Matricaria chamomilla L. was obtained from the plains around the city of Ilam in springtime, then dried and powdered by electric grinders after identification and approval of their scientific name. The obtained powder was mixed with a hydroalcoholic solvent (80% ethanol and 20% distilled water) and placed in an incubator shaker (34°C and 140 rpm) for 3 days. The extract was then purified by Whatman filter paper and concentrated on a rotary. The dried extract was obtained by placing the concentrated extract in dry heat at 30-40°C. It was then stored in the refrigerator for subsequent use.

In order to determine the lethal dose of M. chamomile hydroalcoholic extract (MCHAE), 50 to 6400 mg/kg (expanded dosage increase) were injected intraperitoneally into 8 groups of animals. After 24 h, the mortality rates of each group was measured. LD₅₀ was calculated (220 mg/kg) using computer techniques, and 55 and 110 mg/kg hydroalcoholic extract of chamomile (25 and 50% LD₅₀) were used in this study.

For this experimental study, 30 male mature Wistar rats weighing 150-180 g were used. Animals were purchased from the Animal Breeding Center of the Pasteur Institute of Tehran and transferred to the Animal Maintenance Center of Ilam University of Medical Sciences. They were kept in special cages (temperature of 25±2°C and with 12-h light cycles). Urban water and compressed foods were provided without any limitations. In order to adapt to the laboratory environment, animals were placed in these conditions 1 week before the study.

Group 1 or sham group received a regular daily diet and 2 cc normal saline per day. Group 2 (i.e. control group) received a high cholesterol diet (2%) and also 2 cc normal saline per day. Groups 3 and 4 were treated with a high cholesterol diet (2%) together with 55 and 110 mg/kg of MCHAE, respectively. Finally, Group 5 received a high-cholesterol diet (2%) plus 10 mg/kg of lovastatin (OSVAH Pharm. Co., Tehran, Iran).

Animals were put on fasting state for 12 h before the beginning of the diet and after completion of the study. Blood samples were collected twice- once at the beginning of the experiment and once at the end of the experiment. On the last day of the 8th week, and before each blood sampling, animals were kept at a fasting state for 12 h. The blood was placed in the laboratory for half an hour to form a clot; then, tubes containing the clot were put in a centrifuge device and centrifuged for 5 min at 4500 rpm to separate the serum. Finally, a biochemical kit was used to determine the concentration of malondialdehyde (MDA), superoxide dismutase (SOD), red blood cell (RBC) count, levels of inflammatory markers (e.g. Interleukin (IL)-6), C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and fibrinogen), and lipid profile. ELISA was used to measure MDA, SOD, IL-6, CRP, and fibrinogen using kits (ZellBio GmbH, Ulm, Germany). TNF-α was measured using Diaclone kit (Besancon, France). Lipid profile was also measured using a Biochemistry Enzymatic Kit (Pars Azmun Co., Tehran, Iran) and Hitachi's Automatic Analyzer 902 (Hitachi, Ltd. Tokyo, Japan).

The catalog numbers of the kits used in the experiments were:

MDA Cat. No: ZB-MDA96A, V405

SOD Cat. No: ZB-SOD96A, V407

IL-6 Cat. No: ZB-10135S-R9648

CRP Cat. No: ZB-10167-R9648

Fibrinogen Cat. No: ZB-12006S-H9648

TNF-α Cat. No: 1x96 tests: 865.000.096

HDL-c Cat. No: 1050012

LDL-c Cat. No: 123 051 H912

Cholesterol Cat. No: 1500010

TG Cat. No: 1006132

After blood sampling at the end of the 8^th week, the heart and aorta of each rat were isolated and placed in 10% formalin solution for staining with hematoxylin and eosin (H & E), according to the ethical guidelines of working with laboratory animals and using sterile cuts. After staining, tissue slides were prepared and examined histologically by optical microscopy.

SPSS 16 software and one-way ANOVA were used to analyze the results. The mean level of variables were calculated as mean ± SD for each group of rats. The statistical significance level for all the tests was set as P<0.05.

