Leaf Aqueous Extract of Argania spiniosa Exhibits Antihyperglycemic Effect in Diabetic Rats.

INTRODUCTION: Argania spiniosa L. (Sapotaceae) is an endemic species from south-western Morocco. This plant has many traditional uses including its use in the treatment of diabetes.
OBJECTIVE: The objective of the study was to evaluate the antidiabetic activity of Argania spiniosa Leaf Aqueous Extract (A.S.L.A.E).
METHODS: The antidiabetic effect of A.S.L.A.E was evaluated in both normal and streptozotocin (STZ)-induced diabetic rats treated at a dose of 20 mg/kg body weight for 15 days. The histopathological changes in the liver were evaluated. In addition, the antioxidant activity of this extract was also studied.
RESULTS: Single oral administration of A.S.L.A.E (20 mg/kg) showed no significant change in blood glucose levels in both normal and STZ induced diabetic rats after 6 hours of administration. Furthermore, in normal rats, repeated oral administration of A.S.L.A.E reduced blood glucose levels. Moreover, blood glucose levels decreased in STZ diabetic rats after fifteen days of treatment. According to the oral glucose tolerance test, the A.S.L.A.E (20 mg/kg) was shown to prevent significantly the increase in blood glucose levels in normal treated rats. Moreover, A.S.L.A.E showed antioxidant activity.
CONCLUSION: The results show that Argania spiniosa leaf aqueous extract possesses significant antihyperglycemic activity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.

INTRODUCTION

Traditional herbal medicines have an advantage in the prevention and treatment of diabetes, as they have fewer side effects [1, 2].

The Argan tree, called Argania Spinosa (L.) Skeels is an endemic tree of the southwestern region of Morocco, belonging to the Sapotaceae family. Populations of Morocco traditionally use the fruits of Argania spinosa (A. spinosa) to prepare edible oil [3]. Traditionally, the Argan tree is used in cosmetics for skin and hair care and against acne, gastritis, diarrhea, fever, urticaria and headaches hypercholesterolemia [4-10].

According to a previous ethnobotanical survey which was conducted in Chtouka Ait Baha and Tiznit (Western Anti-Atlas) Morocco, the seed of A. spinosa was used in the traditional medicine by the local population for the treatment of diabetes [11]. Our previous investigation has demonstrated that the fruits’ aqueous extract of A. spinosa possesses antidiabetic effect [12]. In addition, we demonstrated that Arganimide A extracted from A. spinosa had lipid and glucose lowering effect [13]. Nevertheless, no previous pharmacological or clinical study has been carried out to test the anti-diabetic activity of Argania spinosa leaf aqueous extract (A.S.L.A.E). Therefore, the present study was designed to evaluate the effect of A.S.L.A.E in normal and diabetic rats. We have also evaluated the histopathological changes induced by this aqueous extract in the liver both in normal and STZ rats. Additionally, an oral glucose tolerance test was realized and the antioxidant potential of the A.S.L.A.E was also demonstrated.

MATERIAL AND METHODS

Plant Material

Leaves of Argania spinosa (Sapotaceae) were collected from the Souss region in Agadir (Morocco) in 2016-2017 and air-dried at 40°C. The plant was taxonomically identified and a voucher specimen was deposited at the herbarium of the Faculty of Sciences and Techniques, Errachidia.

Preparation of the Aqueous Extract

Plant material was prepared according to the traditional method used in Morocco (decoction), the dose administered was 20 mg of lyophilized aqueous extract per kg of body weight [12-14].

Experimental Animals

The antidiabetic activity of the A.S.L.A.E was studied in adult male Wistar rats weighing about 190-230 g. The animals were housed under standard environmental conditions and maintained with free access to water and ad libitum standard laboratory diet.

Effect of A.S.L.A.E on Glucose Tolerance Test

Fasted normal rats were randomly assigned to three different groups containing five rats each. The duration of the fast was 12 hours with free access to water.

