and Drug 11
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Administrations. 12
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2.3. Administration 13
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C. cyminum’s essential oil was prepared for gavage by dissolving it in olive oil to 14
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reach the volumes and concentrations necessary for the doses for each treatment 15

group. For example, to prepare a dosing solution that provided a 250 mg/kg/d of C. 16
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cyminum’s essential oil for a rat with a body weight of 200 g, 50 mg of the essential 17

oil was dissolved in 50 mg of olive oil to reach a volume of 0.1 mL. The dosing 18
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solutions were prepared weekly and the needed amounts of them were administered to 19
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rats by oral gavage, once per day, without fasting. The stability test for the solution of 20

the C. cyminum’s essential oil in olive oil was performed under the current study 21

storage conditions (such as the temperature of 24°C , 12-h-light/12-h-dark cycle, etc.) and 22

based on the standard protocol during 6 months. The test was repeated at 1st, 2nd, 3rd 23

and 6th month. Based on the results of gas chromatography analysis, there were not 24

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any significant changes in components and indices of C. cyminum’s essential oil 1

during 6-month period. 2

2.4. Study Design 3

Present study assessed sub-chronic oral toxicity of C. cyminum’s essential oil in 4

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Wistar female rats. The study included two intervals, one of which evaluated sub- 5

chronic toxicity of C. cyminum’s essential oil after 23 days and the other assessed it 6

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after 45 days. A total of 80 healthy female Wistar rats with mean weight of 58±7.6 g 7

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randomly divided into 4 groups (20 animals in each group) based on the computer 8

randomization. Based on our previously unpublished study in terms of acute toxic 9

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effects of C. cyminum’s essential oil, the rats received it at dose levels of 0 (control), 10
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250, 500 and 1000 mg/kg/day. Control group was administered sterile water by 11

gavage. To collect blood and organ tissue samples in mid-time of the intervention, 10 12
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rats from each group were euthanized under anesthesia after 23 days passed from the 13
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start of the study. The remaining ones continued receiving their allocated treatments 14
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until 45 days after the start of the intervention and then sacrificed. The following 15

parameters examined during and after the experimental period: clinical signs, body 16
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weight, food and water consumption, hematology, serum biochemistry and organ 17

histopathology. 18
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2.5. Clinical signs 19

All rats were observed once a day during the experimental period to recognize any 20

immediate signs of toxicity, number of mortality and changes in behavior such as 21

motor activity, grooming, pain response, touch response, convulsion, tremors and 22

righting reflex. 23

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2.6. Body weight, food and water consumption 1

The animal body weights were measured before the start of treatment and every week 2

thereafter during the course of the study. Also, the amount of food and water was 3

measured before its supply to each cage and the remaining amount was measured at 4

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the end of the day to calculate the daily food and water consumption. 5

2.7. Hematologic and serum biochemical assessment 6

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At the end of each interval (23-day and 45-day), after overnight fast, the animals were 7

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anesthetized using diethylether and euthanized by exsanguinations from cardiac 8

puncture. Then blood samples were collected through intra-cardiac puncture. Each of 9

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the collected blood samples was divided into two aliquot portions. One portion was 10
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treated with EDTA for hematologic assessment while the other was untreated and 11
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used for serum separation. Also, two blood smears were prepared following sample 12

collection. 13
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Hematologic parameters that were measured using hematological autoanalyzer 14

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(Biotecnica, Model BT 1000, Italy) included total white blood cell count (WBC), total 15
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red blood cell count (RBC), hemoglobin concentrations (HGB), mean corpuscular 16

volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular 17

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hemoglobin concentration (MCHC), hematocrit (HCT) and platelet count. 18

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The untreated blood portions were centrifuged at 3000 rpm for 10 minutes to separate 19

serum samples for biochemical analysis. The serum obtained was evaluated for the 20

following parameters using laboratory commercial kits and a biochemical 21

autoanalyzer (Biotecnica, Model BT 1000, Italy): fasting blood sugar (FBS), blood 22

urea, creatinine (Cr), total cholesterol (TC), triglyceride (TG), sodium (Na), 23