The results of this study showed that at the end of the 1^st and 2^nd week, the groups had no significant differences in terms of mean weight. At the end of the 4^th week, the mean weight of animals in group 2 (control) showed a significant increase compared to group 1 (sham) (P<0.01). At the end of the 6^th week, as observed, groups 3 (55 mg/kg dose of MCHAE) and 2 (control) had a significant increase in weight gain, as compared to group 1 (P<0.01). At the end of the study (8^th week), similar to the 6^th week, there was a significant increase in these two groups as compared to group 1 (P<0.001 and P<0.01, respectively) (Figure 1).

The results of this study showed that atherogenic lesions and the symptoms of atheroma plaque formation were observed in group 2 (control, Figure 2B) which received the hypercholesterolemic diet (2%). However, formation of plaques and its symptoms were not observed in group 1 (sham, Figure 2A) with normal diet. In groups 3 (Figure 2C) and 4 (Figure 2D) that received high cholesterol diet and MCHAE with the doses of 55 and 110 mg/kg body weight per day, as well as group 5 that received lovastatin at a dose of 10 mg/kg body weight per day in addition to the high cholesterol diet (Figure 2E), there were no signs nor symptoms of plaque formation (Figure 2).

Several slides were prepared from coronary artery, but symptoms of plaque formation were not observed in any of the groups.

Figure 2

The results of this study showed that high cholesterol diet (2%) significantly increased the level of serum cholesterol, TG and LDL-c in group 2 (control) as compared to group 1 (sham) (P<0.001). This significant increase (P<0.001) was also observed in group 3 (55 mg/kg dose of MCHAE) as compared to group 1. However, groups 4 and 5 had no significant change in serum cholesterol, TG or LDL-c levels in comparison with group 1 (normal diet) (Table 1).

Serum concentration of HDL-c showed a significant decrease in group 1 (sham) as compared to group 2 (control), group 3 and group 5 (P<0.001). However, group 4 showed no significant change in serum HDL-c level as compared to group 1 (sham), despite the high cholesterol diet (Table 1).

Table 1

Lipid profile	Exp. Group 1 (Sham) (mg/dl)	Exp. Group 2 (Control)	Exp. Group 3 (MCHAE 55 mg/kg)	Exp. Group 4 (MCHAE 110 mg/kg)	Exp. Group 5 (Lovastatin 10 mg/kg)	P-value *: P<0.05
Cholesterol	84.81 ± 5.26	141.78 ± 6.76***	133.08 ± 4.22***	88.98 ± 5.07	84.33 ± 5.92	vs. Exp. Group 1 (Sham)
TG	71.23 ± 4.22	107.85 ± 5.65***	102.95 ± 3.54***	71.41 ± 4.07	70.61 ± 6.00	vs. Exp. Group 1 (Sham)
LDL-c	46.40 ± 2.59	63.31 ± 4.91***	59.56 ± 3.79***	46.90 ± 4.30	49.93 ± 4.26	vs. Exp. Group 1 (Sham)
HDL-c	34.80 ± 1.85	21.55 ± 1.57***	23.61 ± 1.60***	32.90 ±2.15	25.35 ±1.88***	vs. Exp. Group 1 (Sham)

The mean difference was deemed significant at the 0.05 level (*: P<0.05)

At the end of the study, a comparison of the mean inflammatory factors shows that for groups 4 and 5, no statistical significant difference was observed when compared to group 1 (sham), which received only the usual diet (P>0.05). However, in groups 2 (control) and 3, all inflammatory factors increased significantly as compared to group 1 (P<0.001 and P<0.05, respectively) (Table 2).

Table 2

Inflammatory factors	Exp. Group 1 (Sham)	Exp. Group 2 (Control)	Exp. Group 3 (MCHAE 55 mg/kg)	Exp. Group 4 (MCHAE 110 mg/kg)	Exp. Group 5 (Lovastatin 10 mg/kg)	P-value *: P<0.05
Fibrinogen	2.27 ± 0.07(mg/mL)	3.70 ± 0.08***	2.68 ± 0.09*	2.33 ± 0.07	2.41 ± 0.10	vs. Exp. Group 1 (Sham)
TNF-α	10.97 ± 0.21(pg/mL)	17.81 ± 0.30***	13.06 ± 0.14*	11.32 ± 0.25	11.19 ± 0.15	vs. Exp. Group 1 (Sham)
CRP	1.92 ± 0.07(ng/mL)	3.18 ± 0.19***	2.32 ± 0.07*	2.01 ± 0.10	1.98 ± 0.12	vs. Exp. Group 1 (Sham)
IL6	2.95 ± 0.04(ng/L)	3.78 ± 0.09***	3.38 ± 0.14*	3.10 ± 0.06	3.06 ± 0.09	vs. Exp. Group 1 (Sham)

The level of SOD of RBCs in groups 2 (control) and 4 showed a significant increase when compared to that of group 1 (sham) (P<0.001 and P<0.05, respectively). However, in groups 3 and 5, a significant increase in RBC SOD rate was observed in comparison with the sham group, despite receiving a cholesterol-rich diet (Figure 3).