Group I served as control received only vehicle (distilled water);

Group II received the A.S.L.A.E. at a dose of 20 mg/kg;

Group III received a reference drug; glibenclamide at a dose of 5 mg/kg;

Glucose 2 g/kg was fed 30 min after the administration of different doses of A.S.L.A.E and glibenclamide.

Blood was withdrawn from the tail vein at 0, 30, 60, 90 and 120 min [13]. Moreover, glucose levels were estimated by using a reflective glucometer (Contour™TS) [14].

Induction of Diabetes

Diabetes was induced and groups were randomly assigned as it has been previously described [12-15].

Determination of Parameters

Blood glucose levels were determined by the glucose oxidase method using a reflective glucometer (Contour™ TS) from Bayer Diabetes Care.

Histopathological Changes in the Liver, Morphometric Analysis

Histopathological and morphometric analysis of the liver followed the protocol used in our previous study [12-16].

Determination of DPPH (1-1-diphenyl 2-picryl hydrazyl) Radical Scavenging Activity

The free radical scavenging activity of A.S.L.A.E was analysed as it has been described previously [12-17].

Statistical Analysis

Data were expressed as mean ± S.E.M. Statistical differences among the means studied were assessed by two-way ANOVA followed by Bonferroni multiple comparisons test with GraphPad Prism 6 software. Differences were considered to be significant when p < 0.05.

RESULTS

Single Oral Administration

In normal and diabetic rats, a single administration of A.S.L.A.E had no effect on reducing blood glucose levels. On the other side, the glibenclamide revealed a significant decrease (p<0.0001) in both normal and STZ induced diabetic rats after four and six hours after administration (Fig. ).

Repeated Oral Administration

In normal rats, a significant reduction in blood glucose levels was observed after 15 days of A.S.L.A.E oral administration (p<0.01). In STZ rats, a significant reduction in blood glucose levels was observed at the second day of oral treatment (p<0.0001) with A.S.L.A.E (20 mg/kg) and the decrease in blood glucose levels became more significant at the fifteenth day of treatment (p<0.0001). On the other hand, the normal and the diabetic rats treated with glibenclamide showed a significant diminution in blood glucose levels at the end of treatment (p<0.0001) (Fig. ).

Body Weight

In normal and STZ rats, treated with A.S.L.A.E for 15 days, no significant change in body weight was observed. The same result was also observed in glibenclamide-treated group (Fig. ).

Effect of A.S.L.A.E on Glucose Tolerance

A.S.L.A.E has prohibited the increase in blood glucose levels significantly 30 min (p<0.0001), 60 min (p<0.05) and 120 min (p<0.05) after glucose administration when compared to the control group (Fig. ). Glibenclamide treatment has prohibited the increase in blood glucose levels significantly 30 min (p<0.01), 60 min (p<0.0001), 90 min (p<0.0001) and 120 min (p<0.0001) after the administration of 2 g/kg of glucose.

Histopathological Changes in the Liver and Morphometric Analysis

The results show that the diameter of the core of diabetic rats treated with A.S.L.A.E (3.42 ± 0.20) was larger than the diameter of the core of control diabetic rats (3.13 ± 0.16) (Table ). In addition, the number of hepatocytes counted in an area of 40000 µm2 in diabetic rats treated with A.S.L.A.E was equal to 25 hepatocytes. Fig. () illustrates the histopathological changes in the liver of diabetic rats fifteen days after oral administration of A.S.L.A.E (20 mg/kg) or glibenclamide (5 mg/kg).

In diabetic untreated rats (Fig. ), as compared to diabetic treated rats with A.S.L.A.E (Fig. ) or glibenclamide (Fig. ), the hepatocytes were observed to be disorganized with noticeable hepatocellular damages along with the disordered liver architecture. Sinusoids were enlarged with the wall of veins thickened (Fig. ). After 15 days of treatment by A.S.L.A.E or glibenclamide, STZ diabetic rats showed more progressive changes, improvement of liver architecture and lack of central hemorrhagic necrosis. The results showed that the rat liver histopathology of A.S.L.A.E was almost similar to the glibenclamide-treated group (Fig. and Fig. ).