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potassium (K), calcium (Ca), alanine aminotransferase (ALT), aspartate 1

aminotransferase (AST), lactate dehydrogenase (LDH) and total protein. 2

2.8. Histopathological examination 3

After the end of each interval, the sample tissues were obtained from liver, spleen, 4

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kidneys and lungs of all the animals. Tissues were fixed with 10% formalin solution 5

and imbedded in paraffin blocks. Then the paraffin blocks were sectioned at 5 µm 6

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thicknesses. The prepared sections were stained by standard method using 7

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hematoxylin and eosin for microscopic examination. 8

2.9. Statistical Analysis 9

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The data were reported as mean±standard deviation. One way analysis of variance 10

(One way ANOVA) was used to compare mean differences of biochemical and 11
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hematologic parameters between study groups at the end of the intervention. In this 12

analysis, the homogeneity of variance was evaluated and Bonferroni correction was 13
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used as post-hoc test. To compare the mean changes of variables such as weight, food 14
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and water consumption between study groups during the intervention, repeated 15
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measure one way ANOVA test was used. Moreover analysis of covariance 16

(ANCOVA) was applied to compare endpoints of variables between the groups, 17

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adjusted for baseline values. To identify any trends in values for the variables across 18
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the study groups, we conducted linear regression analysis. Analysis was carried out 19

using SPSS software version 16 and two sided P-values < 0.05 was considered 20

significant. 21

3. RESULTS 22

3.1. Gas chromatography analysis 23

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Based on the results of GC analysis showed in supplementary files 1 and 2, the major 1

compounds of the C. cyminum’s essential oil in the current study were cuminyl 2

aldehyde (38.9%), g-terpinene (18.3%), p-cymene (15.4%), b-pinene (11.7%), p- 3

mentha 1,3 (5.3%), p-mentha 1.4 (2.8%) and a-pinene (0.7 %). 4

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3.2. Clinical signs 5

In current study, there was not seen any mortality in treatment and control groups. 6

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Moreover, neither abnormal clinical signs, nor any unusual behavior were found in 7

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rats of the control and treatment groups during the 23-day and 45 day study 8

interventions. 9

3.3.
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Body weight, food and water consumption 10
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The changes in mean body weight of the rats in control and C. cyminum’s essential oil 11
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-treated groups throughout 45 days have been shown in figure 1. The results of the 12

repeated measure one way ANOVA test revealed a significant difference between the 13
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times. In other words, the mean body weight of the rats for all the study groups 14
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significantly increased from the baseline until the end of the study. At baseline the 15
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mean body weight of the rats in control group was significantly lower than those of 16

the C. cyminum’s essential oil -treated groups (P < 0.05). However during the period 17
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from 23 to 45 days, the results of the ANCOVA test, adjusted for baseline values, 18
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showed that the mean weights of the rats in C. cyminum’s essential oil -treated groups 19

were significantly lower than that of the control one (P < 0.05). In other words, the 20

rate of weight gain in rats of C. cyminum’s essential oil -treated groups was 21

significantly lower than that of control one. 22

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The mean amount of water and food consumed by the animal study groups has been 1

shown in figure 2 and 3, respectively. There were no significant differences in water 2

and food consumption between any of the C. cyminum’s essential oil treated-groups 3

and control one. Only there was a significant increase in water consumption as from 4

day 30 in all the study groups. 5

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3.4. Hematologic and serum biochemical assessment 6

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The mean values for hematologic parameters of rats after 23 days and 45 days 7

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intervention, have been shown in table 1 and table 2, respectively. These values did 8

not significantly differ between control and C. cyminum’s essential oil -treated groups 9

