Introduction

Nowadays, many patients  have  resorted  to the herbal medicine for treatment. On the other hand, new medicine despite its capa- bilities and merits, has problems in treating some diseases, especially chronic ones. In fact, there has been remarkable data on the use of herbal medicine in recent years. Gen- erally speaking and disregarding exceptions, these medicines have less side effects than other drugs (Dolatkhahi et al. 2010). Dia- betes mellitus whose prevalence –based on predictions– will increase in human beings’ societies in the future is clinically one of the most common endocrine gland diseases. Fail- ure to properly control diabetes would cause disorders such as Nephropathy, Retinopathy, Neuropathy, and heart disease (Nasri, Hajial- izadh, and Esmaeili-Mahani 2014). Although the use of insulin and chemical medicines  are already the main and effective treatment for diabetes mellitus, these compounds have plenty of side effects such as increase of lip- id storage (lipotrophy), loss of fat tissue in the injection site and outbreak of hypoglyce- mic shock, and they do not influence on the developmental process of diabetic disabling complications in the long term. Considering the growth of human’s knowledge  around the heterogeneity of this disease, the need of finding effective compounds with minimum side effects for the treatment of diabetes and its resulting disorders is felt (Musazadeh and Motavally 2009; Nasri, Hajializadh, and Es- maeili-Mahani 2014). Many drugs have also been used to prevent and treat hyperlipid- emia in humans and in other species. They include Atorvastatin, fish oil containing omega-3 fatty acids, Gemfibrozil, Niacine and garlic. Among them Atorvastatin with a daily dosage of 5 mg/kg intake for 6 weeks has been significantly effective in decreasing

total cholesterol, phospholipid and triglycer- ide levels in dogs.

Atorvastatin has anti-fatty effects in the blood through the competitive inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme a reductase enzyme. This is an early stage in the biosynthesis of cholesterol. Hepatotoxic effects including increase in liver enzymes such as Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Al- kaline Phosphatase (ALP), and also  ceas-  ing the outflow of bile are amongst the side effects of this  drug  in  dogs  (Mosallanejad et al., 2018). Unfortunately, the overuse of chemical drugs for treatment of these cases is the great disadvantage of modern medicine, therefore dangerous side effects are conse- quences of the frequent use of these drugs. For example, the most prevalent side effects of Atorvastatin in humans are namely diges- tive problems, dyspepsia, abdominal pain, headache and myopathic muscular compli- cations, and hepatotoxic effects (increase in enzymes levels of liver such as ALT, AST) and less prevalent reported side effects are damage to optic nerves and intracranial hemorrhage (Haroun, Mahmoud, and Adam 2002). The herb we are taking into consid- eration in this study is Thymus daenensis Cleak. Thymus is a type of indigenous Irani- an herb that is commonly found in the slope of hills and mountains or in gardens. This herb grows short and shrubby and has many medicinal and nutritional uses. All of the ae- rial parts of this herb –top branch trimmings in particular– have medicinal use. The most important species of thymus in Iran are Thy- mus daenensis, Thymus serpllum, Thymus vulgaris, Zataria multiflura  Boiss.  Thymus is a fragrant (aromatic) herb from the family of Lamiacea.  Thymus  daenensis  is a lamia-

ceae herb and contains compounds of tannin, flavonoids, glycosides, caffeine and Rose- mary acids (Lee et al. 2005; Mahmoudabadi, Dabbagh, and Fouladi 2007; Mokhberi et al. 2010) This herb has strengthening effect and digestive, antispasmodic, carminative, anti- fungal, antibacterial, antiseptic, anticonvul- sant, vermicide, anti-rheumatic, expectorant, anti-rheumatism and antioxidant properties. Thymus extract has an extremely antioxidant effect which in addition to lowering blood lipids, can play a role in inhibiting LDL ox- idation process. The thymus also reduces triglycerides and cholesterol levels of blood (Nazari et al. 2013).

Considering that studies on the hypogly- cemic-hypolipidemic effects of Thymus daenensis cleak and on general changes in serum biochemical factors resulting  from  the use of its extract in the animal type sam- ples (i.e. dogs) are limited; therefore, the present study aims at investigating the ef- fects of thymus on the biochemical factors including Fasting Blood Sugar (FBS), Cho- lesterol (CHOL), Triglyceride (TGL), Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL), Alanine Aminotransfer- ase (ALT), and Aspartate Aminotransferase (AST). The results are probably extendable  to human beings.

