Alpha-glucosidase inhibitory and antioxidant activities of various extracts of aerial parts of Fagonia indica Burm .

Purpose: To investigate in vitro antioxidant and anti-diabetic potentials of Fagonia indica Burm.f. Var. indica in order to provide scientific evidence for its traditional use. Methods: Cold maceration method was employed for the preparation of extracts of aerial parts of the plant using chloroform, n-hexane, methanol and water as solvents. Total flavonoid and polyphenolic content of various extracts were determined by standard methods. The antioxidant and anti-diabetic activities of the extracts were determined using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and in vitro αglucosidase (maltase) inhibitory assays, respectively. Results: Preliminary results indicated the presence of various phytochemicals in the extracts. The chloroform extract exhibited the highest contents of total flavonoids (65.98 ± 1.63 mg QE/g) and polyphenols (26.75 ± 1.09 mg GAE/g). This extract also showed the highest free radical scavenging (64.74 ± 1.43 %) with IC50 value of 34.18 ± 5.57 μg/mL while the methanol extract exerted the highest αglucosidase (maltase) inhibitory activity (45.22 ± 0.46 %) with half-maximal concentration (IC50) of 220.4 ± 0.41 μg/mL. Conclusion: The extracts of the aerial parts of Fagonia indica possess significant anti-diabetic and antioxidant effect, thus justifying the traditional use of the plant for treatment of diabetes.


INTRODUCTION
Diabetes mellitus (DM) is a chronic metabolic disorder which is characterized by an increase in blood glucose level (hyperglycemia) and causes disturbances in fat, protein and carbohydrate metabolism. It causes decline in insulin secretion or its action or both [1]. For the past few years the increase in the occurrence of diabetes has been observed [2]. The prevalence of diabetic patients has increased from 171 million to 366 million since 2000 and it is expected to rise even more by 2030 [3]. In type 2 DM, starch hydrolysis by the pancreatic alpha-amylase and the absorption of glucose by the intestinal alpha-glucosidases like maltase and sucrase results in postprandial hyperglycemia (PPHG) [4].
Alpha-glucosidase inhibitors suppress the PPHG effect by suppressing the hydrolysis of disaccharides into glucose monomers leading to decreased glucose absorption [5]. However, synthetic α-glucosidase inhibitors like acarbose are often reported with gastrointestinal side effects which include flatulence, abdominal pain and diarrhea [6]. Therefore, it is the need of hour to search for effective and safe alternative αglucosidase inhibitors showing no side effects. One of the potential approaches to find out a new agent of drug for treating diabetes, especially type 2 DM is the mechanism of α-glucosidase inhibition [7]. Diabetes contributes in causing complications due to free radical reactions [8]. The use of antioxidants in diabetes therapy may prevent diabetic complications [9]. Natural inhibitors of α-glucosidase and α-amylase obtained from the plants can be used effectively in the management of postprandial hyperglycemia having minimum side effects [10]. F.
indica Burm.f. Var. indica (family: Zygophyllaceae) is pruinose glandular or glabrous pale green shrublet having 55 cm height. It is distributed in Pakistan and westwards to North and East tropical Africa [11,12]. It is used as a hypoglycemic agent in a folklore system [13]. Anti-diabetic activity of this plant with a well-defined mechanism has not been evaluated and studied so far. Moreover, no report has shown the antioxidant properties of the plant extracts. This study tends to investigate phytochemicals, the total content of flavonoids, polyphenols, in vitro antioxidant and antidiabetic potential of F. indica Var. indica to provide the scientific evidence for the folkloric/traditional use of this plant in managing diabetes.

Plant collection and authentication
The aerial parts of the plant were collected from Cholistan desert; Bahawalpur, Pakistan and the plant were authenticated by a plant taxonomist (Prof Dr Zaheer-ud-din Khan, Botany Department, Government College University, Lahore, Pakistan). A voucher specimen (GC-Herb-Bot. 2967) was deposited in Dr Sultan Ahmad Herbarium of the same university.

Sample preparation and extraction
The aerial parts of the plant were air dried and crushed into coarse powder. After drying the powder was extracted sequentially with solvents n-hexane, chloroform, methanol and water of increasing polarity by a cold maceration method with frequent stirring. The filtrates of various extracts were concentrated using a rotary vacuum evaporator (Heidolph, Laborata 4002, Germany) under reduced pressure at 40 ˚C and were stored at 0 -4 ˚C for further use.

Phytochemical evaluation (qualitative chemical examination)
Various extracts of the plant were subjected to a qualitative phytochemical screening to identify phytoconstituents (flavonoids, saponins, terpenoids, phenols, glycosides and alkaloids) responsible for antioxidant and anti-diabetic activities using the standard procedures as described by Trease and Evan, and Sofowara [14,15].

