The Antioxidant and Hypoglycemic Properties and Phytochemical Profile of Clusia latipes Extracts

Diabetes mellitus has become one of the primary threats to human health, with its rapidly increasing prevalence, and gravely debilitating clinical complications1. Over the past decade, diabetes prevalence has risen faster in low and middle-income countries than in high-income countries. The World Health Organization (WHO) has projected that DM will be the 7th leading cause of death in 2030. Diabetes is a chronic disease that occurs when the pancreas does not produce sufficient insulin or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar. Hyperglycemia or high blood sugar is a common effect of uncontrolled diabetes, which might lead to serious heart, blood vessel, eye, kidney, and nerve damage over time 2,3. Type 2 diabetes mellitus (T2DM), which is the most common form of diabetes, is produced by the ineffective use of insulin in the body and is characterized by disorders of insulin action and secretion. Either one of these mechanisms could be the predominant feature, but generally, when diagnosed, both are manifested1. In T2DM, particularly, it is believed that oxidative stress caused by overproduction of reactive oxygen species (ROS) is the cause of vascular complication development4. Hyperglycemic condition of DM patients will produce tissue damage via the formation of ROS through five major mechanisms that have been shown to be activated by mitochondrial ROS overproduction: increased flux of glucose and other sugars through the polyol pathway; increased formation of advanced glycation end products; increased expression of the receptor for advanced glycation endproducts (RAGE) and its activating ligands; activation of protein kinase C isoforms; and over-activity of the hexosamine pathway5-7.


INTRODUCTION
Diabetes mellitus has become one of the primary threats to human health, with its rapidly increasing prevalence, and gravely debilitating clinical complications 1 . Over the past decade, diabetes prevalence has risen faster in low and middle-income countries than in high-income countries. The World Health Organization (WHO) has projected that DM will be the 7th leading cause of death in 2030. Diabetes is a chronic disease that occurs when the pancreas does not produce sufficient insulin or when the body cannot effectively use the insulin it produces. Insulin is a hormone that regulates blood sugar. Hyperglycemia or high blood sugar is a common effect of uncontrolled diabetes, which might lead to serious heart, blood vessel, eye, kidney, and nerve damage over time 2,3 . Type 2 diabetes mellitus (T2DM), which is the most common form of diabetes, is produced by the ineffective use of insulin in the body and is characterized by disorders of insulin action and secretion. Either one of these mechanisms could be the predominant feature, but generally, when diagnosed, both are manifested 1 . In T2DM, particularly, it is believed that oxidative stress caused by overproduction of reactive oxygen species (ROS) is the cause of vascular complication development 4 . Hyperglycemic condition of DM patients will produce tissue damage via the formation of ROS through five major mechanisms that have been shown to be activated by mitochondrial ROS overproduction: increased flux of glucose and other sugars through the polyol pathway; increased formation of advanced glycation end products; increased expression of the receptor for advanced glycation endproducts (RAGE) and its activating ligands; activation of protein kinase C isoforms; and over-activity of the hexosamine pathway [5][6][7] .
Alpha-glucosidase is a critical enzyme that catalyzes the cleavage of absorbable monosaccharides starting with disaccharides and oligosaccharides 8 . In this manner, α-glucosidase inhibitors reduce postprandial hyperglycemia by slowing intestinal carbohydrate digestion 9 . Alpha-glucosidase inhibitors are capable of suppressing postprandial hyperglycemia; they are generally used to prevent or treat type II diabetes 10 .
Plants play an important role in health care and are an important source of potentially bioactive substances 11 . The genus Clusia is widely distributed in the tropical and subtropical regions of Central and South America 12 . In the species of this genus, a great variety of biological activities have been found: broad-spectrum antimicrobial activity; chemopreventive cancer effects and antioxidant activity 13 ; anti-inflammatory and anti-hepatotoxic activity and inhibitory action of the human immunodeficiency virus (HIV) [14][15][16] ; and cytotoxic activity 17,18 . In the present study, both the antioxidant and anti-glycemic activity of leaf and stem extracts of C. latipes were evaluated.

Plant material
The leaves and stems of Clusia latipes Planch. & Triana, Clusiaceae were collected in Gonzanama-Quilanga in the Loja province of Ecuador, and species identification was made by Fani Tinitana, PhD. A voucher specimen (PPN-CI 002) was deposited at the Herbarium of the Universidad Técnica Particular de Loja, Ecuador.

