Inhibitory effect of aqueous extract of different parts of unripe pawpaw (Carica papaya) fruit on Fe²⁺-induced oxidative stress in rat pancreas in vitro

Abstract

Context: Carica papaya L. (Caricaceae) is widespread throughout tropical Africa; it is cultivated for its fruits and it is eaten in various ways.

Objective: This study sought to investigate the inhibitory effect of the aqueous extract of different parts of unripe pawpaw fruit on Fe²⁺-induced lipid peroxidation in rat’s pancreas in vitro.

Materials and methods: The aqueous extract of the unripe pawpaw fruit parts; peel (PG), seed (SG), flesh (FG), flesh with peel (FPG) and a combination of equal amount of all parts (CG) were prepared, the total phenolic content and the antioxidant activities of the extracts were then evaluated using various spectrophotometric methods.

Result: PG had the highest total phenol content (1.24 mg GAE/g), flavonoid content (0.63 mg QUE/g), reducing power (7.07 mg AAE/g) and Fe²⁺ chelating ability while the SG had the highest 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability. Furthermore, all the extracts caused a significant decrease (p < 0.05) in the malondialdehyde contents in the pancreas with SG (IC₅₀ = 4.25 mg/mL) having the highest inhibitory effect on Fe²⁺-induced lipid peroxidation.

Discussion and conclusion: This protective effect of the extracts on Fe²⁺-induced lipid peroxidation in rat pancreas could be attributed to their phenolic compounds and, the possible mechanism may be through their antioxidant activities. However, the effect of combination of different parts of unripe pawpaw fruit in equal amount (w/w) on the inhibition of Fe²-induced lipid peroxidation in rat pancreas exhibited additive properties.

• Antioxidant activities
• lipid peroxidation
• malondialdehyde
• phenolic compounds

Introduction

Oxidation of biological molecules has been postulated to induce a variety of pathological events such as diabetes, pancreatitis and ageing (Finkel & Holbrook, 2000). Evidence has shown that these damaging events are caused by free radicals (Halliwell & Gutteridge, 1993). Oxidative stress results from either a decrease of natural cell antioxidant capacity or an increased amount of reactive oxygen species (ROS) in organisms. It is well established that free radicals are associated with process that leads to cell degeneration, especially in organs such as liver, brain and pancreas (Shulman et al., 2004).

In the pancreas, Fe accumulates in acinar cells and in the Islets of Langerhans, thereby resulting in the destruction of β-cells associated with diabetes mellitus (Shah & Fonseca, 2011). High levels of both Cu and Fe, with low levels of Zn and Mn play a crucial role in the progression of several degenerative diseases (Johnson, 2001). Although free Fe in the cytosol and mitochondria may be transported and stored using Fe transport and storage proteins like transferin and feritins and so on, excess free Fe could cause considerable oxidative damage by acting catalytically in the production of ROS which have the potential to damage cellular lipids, nucleic acids, proteins and carbohydrate resulting in wide-ranging impairment in cellular function and integrity (Britton et al., 2002). ROS can directly attack the polyunsaturated fatty acids of the cell membranes and induce lipid peroxidation. Malondialdehyde (MDA) is the end product of lipid peroxidation, which is a process where ROS degrade polyunsaturated fatty acids. This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form advanced glycation end products. The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism (Murray et al., 2000).

