In vitro antidiabetic, anti-inflammatory and antioxidant potential of the ethanol extract of Uromastyx hardwickii skin

Purpose: To evaluate the in vitro antidiabetic, anti-inflammatory and antioxidant potential of the ethanol extract of Uromastyx hardwickii Skin (UHSEE). Methods: The in vitro effects of UHSEE at various concentrations (10 250 μg/mL) on the activities of -amylase, -glucosidase and glucose uptake by yeast cells were used to evaluate its antidiabetic potential. Nitric oxide (NO), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide inhibitory assay were employed to determine its antioxidant effects, while the anti-inflammatory effects were evaluated using human red blood cell (HRBC) membrane stabilization assay. Results: UHSEE inhibited -amylase and -glucosidase enzymes but increased glucose uptake by yeast cells in a concentration-dependent manner (p < 0.05). It also inhibited NO, DPPH, hydrogen peroxide and HRBC hemolysis in a concentration-dependent manner (p < 0.05). Conclusion: Uromastyx hardwickii skin exhibits promising good antidiabetic, antioxidant and antiinflammatory properties in vitro. However, its true potentials in this regard needs to be evaluted in vivo.


INTRODUCTION
Diabetes mellitus is a chronic metabolic and hormonal disorder characterized by hyperglycemia due to reduced insulin release or insulin resistance [1]. Effective management of hyperglycemia, especially postprandial hyperglycemia in diabetic patients has been a major challenge in the clinical treatment of diabetes mellitus. Postprandial hyperglycemia is regulated by α-glucosidase and α-amylase, which catalyze the digestion of carbohydrate in the gut [2]. The down-regulation of α-glucosidase and α-amylase reduces the rate of glucose digestion and absorption, which ultimately diminishes the magnitude of postprandial hyperglycemia in both hyperglycemic and normoglycemic patients [3].
Hyperglycemia is associated with non-enzymatic glycation of protein molecules that lead to elevated production and release of reactive oxygen species (ROS) [4]. The ROS are implicated in the pathogenesis of diabetes mellitus and its complications as well as other disease conditions. They elicit release of inflammatory mediators, predisposes to insulin resistance and cardiovascular diseases. Diabetes has been described as a chronic inflammatory condition [5,6]. Owing to the above fact, the roles of anti-inflammatory agents and antioxidants in treating diabetes should not be under-estimated.
The anti-inflammatory properties of common hypoglycemic drugs used in the clinical treatment of diabetes have been reviewed [6]. Their effects are not just via the reduction of plasma glucose level but also through the modulation of potent inflammatory mediators [6,7].
The use of natural products of either plant or animal origin in the traditional management of diabetes and other disease conditions is popular among Asians and Africans [8,9]. This is due to the high cost and unavailability of orthodox medicines as well as the perceived low sideeffects, cultural acceptance and availability of traditional medicines [10,11]. One of the commonly used animal products in the ethnomedical treatment of diabetes in Pakistan is the skin of Uromastyx hardwickii [12].
Uromastyx hardwickii, popularly known as Hardwicke's spiny-tailed lizard or the Indian spiny-tailed lizard is a species of lizard belonging to the family Agamidae. It is a desert lizard found in both rocky and sandy environs of North Africa and the Middle East, especially India and Pakistan [13]. It is called "Barti chpkali" in Pakistan and the skin is used in the ethnomedical treatment of diabetes mellitus [12].
There is dearth of scientific evidence of its antidiabetic potential, hence this study evaluates the in vitro antidiabetic, antioxidant and antiinflammatory properties of the ethanol extract of Uromastyx hardwickii skin.

Collection of Uromastyx hardwickii
Four of Uromastyx hardwickii lizards were collected from desert and semi-desert areas of Pakistan. They were identified and the specimens were kept in the museum at

Preparation of the extract of Uromastyx hardwickii skin
Uromastyx Hardwickii were anesthetized with diethyl ether and then sacrificed. The skins were excised as described by Brito-Casillas et al [15]. They were dried under shade and ground into powder. About 200 g of the powdered sample was extracted with ethanol using Soxhlet apparatus for 8 h. The ethanol extract of Uromastyx hardwickii skin (UHSEE) was concentrated in vacuo using rotary evaporator at 40 °C.

α-Glucosidase inhibitory activity
The α-glucosidase inhibitory activities of UHSEE and acarbose (10 -250 µg/mL concentration) were determined as described by Mopuri et al [16] without modification. The acarbose was used as the reference standard and the results were expressed as percentage inhibition of αglucosidase activities. Same volume (0.25 mL) of different concentrations (10 -250 µg/mL) of UHSEE or acarbose were incubated with 0.50 mL α-glucosidase solution (1.0 U/mL in 0.1 M phosphate buffer of pH 6.8) at 37 °C for 15 min. Later, 0.25 mL of 4-nitrophenyl β-Dglucopyranoside (pNPG) solution (5 mM in 0.1 M phosphate buffer of pH 6.8) was added and further incubated at 37 o C for 20 min. The absorbance of the mixture was measured spectrometrically at 405 nm and the inhibitory activity was expressed in percentage.

