Inhibitory Effects of Colocasia esculenta (L.) Schott Constituents on Aldose Reductase

The goal of this study was to determine the rat lens aldose reductase-inhibitory effects of 95% ethanol extracts from the leaves of C. esculenta and, its organic solvent soluble fractions, including the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) layers, using dl-glyceraldehyde as a substrate. Ten compounds, namely tryptophan (1), orientin (2), isoorientin (3), vitexin (4), isovitexin (5), luteolin-7-O-glucoside (6), luteolin-7-O-rutinoside (7), rosmarinic acid (8), 1-O-feruloyl-d-glucoside (9) and 1-O-caffeoyl-d-glucoside (10) were isolated from the EtOAc and BuOH fractions of C. esculenta. The structures of compounds 1–10 were elucidated by spectroscopic methods and comparison with previous reports. All the isolates were subjected to an in vitro bioassay to evaluate their inhibitory activity against rat lens aldose reductase. Among tested compounds, compounds 2 and 3 significantly inhibited rat lens aldose reductase, with IC50 values of 1.65 and 1.92 μM, respectively. Notably, the inhibitory activity of orientin was 3.9 times greater than that of the positive control, quercetin (4.12 μM). However, the isolated compounds showed only moderate ABTS+ [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] activity. These results suggest that flavonoid derivatives from Colocasia esculenta (L.) Schott represent potential compounds for the prevention and/or treatment of diabetic complications.


Introduction
According to the World Health Organization (WHO), approximately 200 million people worldwide suffer from diabetes, and it is estimated that this disease will have a severe impact on human health by 2025 [1]. Aldose reductase (AR) is a key enzyme in the polyol pathway that plays important roles in cataract formation and the pathogenesis of diabetic complications such as neuropathy, nephropathy, and retinopathy [2]. Therefore, there is a growing interest in searching for drugs to alleviate the symptoms of diabetic complications. AR is an NADPH-dependent oxidoreductase and it is an important enzyme in the polyol pathway, which catalyses the reduction of glucose to sorbitol, which is further metabolized to fructose by sorbitol dehydrogenase [3]. Thus, AR inhibition represents a key point for the prevention and attention of long-term diabetic complications [4].
As an abundant source of bioactive chemicals plants are an important resource for the development of new drugs [5]. Many flavonoids and polyphenols effectively inhibit AR [6][7][8], and in most cases, herbal medicines lack toxicity and side effects [9][10][11]. Oxidative stress contributes to the progression of diabetes and its complications. Diabetes is usually accompanied by free radical production and impaired antioxidant defenses [12].
Colocasia esculenta (L.) Schott, commonly known as taro, is a tropical perennial plant that is native to Asia and the Pacific, and widely distributed in tropical latitudes [13]. It is a starchy root crop with wide leaves, that are edible. Taro is the main food source for approximately 500 million people living in Asia, Africa, Middle America, and the Pacific Islands [14]. C. esculenta is reported to display anti-diabetic, anti-inflammatory, anti-oxidant and anti-cancer activities [15]. Studies on the chemical constituents of C. esculenta have reported the presence of pelargonidin-3-glucoside, cyanindin-3-rhamnoside, cyanidin-3-glucoside, orientin, isoorientin, vitexin, isovitexin and luteoin-7-O-sophoroside [16]. However, no studies have investigated the AR inhibitory activity constituents from C. esculenta leaves.
The aim of this study was to investigate the in vitro inhibitory effects of Colocasia esculenta (L.) Schott extract and its isolated constituent on AR enzyme activity. We assessed the ability of the major compounds to decrease galactitol accumulation in the lens of a galactosemic rat model ex vivo and their antioxidant effects.