However, the most likely and practical way to fight degenerative diseases is to improve body antioxidant status, which could be achieved by higher consumption of fruits and vegetables. Foods of plant origin usually contain natural antioxidants such as phenolic compounds that can scavenge free radicals (Alia et al., 2003; Oboh, 2005; Oboh & Akindahunsi, 2004; Sun et al., 2002). Phenolic compounds are an important group of secondary metabolites, which are synthesized by plants because of plant adaptation to biotic and abiotic stress condition such as infection, water stress and cold stress (Oboh & Rocha, 2007). In recent years, phenolic compounds have attracted the interest of researchers because of their antioxidants capacity; they can protect the human body from free radicals, whose formation is associated with the normal natural metabolism of aerobic cells. The antiradical activity of flavonoids and phenols is principally based on the structural relationship between different parts of their chemical structure (Rice-Evans et al., 1996). Polyphenols are common constituents of the human diet, present in most foods and beverages of plant origin. They are considered to contribute to the prevention of various degenerative diseases. This assumption originally came from in vitro studies, showing the antioxidant properties of several polyphenols and their ability to modulate the activity of various enzymes. Research suggests that many flavonoids are more potent antioxidant than vitamins C and E (Oboh, 2005; Oboh & Akindahunsi, 2004).

The pawpaw plant [Carica papaya L. (Caricaceae)] is widespread throughout tropical Africa; the plant can be monoecious, dioecious, or hermaphroditic (Janick, 1988). C. papaya is cultivated for its fruits; it is favored by the people of the tropics – as breakfast, and as ingredients in jellies, preserves, or eaten in various ways. The juice makes a popular beverage; young leaves, shoots and fruits cooked as vegetable. It is locally known as Ibepe, Gwanda and Okwere in Yoruba, Hausa and Igbo languages, respectively (Gill, 1992). Phytochemical studies have shown that C. papaya contains alkaloids, carpain, nicotine, flavonols, tannins and terpinenes (Tona et al., 1998). Different parts of the plant are employed in the treatment of different human and veterinary diseases in various parts of the world. For example, in Asian folk medicine, the latex is employed as an abortificant, antiseptic for wound dressing and as a cure for dyspepsia (Reeds, 1976) while in Africa the root infusion is reputed for treating venereal diseases (Duke, 1984). The plant extracts are also reported to have sedative and muscle relaxant (Gupta et al., 1990), reversible antifertility (Chinoy et al., 1997; Harsha & Chinoy, 1996) and purgative properties (Akah et al., 1997). Several independent animal and human studies have reported the anti-diabetic effects of the unripe mature fruits of C. papaya (Olagunju et al., 1995; Olapade, 1995; Salau et al., 2003). Also, the in vivo antioxidant activities of unripe pulp of C. papaya have been studied (Oloyede et al., 2011). Although several works have been reported on the chemical characterization of phyto-constituents and antioxidant properties of C. papaya fruit (Oloyede et al., 2011; Tona et al., 1998), there is, however, limited information on its potential in the prevention of oxidative stress pancreas. Hence, the objective of this study was to investigate the inhibitory effect of water extractible phytochemicals from different parts of unripe pawpaw fruit on Fe²⁺-induced lipid peroxidation in rat’s pancreas in vitro.

Materials and methods

Materials

Fresh unripe pawpaw fruit was obtained from a farmland at Obakekere, Akure, Ondo State. Authentication of the samples was carried out by Mr. K. Oladunjoye at the Department of Biology, Federal University of Technology, Akure, Nigeria, where voucher specimen (no 360) was deposited at the Herbarium. Ten adult male Wistar strain albino rats were purchased from the Animal Production and Health Department, Federal University of Technology, Akure, and acclimatized for 2 weeks, during which period they were maintained ad libitum on commercial diet and water. The handling of animals was carried out in accordance with the recommended international standard (National Research Council, 1988). A UV-visible spectrophotometer (Model 6305; Jenway, Barlo world Scientific, Dunmow, United Kingdom) was used to measure absorbance.

Chemicals and reagents

Chemicals and reagents used such as thiobarbituric acid (TBA), 1,10-phenanthroline, deoxyribose, gallic acid, quecertin, ascorbic acid, Folin-Ciocalteau’s reagent were procured from Sigma-Aldrich, Inc. (St. Louis, MO). Trichloroacetic acid (TCA), MDA, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were sourced from Sigma-Aldrich, Chemie GmbH (Steinheim, Germany). Hydrogen peroxide, methanol, acetic acid, and HCl were sourced from BDH Chemicals Ltd. (Poole, England). Sodium carbonate, AlCl₃, potassium acetate, Tris-HCl buffer, sodium dodecyl sulphate (SDS), FeSO₄, potassium ferricyanide and ferric chloride were of analytical grade while the water was glass distilled.

