Antioxidant, Antidiabetic, and Anti-obesity Potential of Ipomoea reptans Poir Leaves

Ipomoea reptans Poir or kangkung is a popular leafy vegetable, a favorite to people in Asian countries. However, limited information is available on their bioactivities. In the present study, the antioxidant, antidiabetic, and anti-obesity potential of I. reptans leaves were investigated. Different fractions (ethanol, ethyl acetate, and hexane) of I. reptans leaves were evaluated for their scavenging activity on DPPH radicals, whereas their reducing potential was investigated by cupric reducing antioxidant capacity (CuPRAC), total antioxidant, and reducing power assays. The antidiabetic potential was investigated by their inhibition effect on α-glucosidase. Total phenolic and flavonoid contents of I. reptans leaves were solvent dependent. Ethyl acetate contained the highest phenolic content, followed by ethanol and hexane fractions. However, for flavonoid content, the order was ethanol > ethyl acetate > hexane. All fractions showed DPPH scavenging activity in a concentration-dependent manner, with activities weaker than standards ascorbic acid and BHT, in the order of ethanol > ethyl acetate > hexane. All fractions showed reducing capacity, but only hexane and ethanol fractions of I. reptans leaves showed inhibition on α-glucosidase, with hexane showed more potent inhibition compared to acarbose. The study also found that fractions of I. reptans inhibit lipase and trypsin, enzymes related to lipid metabolism. Findings in this study offer a prospect for I. reptans leaves as a functional food source for antioxidant, antidiabetic, and antiobesity purposes.


INTRODUCTION
Ipomoea reptans Poir or kangkung is a popular leafy vegetable, a favorite to people in Asian countries. However, limited information is available on their bioactivities. In the present study, the antioxidant, antidiabetic, and anti-obesity potential of I. reptans leaves were investigated. Different fractions (ethanol, ethyl acetate, and hexane) of I. reptans leaves were evaluated for their scavenging activity on DPPH radicals, whereas their reducing potential was investigated by cupric reducing antioxidant capacity (CuPRAC), total antioxidant, and reducing power assays. The antidiabetic potential was investigated by their inhibition effect on α-glucosidase. Total phenolic and flavonoid contents of I. reptans leaves were solvent dependent. Ethyl acetate contained the highest phenolic content, followed by ethanol and hexane fractions. However, for flavonoid content, the order was ethanol > ethyl acetate > hexane. All fractions showed DPPH scavenging activity in a concentration-dependent manner, with activities weaker than standards ascorbic acid and BHT, in the order of ethanol > ethyl acetate > hexane. All fractions showed reducing capacity, but only hexane and ethanol fractions of I. reptans leaves showed inhibition on α-glucosidase, with hexane showed more potent inhibition compared to acarbose. The study also found that fractions of I. reptans inhibit lipase and trypsin, enzymes related to lipid metabolism. Findings in this study offer a prospect for I. reptans leaves as a functional food source for antioxidant, antidiabetic, and antiobesity purposes.
degenerative diseases, such as cardiovascular diseases and diabetes mellitus. However, the harmful effects of oxidative stress can be prevented by the consumption of antioxidants (Ayala et al., 2014;Lobo et al., 2010). In this case, antioxidant compounds may reduce oxidative stress conditions by stabilizing free radicals by donating protons or electrons or chelating pro-oxidant metal ions (Tan et al., 2018;Kurutas, 2016).
Type 2 Diabetes mellitus (T2DM) is a metabolic disorder characterized by a high level of postprandial blood glucose. This condition can be due to insufficient insulin secretion, or resistance to insulin action, or a combination of both (American Diabetes Association, 2009). It has been known that persistent hyperglycemia induces oxidative stress through multiple interacting pathways, including activation of protein kinase C, activation of the polyol pathway, and increased formation of the advanced glycation end product (Giacco & Brownlee, 2010;Mohora et al., 2007). Besides, the resulting oxidative stress may further damage the pancreatic β-cells, which produce insulin. In addition to oxidative stress, studies have shown that obesity could also increase the risk for T2DM (Stokes et al., 2018). Current management in T2DM and obesity include inhibition of key enzymes related to carbohydrate and lipid metabolisms. Examples of this type of medication include acarbose and orlistat for T2DM and obesity, respectively (Vieira et al., 2019). However, these synthetic inhibitors seem to exert significant adverse side effects that potentially interfere with their clinical uses, such as abdominal discomfort, liver problems, and lactic acidosis (Saha & Verma, 2012). Consequently, there is a need for other alternatives. One possible option could come from natural inhibitors of plant origin, including vegetables and fruit. They have gained global considerations for screening bioactive compounds of medicinal attributes, including antioxidant, antidiabetic, and anti-obesity activities (Choudhury et al., 2018). Besides, high consumption of vegetables and fruit has been recognized to positively correlate with decreased risk of chronic and nondegenerative diseases, such as cardiovascular disease, cancer, and diabetes mellitus (Carter et al., 2010;van't Veer et al., 2000).
Ipomoea reptans (synonym: Ipomoea aquatica), locally knows as 'kangkung,' in Indonesia is a green leafy vegetable distributed widely in the South and Southeast Asia region Indonesia, Malaysia, and India. It belongs to the family Convolvulaceae. Ipomoea reptans is an aquatic plant, easily cultivated in muddy or moist soil. It has a long, hollow, and tender shoot. The leaves are long, heartshaped, and rich with high nutrients, including vitamin A and C and essential minerals such as calcium and iron (Dewanjee et al., 2015;Rahman & Parkpain, 2004).  (Saha et al., 2008). A recent study showed that the antidiabetic activity could be due to the protective effect of I. reptans on the pancreatic β-cells (Hayati et al., 2017). Furthermore, I.
reptans extract was prepared as a nano-emulsifying drug and showed antihyperglycemic activity using the zebrafish (Danio rerio) model (Hayati et al., 2018). The present study sought to investigate antioxidant activity and possible inhibition on α-glucosidase, lipase, and trypsin by I. reptans leaves and its fractions in several solvent systems using in vitro methods.