Determination of α-amylase inhibitory activity
The inhibitory activity of various concentrations (10 -250 µg/mL) of UHSEE on α-amylase was measured using a spectrophotometer [17]. A known volume (0.25 mL) of different concentrations of UHSEE and miglitol were incubated with 0.50 mL of porcine pancreatic amylase (2 U/mL in 0.10 M phosphate buffer of pH 6.8) solution at 37 °C for 20 min. Later, 0.25 mL of starch (10 mg/mL in 0.10 M phosphate buffer of pH 6.8) was added to the mixture, which was incubated at 37 °C for 60 min. 1 mL of dinitrosalicylate colour reagent was added and boiled for 10 min. The absorbance was determined at 540 nm.
The results were presented as percentage inhibition of α-amylase activities and miglitol was used as reference standard. The percentage inhibition of αglucosidase and α-amylase activities were calculated using Eq 1.
where XA = absorbance of control, YA = absorbance of test substance

Effect of UHSEE on glucose uptake by yeast cells
The effects of UHSEE and metformin (10 -250 µg/mL) on glucose uptake by yeast cells (Saccharomyces cerevisiae) (Malteserkors, Denmark) were evaluated as described by Bhutkar and Bhise [18]. The yeast cells were washed by repeated centrifugation (4200 rpm, 5 min) in distilled water until the supernatant fluid were clear. 10 % (v/v) suspension of yeast was prepared in distilled water. 1 mL of glucose solution (25 mmol/L) was added to varied concentrations (10 -250 µg/mL) of UHSEE and the mixture was incubated at 37 °C for 10 min.
After 10 min, 0.1 mL of the yeast suspension was added to the mixture, which was incubated (after thorough mixing) at 37 °C for 60 min. Later, the tubes containing the mixture were spun at 3800 rpm for 5 min in a centrifuge and the glucose concentration in the supernatant was determined. Each concentration was determined in triplicate and metformin was used as reference standard. The result is expressed as increase in glucose uptake (G) s shown in Eq 2 [18].
where X = absorbance of control, Y = absorbance of test substance

Nitric oxide (NO) radical scavenging assay
The NO radical scavenging assay of UHSEE was performed by monitoring the intensity of chromophore formed by reacting Griess reagent and nitrite liberated from sodium nitroprusside. Varied concentrations (10 -250 µg/mL) of UHSEE were tested in triplicate as described by Bryan and Grisham [19]. The results are expressed as inhibition (H). Gallic acid was used as standard.

DPPH radical scavenging assay
The abilities of UHSEE to electron donation was evaluated with 1 mM solution of 2,2-diphenyl-1picrylhydrazyl (DPPH) in ethanol as described by Ak and Gulcin [20]. This protocol estimated the DPPH radical scavenging potential of UHSEE at various concentrations (10 -250 µg/mL). The bleaching of the purple colour of DPPH solution to yellow was monitored spectrophotometrically at 517 nm after 30 min incubation in the dark at room temperature. Ascorbic acid was used as reference and the result was expressed as percentage free radical scavenging of DPPH.
where Ao = absorbance of control; Ai = absorbance of test substance

Hydrogen peroxide scavenging assay
The ability of UHSEE to decompose hydrogen peroxide (H 2 O 2 ) into water and oxygen gas was determined as described by Oktay et al [21] without modification. About 2. where Xo = absorbance of control, Xi = absorbance of test substance

Human red blood cell (HRBC) membrane stabilization assay
The effects of UHSEE on the hemolysis of human red blood cell (HRBC) in hypotonic saline solution was evaluated as described by Anosike et al [22]. Blood sample (5 ml) was collected from a healthy male donor (that has not received antiinflammatory drug in the past 10 days) into EDTA sample bottle. The HRBC was repeatedly washed with normal saline by centrifugation as described by Anosike et al [22] until the supernatant was clear. Thereafter, 0.5 mL of 10 % suspension of the HRBC was added to test tubes containing different concentrations (5 -250 µg/mL) of UHSEE dissolved in hypotonic saline solution in triplicate. The mixtures were incubated for 30 min at 37 °C and later centrifuged at 3000 rpm for 5 min. The absorbance of the supernatants was recorded at 560 nm with spectrophotometer. Hypotonic solution was used as control while diclofenac (250 µg/mL) was used as reference standard. Inhibition (H) was calculated as in Eq 5.