DPPH (1-1-diphenyl 2-picryl Hydrazyl) Radical Scavenging Activity

The different concentrations of the A.S.L.A.E (31.25, 62.5, 125, 250 and 500 μg/ml) showed antioxidant activities in a dose-dependent manner (23.26%, 33.64%, 40.60%, 60.33% and 67.60% inhibition, respectively) on the DPPH radical scavenging assay. On the other hand, the synthetic antioxidant BHT gave the following values: 49.17%, 52.89%, 59.67%, 68.66% and 82.87% inhibition (Fig. ). Linear regression analysis was used to calculate IC50 values. A.S.L.A.E revealed inhibitory concentrations of 50% of free radicals (IC50) of 248.27 μg/ml. In contrast, the synthetic antioxidant butylhydroxytoluene (BHT) showed an IC50 equal to 13.65μg/ml.

DISCUSSION

In the present study, the results showed that A.S.L.A.E (20 mg/kg) induced a significant decrease in blood glucose

H = Hepatocytes, CV = Central Vein, S = Sinusoid, N= Necrosis.

levels both in normal and STZ induced diabetic rats after fifteen days of treatment. Our results are in agreement with our previous study which demonstrated a strong antihyperglycemic effect on diabetic rats of Argania spinosa fruits aqueous extract [12, 18]. Concerning body weight, no significant reduction was observed in normal and diabetic rats treated with A.S.L.A.E after 15 days of treatment. Streptozotocin as an antibiotic agent has been widely used for inducing type I diabetes in a variety of animals by affecting degeneration and necrosis of pancreatic beta cells [19]. STZ injection also led to increased oxidative stress apart from inducing liver damage. In STZ induced diabetic rats, A.S.L.A.E was found to provide significant protection from all the serious effects of STZ after 15 days of treatment. Concerning the oral glucose tolerance test, conduced on normal rats A.S.L.A.E prevented the increase in blood glucose levels significantly after glucose administration (2 g/kg). The results suggest that increased levels of glucose tolerance may be due to increased secretion of insulin, inhibition of α-amylase and α-glucosidase. Otherwise, the ability of A.S.L.A.E to lower the blood glucose levels in the oral glucose tolerance test suggests that rats treated with this extract had an increased glucose uptake [16, 20, 21]. Additionally, A.S.L.A.E revealed an antioxidant capacity. On the other hand, polyphenols from the leaves of A. spinosa belong to the flavonoid (17%) and catechin tannin (14%) family [22, 23]. Furthermore, flavonoid extract A. spinosa has been demonstrated to protect the skin against UV-light [24]. Consequently, the properties of the flavonoids were further studied leading to the discovery of the anti-acne properties of the flavonoid extract and inhibitory properties of their matrix metalloprotease (MMP) [25-29]. MMP constitutes a group of more than twenty enzymes responsible for the degradation of the extracellular matrix. Collagenases and elastases are well known as enzymes involved in the skin-aging process. This may be attributed to the important role of flavonoids as antioxidants. Thus, it is possible that these active compounds are responsible for the antidiabetic activity observed in A.S.L.A.E.

CONCLUSION

The study shows that A.S.L.A.E exerts antidiabetic and antioxidant activity in STZ-induced diabetic rats. More investigations are needed, such as to isolate the active(s) principle(s) of this plant and to clarify its mechanism of action, in addition to toxicological studies.

Table 1

Morphometric analysis of hepatocytes of diabetic rats treated with A.S.L.A.E (20 mg/kg) and the controls groups. Values are expressed as means ± SEM. No significant when compared to control diabetic.

Groups	Diameter of the Core (µm)	Number of Hepatocytes
Control normal	3.23 ± 0.15	29
Control diabetic	3.13 ± 0.16	24
Diabetic rats treated with A.S.L.A.E	3.42 ± 0.20	25
Diabetic rats treated with glibenclamide	3.32 ± 0.26	25