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both after 23 and 45 days passed from the start of the intervention. 10
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The mean values for serum biochemical variables of rats after 23 days and 45 days 11
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intervention, have been shown in table 3 and table 4, respectively. Among these 12

variables, serum levels of TC and TG were significantly lower in C. cyminum’s 13

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essential oil-treated groups than control after 23 days of intervention. The significant 14
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lower levels of TC were found in 250 mg/kg and 1000 mg/kg C. cyminum’s essential 15

oil -treated groups compared with controls. For serum TG, all the three C. cyminum’s 16
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essential oil -treated groups had significantly lower levels when compared with 17
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controls. Moreover, based on the results of the regression analysis, there was a 18
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significant decreasing trend in serum levels of TG across the increase in doses of C. 19

cyminum’s essential oil (P for trend < 0.001). 20

The serum levels of ALT had significantly higher values than those of the controls 21

only in 1000 mg/kg C. cyminum’s essential oil-treated groups after 23 days passed 22

from the start of intervention. 23

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For serum LDH, all the three C. cyminum’s essential oil -treated groups had 1

significantly higher levels when compared with controls after 23 days of intervention. 2

Also, there was a significant increasing trend in serum concentrations of LDH across 3

the increase in doses of C. cyminum’s essential oil (P for trend < 0.001). There were 4

not any significant differences between control and intervention groups in terms of 5

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other biochemical parameters after 23-day intervention. 6

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As shown in table 4, after 45 days from the start of the study, a significant difference 7

only was found between control and C. cyminum’s essential oil-treated groups 8

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regarding the serum levels of LDH. The mean values for serum LDH were 9

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significantly lower in 250 and 500 mg/kg C. cyminum’s essential oil-treated groups 10
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than controls. However the trend was not significant across the doses. Comparisons of 11

the mean concentrations of other biochemical variables between the groups of the 12
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study revealed no significant differences after 45 days intervention. 13

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3.5. Histopathological examination 14

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3.5.1. 23 days after the intervention 15

Minor histological changes were seen in livers of all the C. cyminum’s essential oil- 16
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treated rats that included mild infiltration of mononuclear cells and mild dilation of 17
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sinusoids. Significant changes were not seen in spleen tissues of the C. cyminum’s 18
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essential oil-treated groups. Only the number of the red blood cells slightly increased. 19

Furthermore, the tissues of the left and right kidneys were normal in all the groups of 20

the study. Very mild histological changes have been found in lungs of the C. 21

cyminum’s essential oil-treated rats that included mild inter-cellular edema and mild 22

infiltration of mononuclear cells. 23

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3.5.2. 45 days after the intervention 1

In C. cyminum’s essential oil-treated rats, the histological examination of the liver 2

revealed mild changes that included irregular hepatic lobules and hepatocytes with 3

small vesicles. Significant changes were not seen in spleen tissues of the C. 4

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cyminum’s essential oil-treated group. Only a little hyperemia has been found in 5

spleen sample tissues from all the study groups that could be due to environmental 6

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factors. Moreover, there were no any specific histological changes in kidneys of the 7

C. cyminum’s essential oil-treated rats. A very mild infiltration of mononuclear cells 8

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and inflammation were seen in lungs’ tissues of the C. cyminum’s essential oil-treated 9

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rats. 10
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4. DISCUSSION 11
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In recent decades, much interest has been focused on investigating the therapeutic 12

effects of C. cyminum. By searching medical databases such as pubmed, over 120 13

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articles from the year 2000 until now have been published in regard to pharmaceutical 14
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effects of C. cyminum. in cellular and animal models of the diseases. It is the time to 15

examine the clinical application of C. cyminum. in human subjects. However before 16

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the use of it in clinical trials it is necessary to know the safe doses and adverse effects 17
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of the C. cyminum. Present study was performed to provide this information. We 18

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compared clinical, hematologic, biochemical and histopathological features of female 19