Total flavonoid content (TFC)
TFCs in various extracts were determined following the method of Chang et al [16] with minor modification. Stock solutions of quercetin (1 mg/mL) and the extracts (1 mg/mL) were prepared in methanol. Different concentrations (10 -120 µg/mL) of the standard quercetin were prepared in methanol. In order to prepare the working solution, 200 µL was taken in the test tubes for each of the standard and sample, and finally the volume was made up to 1 mL by the addition of methanol. After, 1 M potassium acetate (100 µL), 10 % w/v of aluminium nitrate solution (100 µL) and 4.6 mL of distilled water were added to these test tubes. The contents were mixed thoroughly and incubated at room temperature for 45 minutes. The absorbance was measured at λ max of 415 nm using a UV spectrophotometer (UV-1800, Shimadzu, Japan). Blank was prepared similarly as the sample but no sample/standard was added to it. TFC was determined from the standard calibration curve of quercetin (milligrams of quercetin equivalent/g of plant extract) using linear regression equation.

Total polyphenolic content (TPC)
TPCs of the extracts were investigated following the method described by Slinkard and Singleton [17] with minor modification. Stock solutions of gallic acid (1mg/mL) and the extracts (1 mg/mL) were prepared using methanol as solvent. Concentrations in the range of 10 -120 µg/mL of the standard gallic acid were prepared to draw the standard calibration curve. 200 µL was taken separately in the test tubes for standard, sample and the volume was made up to 1 mL with methanol. After, 200 µL of FC reagent was added to and the contents, after mixing thoroughly were kept for 4 minutes. Later, the solution of sodium carbonate (15 % w/v, 1 mL) was added and stored at 25˚C for 2 h. The absorbance was measured at λ max of 760 nm wavelength using a spectrophotometer. Blank was prepared similarly, but no standard/sample was added to it. TPC was determined by using calibration curve of standard (milligrams of gallic acid equivalent/g of plant extract) using linear regression equation.

Evaluation of DPPH free radical scavenging activity
The DPPH free radical scavenging activity of the extracts was investigated using DPPH (1, 1diphenyl-2-picrylhydrazyl) assay as described by Lee and Shibamoto [18] and compared with a standard antioxidant BHT. Samples at concentrations (15 -75 µg/mL) were dissolved in a methanolic solution of DPPH (3 mL, 0.1 mM). The solutions after shaking vigorously were kept at room temperature for 1 h. Control contained 3 mL of the DPPH solution and was treated like the standard/sample. The absorbance of all extracts, standard and control was measured at λ max of 517 nm. Methanol was used as blank. Regression equation was applied to calculate IC50 values. Percent free radical scavenging activity (% FRSA) was calculated as in Eq 1 [19]. (1) where Ac = the absorbance of control and Ae = the absorbance of extract.
For each extract the assay was repeated thrice and the mean of three values was taken.

Alpha-glucosidase inhibitory assay
The anti-diabetic activity of various extracts was determined by alpha-glucosidase (maltase) inhibitory assay following the standard method of Dahlqvist [20] with minor modification. Briefly, rat intestinal acetone powder (1 g) was mixed with 10 mL sodium phosphate buffer (pH 7, 0.1 M) and was sonicated for 30 seconds (12 times) with a break of 15 second to prevent heat buildup. Later, centrifugation at 10,000 g at 4 ˚C for 10 minutes, the resultant supernatant collected was labeled as rat intestinal αglucosidase (Maltase enzyme). Enzyme inhibition was determined by incubating the solution of an enzyme (20 µL), phosphate buffer (100 µL, 0.1 M) at pH 7.0, maltose solution (37 mM) and the solutions of various extracts of the plant with varying concentrations of 20 -100 µg/mL at 37 ˚C for 30 min. Acarbose (a reference standard) at various concentrations (20 -100 µg/mL) was used as α-glucosidase inhibitor. The mixtures were placed in boiling water for 5 minutes to terminate the reaction. Using the glucose oxidase method of Bergmeyer and Bernt [21], the amount of glucose released was determined. Alpha-glucosidase (maltase) inhibition (H) was calculated using Eq 2 [22]. (2) where Ac = the absorbance of control and Ae = the absorbance of extract.

Statistical analysis
Assays were conducted thrice and the results are presented as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparison test (Graph Pad Prism 5.0). Differences were considered to be statistically significant at p < 0.05.

Phytochemical profile
Screening of phytochemical constituents of various extracts showed the presence of phytochemicals as shown in Table 1.

Total flavonoid content (TFC)
TFCs of the extracts were determined from the standard quercetin calibration curve using linear regression equation (Y = 0.001x + 0.0071) with a correlation coefficient (R) = 0.998574. The results of TFCs of the extracts are presented in Table  2. It was between 18.891 ± 0.54 mg QE/g to 65.98 ± 1.63 mg QE/g of dried extract. According to Table 2, the chloroform extract showed the highest TFC of 65.98 ± 1.63 mg of QE /g of dried extract. It was followed by n-hexane, methanol and water extracts.

Total polyphenol content (TPC)
TPCs of the extracts were determined from the standard calibration curve using linear regression equation (Y = 0.0037x + 0.0403) with a correlation coefficient (R) = 0.99736. The results of TPCs of the extracts are presented in Table 2 and were ranged from 4.96 ± 1.20 to 26.75 ± 1.09 mg of GAE/g of the extract. The chloroform extract showed the highest TPC of 26.75±1.09 mg of GAE/g of the extract which was followed by methanol, n-hexane and water extracts.

