Evaluation of antidiabetic properties of cactus pear seed oil in rats

Abstract

Context: Cactus pear (Opuntia ficus-indica (L.) Mill. (Cactaceae)) is a medicinal plant widely used to treat diabetes.

Objective: This work investigates the hypoglycemic and antihyperglycemic effect of cactus pear seed oil (CPSO), its mechanism of action, and any toxic effects.

Materials and methods: The hypoglycemic effect of CPSO was evaluated in groups of six healthy Wistar rats given 1 or 2 ml kg⁻¹ orally and compared with groups receiving glibenclamide (2 mg kg⁻¹) or water. Glycemia was determined after 30, 60, 120, 240, and 360 min. The antihyperglycemic effect of CPSO was determined in healthy rats and in streptozotocin-induced diabetic rats (STZ); normal rats received 0.8 ml kg⁻¹ CPSO, while diabetic rats received 1 ml kg⁻¹ CPSO, their controls received water or 2 mg kg⁻¹ glibenclamide. For the antihyperglycemic effect evaluation, all the animals were fasted for 16 h before treatment and received glucose orally at 1 g kg⁻¹ 30 min after treatment; blood was taken after 30, 90, 150, and 210 min. Intestinal glucose absorption was estimated in rat jejunum perfused with a solution containing 5.55 mmol l⁻¹ glucose. Acute toxicity was determined in albino mice that received oral or intraperitoneal doses of 1, 3, or 5 ml kg⁻¹ CPSO.

Results: CPSO (p.o.) decreased postprandial hyperglycemia (60 min after glucose loading), 40.33% and 16.01%, in healthy and STZ-diabetic glucose-loaded rats, respectively. CPSO, also, significantly decreased intestinal glucose absorption by 25.42%. No adverse effects were seen in mice administered CPSO at up to 5 ml kg⁻¹.

Conclusion: CPSO is antihyperglycemic. The effect can be explained partly by inhibition of intestinal glucose absorption.

• Antihyperglycemic
• diabetes
• glucose tolerance
• intestinal glucose absorption
• Opuntia ficus-indica
• postprandial hyperglycemia
• streptozotocin

Introduction

The International Diabetes Federation (IDF) estimated that 382 million people have diabetes in 2013; by 2035 this will rise to 592 million (IDF, 2013). Diabetes mellitus is a complicated metabolic disorder resulting from heterogeneous factors. Impaired glucose metabolism is the most known diabetes’ symptom, which is due to a lack of insulin secretion, impaired insulin activity or both (Dinneen, 2006). Generally, metabolic impairment caused by diabetes induces harmful complications, such as blindness, limb amputation, and finally death (Marles & Farnsworth, 1995), which are exacerbated by glucotoxicity due to a permanently elevated blood glucose level (Bensellam et al., 2012; Rosenthal, 2007). Improving glucose tolerance may ameliorate the patient’s condition and delay or prevent complications, and preventing post-prandial hyperglycemia can improve glucose tolerance. Not only common hypoglycemic agents but also the quality of the diet can inhibit post-prandial hyperglycemia in both diabetic and normal people. Therefore, functional foods, such as plants extracts, administered before meals can have a beneficial effect on hyperglycemia. Vegetable oils can improve cellular glucose tolerance and thus decrease blood glucose level (Foster et al., 2009).

Cactus pear [Opuntia ficus-indica (L.) Mill. (Cactaceae)], Barbary fig or prickly pear is endemic to Mexico (Griffith, 2004) and is popular throughout the Mediterranean area and Morocco. The fresh fruits are widely consumed, and parts are used as functional foods, therapeutics, and animal food (Feugang et al., 2006). Many studies (Deters et al., 2012; Feugang et al., 2010; Kim et al., 2012; Tesoriere et al., 2004; Zou et al., 2005) have shown that cactus pears can reduce oxidative stress and may prevent cancer.