Furthermore, plasma MDA levels of groups 2 (control) and 4 had a significant increase in comparison with group 1 (sham) (P<0.01 and P<0.05, respectively). However, no significant change was observed in groups 3 and 5 as compared to the sham group (Figure 4).

As mentioned earlier, hyperlipidemia is known as a major risk factor for cardiovascular diseases, especially atherosclerosis 10. Therefore, controlling and correcting changes in lipid profiles can play a significant important role in reducing cardiovascular diseases. For this purpose, in a part of this study, the effects of M. chamomile hydroalcoholic extract on the level of inflammatory indicators and lipid profiles were studied.

In this study, administration of MCHAE caused a significant decrease in serum levels of triglyceride, cholesterol and LDL-c, and caused a significant increase in serum HDL-c levels in treated rats as compared to the control group. Interestingly, 110 mg/kg of chamomile extract resulted in a more significant increase in HDL-c levels than lovastatin, with a dose of 10 mg/kg as compared to the control group. The positive effects of MCHAE on lipid profile in this study are consistent with previous studies. For example, in a study conducted by Khan et al. on rats that received high cholesterol diet, it was found that phenolic compounds inhibited the activity of HM-COA reductase and reduced the storage of liver cholesterol 30.

One of the most important disruptive effects of free radicals is the beginning of lipid peroxidation, which leads to destruction of the cell membrane 31. Lipid peroxidation results in changes in the membrane organization and changes in the activity of its dependent enzymes and other proteins. These events are potentially toxic to the cell, together with the release of hydroperoxyl radicals and alkoperoxyls 32. Re-establishing the equilibrium between pro-oxidants and antioxidants allows the cell to regain its physiological activity 33. Free radicals and ROS cause damage to cells and tissues, and promote the development of various diseases, such as cardiovascular diseases, atherosclerosis, cancer, rheumatoid arthritis, and even early aging. Given the fact that these compounds are continuously produced in living cells, there is a need for protective mechanisms against them 34. In hyperlipidemic patients, the levels of lipid peroxidation and LDL oxidation increased, and as a result, the need for antioxidants increased 35.

Antioxidants are effective in the improvement of cardiac lesions, infarction and cardiac arrhythmias by reducing the effect of free radicals and coronary artery thrombosis 36. Natural antioxidants with plant origin, especially oral herbs, in addition to having fewer side effects than chemical antioxidants, can help in protecting the body against free radicals.

The results of this study showed that chamomile hydroalcoholic extract has suitable antioxidant effects and due to the different effects of doses used in this study, it seems that this effect is not dose-dependent. Thus, 55 mg/kg chamomile extract with higher antibiotic effect as compared to 110 mg/kg, prevented an increase in plasma MDA and SOD globules. The results of antioxidant activity of chamomile extract in this study are consistent with those of Ranjbar et al. that studied the protective effect of chamomile hydroalcoholic extract on paraquat toxicity in male rats 37.

Hypolipidemic compounds and antioxidants prevent atherosclerosis. Studies have shown that increased lipid oxidation leads to normal vascular endothelial dysfunction and atheroma plaque formation 12. Lipid peroxidation results in the production of a wide range of substances, such as free oxygen radicals, ketones, ethers, aldehydes and foam cells, which in turn, interfere with endothelial cell attachment and plate formation 38. In a study conducted by Asghari et al., the use of chamomile extract resulted in a significant increase in the activity of antioxidant indicators in rat liver cells exposed to oxidant compound 1. Therefore, it seems that the hydroalcoholic extract of chamomile can play a strong protective role in the prevention of cardiovascular disease because it contains flavonoids and polyphenolic, and reduces lipid peroxidation and substrates of antioxidant enzymes, such as superoxide dismutase and malondialdehyde.

