Antioxidative properties and inhibition of key enzymes linked to type-2 diabetes by snake tomato ( Tricosanthes cucumerina ) and two tomato ( Lycopersicon esculentum ) varieties

This study sought to compare the antioxidant properties [1,1-diphenyl–2 picrylhydrazyl (DPPH) and hydroxyl (OH) radicals scavenging abilities] and inhibition of Fe 2+ -induced lipid peroxidation and two key enzymes relevant to type-2 diabetes (α-amylase and α-glucosidase) of snake tomato (Trichosanthes cucumerina) with two tomato varieties [Lycopersicon esculentum Mill. var. esculentum (ESC) and Lycopersicon esculentum Mill. var. cerasiforme (CER)]. Snake tomato (0.84 mg/g) and CER (0.87 mg/g) had significantly (P < 0.05) higher total phenolic content than ESC (0.27 mg/g). However, CER had the highest total flavonoid content of 0.48 mg/g, compared to snake tomato (0.27 mg/g) and ESC (0.15 mg/g). In consonance with the phenolic content, CER and snake tomato had higher DPPH and OH radicals scavenging abilities than ESC. The inhibition of Fe 2+ induced malondialdehyde (MDA) production in rats pancreas revealed that snake tomato had significantly lower inhibitory effect than CER. Furthermore, snake tomato and CER showed stronger inhibition of α-glucosidase [snake tomato (EC50 = 1.65 mg/ml), CER (EC50 = 1.32 mg/ml)] than α-amylase [snake tomato (EC50 = 2.15 mg/ml), CER (EC50 = 2.39 mg/ml)] activity. The antioxidant properties of snake tomato favourably compared with the cultivars of tomatoes, and its stronger inhibition of α-glucosidase activity than α-amylase activities suggests that snake tomato could be an alternative or complement to the use of lycopersicon tomatoes.


INTRODUCTION
Tricosanthes cucumerina (commonly called snake tomato), snake gourd, viper gourd or long tomato is rich in chemical constituents like flavonoids, carotenoids, phenolic acids (Adebooye, 2008;Ojiako and Igwe, 2008) which makes the plant pharmacologically and therapeutically active.Even though some systems of medicine have been exploring some pharmacological potentials of the snake tomato, such as antidiabetic, *Corresponding author.E-mail: goboh2001@yahoo.com.
hepatoprotective, cytotoxic, anti-inflamatory and larvacidal effects, the 'tomato' still remains underutilized as food or as medicinal plant.Snake tomato has been used as a substitute for the common lycopersicon tomatoes in the tropics especially when the prices of the lycoper-sicon tomatoes go up in the off-season.The common tomatoes commonly used as diet almost all over the world, are a major source of antioxidants, and the consumption of fresh lycopersicon tomatoes has been reported to have health benefits such as cancer preven-tion and inhibition of lipid peroxidation (Bub et al., 2000;Ziegler and Vogt, 2002).However, there is dearth of information on some health benefits of snake tomato to justify its use as a substitute to the lycopersicon tomatoes.
The link between free-radicals and development of diabetes has been well established (Ceriello, 2006;Maritim et al., 2003), more so free-radical damage to the pancreas has been implicated in the diabetogenic process (Akbarzadeh et al., 2007).Diabetes is a major health problem worldwide along with its associated complications (Zimmet et al., 1997) and this could be linked to changes in the dietary patterns in both developing and developed countries.The prevalence of type II diabetes is growing at an exponential rate (Zimmet and Lefebvre, 1996) and a lot of attention is been given to natural products for the management of the disease (Covington, 2001).
This study therefore investigated the antioxidant properties and inhibition of key enzymes linked to type-2 diabetes (α-amylase and α-glucosidase) by the underutilized snake tomato and compare with the common tomatoes so as to find a basis, if any, for the use of snake tomato as a substitute to common tomatoes and also explain the mechanism of action by which the 'tomatoes' can be used in the management of type-2 diabetes.

Sample collection and preparation
Snake tomato (T.cucumerina) and two cultivars of common tomatoes (Lycopersicon esculentum Mill.);Ibadan Local (CER) and Roma VF (ESC) were collected from the main market, identified, washed, weighed and then homogenized by using a blender after distilled water was added (1:3 w/v).The homogenate was centrifuged at 4500 g for 15 min.The supernatant (juice fraction) was recovered and kept in the freezer for subsequent analysis.