Materials
Spectrophotometer measurements were carried out using a Biochrom Libra-S22 (Cambridge, UK

Plant material and extract preparation
The leaves of I. reptans were collected from the

Estimation of total phenolic content
Total phenolic content (TPC) of each fraction was determined based on a Folin-Ciocalteu's method reported previously (Khatoon et al., 2013). Gallic acid (12.5 -200 μg/mL) was used to generate a standard curve. Results were presented as mg gallic acid equivalent (mgGAE)/g dried biomass.

Total flavonoid content
Total flavonoid content (TFC) of each fraction was determined based on an AlCl3 colorimetric method, as reported previously (Simamora et al., 2018a). Quercetin (3.20 -200 μg/mL) was used to generate a standard curve. Results were presented as mg quercetin equivalent (mgQE)/g dried biomass.

DPPH radical scavenging assay
The ability of different I. reptans fractions to scavenge DPPH radicals were evaluated based on a reported method (Simamora et al., 2018b)

CuPRAC assay
Cupric ion reducing antioxidant capacity (CuPRAC) assay was carried out based on a method described previously (Aktumsek et al., 2013). A reaction mixture was prepared to contain 1 mL of 10 mM CuCl2, 1 mL of 7.5 mM neocuproine in ethanol, and 1 mL of 1 M NH4OAc buffer (pH 7.0). Into this mixture was added 0.5 mL extract solution and 0.6 mL water to make a total volume of 4.1 mL. The reaction mixture was incubated at room temperature for 30 minutes, and the absorbance was measured at 450 nm. Trolox (10 -320 μg/mL) was used to prepare a standard curve, and results were reported as mg Trolox equivalent (mgTE)/g dried material.

Total antioxidant assay
Each fraction's total antioxidant activity was determined by a phosphomolybdenum method described previously (Prieto et al., 1999 Reducing power assay Reducing power assay was carried out based on a ferric thiocyanate method reported previously (Gülçin et al., 2012). Reaction mixture was prepared containing 1 mL test solution, 2.5 mL of 0.2 M phosphate buffer pH 6.6, and 2.5 mL of 1% (w/v) potassium ferric cyanide K3[Fe(CN)6]. The reaction mixture was incubated in a water bath at 50°C for 20 minutes and then cooled at room temperature. This mixture was added with 2.5 mL of 10% (w/v, water) trichloroacetic acid, followed by centrifugation of the mixture at 3000 rpm for 10 minutes.
The upper layer (2.5 mL) was taken out and mixed with 0.5 mL of 1% (w/v, water) FeCl3 and 2.5 mL water. The absorbance was measured at 700 nm on a spectrophotometer. Ascorbic acid (1.56 -100 μg/mL) was used to generate a standard curve, and results were reported as mg ascorbic acid equivalent (mgAAE)/g dried biomass.