Antioxidant activity
Various extracts of the plant show % DPPH free radical scavenging ( Table 3). The results showed that the antioxidant activity increases with increase in concentration of the extract/ sample. Chloroform extract exhibited the highest percentage of scavenging DPPH free radical as compared to other extracts (64.74 ± 1.43 % inhibition at the concentration of 75 µg/mL).
The IC 50 (concentration of a plant extract that is required to scavenge 50 % of DPPH free radical) of the extracts are presented in Table 4. Lower the value of IC 50 , greater will be free radical scavenging activity. The chloroform extract exhibited the lowest value of IC 50 , i.e., 34.185 ± 5.57 µg/mL compared to the other extracts. The aqueous extract showed low antioxidant activity, compared to standard (BHT).  Values are expressed as mean ± SD (n = 3). Values are relative to the standard (BHT); . a* p ˂ 0.05 and c*** p < 0.001 in comparison with reference standard (BHT)

Maltase inhibition
The assay showed that various extracts of the plant have α-glucosidase (maltase) inhibition. The methanol extract exhibited the highest maltase inhibition followed by aqueous extract at the concentrations of 20 -100 µg/mL in a concentration-dependent manner. n-Hexane and chloroform extracts showed minimum maltase inhibitory activity and percentage inhibition increased at a lower level even by increasing the concentration of extracts used.
The maximum concentration of the methanol extract (100 µg/mL) showed the highest maltase inhibition of 45.22 ± 0.46 % and the standard reference (acarbose) exhibited 67.41 ± 0.92 % inhibition at the same concentration (Table 5). It has been suggested that methanol extract is rich in phytoconstituents that have the potential in managing diabetes. The results were evaluated by comparing with the standard (acarbose) as presented in Table 5. A significant decrease in (p < 0.001) in the levels of glucose in blood was found among various extracts and standard drug in comparison to diabetic control.  The IC 50 of various extracts and standard (acarbose) are shown in Table 6. The results show that the smaller the IC 50 value, the higher the anti-diabetic activity. Methanol extract was found to have smaller IC 50 value in comparison to other extracts. IC 50 values of all the extracts showed significant results (p ˂ 0.001). The maltase inhibitory activity of various extracts and standard (acarbose) is shown in Figure 1. Inhibition of maltase was observed to be increased with increase in the concentration of the extracts.

DISCUSSION
Keeping the blood glucose level at normal values is a crucial strategy in controlling diabetes with its complications [4]. Therapeutic strategy for the management of diabetes is to decrease PPHG. It is done by the hindrance in glucose absorption by inhibiting carbohydrate metabolizing enzymes (alpha-amylase and alpha-glucosidase) in the gastrointestinal tract. Alpha-glucosidase inhibitors block the action of α-glucosidase enzymes in the small intestine, which limit the conversion of oligo and disaccharides into monosaccharides [23].
The plant under study exhibited α-glucosidase (maltase) inhibition. Methanol extract was found to show the most effective anti-diabetic activity among all the extracts analyzed. Although the extracts (chloroform and n-hexane) have DPPH inhibition (antioxidant activity) at a remarkable level but have less hypoglycemic activity. The study suggests that methanol solvent is efficient to extract hypoglycemic constituents from this plant and this extract exhibiting the maximum activity makes it a target for the isolation and characterization of responsible constituents. DPPH, a free radical has a deep violet colour. It accepts a hydrogen (H) atom or an electron and converts into a stable diamagnetic molecule [24]. The value of absorbance decreases due to the presence of phenolic molecule and the reaction of free radical with antioxidant compound. This study showed that the chloroform extract, which contains the highest content of flavonoids and polyphenols, exhibited the highest antioxidant activity.
The phytochemical investigation of this plant exhibited positive results for triterpenoids, saponins, flavonoids, phenolics, alkaloids, steroids and tannins. The bioactive compounds obtained from various plants have been reported to have anti-diabetic effects. Triterpenoids, saponins, flavonoids and phenolics have a positive correlation as anti-diabetic agents [25,26].
Previously, anti-diabetic activity of another variety of this plant has been reported in the literature. Ethanol extract (70 %) prepared from the whole plant was evaluated for this study in alloxaninduced hyperglycemic mice. The plant exhibited a significant blood glucose lowering effect (303.86 ± 11.71 mg/dL) when compared with that of the diabetic-untreated group (521.57 ± 17.84 mg/dL) [27]. It can be hypothesized by the results obtained in correlation with the previous reports that there is a significant enzyme inhibition by alcoholic extract.
The anti-diabetic activity shown by the various extracts may be due to saponins, triterpenoids, polyphenols, flavonoids or other phytocomponents present in the plant. Therefore, further investigations are required to isolate and characterize terpenoids, saponins or other phytoconstituents responsible for α-glucosidase inhibition.

CONCLUSION
F. indica Var. indica possesses anti-diabetic activity. Thus, the plant is a potential source of effective anti-diabetic therapeutic agent. Furthermore, the results lend some support for the traditional use of this plant in the management of diabetes mellitus.