Extraction and partitions of Clusia latipes
For 7 days at 30°C, the plant material was dried in a tray dryer with airflow and then manually pulverized. Leaves and stems were processed separately. The pulverized leaves (1540 g) and stems (1608 g) were extracted by static maceration at ambient temperature with hexane (Hex), ethyl acetate (EtOAc), and methanol (MeOH) sequentially during three days with each solvent 18 . The procedure was repeated three times and concentrated on a rotary evaporator (Buchi R210; Switzerland, Flawil) at 50 mbar and 35°C, to yield a total of six extracts: leaf extracts, 71.00 g (Hex-L), 28.52 g (EtOAc-L), and 283.87 g (MeOH-L); and stem extracts, 39.44 g (Hex-S), 44.23 g (EtOAc-S), and 117.23 g (MeOH-S).
The most active extracts were partitioned. A portion of the dried methanolic leaf (20 g) and stem (20 g) extracts were then dissolved in methanol:water 9:1 v/v and sequentially partitioned three times with 400 mL of each solvent [(Hexano (Hex), dichloromethane (DCM), and ethyl acetate (EtOAc)] using a separatory funnel at room temperature ( Figure 1). The solvents were removed using a rotary evaporator (Buchi R210; Switzerland, Flawil) at 35°C under vacuum. From the Hex leaves fraction (F-Hex-L), 0.59 g was obtained. The Hex stems fraction (F-Hex-S) yielded 0.19 g, the DCM leaves fraction (F-DCM-L) 5.21 g, the DCM stem fraction (F-DCM-S) 10.28 g, the EtOAc leaves fraction (F-EtOAc-L) 1.82 g, the EtOAc stems fraction (F-EtOAc-S) 2.02 g, the aqueous leaves fraction (F-Aq-L) 11.15 g, and the aqueous stems fraction (F-Aq-S) 6.37 g.

Phytochemical screening
Using the most active extracts and their partitions phytochemical tests were done. Phytochemical screening to test for the presence of secondary metabolites (alkaloids, flavonoids, quinones, saponins, tannins, and terpenoids-steroids), carbohydrates, and fats in the extracts and fractions was carried out using standard procedures. Phytochemical screening results from tests on extracts and fractions revealed the presence or absence of the main secondary metabolites and other phytochemicals based on the presence (+) or absence (-) of expected color changes. The tests performed were based on those reported in the literature 19,20 .

Isolation of secondary metabolites
The most active sample, the EtOAc leaves fraction (1.82 g), was separated by column chromatography with C18-reversed-phase silica gel (40-

Characterization and identification of secondary metabolites
The melting point was determined using a Fisher Johns apparatus (Fisher Scientific Company, USA), and the temperature was not corrected. The 1 H (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on Varian 400 MHz-Premium Schelded equipment (Agilent Technologies, USA). CD 3 OD was used as the solvent, and chemical shifts were expressed in parts per million (ppm). Coupling constants (J) were reported in Hz.
DPPH radical scavenging assay DPPH free radical scavenging capacity was evaluated with a microplate analytical assay according to the literature 21,22 , with slight modifications. A 30-μl aliquot of the different sample concentrations and standard were mixed with 270 μl of DPPH • in methanol solution 100 μM (O.D. adjusted to 1.1 at 515 nm). After incubation at 20°C for 60 min, the absorbance of each solution was measured using a microplate reader (EPOCH 2 BioTek; USA, Vermont) at 515 nm. The percentage of scavenging activity was determined by following the equation described by Cheng et al. (2006): (1) The SC 50 value was obtained through interpolation from linear or logarithmic regression analyses according to the behavior of the data, and the sample concentrations required to scavenge 50% of the DPPH radical were determined. Trolox was used as the reference compound.

ABTS radical scavenging assay
For the ABTS assay, the procedure followed the method of Thaipong et al. (2006) with slight modifications. From the different concentrations, a 30-μl aliquot was taken, and a 270 μl of ABTS •+ solution (ABTS 7.4 mM and 2.6 mM persulfate, 1:1 ratio) was added to a 96-well microplate assay. This was incubated at 20°C for 60 min, and the absorbance of each solution was recorded at 715 nm in a microplate reader (EPOCH 2 BioTek; USA, Vermont). The percentage of ABTS •+ scavenging by the sample was calculated using the Eq. (1). Trolox was used as the reference compound.

α-glucosidase inhibitory activity
Ten milligrams of each sample was dissolved in 1 ml of methanol:H 2 O (1:1 ratio). In cases of complete inhibition, dilutions of the sample solution were made in phosphate-buffered saline (PBS; SIGMA).
The α-glucosidase enzyme inhibitory effect was determined using a 96well microtiter plate with p-nitrophenyl-α-D-glucopyranoside (pNPG; SIGMA) as substrate according to the methods described by Tao et al. (2013), with slight modifications 23 . First, 5 μl of the sample was mixed with 75 μl of PBS (SIGMA) and 20 μl of enzyme solution (0.15 U/ml in PBS pH 7.4; SIGMA). This mixture was then pre-incubated at 37°C for 5 min. After preincubation, 20 μl of pNPG (5 mM in phosphate buffer, pH 7.4) was added and then incubated at 37 °C. Acarbose (5 mg/ml) was used as a positive control. The amount of p-nitrophenol (p-NP) released was measured at 405 nm for 60 min, recording the absorbance every 5 min on a spectrophotometer microplate reader (EPOCH 2 BioTek; USA, Vermont). The results were expressed as percentage inhibition using the formula previously described 24 : (2) in which Ao is the absorbance recorded for the enzymatic activity without inhibitor (control) and As is the absorbance recorded for the enzymatic activity in the presence of the inhibitor (sample test). The IC 50 was calculated using GraphPad Prism v 5.0 software.