Determination of total phenol content
The total phenol content was determined according to the method of Singleton et al. (1999).Briefly, appropriate dilutions of the pastes were oxidized with 2.5 ml 10% Folin-Ciocalteau's reagent (v/v) and neutralized by 2.0 ml of 7.5% sodium carbonate.The reaction mixture was incubated for 40 min at 45°C and the absorbance was measured at 765 nm in the spectrophotometer.The total phenol content was subsequently calculated as gallic acid equivalent.

Determination of total flavonoid content
The total flavonoid content was determined using a slightly modified method reported by Meda et al. (2005), briefly 0.5 ml of appropriately diluted sample was mixed with 0.5 ml methanol, 50 µl 10% AlCl3, 50 µl 1 M potassium acetate and 1.4 ml water, and allowed to incubate at room temperature for 30 min.The absorbance of the reaction mixture was subsequently measured at 415 nm; the total flavonoid content was subsequently calculated.The non-flavonoid polyphenols were taken as the difference between the total phenol and total flavonoid content.

1,1-diphenyl-2 picrylhydrazyl radical scavenging ability
The free radical scavenging ability of the pastes against 1,1diphenyl-2 picrylhydrazyl (DPPH) free radical was evaluated as described by Gyamfi et al. (1999).Briefly, appropriate dilution of the extracts (1 ml) was mixed with 1 ml, 0.4 mM methanolic solution containing DPPH radicals, the mixture was left in the dark for 30 min and the absorbance was taken at 516 nm.The DPPH free radical scavenging ability was subsequently calculated.

Hydroxyl radical scavenging ability
The method of Halliwell and Gutteridge (1981) was used to determine the ability of the pastes to prevent Fe 2+ /H2O2 induced decomposition of deoxyribose.The extract 0 to 100 µl was added to a reaction mixture containing 120 µl of 20 mM deoxyribose, 400 µl of 0.1 M phosphate buffer, 40 µl of 500 µM of FeSO4, and the volume were made up to 800 µl with distilled water.The reaction mixture was incubated at 37°C for 30 min and the reaction was then stopped by the addition of 0.5 ml of 28% trichloroacetic acid.This was followed by addition of 0.4 ml of 0.6% thiobarbituric acid solution.The tubes were subsequently incubated in boiling water for 20 min.The absorbance was measured at 532 nm in a spectrophotometer.

Preparation of tissue homogenates
Adult male rats weighing 220 to 240 g (10 to 12 weeks old) were decapitated under mild diethyl ether anaesthesia and the pancreas was rapidly isolated and placed on ice and weighed.This tissue was subsequently homogenized in cold saline (1/10 w/v) with about 10-up-and -down strokes at approximately 1200 rev/min in a Teflon glass homogenizer.The homogenate was centrifuged for 10 min at 3000 × g to yield a pellet that was discarded, and a low-speed supernatant (S1) was kept for lipid peroxidation assay (Belle et al., 2004).

Lipid peroxidation and thiobarbibutric acid reactions
The lipid peroxidation assay was carried out using the modified method of Ohkawa et al. (1979), briefly 100 µl S1 fraction was mixed with a reaction mixture containing 30 µl of 0.1 M pH 7.4 Tris-HCl buffer, extract (0 to 100 µl) and 30 µl of 250 µM freshly prepared FeSO4.The volume was made up to 300 µl by water before incubation at 37°C for 1 h.The colour reaction was developed by adding 300 µl 8.1% Sodium dodecyl sulphate (SDS) to the reaction mixture containing S1, this was subsequently followed by the addition of 600 µl of acetic acid/HCl (pH 3.4) mixture and 600 µl 0.8% Thiobarbituric acid (TBA).This mixture was incubated at 100°C for 1 h.Thiobarbituric acid reactive species (TBARS) produced were measured at 532 nm and the absorbance was compared with that of standard curve using malondialdehyde (MDA).