Wistar rats between four experimental groups receiving 0, 250, 500 and 1000 mg/kg/d 20

of C. cyminum’s essential oil via oral gavage for 23 and 45 days. There was no 21

mortality, abnormal clinical signs and unusual behavior in all the groups of study 22

during the treatments. There were no significant differences in water and food 23

consumption between any of the C. cyminum’s essential oil treated-groups and control 24

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one. There was a significant increase only in water consumption as from day 30. 1

However, this increase in water consumption occurred in parallel in all the study 2

groups. As a result, it could be due to other factors such as temporary environmental 3

changes rather than treatment effect. 4

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The body weight and food consumption increased in parallel in all groups of rat. 5

Despite the same increase in food consumption during 23 to 45 days after the start of 6

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the intervention, C. cyminum’s essential oil-treated groups had lower body weights 7

than the control one. These effects might suggest a role for C. cyminum’s essential oil 8

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to slow down the weight gain through metabolic pathways. However, clinical studies 9

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are needed to confirm this outcome. Findings from a recent clinical trial showed that 10
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consumption of the C. cyminum and orlistat (an approved medication used for weight 11

reduction) resulted in a similar significant decrease in weight and BMI compared with 12
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placebo in 78 overweight subjects (Taghizadeh et al., 2015). Further studies in this 13

area will reveal more facts. 14

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The hematological variables did not significantly differ between control and C. 15

cyminum’s essential oil -treated groups both after 23 and 45 days intervals. Among 16
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the biochemical parameters, a dose-response effect of C. cyminum’s essential oil on 17

TC and TG was observed only in 23-days interval. However these outcomes were not 18
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obtained after 45 days intervention. Although, some lipid lowering effects have been 19
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proposed for C. cyminum. consumption in animal studies (Dhandapani et al., 2002; 20

Shirke and Jagtap, 2009), current study could not confirm these outcomes because it 21

was organ based and only designed for toxicological evaluation. The pharmacological 22

effects of C. cyminum’s essential oil should be assessed by designing clinical studies. 23

The remaining biochemical and hematological parameters did not significantly differ 24

between the study groups other than serum ALT. The serum levels of ALT had 25

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significantly higher values than those of the controls only in 1000 mg/kg of C. 1

cyminum’s essential oil-treated group in 23-day interval. This might be correlated 2

with the liver histological finding in current study. Minor histological changes were 3

seen in livers of all the C. cyminum’s essential oil-treated rats that included mild 4

infiltration of mononuclear cells and mild dilation of sinusoids in 23-days interval and 5

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irregular hepatic lobules and hepatocytes with small vesicles in 45-days interval. 6

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Although, serum levels of LDH had an increasing trend across the increase doses of 7

the C. cyminum’s essential oil in 23-day interval, it did not have such a trend after 45- 8

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day interval. Inversely, serum levels of LDH decreased significantly in 250 mg/kg 9

and 500 mg/kg doses of C. cyminum’s essential oil-treated groups in 45-day interval. 10

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This inconsistent finding could be due to other variables such as animal differences 11
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and temporary environmental changes. Because there were not any significant 12
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changes in behavioral, and vital signs as well as histopathological examinations, we 13

could not consider these outcomes as treatment effects of C. cyminum’s essential oil. 14
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After evaluation of the organ histopathological tests in current study, no significant 15

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changes were seen in liver, spleen, lungs and kidneys of the C. cyminum’s essential 16
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oil -treated rats in each interval of the study. 17

Present study was the first one of its kind that evaluated the sub-chronic toxicity of C. 18
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cyminum’s essential oil, however, it had some limitations. We aimed to assess the 19
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possible toxicity effects of C. cyminum’s essential oil on major vital organs such as 20

kidneys, liver, lungs and spleen that have more important roles in detoxification of 21

exogenous chemical substances than other organs, however, in current study, due to 22

lack of resources, the toxicity of C. cyminum’s essential oil was not evaluated in other 23

organs such as nervous, reproductive and immune systems. On the other hand, we 24

used female rats for toxicity assessment. Therefore, the present study results should 25