Aqueous extract preparation

The unripe pawpaw fruit was thoroughly washed with water to remove any contaminant. Each of the unripe pawpaw fruit parts (SG, PG, FG, FPG and CG) were blended with distilled water (1:20 w/v). The mixture was filtered and later centrifuged at 400 g for 10 min to obtain a clear supernatant which was then used for subsequent analysis (Oboh et al., 2007).

Determination of phenol content

The extractable phenol content was determined on the unripe pawpaw fruit parts using the method reported by Singleton et al. (1999). Appropriate dilutions of the extracts were mixed with 2.5 mL of 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. The total phenol content was subsequently calculated using gallic acid as standard.

Determination of flavonoid content

The extractable flavonoid contents of unripe pawpaw fruit parts were 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 of 10% AlCl₃, 50 μL of 1 mol/L⁻¹ potassium acetate and 1.4 mL water, and allowed to incubate at room temperature for 30 min. Thereafter, the absorbance of the reaction mixture was measured at 415 nm. The total flavonoid was calculated using quercetin as standard.

Preparation of pancreas homogenates

The rats were decapitated under mild diethyl ether anesthesia and the pancreas tissue was rapidly dissected 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) containing mainly water, proteins and lipids (cholesterol, galactolipid, individual phospholipids, gangliosides) was kept for the lipid peroxidation assay (Belle et al., 2004).

Lipid peroxidation and thiobarbibutric acid reactions

The lipid peroxidation assay was carried out using the method of Ohkawa et al. (1979). Briefly, 100 µL S1 fraction was mixed with a reaction mixture containing 30 μL of 0.1 M Tris-HCl buffer (pH 7.4), extract of unripe pawpaw fruit parts (0–100 μL) and 30 μL of 250 μM freshly prepared FeSO₄. The volume was made up to 300 μL with water before incubation at 37 °C for 1 h. The color reaction was developed by adding 300 μL, 8.1% SDS to the reaction mixture containing S1; this was subsequently followed by the addition of 500 μL of acetic acid/HCl (pH 3.4) and 500 μL, 0.8% TBA. This mixture was incubated at 100 °C for 1 h. The absorbance of thiobarbituric acid reactive species produced was measured at 532 nm. MDA produced was expressed as % Control, The IC₅₀ (extract concentration required to inhibit 50% of MDA produced) values were calculated using non-linear regression analysis.

Fe²⁺ chelation assay

The Fe²⁺ chelating ability of all the extracts were determined using a modified method of Minotti and Aust (1987) with a slight modification by Puntel et al. (2005). Freshly prepared 500 μmol/L⁻¹ FeSO₄ (150 μL) was added to a reaction mixture containing 168 μL of 0.1 mol/L⁻¹ Tris-HCl (pH 7.4), 218 μL saline and the extracts (0–25 μL). The reaction mixture was incubated for 5 min, before the addition of 13 μL of 0.25% 1,10-phenanthroline (w/v). The absorbance was subsequently measured at 510 nm. The Fe²⁺ chelating ability was subsequently calculated with respect to the reference. where Abs_ref = absorbance of the reference (reacting mixture without the test sample) and Abs_sample = absorbance of reacting mixture with the test sample.

