BENEFICIAL EFFECTS OF ROSMARINUS OFFICINALIS AND THYMUS NUMIDICUS ON KEY ENZYMES OF CARBOHYDRATE METABOLISM IN ALLOXAN-INDUCED DIABETIC RATS

rats receiving vehicle (0.5 ml/100 g bw), TN-EA (300 mg/kg b.w), RO-EA (300 mg/kg b.w), respectively. The 06 remaining groups were composed of diabetic rats receiving vehicle (0.5 ml/b.w), TN-EA (150 and 300 mg/kg b.w), RO-EA (150 and 300 mg/kg b.w) Glibenclamide (5 mg/kg b.w), respectively. The process lasted for 21 days. Then we proceeded to the assays. The analysis of the results showed a dose-dependent reduction in glycemia for the anti-hyperglycemic and hypoglycaemic tests of the RO-EA with the dose of 150 and 300 mg/kg b.w, the latter demonstrated a significant increase in insulin and hemoglobin hexokinase enzyme activity, glucose-6-phosphate dehydrogenase and a concomitant effect modulating plasma glucose levels. In addition to decreased plasma HbA1c levels, glucose-6-phosphatase and fructose-1,6-bisphosphatase enzymatic activity in diabetic rats. The administration of this fraction prevented the loss of body weight and led to an improvement in the activity of glycogen synthase and glycogen phosphorylase. In conclusion, the RO-EA fraction at a dose of 300 mg/kg b w has an anti-hyperglycemic power in diabetic rats, these results were confirmed by histological examination of the liver.

Rosmarinus officinalis and Thymus numidicus (RO-EA and TN-EA) was the subject of our study.

Experimental animals
Healthy male Wistar rats of the Rattus novergicus aged 2 months weighing between 200 and 220g with no prior drug treatment, were used just for the present study, were obtained from the Pasteur Institute, and the animals were acclimatized to laboratory hygienic conditions before 10 days to starting the experiment under these conditions (temperature: 23c° ± 2 c° and natural photoperiod: 12h light and 12h dark), they were fed with pellet diet (ONAB-Elharouche, Skikda-Algeria), and water available adlibitum.

Acute toxicity studies
Before starting our study on diabetes an acute toxicity test was done to evaluate our flavonoid fraction, this test was determined according to the guidelines of the OECD n° 420 (organization for economic cooperation and development). Male rats (200-220g) were used and divided into different groups of 6 rats. Each group received different doses of test sample up to 2000 mg/kg b.w, after that the animals were monitored for 14 days to confirm if there are mortalities or detected behavioral response. In the end, no deaths were found up to the dose 2000 mg/kg b.w, Therefore, 300 mg/kg b.w was chosen as the most advanced experimental dose.

Anti-hyperglycemic activity of R.O-EA and T.N-EA in glucose-loaded normal-animals
The antidiabetic activity of the ethyl acetate fraction was evaluated according to the method of (Jarald et al., 2009) with a slight modification. The animals were randomly divided into 6 groups, 6 rats for each, the negative control received only the vehicle solution 1% Tween 80 dose 0,5 ml/100g b.w, and the positive control received a dose of glibenclamide 5 mg/kg b.w. The other groups were treated with the extract of RO-EA and TN-EA with different doses (150 mg/kg and 300 mg/kg b.w). The animals received their doses orally with a gavage feeding tube number 7. The blood glucose level of the animals was taken after a period of overnight fasting at a time 0. Just after the first measurement, the animals have received all the treatment and remained to receive the dose of glucose 4 mg/kg b.w, and the other blood glucose measurement was taken at ½, 1, 2, 3h after a glucose dose. Blood glucose was estimated with a glucometer (DIAGNO-CHECK smart) from the tail tips of rats.

Hypoglycemic activity of R.O-EA and T.N-EA in normal fasting animals
The same steps done in the previous test were repeated. However, the rats fasted overnight for 10h, the first group which remained as negative control has received a dose of 0,5 ml/100g b.w of vehicle. The second group received glibenclamide as a reference dose of 5 mg/kg dissolved in water saline (0.9 w/v NaCl). Group 3 up to 6 received the extract dose of RO-EA and TN-EA (150 mg/kg and 300 mg/kg b.w), as mentioned in the table: 2, the blood sample was taken from the tail tip at a time 0 (before oral administration) and ½, 1, 2 and 3h after vehicle, extract and glibenclamide administration. The blood glucose level was done like the previous test.