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be treated with cautious in terms of the other organs' toxicity and male gender. In 1

addition, current study had shorter duration than typical pre-chronic toxicity studies 2

recommended by regulatory guidelines. Therefore, it should be taken into account that 3

longer exposure periods (eg., 90 day) may also yield different results. Another 4

limitation of present study was using sterile water in place of olive oil (vehicle) for 5

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gavage in control group. However the aim of gavaging in control group is to create 6

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the similar stress conditions that affect intervention groups, although using olive oil 7

for gavage would be a better choice and help to strengthen the current study design. 8

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Despite all these limitations, present study was the first one of its kind that evaluated 9

the short term sub-chronic toxicity of C. cyminum’s essential oil with repeated-dose 10

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design. Data from current research would be a basis for designing future chronic 11
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toxicity studies with longer durations and stronger designs. 12
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CONCLUSION 13
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Overall, the results of current study showed that oral administration of C. cyminum’s 14
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essential oil had no obvious adverse effects on clinical signs, body weight, food and 15

water consumption, hematology, biochemistry and organ histology in a sample of 16

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healthy female Wistar rats after 23 days and 45 days from the start of the study. 17

However, we found an increase in serum levels of ALT only at dose level of 1000 18
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mg/kg/d after the 23-days interval. Although, such increase in serum ALT levels was 19
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not observed in 45-day interval and the histological changes that were seen in liver 20

samples of rats were mild, to protect against any possible toxicity of C. cyminum in 21

future human studies, we conservatively defined the non-observed adverse effect level 22

(NOAEL) for C. cyminum’s essential oil as 500 mg/kg/d in female Wistar rats. 23

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Acknowledgments 2
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This study was funded by the Research and Development Division of Barij Essence 5

Company, Kashan, Iran. The study protocol was approved by ethics committee of 6

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Tehran University of Medical Sciences. 7

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Conflict of interest statement 9

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The authors have no conflicts of interest to declare. 10

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Highlights

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• Toxicity of C.cyminum’s essential oil was explored in healthy female Wistar rats

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C.cyminum had no observed adverse effects on some clinical and biological variables

• Serum levels of alanine transaminase increased only at dose level of 1000 mg/kg/day

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• The non-observed adverse effect level for C.cyminum was defined as 500 mg/kg/day

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REFERENCES

Bettaieb, I., Bourgou, S., Wannes, W.A., Hamrouni, I., Limam, F., Marzouk, B., 2010. Essential oils,
phenolics, and antioxidant activities of different parts of cumin (Cuminum cyminum L.). J Agric
Food Chem 58, 10410-10418.

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Chauhan, P.S., Satti, N.K., Suri, K.A., Amina, M., Bani, S., 2010. Stimulatory effects of Cuminum
cyminum and flavonoid glycoside on Cyclosporine-A and restraint stress induced immune-
suppression in Swiss albino mice. Chem Biol Interact 185, 66-72.

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Clevenger, J., 1928. Apparatus for the determination of volatile oil. The Journal of the American
Pharmaceutical Association (1912) 17, 345-349.

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De Martino, L., De Feo, V., Fratianni, F., Nazzaro, F., 2009. Chemistry, antioxidant, antibacterial and
antifungal activities of volatile oils and their components. Nat Prod Commun 4, 1741-1750.

Dhandapani, S., Subramanian, V.R., Rajagopal, S., Namasivayam, N., 2002. Hypolipidemic effect of

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Cuminum cyminum L. on alloxan-induced diabetic rats. Pharmacol Res 46, 251-255.
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Gagandeep, Dhanalakshmi, S., Mendiz, E., Rao, A.R., Kale, R.K., 2003. Chemopreventive effects of
Cuminum cyminum in chemically induced forestomach and uterine cervix tumors in murine
model systems. Nutr Cancer 47, 171-180.
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Johri, R.K., 2011. Cuminum cyminum and Carum carvi: An update .Pharmacogn Rev 5, 63-72.