Degradation of deoxyribose (Fenton’s reaction)

The ability of all the aqueous extracts to prevent Fe²⁺/H₂O₂-induced decomposition of deoxyribose was carried out using the method of Halliwell and Gutteridge (1981). Briefly, freshly prepared aqueous extract (0–100 μL) was added to a reaction mixture containing 120 μL 20 mM deoxyribose, 400 μL 0.1 M phosphate buffer, 40 μL 20 mM hydrogen peroxide and 40 μL 500 μM FeSO₄, and the volume made to 800 μL with distilled water. The reaction mixture was incubated at 37 °C for 30 min, and the reaction was stopped by the addition of 0.5 mL of 2.8% TCA, this was followed by the addition of 0.4 mL of 0.6% TBA solution. The tubes were subsequently incubated in boiling water for 20 min. The absorbance was measured at 532 nm. where Abs_ref = absorbance of the reference (reacting mixture without the test sample) and Abs_sample = absorbance of reacting mixture with the test sample.

1,1-DPPH free radical scavenging ability

The free radical scavenging ability of all the extracts 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 methanol 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 of the extracts was subsequently calculated. where Abs_ref = absorbance of the reference (reacting mixture without the test sample) and Abs_sample = absorbance of reacting mixture with the test sample.

Reducing property

The reducing properties of all the extracts were determined by assessing the ability of the extract to reduce a FeCl₃ solution as described by Pulido et al. (2000). A 2.5 mL aliquot was mixed with 2.5 mL, 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL, 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min, and then 2.5 mL, 10% TCA was added and centrifuged at 650 g for 10 min. A 5 mL of the supernatant was mixed with an equal volume of water and 1 mL, 0.1% ferric chloride. The same treatment was performed to a standard ascorbic acid solution and the absorbance taken at 700 nm. The reducing power was then calculated and expressed as ascorbic acid equivalents.

Characterization of phenolic constituent using gas chromatographic analysis

The qualitative–quantitative analysis of the phenolic compounds of the samples was carried out using the method reported by Kelley et al. (1994). The phenolic compounds were extracted from each sample as described by Kelley et al. (1994). After extraction, the purified phenolic extracts (1 µL: 10:1 split) were analyzed for composition by comparing with phenolic standards (Aldrich Chemical Co., Milwaukee, WI) and a cochromatography with standards on a Hewlett-Packard 6890 gas chromatograph (Hewlett-Packard Corp., Palo Alto, CA) equipped with a derivatized, non-packed injection liner, a Rtx-5MS (5% DIPHENTL-95% dimethyl polysiloxane) capillary column (30 m length, 0.25 mm column id., 0.25 µm film thickness), and detected with a flame ionization detector (FID). The following conditions were employed phenolic acid (PA) separation; injector temperature, 230 °C; temperature ramp, 80 °C for 5 min then ramped to 250 °C at 30 °C/min; and a detector temperature of 320 °C.

Purification of extracted phenolic acids for GC analysis

After extraction, an aliquot (5–15 mL) of the supernatants was passed through a conditional Varian (Varian Assoc., Harbor City, CA) Bond Elut PPL (3 mL size with 200 mg packing) solid-phase extraction tube at ∼5 mL/min attached to a Visiprep (Supelco, Bellefonte, PA). The tubes were then placed under a vacuum (−60 KPa) until the resin was thoroughly dried after which the phenolic acids were eluted with 1 mL of ethyl acetate into GC autosampler vials. The PPL tubes were conditioned by first passing 2 mL of ethyl acetate followed by 2 mL water (pH < 2.0).

Data analysis

The result of three replicate experiments were pooled and expressed as mean ± standard deviation (SD; Zar, 1984). A one-way analysis of variance (ANOVA) and the least significance difference (LSD) were carried out. Significance was accepted at p ≤ 0.05.

Results and discussion

Many plants are rich sources of phytochemicals, and intake of these plant chemicals has protective potential against degenerative diseases (Chu et al., 2002). The total phenol content of the different parts of unripe pawpaw fruit is presented in Table 1. The result revealed that the PG (1.24 mg GAE/g) had the highest total phenol content followed by SG (0.98 mg GAE/g), CG (0.95 mg GAE/g) while the FG (0.39 mg GAE/g) had the least; however, the values obtained are lower than what was reported for some hot peppers (Oboh et al., 2007), green teas (Oboh & Rocha, 2008) and some tropical leafy vegetables (Oboh, 2005; Oboh & Akindahunsi, 2004), but were higher than ginger varieties (Oboh et al., 2012). Phenolic compounds can protect the human body from free radicals, whose formation is associated with the normal metabolism of aerobic cells. They are strong antioxidants capable of removing free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals and inhibit oxidases (Amic et al., 2003).

