C. Europaea Extracts: Comparison
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Caralluma europaea (Guss.) N.E.Br. (C. europaea), is a medicinal plant used traditionally to treat diabetes mellitus (DM) in Morocco. 

  • Caralluma europaea
  • antidiabetogenic effect
  • hyperglycemia
  • glucose diffusion
  • α-amylase
  • antioxidant

1. Introduction

Diabetes mellitus (DM), is a group of metabolic disorders that is characterized by a high blood glucose level (hyperglycemia), mainly caused by insufficient insulin production or unresponsiveness of the body to insulin or both [1,2][1][2]. The postprandial hyperglycemia takes part in the development of numerous diabetes complications such as nephropathy, retinopathy, neuropathy, and diabetic foot ulcer [3,4][3][4]. The control of postprandial hyperglycemia is one of the vital alternatives to the management of chronic hyperglycemia in diabetic patients [5,6][5][6]. This is accomplished by inhibiting the enzymes involved in the digestion of carbohydrates such as α-amylase [6,7][6][7]. Therefore, inhibiting α-amylase could be an effective way to reduce starch digestibility, decrease the rate of glucose absorption, and consequently reduce postprandial hyperglycemia [8,9,10][8][9][10]. In addition to this, it has been well documented that oxidative stress plays a major role in the pathology of diabetes complications of both types of diabetes mellitus [11,12][11][12]. Oxidative stress is an imbalance between the production of oxygen reactive species (ROS) and antioxidant defenses [13]. It results from high concentrations of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) [9]. It is believed that oxygen reactive species have been implicated in the etiology and development of vascular complications in diabetes [14,15,16][14][15][16]. Free radical formation in diabetes leads to damage of the biomolecules such as lipids, proteins, and nucleic acids, and induces the appearance of long-term diabetic complications [17,18][17][18]. There are various approaches to treat and prevent diabetes and its vascular complications; one of them is herbal remedies. Therefore, there is an increasing interest in evaluating herbal medicines, which are seen to be safer and to have negligible side effects [19]. The most popular natural antioxidants are vitamin C, vitamin E, flavonoids, carotenoids, and phenolic compounds [20]. Antioxidants, and vitamins such as vitamin C and E, are effective in reducing oxidative stress in experimental diabetes [21,22][21][22]. Moreover, vitamin C has also been shown to reduce glycosylated hemoglobin in the diabetic patient [23]. Over the past decade, evidence has accumulated that plant products have been shown to have an important antioxidant activity [24,25][24][25]. Caralluma europaea (Apocynaceae family) is a leafless, succulent and angular plant distributed in Morocco, Egypt, Spain, Italy, Libya, Tunisia, and Algeria [26,27][26][27]. In Moroccan traditional medicine, it is used for antidiabetic properties [28,29][28][29]. Moreover, various extracts of aerial parts C. europaea showed antidiabetic activities in an animal model for T1DM [30,31][30][31]. Besides, C. europaea was also found to be more effective in inhibiting the activities of α-glucosidase and α-amylase (key target enzymes for T2DM) [30,32][30][32]. 

2. Total Phenolic, Flavonoid, and Tannin Contents of C. europaea

The total phenolic and flavonoid contents of EACe are presented in Table 1. The results were determined from calibration curves of gallic acid (Y = 0.0123x + 0.0004, R2 = 0.993), quercetin (Y = 0.1638x + 0.0193, R2 = 0.9839), andgallic acid (Y = 0.1185x, R2 = 0.9819), respectively. The content of phenolic compounds in ethyl acetate fraction was 7.159 ± 0.35 mg GAE/g, while the total flavonoid content was 1.523 ± 0.01 mg QE/g. A lower quantity of tannin content was observed in EACe, with 0.097 ± 0.002 mg GAE/g Extract.
Table 1. Total phenolic, flavonoid and tannin contents of C. europaea (n = 3).
Extract Species Total Polyphenols (mg GAE/g Extract) Total Flavonoids (mg QE/g Extract) Total Tannins (mg GAE/g Extract)
Ethyl acetate fraction (EACe) C. europaea 7.159 ± 0.35 1.523 ± 0.01 0.097 ± 0.002

