The total phenol content of the extracts was determined by the Folin-Ciocalteu method [24] with some modifications. Firstly, 100 $\mu$ L of each extract at a concentration of 10 mg/mL were mixed with 500 $\mu$ L of the Folin reagent (10%) and at least 6 mL of distilled water. After 1 min and before 8 min, we added to the mixture 1.5 mL of Na ${}_2$ CO ${}_3$ (20%). The volume was adjusted to 10 mL with distilled water. The absorbance was determined at 760 nm. A standard curve was evaluated using gallic acid concentrations ranging from 15.62 to 500 $\mu$ g/mL. The total phenol content was expressed as $\mu$ g gallic acid equivalents per mg of dry weight of extract ( $\mu$ g GAE/mg of extract). All determinations have been carried out in triplicate.

Total flavonoid content was assessed according to the aluminum chloride colorimetric method [25]. Briefly, 2 mL of distillated water and 100 $\mu$ L of AlCl ${}_3$ (5%) were added to 300 $\mu$ L of extract at a concentration 10 mg/mL. The volume is adjusted to 5 mL with distilled water. This solution was well mixed before being let to stand at room temperature for 30 min. The absorbance was measured at 425 nm. A series of concentration of quercetin (15.62–1000 $\mu$ g/mL) was used as the standard to obtain the calibration curve. Total flavonoid content was expressed as $\mu$ g quercetin equivalent per mg of dry weight of extract ( $\mu$ g QrE/mg extract). All measurements were performed in triplicate.

The method described by Julkunen-Tiitto (1985) with slay modification is used to determine the condensed tannins content [26]. 50 $\mu$ L of each extract or standard solution was mixed with 1.5 mL of vanillin (4% solubilized in methanol), and then 750 $\mu$ L of concentrated HCl was added. The well-mixed solution was incubated at ambient temperature in the dark for 20 min. The absorbance was read at 500 nm. Catechin (1.95–1000 $\mu$ g/mL) was used to make the standard curve and the results were expressed as $\mu$ g Catechin equivalents per mg of extract dry weight ( $\mu$ g CE/mg extract). All determinations have been carried out in triplicate.

The antiradical scavenging activity of sample was evaluated using a radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) [27]. The sample extract (200 $\mu$ L) at range of concentration (25–1000 $\mu$ g/mL, the choice of concentration is based on our previous study, which demonstrated that 1 mg/mL has a maximum in vitro antispasmodic effect [17]. For that, we made dilutions from a concentration of 1 mg/mL) was mixed with an 1800 $\mu$ L of DPPH solution 0.004% solubilized in methanol. After 30 min, the absorbance was measured at 517 nm by the spectrophotometer. Ascorbic acid, a standard antioxidant, was used as a reference. All tests were performed in triplicate. Radical-scavenging activity of DPPH was calculated as the following percentage

(1) $$\%\text{Radical scavenging activity}=\frac{\text{A0}-\text{A1}}{\text{A0}}\times 100$$

A0: Absorbance of DPPH alone

A1: Absorbance of sample mixed with DPPH solution

The result is presented as IC ${}_{50}$ (The concentration of the samples inhibiting the radical scavenging activity by 50%).

The ferric ion (Fe ${}^{3+}$ ) reducing power assay was carried out according to the method described by Amarowicz et al. [28]. 2.5 mL of 0.2 M of phosphate buffer (pH 6.6) and 2.5 mL of potassium ferricyanide (1% w/v) were added to 1 mL of the samples dissolved in distilled water (AcAE or AcEE) at range of concentration (62–1000 $\mu$ g/mL). The mixture obtained was then incubated at 50 °C for 20 min. After the incubation 2.5 mL of Trichloroacetic acid (10% w/v) was added. The mixture was centrifuged at 3000 rpm for 10 min and 2.5 mL of the supernatant was collected and mixed with distilled water (2.5 mL) and 0.5 mL of FeCl ${}_3$ (0.1% w/v). The absorbance was then measured at 700 nm against blank sample.

Ascorbic acid was used as a reference compound. All of the tests were performed in triplicate. The result is presented as IC ${}_{50}$ (The concentration of the samples caused 50% of inhibition).