Materials and Methods

To do this investigation, 8 adult male Per- sian Shepherd dogs with a weight range of 30-35 kg were used. During the experiment, the dogs were kept in separate cages under standard conditions at 25-30 °C and in the 12-hour light-dark cycle, so that they could easily access water and food. The dogs were fed with the same invariant diet (cooked chicken, i.e. chicken heads and paws) for 1 month. Also,  dogs  were  given anti-parasitic

medications of Mebendazole and Praziquan- tel (given based on recommended dosage of them) two weeks before the start of the study to remove any digestive parasitic disease, they have also been cleansed of skin parasites through being injected twice with Ivermectin and their skin was washed with anti-parasitic shampoo. Then their health was thoroughly examined before starting the study. All of their vital signs, such as heart rate, respira- tory rate, body temperature, and blood pres- sure, were checked and recorded. All of the dogs were examined at least twice a day and were kept in separate cages; the place they were kept was rinsed every day; its floor and the cages were disinfected once every 4 days and all of the reports were recorded (Mosal- lanejad et al. 2018; Vahid Dastjerdi, Jafarian Dehkordi, and Torkan 2017). This study was carried out at Veterinary Hospital  of Islam- ic Azad University  of  Shahrekord,  located in Kian, Iran. Dogs were randomly divided into two groups of four. The first group was  a control group that did not receive any thing to eat other than water and food. The group  2 was the receiver of 200mg/ml and admin- istered as 1ml/kg amount of Thymus aenen- sis Cleak extract twice a day  (every  time 100 mg/kg) for 7 and 14 days. The dosage assigned was based on the dose prescribed for lab rats rat (Yaghmaei, Heydarian, and Poorbahman 2012). After Thymus daenensis Cleak was provided for this research from Shaheed  Fozveh  research   station   located in Najaf Abad in Isfahan province, its type and species were identified  and  confirmed by the Agricultural and Natural Resources Research Center of Isfahan Province. Then its specimen was kept in Herbarium unit of the aforementioned center (Herbarium num- ber 13713). The collected samples dried in the shadow were then powdered by a   grind-

er. Thymus powder was poured into a glass container; 96% medical alcohol was added; its lid was tightly closed, following this the mixture was given 72 h to soak  well. Af-  ter the specified time passed, the extract of this mixture was taken through percolation method (extraction under pressure). It was condensed by Rotary Evaporator and then desiccated as much as possible by Desicca- tor. For use, some sterilized distilled water according to desired density was added to  the solid extract. Finally, the animals under investigation were fed with a specified dose of the acquired extract twice during 24 h and for 14 days. To blood let, blood vessels of front paw (Cephalic veins) were used in this manner: the dogs were restrained; hairs cov- ering the target place were shaved; then it was disinfected with some cotton and alco- hol; and blood letting was done gently. Col- lecting blood specimens was done for each dog individually three times on the 1st , 7th and 14th day during the research using sy- ringes of 5 cc blood capacity. Afterwards the specimens were transferred to the separate and marked test tubes. When the specimens had been collected, they were immediately transferred to the laboratory. They were cen- trifuged inside the laboratory, frozen  after  the serum separation by sampler and then transferred to separate and specific micro tubes.  The  intended  factors  —comprised of FBS, cholesterol (CHOL), triglyceride (TGL), low density lipoprotein (LDL), high density lipoprotein (HDL), alanine amino- transferase (ALT) and aspartate aminotrans- ferase (AST)— were measured via an enzy- matic and photometric method (Pars Azmon kits) using Italian Eco-Plus Autoanalyzer (2006) with serial number of GD100139, in  a laboratory equipped with a spectrophotom- eter. Finally, the collected data were   entered

into SPSS (V20) software and because the results obtained from the Shaipro-Wilk test represented the normal distribution of the data, independent t-test  was  used  in  order to compare the means of blood parameters between the two groups at times, and ANO- VA test was employed to compare the mean changes in blood parameters in each of the two groups after 14 days. In all stages of data analysis, the significance level less than 0.05 was taken into consideration

Results

In the present study, FBS level was not significantly different in two groups before intervention (P value > 0.05) but 14 days af- ter the intervention, the average level of FBS (42.50 ± 9.27 mg/dl) in  the  group  receiv- ing the Thymus daenensis celak (treatment group) was significantly lower than its aver- age level (59.50 ± 9.21 mg/dl) in the control group (P value = 0.045). In addition, after 14 days, the average level of FBS in the control group did not change significantly  (P   value

= 0.760), but it had a significant decrease in the treatment group (decrease FBS mg/ml: 16.75; P value = 0.001, Table 1).

With respect to average levels of  AST and ALT factors of the two groups under investigation, although there was no sig- nificant difference before the intervention (the starting point of this experiment) and 7 and 14 days after the intervention (P   value

> 0.05), there was a significant decrease in the AST level of the treatment group over 14 days (AST reduction: 18,000 u / l; P val- ue = 0.035, Table 2).