3. Antioxidant Activity

3.1. DPPH-Radical Scavenging Activity

The antioxidant capacity of the ethyl acetate fractions from C. europaea was determined by measuring their ability to scavenge DPPH radicals (Figure 1). The results were expressed as IC50 (mg/mL), which is the amount of antioxidant necessary to decrease the initial DPPH radical by 50% [40][33]. A lower IC50 value corresponds with a higher antioxidant power. An excellent antioxidant activity was found in the ethyl acetate fraction, with an IC50 = 0.22 ± 0.01 mg/mL. By comparison, the standard ascorbic acid had free-radical scavenging activity of IC50 = 0.17 ± 0.003 mg/mL. In this study, the reduction of DPPH occurred in a concentration-dependent manner (dose–response), as observed from the high reduction of DPPH (higher radical activity) at 2.5 mg/mL concentrations. The scavenging activity increased significantly with increasing EACe concentrations up to a certain dose, 2.5 mg/mL, and then leveled off with a maximum of 98% of the antioxidant effect.
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Figure 1. DPPH-scavenging activity of ethyl acetate fraction of C. europaea (EACe). Each value is expressed as mean ± SEM (n = 3).

3.2. β-Carotene Bleaching Method

The β-carotene bleaching technique is based on the loss of the orange color due to its reaction with radicals formed by linoleic acid oxidation in an emulsion. Therefore, the rate of β-carotene bleaching can be slowed down in the presence of antioxidants [41][34]. The ability of EACe to inhibit the bleaching of β-carotene is presented in Figure 2. Ascorbic acid was used as the standard drug for the evaluation of antioxidant activity. The IC50 value of EACe indicated that C. europaea extract possessed stronger antioxidant potential (IC50 = 1.153 ± 0.07 mg/mL). A similar antioxidant activity was found in the ethyl acetate fraction (EACe) in comparison with the results obtained for ascorbic acid (IC50 = 1.166 ± 0.104 mg/mL).
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Figure 2. Antioxidant activity of ethyl acetate fraction from C. europaea (EACe). Each value is expressed as mean ± SEM (n = 3).

3.3. Reducing Power

The ferric-reducing antioxidant power method is based on the reduction of Fe3+ to Fe2+ ions in the presence of antioxidants in an acidic medium, which has an intense blue color and can be monitored by measuring the change in absorption at 593 nm. Increased absorbance is proportional to the antioxidant potency. The results obtained for the FRAP assay are presented in Figure 3. As shown, the ethyl acetate fraction from C. europaea (EACe) showed the highest reducing ability. It was found that the reducing power increased with concentrations of the EACe sample. Significantly, higher reducing power was observed at 1 g/mL. Ascorbic acid was utilized as a reference standard for the evaluation of the antioxidant activity.
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Figure 3. Reducing power of EACe at different concentrations. Each value represents a mean ± SD (n = 3).

4. Effects of C. europaea on Glucose Diffusion In Vitro

In this study, the inhibitory effect on glucose movement in vitro was evaluated using a dialysis tube containing the extracts and glucose that had been soaked in NaCl solution. The diffusion of glucose into the external solution was measured by a glucose oxidase kit at 30, 60, 120, and 180 min. The effects of the aqueous extract (AECe) (5 mg/mL) and the ethyl acetate fraction (EACe) (5 mg/mL) from C. europaea on glucose diffusion are represented in Figure 3. The results obtained showed that the effect of AeCe was similar to the control. It did not significantly decrease glucose diffusion. It was evident from the graph that AeCe did not exhibit any significant inhibitory effect on glucose diffusion. However, the ethyl acetate fraction from C. europaea significantly inhibited glucose diffusion at 60, 120, and 180 min, and the glucose concentrations of the external solution were 0.58 ±0.02; 0.71 ± 0.06; and 0.66 ± 0.02 mmol/L, respectively, compared to the control group (Figure 4A). Additionally, the area under the curve for the AeCe group (135.03 ± 8.26 mmol/min) was not significantly different to the control group (139.30 ± 3.64 mmol/min), while the area under the curve for the EACe group was significantly lower (p < 0.05) (104.25 ± 6.18 mmol/min) than the area under the curve forthe control group (139.30 ± 3.64 mmol/min) (Figure 4B).
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Figure 4. Effect of aqueous extract and fractions of C. europaea (5 mg/mL) on glucose diffusion through a dialysis membrane (A), and with a representation of the area under the curve of tested extracts (B). The values are the means ± SEM (n = 6). * p < 0.05, ** p < 0.01 compared to the control group.