The antioxidant activity of AcAE and AcEE was carried out by measuring the inhibition of the oxidative degradation of $\beta$ -carotene according to the protocol described by Miller (1970) [29] and modified by Bekkouch et al. [30]. Briefly, an emulsion of $\beta$ carotene/linoleic acid was prepared by solubilizing 2 mg of $\beta$ -carotene in 1000 $\mu$ L of chloroform. Then 2 mg of linoleic acid and 200 mg of Tween (80) were added. To remove the chloroform, the mixture was then evaporated using a rotavapor. At the end, a volume of 100 mL of distilled water was added with vigorous stirring. In a series of test tubes were added a volume of 50 $\mu$ L of samples or Butylated hydroxyanisole (BHA) at different concentrations (62–1000 $\mu$ g/mL). A volume of 2550 $\mu$ L of the $\beta$ -carotene solution was the introduced into the tubes. Absorbance was measured at 490 nm before and after 2 hours incubation at 50 °C against a blank that contains ethanolfor the BHA. The measurements were made in triplicate.

The percentage of relative antioxidant activity after 2 hours was calculated according to the following formula

(2) $$\begin{array}{c}\hfill \text{Bleaching inhibition}\%=100-\hfill \\ \hfill \left(\frac{\text{initial Absorbance}-\text{Absorbance after}2\mathrm{h}}{\text{initial Absorbance}}\times 100\right)\hfill \end{array}$$

The result is presented as IC ${}_{50}$ (The concentration of the samples caused 50% of bleaching inhibition).

The assay mixtures contained 200 $\mu$ L of porcine pancreatic $\alpha$ -amylase enzyme solution (13 IU), 200 $\mu$ L of phosphate buffer (0.02 M; pH = 6.9) and 200 $\mu$ L of sample (AcAE, AcEE or acarbose) at the following concentrations (0.56, 1.12, 2.25 mg/mL). The mixtures were pre-incubated at 37 °C for 10 min. Then, 200 $\mu$ L of starch (1%) dissolved in phosphate buffer was added to each tube and were incubated for 20 min at 37 °C. Then, 600 $\mu$ L of 3.5-dinitrosalicylic acid (DNSA) color reagent was added to stop the enzymatic reaction. The tubes were then incubated for 8 minutes at 100 °C. After that, they were immersed in an ice-cold water bath for a few minutes. The mixture was diluted by adding 1 mL of distillate water and the absorbance was measured at 540 nm. All tests were performed in triplicate. The inhibition percentage was calculated using the below formula:

(3) $$\text{Inhibitory activity}(\%)=\frac{\text{OD Test}-\text{OD Control}}{\text{OD Test}}\times 100$$

The concentration of the samples inhibiting the enzymatic activity of $\alpha$ -amylase by 50% (IC ${}_{50}$ ) was calculated graphically according to the function:

(4) $$\mathbf{\text{Inhibition percentage}}=\mathrm{f}\text{(log sample concentraction)}$$

The effect of AcAE and AcEE on $\alpha$ -glucosidase activity was calorimetrically quantified by monitoring the glucose release from sucrose degradation. The assay mixtures contained 100 $\mu$ L of sucrose (50 mM), 1000 $\mu$ L of phosphate buffer (50 mM; pH = 7.5) and 100 $\mu$ L of $\alpha$ -glucosidase enzyme solution (10 IU). Then, 20 $\mu$ L of acarbose, AcAE or AcEE at two different concentrations: 328 and 656 $\mu$ g/mL were added to the mixture. The same volume of distilled water was replaced in the control. Then, it was incubated at 37 °C in water bath for 25 min. After, the mixture was heating at 100 °C for 5 min in order to stop the enzymatic reaction, and the release glucose was estimated by glucose oxidase method using a commercially auto-kit (The GOD-POD is a colored glucose indicator, purchased from Biosystems. S.A Barcelona, Spain). The absorbance was measured at 500 nm. All tests were performed in triplicate. The inhibition percentage was calculated using the formula:

(5) $$\text{Inhibitory activity}(\%)=\frac{\text{OD Control}-\text{OD Test}}{\text{OD Control}}\times 100$$

Principe of GOD-POD method: Glucose oxidase enzyme (GOD) oxidizes the specific substrate D-glucose to gluconic acid and hydrogen peroxide (H ${}_2$ O ${}_2$ ) is liberated. Peroxidase enzyme acts on hydrogen peroxide to liberate oxygen (O). Nascent oxygen then couples with 4-aminoantipyrine and phenol to form red quinoneimine dye. The intensity of color is directly proportional to concentration of glucose in plasma. The intensity of color is measured colorimetrically at 500 nm [32].