Dose-dependent effect of extract RO-EA and TN-EA on plasma glucose and insulin level in diabetic rats
Diabetes was induced in the rats (after fasting overnight for 8 hours), with an Alloxan single dose of 120 mg/kg b.w in 0,9 w/v NaCl intraperitoneally. The rats were placed in the cages with bottles filled with 10% glucose for the next 24 h to prevent hypoglycemia. After 72 hours of injection, fasting blood glucose was measured, the rats that showed a blood glucose level more than 300 mg/dl have been accepted for the test. The selected 54 rats were randomly divided into 9 groups, 6 rats for each, as the following table: Group 1: Normal control rats(vehicle 0,5 ml/100g b.w) Group 2: Normal + TN-EA (300 mg/kg b.w) Group 3: Normal + RO-EA (300 mg/kg b.w) Group 4: Diabetic control rats(vehicle 0,5 ml/100g b.w) Group 5: Diabetic + TN-EA (150 mg/kg b.w) Group 6: Diabetic +TN-EA (300 mg/kg b.w) Group 7: Diabetic + RO-EA(150 mg/kg b.w) Group 8: Diabetic + RO-EA(300 mg/kg b.w) Group 9: Diabetic +Glibenclamide(5 mg/kg b.w) The extract was dissolved in vehicle (1% Tween 80), glibenclamide was dissolved in water saline 0.9 w/v NaCl, water and food consumption were monitored throughout the 21-day period at a fixed time for each group and renewed each day, and the weight of the rats was taken before the start of treatment and before the sacrifice. After 21 days of treatment, rats were fasted overnight then anesthetized and sacrificed by cervical decapitation, blood was collected to measure the serum concentration of glucose and insulin, and the treatment group which appeared to show a good improvement in plasma glucose and insulin level was selected to determine the Hb and HbAc1 level and also for histological part and assay of the key carbohydrate metabolism enzyme, the liver was recovered in 10% of formalin solution and immediately sent for histological study, the liver was rinsed with icecold saline, homogenized with a 10% w/v of 0,1 M Tris-HCl buffer (pH 7,4), then centrifuged (10000× g for 15 min at 4° C), the supernatant was used for the determination of hepatic enzyme and glycogen.

Biochemical assays
The

Statistical analysis
The results are expressed as means ± standard deviation, (n =6), Difference between groups was assessed by analysis of variance (One Way ANOVA,) followed by a Tukey post-hoc test using Graph Pad prism 8.0.1. software, the level of significance was set at P <0.05.

Anti-hyperglycemic activity of R.O-EA and T.N-EA in glucose-loaded normal-animals
The rats received its extract at different doses. After 30 min of glucose administration, an increase in serum glucose levels in different groups were observed. The control group reached the level of fasting glucose in 2 hours. On the other hand, the groups treated with glibenclamide and the extract of RO-EA and TN-EA at a dose of 300 mg/kg b.w reached it significantly at the end of the 1st hour compared to the dose of 150 mg/kg b.w (after 2 hours). Comparing the antihyperglycemic power of these extracts with glibenclamide, the extract of RO-EA at the dose of 300 mg/kg b.w proved to be significantly P<0.05 very close to glibenclamide (Table 1).

Hypoglycemic activityof RO-EA and TN-EA in normal fasting animals
The evaluation of the hypoglycemic activity has clearly shown that extract of the RO-EA fraction exhibited a very significant hypoglycemic activity compared to the control group, and a significantly strong p<0.05 at the dose 300 mg/kg b.w compared to the dose 150 mg/kg b.w, this activity is dose-dependent, it was observed after the 1st 30 min. On the other hand, with the extract of the TN-EA fraction no hypoglycemic activity was recorded with the two-doses, no significant difference p≥0.05 compared to the control group (Table 2).

Dose-dependent effect of extract RO-EA and TN-EA on plasma glucose and insulin level in diabetic rats
Analysis of the results showed a significant increase in the level of serum glucose and a significant decrease in the level of plasma insulin in the diabetic control rats.
After 21 days of treatment with RO-EA and TN-EA fraction extract, the rats exhibited good improvement, it prevented the increase in plasma glucose and reversed the serum insulin level when compared to the diabetic rat control, moreover, it was observed that the dose of 300 mg/kg b.w of the RO-EA fraction was more promising compared to the other dose and the other fraction (TN-EA), it significantly prevented the increase in glucose and the decrease in insulin, it was observed that the effect of this dose the most similar to glibenclamide, this dose was fixed as an effective dose for the rest of our study. Regarding the administration of RO-EA and TN-EA fraction extract to normal rats, no significant change was observed in insulin and serum glucose levels when compared to normal control rats (Fig. 1).