Kumar, P.A., Reddy, P.Y., Srinivas, P.N., Reddy, G.B., 2009. Delay of diabetic cataract in rats by the
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antiglycating potential of cumin through modulation of alpha-crystallin chaperone activity. J

Nutr Biochem 20, 553-562.
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Morshedi, D., Aliakbari, F., Tayaranian-Marvian, A., Fassihi, A., Pan-Montojo, F., Perez-Sanchez, H.,
2015. Cuminaldehyde as the Major Component of Cuminum cyminum, a Natural Aldehyde with
Inhibitory Effect on Alpha-Synuclein Fibrillation and Cytotoxicity. J Food Sci 80, H2336-2345.
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Pandey, S., Patel, M.K., Mishra, A., Jha, B., 2015. Physio-Biochemical Composition and Untargeted
Metabolomics of Cumin (Cuminum cyminum L.) Make It Promising Functional Food and Help in
Mitigating Salinity Stress. PLoS One 10, e0144469.
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Shirke, S.S., Jagtap, A.G., 2009. Effects of methanolic extract of Cuminum cyminum on total serum
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cholesterol in ovariectomized rats. Indian J Pharmacol 41, 92-93.

Sowbhagya, H.B., 2013. Chemistry, technology, and nutraceutical functions of cumin (Cuminum
cyminum L): an overview. Crit Rev Food Sci Nutr 53, 1-10.

Taghizadeh, M., Memarzadeh, M.R., Asemi, Z., Esmaillzadeh, A., 2015. Effect of the cumin cyminum
L. Intake on Weight Loss, Metabolic Profiles and Biomarkers of Oxidative Stress in Overweight
Subjects: A Randomized Double-Blind Placebo-Controlled Clinical Trial. Ann Nutr Metab 66, 117-
124.

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Figure legends

Fig 1. The mean weight changes in healthy female Wistar rats treated with Cuminum

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cyminum L.’s essential oil after 45 day toxicological assessment. (*P<0.05, based on the
ANCOVA test adjusted for baseline values)

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Fig 2. Water consumption changes in healthy female Wistar rats treated with C. cyminum’s

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essential oil after 45 day toxicological assessment

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Fig 3. Food consumption changes in healthy female Wistar rats treated with C. cyminum’s
essential oil after 45 day toxicological assessment
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Table 1. Hematology values of healthy female Wistar rats treated with C. cyminum’s
essential oil after 23 day toxicological assessment

Parameters a Dose (mg/kg/d)

0 250 500 1000

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WBC (count/mcL) 5320 ± 1100 4460 ± 900 4760 ± 800 4370 ± 890

RBC (×1012/L) 7.75 ± 0.1 6.89 ± 0.46 7.14 ± 0.23 6.71 ± 0.94

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Hemoglobin (g/dL) 13.5 ± 0.4 14.4 ± 0.15 14.9 ± 0.3 13.1 ± 0.4

Hematocrit (%) 44.9 ± 0.8 48 ± 2.2 50.4 ± 3.7 43.1 ± 5

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M.C.V (fL) 62.3 ± 1.1 69.7 ± 2.3 70.6 ± 6.1 64.2 ± 5

M.C.H (pg) 18.7 ± 0.45 20.9 ± 2.1 20.9 ± 0.9 19.5 ± 0.7

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M.C.H.C (g/dL) 30.1 ± 0.1 30 ± 0.45 29.6 ± 0.45 30.4 ± 0.1
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Platelet Count (×109/L) 818 ± 11 685 ± 86 856 ± 76 745 ± 92
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Table 2. Hematology values of healthy female Wistar rats treated with C. cyminum’s
essential oil after 45 day toxicological assessment

Parameters a Dose (mg/kg/d)

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0 250 500 1000

WBC (count/mcL) 8940 ± 1100 7210 ± 900 10570 ± 1800 6610 ± 920

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RBC 12
(×10 /L) 8.65 ± 0.1 8.73 ± 0.4 8.23 ± 0.44 8.06 ± 0.74