Before intervention, there was no signif- icant difference between the two groups re- garding the levels of LDL and HDL factors (P value > 0.05); however, on the 14th day, the average level of LDL (37.50 ± 3.00 mg/

dl) in the group receiving Thymus daenen- sis celak (treatment group) was significant- ly lower than its average level in the con- trol group (51.13 ± 3.56 mg/dl, P value = 0.001). Furthermore, on the 7th and 14th day, the average amounts of HDL level in the treatment group (216.25 ± 18.84 mg/    dl and 193.25 ± 33.37  mg/dl  respective- ly) were more  than  its  average  amounts  in the control group (138.37 ± 12.46 mg /   dl and 141.62 ± 27.52 mg/dl     respectively,

P value > 0.05). On the other hand, while the changes in LDL and HDL levels over the period of 14 days were not significant   in the control group (P value > 0.05), there was a significant decrease in the LDL level (decrease: 16; P value = 0.046) and there was a significant increase in HDL level (in- crease: 57.75; P value = 0.022) in the treat- ment group in this period compared to the time before intervention (starting point of this experiment).

Table 1. Comparing the average level of FBS between the two groups at different time intervals

Data shown as mean ± SD.

*: Significant level of Independent Sample t-test for comparison between two groups

**: Significant level of repeated measure ANOVA for comparison by passing time in each group

Table 2. Comparing the average level of AST and ALT between the two groups under investigation at different time intervals

Data shown as mean ± SD.

*: Significant level of Independent Sample t-test for comparison between two groups

**: Significant level of repeated measure ANOVA for comparison by passing time in each group

Table 3. Comparing the average level of LDL and HDL between the two groups under investigation at different time intervals

LDL(mg/dl)

HDL(mg/dl)

Data shown as mean ± SD.

*: Significant level of Independent Sample t-test for comparison between two groups

**: Significant level of repeated measure ANOVA for comparison by passing time in each group

Finally, TG and CHOL levels were not sig- nificantly different between the two groups before intervention and seven days after in- tervention (P value > 0.05). However, on the 14th day after intervention, average levels of TG and CHOL in the treatment group being respectively 37.25 ± 2.75 mg/dl and   205.75

± 48.32 mg/dl were remarkably and signifi- cantly lower than their average levels in   the

control group being 47.13 ± 3.54 mg/dl   and

282.13 ± 18.64 mg/dl respectively (P value < 0.05). In addition, after 14 days passed, the changes in these two factors were insignif- icant in the control group (P value > 0.05). On the contrary, in the group receiving Thy- mus daenensis celak (treatment group), the significant decrease of these two factors was observed (P value < 0.05, Table 4).

Table 4. Comparing the average level of TGL and CHOL between the two groups under investigation at different time intervals

CHOL(mg/dl)

Data shown as mean ± SD.

*: Significant level of Independent Sample t-test for comparison between two groups

**: Significant level of repeated measure ANOVA for comparison by passing time in each group

It should be noted that no clinical symp- toms of intoxication or drug intolerance (such as diarrhea, vomiting, loss of appetite, etc.) were perceived in the dogs investigat- ed. All the information in the tables was ex- pressed in terms of the average ± standard deviation. P values less than 0.05 were con- sidered significant.

Discussion

The effects of thymus extract on reducing the level of glucose, lipid and liver enzymes have been approved by several studies. The results of present study demonstrated  that  the level of FBS parameter had a significant decrease subsequent to consumption of Thy- mus daenensis celak extract, so that in the treatment group, it was significantly lower  on the 14th day after intervention than start- ing  point  (before  the  intervention,  P value

<0.05).

In a similar study conducted by Yagh- maie et al. (2011), the effect of thymus ex- tract on hyperlipidemia was considered in male Wistar rats being adult and diabetic. They observed that thymus extract reduced glucose in blood serum and also it effec- tively reduced undesirable lipids in blood serum in those rats (Yaghmael, Heydarian, and Poorbahman 2012). Gholamhossie-  nian et al. in another study (2008) regard- ing the hypoglycemic effect of four herbal extracts affecting α-glucosidase in diabetic rats stated Zataria multiflora caused lower- ing blood glucose level and was effective in controlling glucose in diabetic rats (Ghol- amhoseinian Najar, Fallah, and Sharififar 2015) Nasri et al. (2013), also in their study about the effect of thymus extract on reduc- ing glucose in diabetic rats expressed that blood glucose levels in diabetic animals which received Thymus extract at the doses

of 150 and 100 mg/kg was reduced as com- pared to the pretreatment levels (P<0.05  and P<0.01, respectively)