Heat-induced hemolysis assay
The effects of UHSEE on heat-induced hemolysis of HRBC was evaluated as described by Anosike et al [22]. The blood collection and preparation were as stated in the previous section. Thereafter 0.5 ml of 10 % suspension of the HRBC was added to test tubes containing different concentrations (5 -250 µg/mL) of UHSEE dissolved in saline in triplicate. The mixtures were incubated for 30 min at 54 °C and later centrifuged at 3000 rpm for 5 min. The absorbance (ABS) of the supernatants was determined at 560 nm with spectrophotometer. Hypotonic solution was used as control while diclofenac (250 µg/mL) was used as reference standard. Inhibition (H) was computed as in Eq 6.

Statistical analysis
Data were subjected to ANOVA followed by Tukey's multiple comparison test to determine significant difference using SPSS 22.0 software. P < 0.05 was considered sttisticlly significant.

Effect of UHSEE on α-glucosidase activity
The UHSEE and acarbose produced concentration-dependent inhibition of αglucosidase activities (Figure 1). The αglucosidase inhibitory activity of UHSEE was significantly (p < 0.05) lower compared to that of acarbose.

Effect of UHSEE on α-amylase activity
UHSEE and miglitol inhibited α-amylase activity in a concentration-dependent manner (Figure 2). The inhibitory effects of UHSEE and miglitol against -amylase activities were comparable except at 200 and 250 µg/mL where the former produced lower effects.

Effect of UHSEE on glucose uptake by yeast cells
The UHSEE and standard inhibited glucose uptake by yeast cells in a concentrationdependent manner (Figure 3). The inhibitory effects of UHSEE and metformin were comparable.

Nitric oxide (NO) scavenging activity
Both UHSEE and gallic acid inhibited nitric oxide (NO) in a concentration-dependent manner. Their inhibitory properties against NO were comparable (Figure 4).

DPPH radical scavenging activity
The UHSEE and ascorbic acid produced concentration-dependent increase in the scavenging of DPPH radicals ( Figure 5).

Hydrogen peroxide scavenging
UHSEE and ascorbic acid scavenged hydrogen peroxide in a concentration-dependent manner ( Figure 6). The hydrogen peroxide scavenging ability of UHSEE was lower (p < 0.05) compared to ascorbic acid.

Effect of UHSEE on heat-induced haemolysis
UHSEE inhibited heat-induced hemolysis of HRBC in a concentration-dependent manner ( Table 2).

DISCUSSION
The ethanol extract of Uromastyx hardwickii skin (UHSEE) inhibited the activities of α-glucosidase and α-amylase and increased glucose uptake by yeast cells in a concentration-dependent manner. UHSEE also scavenged reactive oxygen, hydroxyl and NO radicals as well as inhibited the haemolysis of HRBC induced by heat and hypotonic saline.
The inhibition of α-glucosidase and α-amylase activities by UHSEE suggests that it has the capacity to delay carbohydrate digestion and glucose absorption, which will significantly reduce the postprandial increase in blood glucose. The regulation of postprandial hyperglycemia in diabetic patients is severely impaired due to compromised insulin secretion or insulin resistance [23]. Agents such as acarbose and miglitol which inhibit α-glucosidase and αamylase activities are useful in the management of type 2 diabetes mellitus [24]. The results of this study are in agreement with the report of Gopal et al [25] on the antidiabetic activities of Lactuca sativa.
The UHSEE enhanced glucose uptake by yeast cells indicates that it might promote glucose uptake by peripheral cells [18]. Increase in the uptake of glucose by skeletal and hepatic cells is the mechanism of action of metformin. This indicates that UHSEE and metformin may possess similar mechanisms of action.
The inhibitory activity of UHSEE on reactive oxygen species and NO radicals imply that it has antioxidant property that can alleviate oxidative stress implicated in the development and progression of diabetes mellitus and its complications [4]. The finding of this study is in agreement with the report of Da Cunha et al [26] on the antioxidant and antidiabetic potentials of Apis mellifera.
Membrane stabilization test was used to evaluate the anti-inflammatory potential of UHSEE. It significantly inhibited the haemolysis of HRBC in concentration-dependent manner, in both hypotonic saline and under heat stress. This suggests that UHSEE possesses antiinflammatory properties. UHSEE may have stabilized the membrane of HRBC by inhibiting the rupture and release of lysosomal enzymes [22], which normally accompanies the inflammatory process. The anti-inflammatory activity of UHSEE observed in the present study corroborates the earlier work of Ferreirra et al [27] on zoo therapeutics used in Brazillian traditional medicine. The anti-inflammatory property of UHSEE could be useful in the treatment of diabetes, which is essentially an inflammatory process.

CONCLUSION
The ethanol extract of Uromastyx hardwickii skin (UHSEE) possesses good antidiabetic, antioxidant and anti-inflammatory properties in vitro. The findings lend some support for the use of UHSEE for treating diabetes in traditional medicine in Pakistan.