Effects of the extract R.O-EA fraction in a change in body weight, food, and water intake
The evaluation of the results was shown a significant decrease in body weight in diabetic control rats, and a significant increase in water and food consumption, compared to normal rats, all these changes were significantly restored during treatment with the extract of the RO-EA fraction and glibenclamide. Concerning normal control rats and normal rats treated with the RO-EA fraction no significant change was observed between them ( Fig. 2. 3. 4).  Figure 3 Effect of RO-EA on water intake in control and experimental rats. Values were expressed as means ± SEM (n = 6), minimal significant level; P< 0,05, significantly difference; a in respect to N.Control, b in respect to Diabetic control, c in respect to D+gliben, (ANOVA followed with Tukey test), N; normal, D; diabetic, Gliben; glibenclamide, RO; Rosmarinus officinalis, EA; ethyl acetate fraction.

Figure 4
Effect of RO-EA on food intake in control and experimental rats. Values were expressed as means ± SEM (n = 6), minimal significant level; P< 0,05, significantly difference; a in respect to N.Control, b in respect to Diabetic control, c in respect to D+gliben, (ANOVA followed with Tukey test), N; normal, D; diabetic, Gliben; glibenclamide, R.O; Rosmarinus officinalis, EA; ethyl acetate fraction. Table 3 summarizes the blood Hb and HbA1c levels in the control and experimental rats. This table shows that the diabetic rats suffered a significant decrease in the level of Hb and a significant increase in the level of HbA1c when compared with normal control rats. After treatment with RO-EA extract fraction and glibenclamide, a good improvement was observed with diabetic rats, the blood level of Hb and HbA1c was restored. No significant difference was observed in normal rats treated with the RO-EA fraction at the dose of 300 mg/kg b.wt when compared with normal control rats.

Effect of RO-EA fraction extract on activities of carbohydrate metabolic enzymes
The analysis of the results from (Table 4) shows the effect of the RO-EA fraction extract on the activity of carbohydrate metabolism enzymes in the liver of control and experimental rats, we noticed a significant decrease in hexokinase and glucose-6-phosphate dehydrogenase activity and a significant increase in glucose-6phosphatase and fructose-1,6-bisphosphatase activity in diabetic rats compared to normal rats. It was noted that this activity returned almost to normal when applying treatment with RO-EA fraction extract and also the same was observed with glibenclamide drug. No significant difference was noticed in normal rats treated with RO-EA compared to normal rats. Effect of RO-EA fraction extract on glycogen content, glycogen synthase, glycogen phosphorylase, and liver weight Table 5 summarizes the variations in liver weight, glycogen content, and the enzymatic activity of glycogen synthase and glycogen phosphorylase in control and experimental rats, a significant decrease in liver weight, glycogen level, and glycogen synthase activity was observed in diabetic rats, while glycogen phosphorylase activity was increased significantly compared to normal rats, so these altered parameters were restored almost to normal upon treatment with RO-EA fraction extract and the same thing was observed with the drug glibenclamide, no significant difference was observed in normal rats compared to normal rats treated. Liver histomorphometric study