Table 1. Total phenol content, total flavonoid content, and ferric reducing antioxidant property (FRAP) of different parts of unripe pawpaw fruit (C. papaya).

Sample	Total phenol (mg GAE/g)	Total flavonoid (mg QUE/g)	FRAP (mg AAE/g)
SG	0.98^a± 0.04	0.17^a± 0.06	3.92^a± 0.04
FG	0.39^b± 0.04	0.11^b± 0.01	0.66^b± 0.07
PG	1.24^c± 0.10	0.63^c± 0.06	7.07^c± 0.01
FPG	0.39^b± 0.00	0.11^b± 0.00	2.06^d± 0.01
CG	0.95^d± 0.01	0.34^d± 0.01	3.99^e± 0.08

AAE, ascorbic acid equivalent; GAE, gallic acid equivalent; QUE, quercetin equivalent; SG, seed of unripe pawpaw; FG, flesh of unripe Pawpa; PG, peel of unripe pawpaw; FPG, flesh with peel of unripe pawpaw; CG, combination of equal proportion of seed, flesh, and peel of unripe pawpaw.

Values represent mean ± standard deviation of triplicate readings.

Values with the same superscript along the column are not significantly (p < 0.05) different.

Furthermore, the total flavonoid content of the unripe pawpaw parts, as presented in Table 1, revealed that PG (0.63 mg QUE/g) had the highest flavonoid content followed by CG (0.34 mg QUE/g), SG (0.17 mg QUE/g) while the FG (0.11 mg QUE/g) had the least. However, the values obtained are lower than what was reported for tropical clove bud (Ademiluyi et al., 2009) and some tropical green leafy vegetables (Oboh et al., 2008). The presence of derivatives of flavonoids has been found in many spices; moreover, numerous studies have conclusively shown that the majority of the antioxidant activity maybe from compounds such as flavonoids, isoflavones, flavones, anthocyanins, catechin and isocatechin rather than from vitamins C, E and β-carotene (Marin et al., 2004; Oboh et al., 2007). Flavonoids have antioxidant activity and could therefore lower cellular oxidative stress (Oboh et al., 2007). Polyphenols are considered to be strong antioxidants due to the redox properties of their hydroxyl groups (Materska & Perucka 2005).