α-Amylase inhibition assay
This was measured using the dinitrosalicylic acid method adapted from Bernfeld (1955).Appropriate dilution of the pastes (500 μl) and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing Hog pancreatic α-amylase (EC 3.2.1.1)(0.5 mg/ml) were incubated at 25°C for 10 min.Then, 500 µl of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to each tube.The reaction mixtures was incubated at 25°C for 10 min and stopped with 1.0 ml of dinitrosalicylic acid colour reagent.Thereafter, the mixture was incubated in a boiling water bath for 5 min and cooled to room temperature.The reaction mixture was then diluted by adding 10 ml of distilled water, and absorbance measured at 540 nm.The EC50 (the extract concentration inhibiting 50% of the α-amylase activity) of the pastes was calculated.

α-Glucosidase inhibition assay
Appropriate dilution of the pastes (50 μl) and 100 μl of αglucosidase solution (1.0 U/ml) in 0.1 M phosphate buffer (pH 6.9) was incubated at 25°C for 10 min.Then, 50 μl of 5 mM pnitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added.The mixtures were incubated at 25°C for 5 min before reading the absorbance at 405 nm in the spectrophotometer.The α-glucosidase inhibitory activity was expressed as percentage inhibition.The EC50 of the pastes was calculated (Apostolidis et al., 2007).

Data analysis
The results of three replicates were pooled and expressed as mean ± standard deviation (SD).One-way analysis of variance (ANOVA) and least significance difference (LSD) were carried out (Zar, 1984).Significance was accepted at p ≤ 0.05.

RESULTS
The results of the total phenolic and flavonoid contents are presented in Table 1.ESC had significantly lower total phenolic content than snake tomato and CER, while CER had significantly higher total flavonoid content than snake tomato and ESC.Figures 1, 2 and Table 2 reveal that CER and snake tomato had higher radicals scavenging abilities than ESC.The inhibition of Fe 2+ induced MDA production in rats pancreas is presented in Figure 3 and Table 2. Snake tomato had significantly lower inhibitory effect than CER, but higher inhibitory effect than ESC.Furthermore, as presented in Figures 4, 5 and Table 2, snake tomato and CER showed stronger inhibi-Table 1.Total phenol and total flavonoid content of snake tomato and two tomato varieties (ESC and CER) (mg/g).