Hemoglobin (g/dL) 16.3 ± 0.4 16.7 ± 0.2 15.1 ± 0.6 15 ± 0.6

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Hematocrit (%) 49.7 ± 0.8 51.1 ± 2.1 47 ± 3.9 46.7 ± 5.1

M.C.V (fL) 57.5 ± 1.2 58.5 ± 2.1 57.1 ± 7.4 57.9 ± 1.9

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M.C.H (pg) 18.8 ± 0.3 19.19 ± 3 18.3 ± 0.5 18.6 ± 0.5
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M.C.H.C (g/dL) 32.8 ± 0.1 32.7 ± 0.3 32.1 ± 0.5 32.1 ± 0.9

Platelet Count (×109/L) 845 ± 23 1310 ± 286 800 ± 76 887 ± 94

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Table 3. Biochemistry values of healthy female Wistar rats treated with C. cyminum’s
essential oil after 23 day toxicological assessment

Parameters a Dose (mg/kg/d)

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0 250 500 1000

Blood Sugar (mg/dl) 83 ± 16 96 ± 15.5 138±38 106±11.5

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Blood Urea (mg/dl) 49 ± 3 49 ± 7 53±7 78±16

Creatinine (mg/dl) 0.6 ± 0.0 0.7 ± 0.1 0.7±0.1 0.7±0.1

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Cholesterol (mg/dl) 66 ± 2.0 45* ± 6.0 65±4.0 29**±14

Triglycerides (mg/dl) 132 ± 26 60* ± 22.5 79*±11.2 32**±12

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Sodium (mEq/l) 139 ± 1 143 ± 2 143±3 139±4
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Potassium (mEq/l) 3.9 ± 0.1 3.7 ± 0.1 3.7±0.1 4.1±0.2

Calcium (mEq/l) 10.1 ± 0.1 11.8 ± 0.9 10.1±0.1 9.6±0.3

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ALT (IU/l) 47 ± 5.0 49 ± 8.5 44±6.5

AST (IU/l) 160 ± 9 178 ± 12 194±29 195±15

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LDH (IU/l) 661 ± 55 922* ± 148 899±138* 1024±168*

Total Protein (g/dl) 6.3 ± 0.05 6.5 ± 0.1 6.8±0.2 6.2±0.3

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** Significantly different from controls with p < 0.01

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Table 4. Biochemistry values of healthy female Wistar rats treated with C. cyminum’s
essential oil after 45 day toxicological assessment

Parameters a Dose (mg/kg/d)

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Blood Sugar (mg/dl) 82 ± 14 96 ± 14 138±28 106±9

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Blood Urea (mg/dl) 45 ± 4 59 ± 8 52±4 73±12

Creatinine (mg/dl) 0.6 ± 0.0 0.6 ± 0.1 0.7±0.1 0.6±0.1

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Cholesterol (mg/dl) 71 ± 2.0 77 ± 6.0 111±26 46±34

Triglycerides (mg/dl) 81 ± 26 60 ± 22.5 93±22.2 57±9

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Sodium (mEq/l) 141 ± 1 140 ± 3 141±1 137±4
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Potassium (mEq/l) 4.3 ± 0.1 3.9 ± 0.2 3.8±0.2 4.6±0.3

Calcium (mEq/l) 10.6 ± 0.2 10.6 ± 0.3 10.6±0.1 9.9±0.4

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ALT (IU/l) 44 ± 5.0 56 ± 6 44±6 36±13

AST (IU/l) 187 ± 7 168 ± 19 95±32 115±25

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LDH (IU/l) 942 ± 155 598 ± 148 577*±138 713±168

Total Protein 6.9 ± 0.25 6.9 ± 0.1 7.2±0.2 6.5±0.5

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(g/dl)

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*Significantly different from controls with p < 0.05

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