The finding that Fe²⁺ caused a significant increase in the MDA content of the pancreas agreed with earlier report where Fe²⁺ was shown to be a potent initiator of lipid peroxidation (Oboh et al., 2007). The increased lipid peroxidation in the presence of Fe²⁺ could be attributed to the fact that Fe²⁺ can catalyze one-electron transfer reactions that generate ROS, such as the reactive OH˙, which is formed from H₂O₂ through the Fenton reaction. Iron also decomposes lipid peroxides, thus generating peroxyl and alkoxyl radicals, which favors the propagation of lipid oxidation (Zago et al., 2000). In the pancreas, Fe accumulates in acinar cells and in the Islets of Langerhans, thereby resulting in the destruction of β-cells associated with diabetes mellitus (Shah & Fonseca, 2011). Therefore, possible depletion of iron could decrease oxidative stress throughout the body (Minamiyama et al., 2010). However, the aqueous extracts from the different parts of unripe pawpaw fruit caused a dose-dependent significant decrease (p < 0.05) in the MDA content of the Fe²⁺-stressed pancreas homogenates (Figure 1). Nevertheless, aqueous extract of the SG (EC₅₀ = 4.25 mg/mL) had the highest inhibitory effect on Fe²⁺-induced lipid peroxidation in rat’s pancreas followed by CG (EC₅₀ = 4.82 mg/mL) while the PG (EC₅₀ = 6.05 mg/mL) had the least when taking into account the EC₅₀ values in Table 2. However, judging by the result (Table 2), the effect of combination on the inhibition of Fe²⁺-induced lipid peroxidation is additive. The mode of inhibition of Fe²⁺-induced lipid peroxidation cannot be categorically stated. However, there is the possibility that the water extractable phytochemicals could have formed complexes with the Fe²⁺, thereby preventing them from catalyzing the initiation of lipid peroxidation and/or the possibility that the phytochemical could have scavenge the free radical produced by the Fe²⁺-catalyzed reaction (Oboh et al., 2007). Glucose in the presence of transition metals (such as Fe) can be oxidized to generate ROS, which has been implicated in the progression of diabetes and diabetic complications. Excess cytosolic iron is a known factor of gestational diabetes (Lao et al., 2001) and a clear risk factor for the disease in normal populations (Fernández-Real et al., 2002). Lowering iron improves insulin sensitivity (Fernández-Real et al., 2002). Thus, iron status may be a major determinant in the development of type 2 diabetes (Zheng et al., 2008). Fe²⁺ could also take part in the Fenton reaction leading to the generation of OHċ, which later attacks DNA, protein, membrane lipids and several other biomolecules of physiological importance.

Figure 1. Inhibition of Fe²⁺-induced lipid peroxidation in rat’s pancreas by aqueous extract of different parts of unripe pawpaw fruit (C. papaya). Key: SG, seed of unripe pawpaw extract; fg, flesh of unripe pawpaw extract; pg, peel of unripe pawpaw extract; fpg, flesh with peel of unripe pawpaw extract; CG, combination of equal proportion of seed, flesh and peel of unripe pawpaw extract.

Table 2. EC₅₀ of aqueous extract of different parts of unripe pawpaw fruit on Fe²⁺-induced lipid peroxidation in rat’s pancreas.

Sample	EC50 of Fe^2+ induced lipid peroxidation in rat pancreas (mg/ml)
SG	4.25
FG	5.03
PG	6.05
FPG	5.84
CG	4.82

SG, seed of unripe pawpaw extract; FG, flesh of unripe pawpaw extract; PG, peel of unripe pawpaw extract; FPG, flesh with peel of unripe pawpaw Extract; CG, combination of equal proportion of seed, flesh and peel of unripe pawpaw extract.

The Fe²⁺ chelating ability of the aqueous extracts from the different parts of unripe pawpaw fruit as revealed in Figure 2 showed that the extracts were able to chelate Fe²⁺ in a dose-dependent manner. However, aqueous extract of the PG had the highest Fe²⁺ chelating ability followed by SG, CG, FPG and FG in that order. This result is in agreement with the phenolic content (Table 1). The ability of substances to chelate and deactivate transition metals, prevent such metals from participating in the initiation of lipid peroxidation and oxidative stress through metal catalyzed reaction is considered an antioxidant mechanism of action (Oboh et al., 2007). The functional groups present in the extracts such as –OH, –SH, –COOH, PO₃H₂, C=O, –NR₂, –S– and –O– may be responsible for the Fe²⁺ chelating ability of the extract (Gülçin, 2006; Lindsay, 1996; Yuan et al., 2005). Earlier reports revealed that compounds with structures containing two or more of the following functional groups: –OH, –SH, –COOH, PO₃H₂, C=O, –NR₂, –S– and –O– in a favorable structure–function configuration can show metal chelation activity (Gülçin, 2006; Lindsay, 1996; Yuan et al., 2005).

Figure 2. Fe²⁺Chelating ability of aqueous extract of different parts of unripe pawpaw fruit (C. papaya). Key: SG, seed of unripe pawpaw extract; FG, flesh of unripe pawpaw extract; PG, peel of unripe pawpaw extract; FPG, flesh with peel of unripe pawpaw extract; CG, combination of equal proportion of seed, flesh and peel of unripe pawpaw extract.