5. In Vitro Pancreatic α-Amylase Inhibition Assay

The effects of AeCe and EACe on the activity of pancreatic α-amylase in vitro are represented in Figure 5. The α-amylase inhibitory potential of aqueous extract and ethyl acetate fraction from C. europaea was evaluated using starch as the substrate and acarbose as the positive control. Indeed, AeCe, EACe, and acarbose exhibited a dose-dependent enzyme inhibition. The ethyl acetate fraction exhibited a percentage inhibition of 77.30% and 88.29% at concentrations of 50 and 100 µg/mL, respectively. Likewise, the aqueous extract showed a percentage inhibition against α-amylase: 59.57% (50 µg/mL) and 68.79% (100 µg/mL). The standard drug acarbose displayed 85.81% and 94.14% inhibitions of α-amylase activity at concentrations of 0.18µg/mL and 32µg/mL, respectively.
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Figure 5. Inhibitory activity of the enzymes α-amylase by AeCe, EACe, and acarbose in vitro. The values are the means ± SEM (n = 3). AeCe: Aqueous extract of C. europaea; EACe: Ethyl acetate fraction of C. europaea. *** p < 0.001 compared to the control group.

6. In Vivo Pancreatic α-Amylase Inhibition Assay

6.1. Oral Starch Tolerance Test

Nondiabetic Rats

Using normal rats, treated groups receiving 250 mg/kg of AeCe had significantly reduced postprandial hyperglycemia at 30 min (1.31 ± 0.02 g/mL) and 60 min (1.04 ± 0.04 g/mL). Likewise, the group treated with EACe with 50 mg/mL showed significant (p < 0.05, p < 0.01) decrease in blood glucose levels at 30 min (1.29 ± 0.04 g/L) and 60 min (0.95 ± 0.06 g/L), following starch loading. Therefore, no effect was observed at 120 min in all extract-treated groups when compared with the control group pretreated with distilled water (10 mL/kg). In addition, acarbose significantly (p < 0.01 and p < 0.001) reduced postprandial hyperglycemia during the two hours after starch loading at 30 min (0.97 ± 0.03 g/L), 60 min (0.97 ± 0.02 g/L), and 120 min (0.93 ± 0.02 g/L) when compared to the control group of rats pretreated with distilled water (Figure 6A). Additionally, the area under the curve (AUC d-glucose) was significantly (p < 0.05) lower in rats treated with AeCe (119.30 ± 3.07 g/L/min) and EACe (124.21 ± 2.33 g/L/min) than in rats treated with distilled water (136.35 ± 3.28 g/L/min). Additionally, the area under the curve of acarbose was significantly lower (115.08 ± 2.21 g/L/min) than the area under the curve of the water-treated rats (136.35 ± 3.28 g/L/min) (Figure 6B).
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Figure 6. Effect of AeCe, EACe, and acarbose on plasma glucose levels after starch intake in normal rats (A), with a representation of the area under the curves (B). The values are the means ± SEM (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group.

Diabetic Rats

The administration of AeCe at 250 mg/kg to STZ-induced diabetic rats significantly inhibited the blood glucose elevation after starch loading at 60 min (p < 0.05; 1.74 ± 0.10 g/L) and 120 min (p < 0.01; 1.65 ± 0.09 g/L) whencompared to the group of rats pretreated with distilled water in which the starch overloadinduced a remarkable peak in blood glucose at 30 min (2.47 ± 0.10 g/L), 60 min (2.29 ± 0.16 g/L), and 120 min (2.26 ± 0.13 g/L). Furthermore, EACe at 50 mg/kg significantly reducedpostprandial hyperglycemia at 30 min (p < 0.05; 2.07 ± 0.14 g/L), 60 min (p < 0.01; 1.60 ± 0.13 g/L), and 120 min (p < 0.001; 1.22 ± 0.009 g/L) whencompared to the control group. Similarly, acarbose at 10 mg/kg failed to suppress the peak blood glucose significantly (p < 0.001) at 30, 60, 120 min, and the glucose concentrations were 1.68 ± 0.09 g/L, 1.25 ± 0.02 g/L, and 1.14 ± 0.04 g/L, respectively (Figure 7A). Besides, the area under the curve (AUC d-glucose) was significantly (p < 0.001) lower in rats treated with AeCe (210. 55 ± 6.22 g/L/min) and EACe (190.35 ± 6.75 g/L/min) than in rats treated with distilled water (264.60 ± 6.87 g/L/min). Additionally, the curve area of acarbose was significantly (p < 0.001) lower (160.24 ± 4.82 g/L/min) than that of control rats (Figure 7B).
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Figure 7. Effect of AeCe, EACe, and acarbose on plasma glucose levels after starch intake in diabetic rats (A), with a representation of the area under the curves (B). The values are the means ± SEM (n = 6). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group.

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