In this study, the effects of chamomile extract on the level of blood inflammatory indicators were investigated. When cardiovascular diseases (including coronary artery sclerosis) develop, blood inflammatory indicators such as TNF-α, CRP, IL-β1, and IL-6 are altered 3940. The level of these indices increases as a result of inflammation which is caused by increased oxidative stress 41. The results of this study indicate that treatment with 110 mg/kg chamomile hydroalcoholic extract prevents a significant increase in serum levels of TNF-α, CRP, IL-6 and Fibrinogen, as compared to group 1 (sham). These results are consistent with previous studies that emphasized the anti-oxidant and anti-inflammatory properties of chamomile 142. In a study which was conducted to evaluate the effects of chamomile capitulum on xylene-acetic acid-induced inflammation in rats, it was observed that the herb had an anti-inflammatory effect 42.

Chamomile extract consists of 120 types of chemical compounds, with the most important ones being chamazulenes, flavonoids and coumarins 3643. It seems that chamomile has antioxidant properties and the ability to neutralize oxygen radicals due to the presence of flavonoid compounds 4344. The association of terpenoids in plants with strong anti-inflammatory effects has also been proven 45. Flavonoids are also responsible for lowering of blood pressure and anti-inflammatory properties 46. It is evident that the most common intervention in the prevention of cardiovascular diseases is the use of drugs which lower blood lipids- that is, statins 47. The use of statins causes reduction in serum LDL level, coronary artery diseases and finally reduction in mortality rates 4748. But, this set of drugs is known to have many side effects like myopathy, rhabdomyolysis, reduction in hepatic enzymes, nausea, dizziness and digestive tract problems, as well as elevation in cancer risk, liver damage and an increased risk of new-onset diabetes mellitus 4950515253. Life-threatening rhabdomyolysis and idiopathic polyneuropathy represent the most important muscular symptoms 54555657585960. Both conditions are among the inherent properties of statins 60585955. Statins have also been reported to inhibit the biosynthesis of Q10 coenzyme which is required for energy generation 6162. Due to the same reasons, the use of statins is accompanied by clinical constraints. Therefore, the hydroalcoholic extract of chamomile is suitable for reducing the level of inflammatory indicators, correction of lipid profile changes and prevention of cardiovascular diseases, including atherosclerotic plaques in animal samples.

The results of this study revealed that treatment of hypercholesterolemic rats with chamomile hydroalcoholic extract did not only prevent a significant increase of triglycerides, cholesterol and LDL-c levels and inflammatory factors, but it also prevented significant reduction in serum HDL-c levels. However, the suitable dose in this study was 110 mg/kg chamomile hydroalcoholic extract except for the antioxidant activity part. This extract also prevented the development of atheroma plaques in hypercholesterolemic rats. As mentioned earlier, these effects are probably due to the presence of flavonoids in the chamomile. In general, people at risk of cardiovascular diseases are recommended to strengthen their antioxidant potency with the use of natural antioxidants to minimize the risk of free radicals. Of course, understanding the exact mechanism of these reagents requires precise molecular and mechanistic studies in the future.

The authors report no conflicts of interest in this work.

Shahin Nargesi, Ardeshir Moayeri, Ayub Ghorbani and Mansour Amraei: designed the study and performed the experiments

Ehsan Shirzadpour: conducted the data analysis

Mansour Amraei, Ehsan Shirzadpour and Yaser Seifinejad: participated in drafting the paper

Shahin Nargesi, Ardeshir Moayeri and Mansour Amraei: wrote the manuscript. All authors read and approved the manuscript.

The authors thank the Deputy Director of Research and Technology, Ilam University of Medical Sciences.

This study was carried out in accordance with the recommendations of ICLAS (International Council for Laboratory Animal Science). The protocol was approved by the Ethics committee of Ilam University of Medical Sciences (IR.MEDILAM.REC.1394.162).

CRP: C-reactive protein

CVD: Cardiovascular diseases

HDL-c: High-density lipoprotein cholesterol

IL-6: Interleukin 6

IL-β1: Interleukin β1

LDL-c: Low-density lipoprotein cholesterol

MCHAE: Matricaria chamomile hydroalcoholic extract

MDA: Malondialdehyde

ROS: Reactive oxygen species

SOD: Superoxide dismutase

TG: Triglycerides

TNF-α: Tumor necrosis factor