This high Fe²⁺chelating ability is of immense importance in the protective ability of polyphenol against oxidative stress, because it is usually too late to attempt to use OH radical scavengers for therapeutic purposes. The reason for this is that extraordinarily high reactivity of hydroxyl radicals toward most biomolecules would require unreasonably high concentrations of intercepting scavengers to outcompete the biomolecules of interest (Bayir et al., 2006), thereby making Fe²⁺ chelators a better therapeutic alternative. The hydroxyl radical (OH˙) scavenging ability of the aqueous extracts of the different parts of unripe pawpaw is presented in Figure 3. The results revealed that all the extracts were able to scavenge OH˙ produced from the decomposition of deoxyribose in Fenton reaction at the two concentrations (1.09 mg/mL; 2.17 mg/mL) tested. However, at lower concentration (1.09 mg/mL), CG had the highest OH radical scavenging ability while at higher concentration (2.17 mg/mL) there was no significant (p < 0.05) difference among the extracts except the SG which had the least.

Figure 3. OH radical scavenging ability of aqueous extract of different parts of unripe pawpaw Fruit (C. papaya). Key: SG, seed of unripe pawpaw extract; FG, flesh of unripe pawpaw extract; PG, peel of unripe pawpaw extracts; FPG, flesh with peel of unripe pawpaw extract; CG, combination of equal proportion of seed, flesh and peel of unripe pawpaw extract.

Antioxidants carry out their protective role on cells either by preventing the production of free radicals or by neutralizing/scavenging free radicals produced in the body (Alia et al., 2003; Amic et al., 2003; Oboh et al., 2007). The result of the DPPH radical scavenging ability of the extract as presented in Figure 4 revealed that all the extracts scavenged DPPH radicals in a dose-dependent pattern (0–16.67 mg/mL). However, the SG (22.9–52.35%) had the highest DPPH radical scavenging ability while the FG (0.9–17.3%) had the least. The DPPH radical scavenging ability of the water extractible phytochemical could be attributed to the hydrogen donating ability of the hydroxyl groups of the phenolics (Table 3, Figure 5a–e).

Figure 4. DPPH free radical scavenging ability of aqueous extract of different parts of unripe pawpaw Fruit (C. papaya). Key: SG, seed of unripe pawpaw extract; FG, flesh of unripe pawpaw extract; PG, peel of unripe pawpaw extract; FPG, flesh with peel of unripe pawpaw extract; CG, combination of equal proportion of seed, flesh and peel of unripe pawpaw extract.

Figure 5. The chromatographic trace of phenolic constituents in (a) flesh, (b) seed, (c) peel, (d) flesh with peel, (e) combination of flesh, seed and peel in equal amount of unripe pawpaw by GC with FID. The main phenolic constituents are shown in Table 3.

Table 3. The main phenolic constituents in different parts of unripe pawpaw fruit (Carica papaya).

Name of compound (mg/100 g)	FG	SG	PG	FPG	CG
Coumarin	1.78^a	9.20^d	9.00	8.43	7.98
p-Coumaric acid	1.52	6.32^d	9.68	10.97	11.78
o-Coumaric acid	1.73	6.61^d	5.85	7.59	8.94
Vanillic acid	3.64	1.37^d	1.67^e	4.16	4.87
Caffeic acid	1.25	1.58^e	13.35	15.09	18.99
Ferulic acid	6.14^a	2.48^d	15.4	17.03	16.77
Syringic acid	1.63	4.26^d	7.08^d	1.79	2.15
Sinapinic acid	4.81	1.71^d	7.12^e	4.63	5.6
Genistein	1.64^b	3.84	1.56	1.77	2.12
Apigenin	2.38^b	11.89	13.96	14.5	15.31
Naringenein	2.93^c	2.39^c	11.82	13.07	15.73
Kaempferol	4.31^b	1.58^d	18.86	20.48	24.52
Luteolin	2.77^b	15.1	7.77	8.66	8.03
Epicatechin	3.26^d	27.09	18.86	20.48	24.52
Epigallocatechin	9.13^d	7.39	8.33	9.96	11.81
Quercetin	1.07^d	18.24	13.37	14.78	17.42
2-Phenylethyl-beta-D-glucoside	4.38	8.93	4.33	4.31	5.14
Phenyl-6^I-O-maloyl-β-d-glucoside	1.28^d	7.59^e	8.13	8.58	7.67