DISCUSSION
The total phenol content of snake tomato was not significantly (P > 0.05) different from that of CER, but higher than that of ESC.However, the total flavonoid content of snake tomato was significantly (P < 0.05) lower than that of CER, but higher than that of ESC.The antioxidant capacities of the 'tomato' samples were assessed not only for comparison between the species but also because free radicals are involved in the development and complications of diabetes in a number of ways; the white blood cell production of reactive oxygen species mediates the autoimmune destruction of the beta cells in the islets of langerhans in the pancreas (Yoon and Jun, 2005).Abnormalities in transition metal metabolism are postulated to result in the establishment of diabetes (Parameshwar et al., 2012).Diabetes can be induced in animals by the drugs alloxan and streptozotocin; the mechanism of action of these two drugs is different, but both result in the production of reactive oxygen species and scavengers of oxygen radicals have been found to be effective in preventing diabetes in these animal models (Moussa, 2008).
The DPPH* scavenging ability of snake tomato was not significantly (P > 0.05) different form ESC, but lower than that of CER.This trend in the results agree with the total phenolic and flavonoid content of the samples and many earlier research articles, where correlation were reported between phenolic content and antioxidant capacity of some plant foods (Amic et al., 2003;Chu et al., 2002;Oboh and Ademosun, 2011).Furthermore, the trend in the DPPH* scavenging ability agrees with the OH* scavenging ability of the samples.All the samples scavenged OH* produced from the decomposition of deoxyribose in a dose dependent manner but with CER and snake tomato having the highest scavenging abilities as there was no significant (P > 0.05) difference in their EC 50.The antioxidant properties of the samples could be  attributed to a combination of carotenoids and other phenolic compounds.Lycopene, which is the major carotenoid in tomatoes has been shown by cellular and molecular studies to exhibit potent antioxidative properties (Khachik et al., 2002;Sahasrabuddhe, 2011), and correlations have been established between the phenolic content and antioxidant properties of many samples (Jayaprakasha et al., 2008;Kedage et al., 2007).
MDA is increased when Fe 2+ induces lipid peroxidation by catalyzing the decomposition of hydrogen peroxide to generate hydroxyl radical via the fenton reaction (Bayir et    al., 2006;Oboh et al., 2007).Incubation of rat's pancreas in the presence of 250 µM Fe 2+ caused a significant increase in the malondialdehyde (MDA) content of the pancreas.Free-radicals induced pancreatic damage has been linked to the development of diabetes (Akbarzadeh et al., 2007).Nevertheless, the 'tomato' samples significantly (P<0.05)inhibited MDA production in the pancreas in a dose-dependent manner as shown in Table 2.However, CER and snake tomato had significantly (P < 0.05) stronger inhibitory effect on MDA production in the pancreas (in vitro) than ESC.The reason for the higher inhibitory ability of CER and snake tomato cannot be categorically stated, but it could be due to other antioxidant mechanisms, since CER and snake tomato had higher phenolic contents, DPPH* and OH* scavenging abilities.
The MDA inhibitory ability of snake tomato and the lycopersicon tomatoes cannot be ascribed to the lyco-pene content alone as a combination of purified lycopene with a-tocopherol was reported to result in a significant greater inhibition of in vitro low density lipoprotein (LDL) oxidation, than the expected additive individual inhibitions.Purified lycopene was also shown to act synergistically with other natural antioxidants like the flavonoid glabridin, the phenolics rosmarinic acid and carnosic acid, and garlic in inhibiting LDL oxidation in vitro (Furhman et al., 2000).Thus, the combination of lycopene and other natural antioxidants present in the samples may be responsible for the potent inhibition of lipid peroxidation.
α-Amylase hydrolyzes starch to maltose, while αglucosidase enzymes are responsible for the breakdown of oligo-and/or disaccharides to monosaccharides, and an inhibition of these enzymes therefore leads to a decrease in blood glucose level, and this is one of the therapeutic approaches for reducing postprandial blood  glucose values in a bid to prevent/manage diabetes (Shim et al., 2003).The samples inhibited α-amylase activity in a dose-dependent manner, however the EC 50 (Table 2) revealed that the trend of the result was different from the earlier results as ESC had the highest inhibitory effect.It is noteworthy however that snake tomato had higher inhibitory effect on α-amylase activity than CER.Furthermore, the α-glucosidase inhibitory activity showed that the samples inhibited the enzyme activity in a dose-dependent manner.
A comparison of the α-amylase and α-glucosidase inhibition by the samples revealed a stronger inhibition of α-glucosidase activity, but milder inhibition of α-amylase activities by snake tomato and CER.A stronger inhibition of α-glucosidase activity, but milder inhibition of αamylase activities is desirable as this could address the main drawback of currently used α-glucosidase and αamylase inhibitors drugs which is caused by the excessive inhibition of pancreatic α-amylase resulting in the abnormal bacterial fermentation of undigested carbohydrates in the colon (Rao and Jamil, 2011).These results confirms the claim that natural inhibitors from dietary plants could show lower inhibitory effect against α-amylase activity and a stronger inhibitory activity against α-glucosidase and can be used as effective therapy for postprandial hyperglycemia with minimal side effects (Kwon et al., 2006(Kwon et al., , 2007)).Furthermore, Kar et al. (2003) showed that crude ethanolic extract of snake tomato showed significant blood glucose lowering activity in alloxan induced diabetic albino rats and Arawwawala et al. (2009) using hot water extract of aerial parts of snake tomato also noted the improved glucose tolerance and tissue glycogen in non insulin dependent diabetes mellitus induced rats.

Conclusion
The inhibition of key enzymes linked with type-2 diabetes (α-amylase and α-glucosidase) and antioxidative properties of the "tomatoes" used in this study could make them good dietary means for the management and/or prevention of type-2 diabetes.The antioxidant properties of snake tomato which favourably compares with the other lycopersicon tomatoes (except CER), combined with its stronger inhibition of α-glucosidase activity, but milder inhibition of α-amylase activities suggests that snake tomato could be an alternative or complement to the use of lycopersicon tomatoes.

Table 2 .
EC50 of DPPH* and OH* scavenging abilities, inhibition of Fe 2+ induced MDA Production, α-amylase and αglucosidase activities of snake tomato and two tomato varieties (ESC and CER).