Cactus pear seed oil (CPSO) is an organic extract that contains fatty acids, represented by linoleic acid, a major polyunsaturated fatty acid, oleic acid, the dominant monounsaturated fatty acid, and palmitic acid, the major saturated fatty acid (Ennouri et al., 2005; Ramadan & Morsel, 2003; Sawaya & Khan, 1982). CPSO supplementation of the diet of normal rats decreased blood glucose, increased liver and muscle glycogen, and decreased low-density lipoprotein cholesterol (Ennouri et al., 2006a, 2007). Furthermore, O. ficus-indica is reputed to be antidiabetic (El-Hilaly et al., 2003; Marles & Farnsworth, 1995; Tahraoui et al., 2007). Both cladodes extract and stemfruit combinations lowered plasma sugar in a healthy rat model (Butterweck et al., 2011), and cactus pear polysaccharides were beneficial in streptozotocin (STZ)-induced diabetic rats (Liu et al., 2010). Seed powder and seed oil from this plant reduced plasma glucose levels, improved the hepatic and plasma lipid profile and reduced cholesterolemia and hepatic and muscular glycogen in healthy rats treated subchronically (Ennouri et al., 2006a,b, 2007).

The present work investigates the hypoglycemic and antihyperglycemic effects of CPSO, its possible mechanism of action, and any toxic effects in normal and diabetic rats.

Materials and methods

Animals

Healthy adult Wistar rats of each sex weighing 170–230 g were housed in polypropylene cages at the animal house of the Faculty of Sciences, Mohammed I University, Oujda, Morocco. The animals were maintained in environmental conditions and fed standard diet and water ad libitum.

All animals were cared for in compliance with the Guide for the Care and Use of Laboratory Animals, US National Institutes of Health (2012).

Acute toxicity

Oral and intraperitoneal acute toxicities were determined in albino mice weighing 20–30 g. For each route, four groups of six mice (three males and three females) received increasing doses of CPSO (1, 3, and 5 ml kg⁻¹) or distilled water (10 ml kg⁻¹). After treatment, the animals were observed daily for 14 d to assess behavioral and autonomic toxic effects.

Hypoglycemic and antihyperglycemic effects

Experimental diabetes was induced by a single intraperitoneal injection of 60 mg kg⁻¹ streptozotocin (STZ, Sigma-Aldrich, Hamburg, Germany) freshly dissolved in citrate buffer (0.1 mol L⁻¹ phosphate, 1 mol L⁻¹ citrate, pH 4.5). Animals with a fasting blood glucose level >1.5 g L⁻¹ on day 7 after injection were considered diabetic (Wu & Huan, 2008) and were included in the study.

Hypoglycemic and antihyperglycemic effects were assessed in normal and STZ-induced diabetic rats fasted for 16 h with free access to water.

For determination of the hypoglycemic effects of CPSO, blood was withdrawn from the tail vein under light ether anesthesia and centrifuged in a hematocrit centrifuge (Hermle Z 230H, HERMLE, Gosheim, Germany) for 10 min; the plasma was used to determine glucose levels by the glucose oxidase–peroxidase method using a commercial kit (Glucose, Biosystems, Barcelona, Spain). In this experiment, normal rats were divided in four groups of six rats each, which received by oral gavage CPSO (Argan Oil Company, Casablanca, Morocco) at 1 or 2 ml kg⁻¹, glibenclamide (purchased from a local pharmacy as Benclamide 5 mg) at 2 mg kg⁻¹ or distilled water (10 ml kg⁻¹). Blood glucose was estimated at 30, 60, 120, 240, and 360 min after treatment (Bellahcen et al., 2012).

For determination of the antihyperglycemic effects of CPSO, healthy and STZ-induced diabetic rats were divided into three groups of six rats each. Normal rats received CPSO by oral gavage at 0.8 ml kg⁻¹, diabetic rats received CPSO at 1.0 ml kg⁻¹, healthy and diabetic rats received glibenclamide at 2 mg kg⁻¹, and healthy and diabetic controls received distilled water at 10 ml kg⁻¹. All the animals were orally loaded with glucose (1 g kg⁻¹) 30 min after treatments. Blood was sampled by tail vein incision 30, 90, 150, and 210 min after treatment.