RESULTS
Regarding antioxidant capacity, both in the DPPH free radical elimination activity test and ABTS free radical test, we found that the greater the polarity of the extracts, the greater the antioxidant capacity. Thus, leaf and stem methanol extracts had SC 50 values of 6.44 μg/ml and 6.77 μg/ml, respectively. In the ABTS free radical test, the methanolic extracts present SC 50 values of 5.43 μg/ml for leaves and 4.59 μg/ml for stems (Table 1).
On the other hand, the α-glucosidase inhibitory activity was observed in vitro in all samples. The positive acarbose control had an IC 50 of 377 μM (243.39 μg/ml), which was in good agreement with the results reported by Feng et al. (2011) 25 . Similar to the α-glucosidase inhibitory activity, the samples that showed high activities were of methanolic extracts of leaves and stems with IC 50 values of 5.01 μg/ml and 2.30 μg/ ml, respectively (Table 1).
Given the activity of the extracts, the most effective extracts were fractionated according to the scheme presented in Figure 1. The results obtained from the phytochemical examination revealed the presence of alkaloids, carbohydrates, flavonoids/xanthones, quinones, saponins, and tannins ( Table 2).
The antioxidant capacity and α-glucosidase inhibitory activity were evaluated from the fractions of methanol extracts obtained from leaves and stem (Table 3). The antioxidant activities of the DCM, EtOAc, and aqueous leaf and stem fractions increased significantly relative to the Hex fractions. The fractions with the highest biological activity are those obtained in EtOAc (F-EtOAc), both in leaves and stems. The SC 50 of F-EtOAc from leaves was 4.70 μg/ml, and for DPPH and 3.29 μg/ ml for ABTS, thus exhibiting higher antioxidant activity for ABTS. The F-EtOAc stems had a DPPH antioxidant capacity of SC 50 : 3.58 μg/ml and ABTS of 2.27 μg/ml (Table 3).
Similar to the extracts, the fractions with the highest antioxidant capacity also have the highest α-glucosidase inhibitory activity. Thus, the F-EtOAc leaves and F-EtOAc stems exhibited potency, with IC 50 values ranging from 0.90 to 3.88 μg/ml (Table 3).
Based on these results, the F-EtOAc leaf fraction was separated by column chromatography to obtain a flavonoid glycoside, isoquercitrin (7.3 mg); the structure is shown in Figure 2. The structural characterization of this compound was carried out by spectroscopic and spectrometric analyses and by comparison with published data 26,27 .    All values were expressed as means ± standard error (n = 3). Table 3: DPPH and ABTS free radical-scavenging activity and α-glucosidase inhibitory activity of fractions obtained from methanolic extracts of C. latipes leaves and stems.

DISCUSSION
EtOAc fractions from methanolic extracts of leaves and stems had the most active DPPH antioxidant activity and were shown to be more effective than the Trolox positive control. The ranges of antioxidant capacity obtained are within similar ranges to other species of the same family, both for DPPH and ABTS [28][29][30][31] . Likewise, F-EtOAc leaf fractions had α-glucosidase inhibitory activity, similar to other species in the Clusiaceae family 29,32-34 . Flavonoids and xanthones have been isolated from some species of the Clusiaceae family [34][35][36][37][38] . There is a strong relationship between phenolic compounds from natural sources and α-glucosidase inhibition [39][40][41][42][43] . Isoquercitrin is of interest to the food and pharmaceutical industries because of its biological properties as anti-inflammatory, hypotensive, anti-mutagenesis, anti-oxidative, anti-depressant, hypolipidemic, and anti-viral effects 27,44,45 . It has been previously reported that isoquercitrin has several activities that are related to the control and prevention of diabetes 46,47 . Previously, it has been experimentally established that isoquercitrin has an α-glucosidase inhibitory activity with an IC 50 of 0.185 mM 48 .

CONCLUSION
The highest α-glucosidase inhibitory activity was detected in the ethyl acetate fraction obtained from leaf methanol extract, with a halfmaximal inhibitory concentration (IC 50 ) value of 0.90 μg/ml. In this study, we observed an association between α-glucosidase content and the antioxidant activities of the C. latipes fractions.
We propose that the α-glucosidase inhibitory and antioxidant activities of C. latipes could be due to the presence of isoquercitrin.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.