α-Glucosidase inhibition assay
Inhibition of α-glucosidase was assayed by a previously reported method . In this assay, p-

Qualitative test for lipase inhibition
Inhibition on lipase was assayed by a qualitative method of a phenol red agar plate reported previously (Gupta et al., 2015), with some modifications. In this method, agar (2%, w/v) was suspended with phenol red indicator solution was suspended into a circular well in the agar, and the reaction was incubated for ten minutes at 37°C.
Lipolytic degradation releases fatty acids from the substrate, which changes the indicator color from yellow to red.

Qualitative test for trypsin inhibition activity
Inhibition activity of I. reptans leaves fractions on trypsin was evaluated based on a qualitative method as reported before (Vijayaraghavan & Vincent, 2013), with some modifications. An agar plate was prepared by dissolving agar (1.5%, w/v) added with skimmed milk (5%, w/v).
The agar solution was poured into Petri dishes and let to solidify. Wells of 5 mm were punched. The test solution was prepared by mixing trypsin solution (10 mg in 10 mL of 100 mM tris buffer HCl pH 7.6) with fractions of I.
reptans leaves or natrium diclofenac (positive control) in a 1 : 1 ratio. A 50 µL of each test solution was loaded into each well and incubated overnight at 37°C. Trypsin inhibition was observed by a decrease in zone diameter in the presence of inhibitors.

Statistical analysis
All experiments were conducted in three replicates, and results were presented as mean ± SD. The significance of difference among multiple averages was determined by analysis of variance (ANOVA), followed by a Tukey post hoc test at a 5% significance level.

Total phenolic and flavonoid contents
In this study, the leaves of I. reptans were extracted using ethanol, and partitioned by hexane and ethyl acetate.

In vitro antioxidant activities
One of the antioxidant mechanisms of action is removing free radicals, which can be achieved by transferring protons or electrons from antioxidant compounds to the free radicals (Lobo et al., 2010). In the present study, the radical scavenging activity of I. reptans fractions was evaluated using stable DPPH radicals. The use of DPPH radicals may be relevant to represent a lipophilic radical initiated by lipid auto-oxidation (Shukla et al., 2016). It was proposed that the scavenging mechanism for DPPH to form the non-radical DPPH-H is predominantly through proton transfer (Marxen et al., 2007).  Phenolic and flavonoid compounds are known to be strong proton donors (Paixão et al., 2007). Quercetin derivative isolated from I. reptans was shown to have a potent DPPH radical scavenging activity (Prasad et al., 2005 (Choirunnisa et al., 2016). It is known that the ease of each metal ion to be reduced to a lower oxidation state depends on the redox potential of each metal ion.  (Saha et al., 2008). This study used STZ induced diabetic rats and observed a decrease in MDA level and an increase in GSH level in the liver, pancreas, and kidney tissue of extract-treated rats, indicating a lower oxidative stress condition to extract treatment. These results complement those observed in DPPH scavenging activity; thus, I. reptans can act as radical scavengers and reducing agents.

In vitro antidiabetic activity
As α-glucosidase hydrolyzes the catalytic degradation of polysaccharides or oligosaccharides into glucose, this enzyme has become a therapeutic target for regulating blood glucose levels. In vitro antidiabetic activity of ethanol, ethyl acetate, and ethanol fractions of I. reptans was evaluated by examining their inhibition effect on αglucosidase activity. In this study, acarbose, a standard αglucosidase inhibitor, was used as a positive control. showed no inhibition activity on α-glucosidase.  (Saha et al., 2008). The observed hypoglycemic activity in this animal model may be due to the inhibition activity of I.

Results in
reptans leaves on α-glucosidase. Various phenolic and flavonoid compounds have been reported to inhibit αglucosidase in vitro (Limanto et al., 2019;Yin et al., 2014).
However, the lack of activity observed in ethyl acetate indicates that α-glucosidase inhibition activity is not only attributed to phenolic and flavonoid compounds.

In vitro anti-lipase activity
In vitro anti-obesity activity for ethanol and ethyl acetate fractions were conducted based on inhibition activity on lipase. In the present study, a qualitative method using a phenol red agar plate was used, and the results can be seen in Figure 1.

In vitro anti-trypsin activity
Trypsin has been studied for its role in treating obesity