Results and Discussion
In most cases, natural herbal medicines lack toxic and side effects, so there is growing interest in natural products as sources of new drugs [17]. For years, many medicinal plants and their extracts have been demonstrated to effectively treat diabetes [18]. The purpose of this study was to identify new AR inhibitors (ARI) from Colocasia esculenta (L.) Schott for the treatment of diabetic complications.
Flavonoids are reported to dramatically inhibit aldose reductase. In particular, quercetin, quercitrin, and myricitrin are lead compounds that preceded the discovery of tolrestat. In order to confirm the type of rat aldose reductase inhibitory activity caused by compounds 2 and 3, we performed a kinetic study using different concentrations of DL-glyceraldehyde as a substrate (concentration: 0.1-1 mM). Kinetic analysis using Lineweaver-Burk plots of 1/velocity and 1/concentration for compounds 2 and 3 are shown in Figure 2. Changes in substrate concentration resulted in different slopes and x-axis intersects for the uninhibited enzyme and different concentrations of the compounds.
The K m (Michaelis-Menten constant) was unchanged, while the maximum velocity (V max ) decreased. Therefore, compounds 2 and 3 are noncompetitive, indicating that the inhibitor were unable to bind the substrate binding area or the NADPH binding area. Next, we calculated the inhibitory constant (K i ) from secondary Lineweaver-Burk plots, and the compounds display K i values of 3.23 × 10 −6 M and 5.88 × 10 −6 M, respectively. The lower K i value implies tighter binding with the free enzyme AR or the enzyme substrate complex, so compound 2 is expected to be a more effective inhibitor than compound 3 [28]. In diabetes, activation of the polyol pathway leads to the accumulation of sorbitol in various tissues. Excess glucose causes flux through the polyol pathway, which significantly increase AR activity and the accumulation of sorbitol [29]. Galactose produces a greater accumulation of polyol than glucose, because it has a higher affinity for AR [29][30][31]. Thus, we evaluated the effects of compounds 2 and 3 on galactitol accumulation in rat erythrocytes and lenses. Incubation of rat lenses for 6 days in 30 mM galactose increased the intracellular accumulation of galactitol. As shown in Table 3, compound 2 and compound 3 decreased galactitol accumulation in rat erythrocyte by 16.1% and 35.2%, respectively at 5 µg/mL (quercetin = 47.9%). These compounds also inhibited galactitol accumulation in isolated rat lenses by 9.1% and 2.7% at 5 µg/mL, respectively (quercetin = 3.8%).  Oxidative stress plays a key role in the pathogenesis of vascular complications in diabetes, and it provides an early marker of such damage in the development of endothelial dysfunction [32]. Therefore, we evaluated the antioxidant activity of C. esculenta and its isolated constituents. Only the EtOAc fraction showed minor activity in the ABTS + assay ( Table 4). The ABTS + inhibitory activities of the isolated constituents were tested, and trolox used as the positive control. Except for compounds 1, 4 and 5, all compounds exhibited ABTS + inhibitory activity (Table 5).

Chemicals and Reagents
DL-Glyceraldehyde, the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin (BSA), sodium phosphate and quercetin used in this study were purchased from Sigma (St. Louis, MO, USA). Human recombinant aldose reductase was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals and reagents used were of analytical grade.

Plant Materials
Colocasia esculenta (L.) Schott was purchased from Dae Kwang Herb Medicine Co., Ltd. (Chuncheon, Korea) and the voucher specimen (No. RIC-1021) was deposited at Regional Innovation Center, Hallym University, Republic of Korea.

Extraction and Isolation
Fresh Colocasia esculenta (L.) Schott (7.3 kg) leaves were extracted with 95% ethanol (60 L × 2 times) for 2 h at 100 °C. The combined filtrates were concentrated to dryness in vacuo at 40 °C. The extract was suspended in distilled water and partitioned sequentially with n-hexane, methylene chloride (CH 2 Cl 2 ), ethyl acetate (EtOAc) and n-butanol (BuOH), respectively. The EtOAc fraction showed strong inhibitory activity on AR, so this fraction (7 g

Preparation of Aldose Reductase
Crude rat lenses aldose reductase (rAR) was prepared as follows: lenses were removed from Sprague-Dawley rats weighing 250-280 g and frozen at −70 °C until use. The rat lens homogenate was prepared according to the method of Hayman and Kinoshita with some modifications [33][34][35]. Non-cataractous transparent lenses were pooled and homogenate was prepared in 0.1 M phosphate buffer saline (pH 6.2). After centrifugation at 10,000 rpm for 20 min in a refrigerated centrifuge, the supernatant, which was then collected and as the rAR, all procedures were carried out at 4 °C.