Intestinal perfusion

Rats were fasted for 36 h, then anaesthetized by intramuscular injection of sodium pentobarbital (50 mg kg⁻¹) and placed on a homeothermic pad to maintain the body temperature at 37 °C. Laparotomy was performed by a midline incision through an abdominal muscle, and a jejunum segment measuring approximately 10 cm was catheterized with polyethylene tubes at both proximal and distal ends. After catheterization, the incision was sutured and the surgical area covered with cotton imbibed with saline (0.9% NaCl) to prevent dehydration. A solution of 126.1 mmol L⁻¹ NaCl, 2.70 mmol L⁻¹ KCl, 0.4 mmol L⁻¹ NaH₂PO₄·2H₂O, 1.04 mmol L⁻¹ MgCl₂·6H₂O, 7.14 mmol L⁻¹ NaHCO₃, 6.93 mmol L⁻¹ CaCl₂, and 5.55 mmol L⁻¹ glucose (pH 7.5) was perfused through the intestinal lumen with a peristaltic pump (Thermo Fisher Scientific Inc., Waltham, MA) at a constant flow rate (0.53 ml min⁻¹) and temperature (37 °C) for 10 min to clean out any residual material and to allow intestinal adaptation; the main perfusion was carried out for 1 h. An aliquot of the perfusate was collected for glucose determination (Bnouham et al., 2003), the length of the perfused segment was measured, and the rats were euthanized. Intestinal glucose absorption was estimated in mg 10 cm⁻¹ h⁻¹.

Four groups of rats were used: one received the perfusion solution with added CPSO (1 ml kg⁻¹); one received the solution with added emulsified CPSO (0.36% DMSO, 1% Tween 20); one received the solution with added acarbose (3 mg kg⁻¹), a standard inhibitor of d-glucose luminal absorption (Hirsh et al., 1997); and one group rat served as the control, receiving the solution alone.

Statistical analysis

The results are expressed as means ± standard errors of the means (SEMs). Student's t-test was used, and differences were considered statistically significant at p < 0.05.

Results

Acute toxicity

After oral or intraperitoneal administration of CPSO at a dose of 1, 3, or 5 ml kg⁻¹, no mortality was seen, and there were no behavioral or autonomic effects throughout the 14-d period of observation.

Hypoglycemic effect

Oral administration of CPSO at 1 or 2 ml kg⁻¹ did not significantly affect the fasting blood glucose level of healthy rats, although glibenclamide induced a significant (p ≤ 0.001) decrease in blood glucose level (0.60 ± 0.05 g L⁻¹) at 360 min after its administration (Table 1).

Table 1. Changes in blood glucose level variation during 6 h after oral administration of cactus pear seed oil (CPSO) at 1 or 2 ml kg⁻¹ and of glibenclamide at 2 mg kg⁻¹ in healthy rats.

	Time (min)
Treatment	0	30	60	120	240	360
Control	0.89 ± 0.06	1.26 ± 0.06	1.20 ± 0.12	0.97 ± 0.04	0.91 ± 0.02	0.91 ± 0.04
CPSO (1 ml kg^−1)	0.92 ± 0.01	1.32 ± 0.07	1.51 ± 0.14	1.11 ± 0.06	0.99 ± 0.03	0.94 ± 0.03
CPSO (2 ml kg^−1)	0.90 ± 0.02	1.16 ± 0.08	1.07 ± 0.07	1.01 ± 0.04	0.98 ± 0.05	0.95 ± 0.05
Glibenclamide (2 mg kg^−1)	0.93 ± 0.03	1.30 ± 0.09	1.17 ± 0.09	0.99 ± 0.07	0.80 ± 0.06	0.60 ± 0.05*

Values are means ± SEM (n = 6).

*p ≤ 0.001 compared with the control group.

Antihyperglycemic effect

Oral administration of CPSO at 0.8 ml kg⁻¹ to healthy rats significantly inhibited (p ≤ 0.001) the hyperglycemia that follows glucose loading, 1.35 ± 0.14 g L⁻¹ at 90 min for treated rats and 2.27 ± 016 g L⁻¹ for a normal control group. Furthermore, the area under the curve (AUC_glucose) was significantly smaller (286.3 ± 24.18 g L⁻¹min⁻¹) than that of normal controls (390.1 ± 22.59 g L⁻¹min⁻¹) (Figure 1). In the STZ-diabetic rats, oral administration of CPSO improved the glucose tolerance in the STZ-diabetic treated group (3.56 ± 0.20 g L⁻¹) at 90 min, compared with the diabetic control group (4.24 ± 0.14 g L⁻¹), and the AUC_glucose of CPSO-treated diabetic rats (789 ± 15.79 g L⁻¹min⁻¹) was significantly smaller than that of diabetic controls (798.9 ± 15.79 g L⁻¹min⁻¹) (Figure 2).

Figure 1. Effects of cactus pear seed oil (CPSO) on glucose plasma level and AUC_glucose in normal rats. Values are means ± SEM (n = 6). *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001 compared with normal controls.