DISCUSSION
Diabetes mellitus is now a serious metabolic disease threatening most people around the world therefore we used an experimental model of diabetes induced by Alloxan (Ajiboye et al., 2020), this model is well known to study different hypoglycemic agents (Sekar et al., 2020), which have a crucial role in the prevention of complications related to diabetes through the good control of blood glucose (Preshaw et Bissett, 2019). On the basis of this statement, we used normal rats loaded with glucose to obtain a hyperglycemic model which will allow us to reveal the anti-hyperglycemic activity of certain plant extracts (Ogar et al., 2018).
To determine the ability of an extract to prevent hyperglycemia, we used a hyperglycemic model loaded with glucose (Krishnan et al., 2021), from this model the 2 fractions of the extract RO-EA and TN -EA revealed an antihyperglycemic activity, and more significant at a dose of 300 mg/kg b. w of the RO-EA fraction, we know that a high blood glucose level will stimulate the secretion of insulin which will promote the entry of glucose to the peripheral tissue and are controlled through several mechanisms (Campbell et Newgard, 2021). So from our study (control glucose), it seems that insulin requires 2 hours to reach the fasting glycemia level. On the other hand, in the RO-EA fraction and glibenclamide only 1 hour, which gives an idea of the strong implication of the activity of RO-EA and glibenclamide in the use of glucose (Mendes et al., 2021). During a state of glucose tolerance, glibenclamide plays a crucial role in the stimulation of the β cells of the pancreas for the high release of insulin (Stožer et al., 2021), therefore it seems that the anti-hyperglycemic activity of the flavonoid fraction RO-EA involves an insulin-like effect (Etsassala et al., 2021), probably either through the improvement of peripheral glucose consumption, or enhanced the sensitivity of β cells to glucose, which will promotes the release of insulin (Pereira et al., 2019), and also either by inhibition of gluconeogenesis enzymes (Hasanpour et al., 2020). Several different plant extracts with anti-hyperglycemic activity have been described in several literatures (Jacob et Narendhirakannan, 2019), yet the RO-EA fraction exhibited hypoglycemic activity. This activity probably due to the involvement of the RO-EA fraction in the increased release of insulin. This effect seems similar to the mechanism of action of the drug sulphonylureas (Chinsembu, 2019; Blahova et al., 2021). On the other hand, the extract of the TN-EA fraction did not exhibit any hypoglycemic activity in normal rats or treated diabetic ones and although it has previously exhibited antihyperglycemic activity in the glucose-loaded model (Honari et al., 2018), it is proposed that this mechanism is similar to the action of biguanide drug (Kifle et al., 2022) which does not promote insulin release, it is involved in the enhancement of glucose uptake by peripheral tissues, reduce hepatic gluconeogenesis (Herrera -Balandrano et al., 2021), moreover it has antihyperglycemic and nonhypoglycemic activity (Haile et al., 2021). Alloxan induces diabetes mellitus through selective necrosis of β cells of the pancreatic islets, therefore the amount of insulin decreases an imbalance in glucose control (Papuc et al., 2021). The persistence of high glucose levels will trigger a glycation reaction between plasma proteins and glucose (Toma et al., 2020), Hb is one of these proteins, a high level of its glycated form HbA1c is a reliable index in the control of glycemia and diagnosis of diabetes. The high amount of HbA1c is proportional to the fasting glucose level in diabetic patients (Lundholm et al., 2020), the administration of the RO-EA fraction and glibenclamide for 21 days significantly reversed the insulin and glucose levels and restored the levels of Hb and HbA1c to almost normal in treated diabetic rats (Gerges et al., 2021). We can conclude that the restoration of insulin level is produced by the effect of the RO-EA fraction that leads to an improvement in glucose control (Rodrigues et al.,  2019), probably by inhibition gluconeogenesis or improvement of peripheral glucose consumption (He et al., 2019; Momtaz et al., 2019). Several studies have cited the side effects of diabetes induced by Alloxan in rats, among these effects are polyphagia, polydipsia, wasting, and muscle loss (Elangovan et al., 2019), the latter due to insulin deficiency which leads to increased protein catabolism, the increase in proteolysis aims to compensate for the role of carbohydrates in the production of energy (Arcaro et al., 2021). The administration of the RO-EA fraction and glibenclamide improves the recovery of body weight, consumption of water and food, the restoration body weight in treated diabetic rats probably due to increased glycemic control which in turn will lead to decreased proteolysis (Rodrigues et al., 2019; Salles et al., 2021; Saravanakumar et al., 2020). Diabetes mellitus has a direct or indirect impact on glucose control, generally by the decrease in insulin release, insulin-dependent enzymes such as hexokinase, glucose-6-phosphate dehydrogenase, and glycogen synthase will be inhibited (Kalaivani et Sankaranarayanan, 2021; Mabate et al., 2021), which will promote the activity of enzymes of gluconeogenesis and glycogenolysis (glucose-6-phosphatase, fructose-1,6-bisphosphate, glycogen phosphorylase) (Sundaram  et al., 2019), and consequently, hepatic glycogen will be depleted, and proteolysis increases, which will lead to a decrease in liver weight (Balakrishnan et al., 2019). In our study, we recorded all these alterations in diabetic rats induced by Alloxan, the administration of the RO-EA fraction and glibenclamide reversed all these alterations, the weight of the liver was recovered following the increase in the enzymatic activity of hexokinase, and glucose-6-dehydrogenase and glycogen synthase (Zangeneh et al., 2018; Vinayagam et al., 2018; Krishnan et al., 2020), which are insulin-dependent enzymes, play a role in the control and metabolism of glucose, therefore the enzymatic activity of glucose-6-phosphatase, fructose-1,6bisphosphatase and glycogen phosphorylase was decreased compared to untreated diabetic rats (Amadi et al., 2021), which in turn leads to an increase in hepatic glycogen levels, this improvement in glucose control probably due to the action of the RO-EA fraction and glibenclamide on the regeneration of pancreatic β cells which increases the insulin level (

CONCLUSION
In conclusion, the administration of the extract of the RO-EA fraction (300 mg/kg b.w) to diabetic rats induced by Alloxan has an appreciable effect, it significantly modulates plasma glucose and insulin levels, body weight, food consumption, Hb and HbA1c levels, and hepatic glycogen levels, thus this fraction significantly restored the altered activity of key carbohydrate metabolism enzymes almost to normal, this fraction of the RO extract -EA is considered a therapeutic virtue and can be developed as a treatment for the complication of diabetes mellitus. Further studies are needed to reveal the exact mechanism of action of the RO-EA fraction on key carbohydrate metabolism enzymes.

Conflicts of interest/competing interests:
The authors declare that they have no conflict of interest.