Determination of Aldose Reductase Inhibition in Vitro
AR activity was assayed spectrophotometrically by measuring the decrease in the absorption of NADPH at 340 nm over a 4-min period according to the method of Hayman and Konoshita with some modifications, using DL-glyceraldehyde as the substrate. Each 1.0 mL cuvette contained equal units of the enzyme, 0.10 M sodium phosphate buffer (pH 6.2), 0.3 mM NADPH, with or without 10 mM of the substrate and an inhibitor [10,36]. The concentration of inhibitors giving 50% inhibition of enzyme activity (IC 50 ) calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity.

Kinetics of Recombinant Human Aldose Reductase
Reaction mixtures consisted of 0.1 M potassium phosphate, 0.16 mM NADPH, 2 mM of recombinant human aldose reductase (rhAR) with varied concentrations of substrate DL-glyceraldehyde and AR inhibitor in a total volume of 200 µL. Concentrations were ranged from 0.1 to 1 mM for DL-glyceraldehyde, from 0.1 to 1 mM for active compound. Recombinant human aldose reductase activity was assayed spectrophotometrically by measuring the decrease in absorption of NADPH at 340 nm after substrate addition using a Bio Tek Power Wave XS spectrophotometer (Bio Tek Instruments, Winooski, VT, USA) [34].

Lens Culture and Intracellular Galactitol Measurement
Lenses isolated from 10-week-old male rats were cultured for 6 days in TC-199 medium that contained 15% fetal bovine serum 100 units/mL penicillin and 0.1 mg/mL streptomycin under sterile conditions in an atmosphere of 5% CO 2 and 95% air at 37 °C. Samples were dissolved in dimethyl sulfoxide. The lenses were divided into five groups and cultured in medium containing 5 mM glucose, 30 mM galactose and rosmarinic acid or caffeic acid ethylene ester. Each lens was placed in well containing 1.0 mL of medium. Galactitol was determined by HPLC after its derivatization by reaction with benzoic acid to a fluorescent compound [37].

Blood Culture and Intracellular Galactitol Measurement
Blood sample was collected in heparin containing polypropylene tube from 10-week-old male rats. For sugar and sugar alcohol analysis, erythrocytes from heparinized blood were separated from the plasma and buffy coat by centrifuging at 2000× g for 10 min. The cells were washed thrice with normal saline (0.9% NaCl) at 4 °C. In the final washing, the cells were centrifuged at 2000× g for 10 min to obtain a consistently packed cell preparation. The packed cells (1 mL) were then incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) containing 30 mM galactose in the presence or absence of samples at 37 °C in 5% CO2 for 3 h. The erythrocytes were washed with cold saline by centrifuging at 2000× g for 10 min, precipitated by adding 6% of cold perchloric acid (3 mL), and centrifuged again at 2000× g for 10 min. The supernatant was neutralized with 2.5 M K 2 CO 3 at 4 °C and used for galactitol determination [38]. HPLC analysis for sugar and sugar alcohol in blood was performed with this supernatant of red blood cell homogenate after being benzoylated.

ABTS + Assay
The method of Re et al., [39] was used with slight modifications. ABTS diammonium salt (2 mM) and potassium persulfate (3.5 mM) were mixed, diluted in distilled water and kept in the dark at room temperature for 24 h before use. After addition of ABTS + solution to 10 µL of antioxidant compounds were recorded at after 10 min reaction. The percentage inhibition of absorbance at 750 nm is calculated and potted as a function of concentration of antioxidants. Trolox was used as positive control.

Conclusions
The present study isolated ten compounds from the leaves of C. esculenta. Among the isolated compounds orientin (2) and isoorientin (3) significantly inhibited rat lens aldose reductase with IC 50 values of 1.65 and 1.92 μM, respectively. Specifically, the inhibitory activity of compound 2 was 3.9 times greater than that of the positive control (quercetin = 4.12 μM). Kinetic analysis using Lineweaver-Burk plots of 1/velocity and 1/concentration indicate that compounds 2 and 3 are noncompetitive inhibitors. Our results indicate that flavonoids 2 and 3 isolated from C. esculenta leaves prevented the accumulation of sorbitol in rat lenses. On the basis of AR inhibition, we conclude that compounds 2 and 3 have therapeutic potential for preventing and treating diabetic complications, although further clinical research is needed.