The results showed that the AcAE and AcAE (250 mg/kg) decreased postprandial glycaemia in the normal rats at time 30 and 60 min in a very significant manner (p 0.001) compared to the control which increased the glycaemia level from 0.9 g/L up to 1.5 g/L after starch administration. However, the blood glucose level at time 120 min showed no significant difference between the both extracts group and the control group. Concerning the group received the acarbose (10 mg/kg) show similar effect compared with the AcAE and AcEE at time 30 and 60 min (Fig. 6A). In the control group of alloxane-diabetic rats (Fig. 6B), the glycaemia increased from 3.67 g/L to 4.66 g/L at 30 min. This glycaemia decreases significantly in presence of the both extract (p 0.01) and very significantly in the acarbose group (p 0.01).

Fig. 6.

Effect of Artemisia campestris L. Aqueous extract (AcAE) and hydro-ethanolic extract (AcEE) on blood glucose level in normal (A) and diabetic rat (B) after consumption of starch as an $\alpha$ -amylase substrate. Data represent mean $\pm$ SEM (n = 5). The difference between the groups was calculated with a two-way analysis of variance (ANOVA). Each value represent means $\pm$ SEM. * p 0.05, ** p 0.01, and *** p 0.001 compared to the control group.

The results of the effect of the extracts of Artemisia campestris L. on the inhibition of $\alpha$ -glucosidase in vivo in normal rats was illustrated in Fig. 7A. After sucrose administration in the control group, the glycaemia increased from 0.94 g/L to 1.56 g/L at 30 min and continued to rise until 1.59 g/L. In the presence of AcAE and AcEE (250 mg/kg), the blood sugar decrease significantly (p 0.001) at 30 min compared with the control group. At 60 and 120 min, the glycaemia level continues to decrease very significantly (p 0.001) compared to the control group and until more than acarbose group. However, in alloxane diabetic rats, the postprandial glucose concentrations increase in the control group from 3.45 g/L to 4.086 g/L after 30 min. On the other hand, in the group treated with acarbose, blood sugar levels were reduced by 3.21 g/L to 2.61 g/L at 30 min, the same remark observed in the AcAE and AcEE groups (Fig. 7B).

Fig. 7.

Effect of Artemisia campestris L. Aqueous extract (AcAE) and hydro-ethanolic extract (AcEE) on blood glucose level in normal (A) and diabetic rat (B) after consumption of sucrose as an $\alpha$ -glucosidase substrate. Data represent mean $\pm$ SEM (n = 5). The difference between the groups was calculated with a two-way analysis of variance (ANOVA). Each value represent means $\pm$ SEM. * p 0.05, ** p 0.01, and *** p 0.001 compared to the control group.

The results of the intestinal glucose absorption study show that the amount of glucose absorbed in the control group is 12.18 $\pm$ 0.61 mg/10 cm/h. This amount decreases significantly in presence of AcAE (p 0.001) and AcEE (p 0.001) with amount of 6.53 $\pm$ 0.57 and 5.34 $\pm$ 0.64 mg/10 cm/h respectively. These results are comparable with the result obtained in phlorizin group with an amount of glucose-absorbed equal a 4.71 $\pm$ 0.24 mg/10 cm/h (Fig. 8).

Fig. 8.

Effect of AcAE and AcEE on intestinal glucose absorption in Wistar rat. Data represent mean $\pm$ SEM (n = 5). The difference between the groups was calculated with a one-way analysis of variance (ANOVA). Each value represent means $\pm$ SEM *** p 0.001 compared to the control group.