SG, seed of unripe pawpaw; FG, flesh of unripe pawpaw; PG, peel of unripe pawpaw; FPG, flesh with peel of unripe pawpaw; CG, combination of equal proportion of seed, flesh, and peel of unripe pawpaw.

^a= ×10⁻¹, ^b= ×10^{−2, c}= ×10^{−3, d}= ×10^{−4, e}= ×10⁻⁵.

Reducing power is a potent antioxidation defense mechanism. The two mechanisms that are available to affect this reducing power are by electron transfer and hydrogen atom transfer (Dastmalchi et al., 2007). This is because the ferric-to-ferrous ion reduction occurs rapidly with all reluctants with half reaction reduction potentials above that of Fe³⁺/Fe²⁺, the values in the Ferric reducing antioxidant property (FRAP) assay will express the corresponding concentration of electron-donating antioxidants (Halvorsen et al., 2002). The results revealed that the PG (7.07 mg AAE/g) had the highest reducing power followed by CG (3.99 mg AAE/g), SG (3.92 mg AAE/g) while FG (0.66 mg AAE/g) had the least. This trend is in agreement with the phenolic content and the Fe²⁺ chelating ability. Phenolic compounds have been reported to possess a higher reducing power than classical antioxidants such as BHA, BHT, tocopherol and Trolox (Gülçin, 2006). The high reducing power of the unripe pawpaw extracts will be of immense advantage in neutralizing free radicals generated in hyperglycemic condition associated with diabetes thus slowing down the development of diabetic complications arising from oxidative stress.

The antioxidant properties of plant foods have been linked to the presence of an array of important phenolic and non-phenolic phytochemicals including phenolic acids, flavonoids and alkaloids (Cheplick et al., 2007; Chu et al., 2002). However, characterization of the extract with GC revealed that the major constituent of the flesh, seed and peel extract of unripe pawpaw are sinapinic acid, 2-phenyl-6^I-O-β-d-glucoside, O-coumaric acid, epicatechin, quercetin, luteolin, ferulic acid, kaempferol and caffeic acid (Table 3, Figure 5a–e). Phenolic compounds can protect the human body from free radicals, whose formation is associated with the normal metabolism of aerobic cells. They are strong antioxidants capable of removing free radicals, chelate metal catalysts, activate antioxidant enzymes, reduce α-tocopherol radicals and inhibit oxidases (Amic et al., 2003).

Therefore, the protection of pancreas tissue from Fe²⁺-induced lipid peroxidation by the extract of different parts of unripe pawpaw fruit (C. papaya) could be attributed to their phenolic compound and, the mechanism through which they possibly do this, is by their Fe²⁺ chelating ability, radical scavenging abilities and reducing power.

Conclusion

The aqueous extract of the different parts of unripe pawpaw fruit (C. papaya) is able to protect the pancreas from Fe²⁺-induced lipid peroxidation in vitro. Therefore, the protection of pancreas tissue from Fe²⁺-induced lipid peroxidation by the extract of different parts of unripe pawpaw fruit (C. papaya) could be attributed to their phenolic compound and, the mechanism through which they possibly do this, is by their Fe²⁺-chelating ability, radical scavenging abilities and reducing power. However, the effect of the combination of different parts of unripe pawpaw fruit in equal amount (w/w) on the inhibition of Fe²-induced lipid peroxidation in rat pancreas exhibited additive properties.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.