As it is the case about other lamiaceae compounds, the effects of consumption of Thymus daenensis celak on lowering blood sugar was attributed to terpenoid and fla- vonoid compounds. So, considering all of the similar studies performed, we can con- clude that they were consistent with the re- sult of this study (Nasri, Hajializadh, and Esmaeili-Mahani 2014). Concerning LDL factor, on the 14th day, it was clear that it was significantly lower in the group re- ceiving Thymus daenensis celak (treatment group) than the control group (P value = 0.001). Also, HDL level was higher in the treatment group than in the control group  on 7th and 14th days (P value < 0.05). So, there was a significant decrease in LDL level and a significant increase in HDL  level in the treatment group. Likewise, TG and CHOL levels in the treatment group were significantly lower than the control group on the 14th  day  after  intervention  (P value < 0.05). There was significant de- crease in the group receiving the Thymus daenensis celak extract  (treatment  group, P value < 0.05). Illustrating more similar studies we refer to the study conducted by Nazari et al. (2013) on the effect of Thymus daenensis Cleak and  Satureja  Bakhtiari-  ca on plasma’s lipoproteins of gerbils fed with high-fat diet. They found that Thy-  mus daenensis  reduces  LDL and  TGL but it increases the HDL factor. They also ex- pressed that the decrease in LDL in the re- cipient group was related to the compounds of this herb including flavonoids and phe- nols (Nazari et al. 2013)

According to a study done by Akhtar et al. in 2014 on evaluating the effect of   Thymus

linearis Benth on reducing blood pressure in mice with normal and high blood pres- sure, it has been concluded that the thymus leads to reduction in AST, Chol, TG, LDL and increase in HDL (Akhtar et al. 2014). Zarei et al. (2011) in their study investigat- ing the level of lipids in blood serum in rats which had high-cholesterol and consumed the extract of Zataria multiflora found that the extract of Zataria multiflora decreased blood lipids (Zarei, Eftekhary, and Aqhaba- ba 2014). Therefore, the results of present study are consistent with most of the stud- ies in this respect. Nonetheless, it does not conform to the study by Nik and colleagues (2012) who investigated the use of Silybum marianum and thymus and measured the blood parameter levels of broiler chicks. While they found that thymus increased LDL and decreased HDL in the blood se- rum of broiler chicks, we observed just the opposite in the present study: an increase    in the HDL factor and decrease in LDL in blood serum (Nik et al. 2014).

The incidence of liver injuries due to herbal and dietary supplements have risen largely over the last decade probably in part due to their growing consumption (Mad- dukuri and Bonkovsky 2014). Thus, it is necessary to carefully determine the influ- ence of various herbal medicines on liver function, in both normal and pathological conditions (Rašković et al. 2015). The ex- tent of hepatocellular damage was assessed by measuring activities  of AST  and ALT  in serum, in the present study . Ultimately, there was no significant difference between the two groups in terms of average levels  of AST and ALT before intervention, on the seventh, and on the fourteenth days after the intervention (P value < 0.05) but the AST level had a significant decrease in the  treat-

ment group over 14 days.

During the investigation of the effect of pharmaceutical formulation of a syrup con- taining thymus on liver injuries caused by carbon tetrachloride in gerbils, Raskovic  et al. (2015) found that the Thymus syrup con- taining Thymol reduced AST in blood se- rum of gerbils (Rašković et al. 2015). While studying the effect of Caraway fruits and Thymus vulgaris’ leaves or  the  combina- tion of the two (containing 2% caraway and 10% Thymus vulgaris) on rats, Haroun et al. (2002) reached the conclusion that the com- bination reduced the AST factor in rats. This conclusion is in agreement with the results obtained from the present study (Haroun, Mahmoud, and Adam 2002).While Grespan et al. (2014) were examining the pretreat- ment effect of Thymus vulgaris’ essential oil on the experimental model of liver injuries induced by Acetaminophen in rats, they re- lied on the  hepatoprotective  (TEO)  effects of Thymus vulgaris’ essential oil and found that the amount of AST has decreased due   to the use of Thymus vulgaris. The results of this study are in agreement with those of the present study (Grespan et al. 2014). It can be said that regarding the ALT factor shown in Table 2 and the decreasing flow of the en- zyme, if the duration of the intervention or dosage of the medicine was increased, the results of this parameter would probably be- come meaningful. In the same respect and in a similar study on rats receiving thyme syr- up, Raskovic et al. in 2015 found a signifi- cant reduction of the AST parameter in the group taking carbon tetrachloride. Eventu- ally, the researchers attributed the effects of thyme consumption on reduction of ALT and AST factors to compounds such as thymol and carvacrol.

Conclusion:  Finally,  considering the facts