Figure 2. Effects of cactus pear seed oil (CPSO) on glucose plasma level and AUC_glucose in STZ-induced diabetic rats. Values are means ± SEM (n = 6). **p ≤ 0.001; ***p ≤ 0.0001 compared with diabetic controls.

In situ single-pass intestinal perfusion

The absorption of d-glucose by the perfused intestinal jejunum segment is shown in Figure 3. Perfusion of emulsified CPSO significantly decreased (p ≤ 0.05) glucose absorption through the perfused jejunum segment (8.44 ± 0.92 mg 10 cm⁻¹ h⁻¹) compared with normal control group (11.32 ± 0.97 mg 10 cm⁻¹ h⁻¹).

Figure 3. Effect of cactus pear seed oil (CPSO) on free d-glucose absorption assessed by in situ single-pass intestinal perfusion. Data are means ± SEM (n = 6). *p ≤ 0.05; **p ≤ 0.001 compared with normal controls.

Acarbose (3 mg kg⁻¹) induced a greater inhibitory effect (p ≤ 0.001).

Discussion

The results show that oral administration of CPSO can improve glucose tolerance by decreasing postprandial hyperglycemia in healthy glucose-loaded rats and STZ-induced diabetic glucose-loaded rats, but it has no effect on the basal glucose level of healthy rats fasted overnight. Also, CPSO partially decreased d-glucose intestinal absorption. The result of the acute oral toxicity test shows that the product is safe at doses up to 5 ml kg⁻¹.

It has been reported that the lipid fraction of O. ficus-indica seed oil contains polyunsaturated fatty acids (linoleic acid) and monounsaturated fatty acids (oleic acid), and the unsaponifiable fraction contains liposoluble vitamins (tocopherols, vitamin K1), sterols, and carotenoids (β-carotene) (Ennouri et al., 2005; Mannoubi et al., 2009; Oguzhan et al., 2006; Ramadan & Morsel, 2003). The omega-3 fatty acids increase the insulin secretion stimulated by d-glucose by contact with Langerhans islet beta cells (Oguzhan et al., 2006). In addition, polyunsaturated fatty acids enhance cell membrane fluidity and GLUT4 transporter expression (Manco et al., 2004). Moreover, polyunsaturated fatty acids improve insulin-induced glucose uptake on insulin-sensitive cells (3T3-L1 adipocytes) by increasing GLUT1 and GLUT4 plasma membrane density (Nugent et al., 2001). This mechanism may be responsible for enhanced glucose-induced insulin secretion. An amplified insulin reaction might be involved in the rapid inhibition of postprandial hyperglycemia by CPSO.

It has been reported that antioxidants not only affect the plasma stability of unsaturated fatty acids but also improve the insulin sensitivity of L6 muscle cells (Vinayaga Moorthi et al., 2006). The β-carotene plasma level is inversely correlated to the fasting plasma glucose level, and carotenoids are involved in diabetes control (Abahusain et al., 1999). All these consideration lead us to suggest that the acute postprandial glucose-lowering action of CPSO can be explained by increased intra-pancreatic glucose-induced insulin release from β-cells, extrapancreatic enhancement of glucose uptake by insulin sensitive tissues, or both. This extrapancreatic effect of CPSO may be due to the presence of fatty acids of various degrees of unsaturation (Barra et al., 1995). It is known that fatty acids disturb the absorption function of enteric cells when they are present in the luminal space.

Conclusion

The present study confirms that CPSO has an anti-postprandial hyperglycemic effect in normal and STZ-induced diabetic rats. This effect should prevent the complications of glucotoxicity and is due, in part, to inhibition of intestinal d-glucose absorption. Further in vitro tests and subchronic and chronic assays are required to confirm this acute effect on glucose metabolism and to assess the effect of CPSO in primary or secondary prevention of diabetes mellitus of both types.

Declaration of interest

The authors declare that there is no conflict of interest and they are alone responsible of the content of this article. This work was supported by grants from CNRST, Morocco [Project URAC-40] and from Belgium [Program 3, CUD Project].

Acknowledgements

The authors are thankful to Badraoui Mustapha and Ramdaoui Karim for technical help and animal breeding. The authors are thankful to Pr. Elizabeth Heseltine (editor for WHO and various other international organizations and national institutes in United Kingdom), for English correction.