Profiles and Antioxidant Activity in
Fruit Portions of Marx Red Bartlett and
Starkrimson Pear Cultivars

Z. Chikwambi^*and M. Muchuweti

Department of Biochemistry, Faculty of Science,

University of Zimbabwe, Harare, Zimbabwe

ABSTRACT

Plant secondary metabolites, for example phenolic compounds and DNA polymorphisms constitute biochemical markers, used in the evaluation of germplasm for different purposes. In this study, an evaluation of the phenolic content, HPLC phenolic profiles, 1, 1–Diphenyl–2–picryl hydrazyl (DPPH• scavenging activity, and β-carotene-linoleic acid antioxidant activity, in common pear (Pyrus communis L.) cultivars Starkrimson, Marx red Bartlett (MRB), Clapp’s favourite and their reverted spots was done. The distribution of the phenolic content in pear fruit showed compartmentalization of the secondary metabolites, with higher content in the peel than the pulp. Clapp’s favourite had the highest total phenolic content while MRB mutant A had the least. The HPLC profiles however showed variations in composition, quantities and distribution of phenolic compounds in peel and pulp before and after acid hydrolysis. DPPH• free radical scavenging activity of the methanolic extracts from the peel and pulp showed positive correlation with total phenolic content. The β-carotene-linoleic acid antioxidant activity did not show a direct relationship with total phenolic content. Presence or absence of secondary metabolites alone can not be used as a marker in pear breeding but would require complementing with genetic information. The results show some of the challenges in finding markers linked to color of the peel among different pear cultivars, which have a high degree of similarity to one another.

Keywords:Pyrus communis L., Starkrimson, Clapp’s favourite, Marx red Bartlett, Antioxidant activity, Scavenging activity, Phenolic compounds.

Introduction

Common pear (Pyrus communis L.) is a deciduous pome fruit of the Rasaceae family, subfamily Maloideae. Pears thrive best in temperate climates and have a higher chilling requirement than apple, confining their production to the coolest areas with more than 400 chilling hours (Rehm and Espig, 1991; Rice et al., 1986). In Zimbabwe production is mainly in the eastern highlands.

The ultimate objective of the production, handling and distribution of fresh fruits and vegetables is to satisfy consumer’s requirements (Escarpa and Gonzalez, 2001). In general the attractiveness of fruits and vegetables to consumers is determined by sensory quality attributes such as colour, astringency, bitterness and aroma and to different aspects of fruits including health benefits (Macheix et al., 1990; Mozetic et al., 2002; Kuti, 2004; Barberan and Espin, 2001). Flavonoids and hydroxycinnamic acid derivatives, secondary metabolites, contribute largely to both fruit colour and through fruit consumption, to human health (Hamauzu, 2006; Mozetic et al., 2002).

There is considerable evidence for the role of antioxidant constituents of fruits and vegetables in the maintenance of health and disease prevention (Veberic et al., 2005; Cao et al., 1998; Goh et al., 2003). Recent studies have shown that the majority of the antioxidative and possible anticarcinogenic activity of fruits and vegetables may originate from the flavonoids and other phenolic compounds (Vallejo et al., 2003; Yang et al., 2001). The term phenolic antioxidant refers to both simple phenolic acids and flavonoids (Harborne, 1998). They are products of secondary plant metabolism and are ubiquitous natural components of plants (Becker et al., 2004), whose evolution, accumulation, content and composition in fruit tissues is invaluable in breeding for improved functional fruits.

Consumption of different pear cultivars contributes to different antioxidant phenolics levels in the diets (Veberic et al., 2005). Characterization of major phenolic families involved is invaluable to the informed recommendation of particular diets and markets with an optimal knowledge of the contents and activity of these natural antioxidants (Chun et al., 2005; Rooyen and Bower, 2003; Miller, 1998).

Colour differences were noted in 2004-2005 season, on the fruits of Starkrimson (red) and Marx red Bartlett (red) common pear trees at Nyanga Experiment Station. New green coloured pear bud mutants were identified on these trees. In this study phenolic compound HPLC profiles, free radicals scavenging activity, and antioxidant activity of the of the phenolic compounds in the wild types and their bud mutants were evaluated and used to discriminate between wild type pear cultivars and their bud mutants with different peel colour. The chemical composition and activities constitute a biochemical marker, which can be used in finger printing cultivars for proper referencing of the germplasm.

Materials and Methods

Pear Samples

The pears investigated in the study are described in Table 18.1. The fruit samples were harvested ripe from Rhodes Inyanga Experiment Station, February 2006. The collected fruits samples were stored at –20 °C and analysed at the University of Zimbabwe’s Department of Biochemistry, in February to May 2006. Peel and pulp portions of the fruit were used as fresh samples.

Table 18.1: Pears Investigated in the Study

NES: Nyanga Experiment Station.

Chemicals

Ethylenediaminetetraacetic acid (EDTA); 1, 1–Diphenyl–2–picryl hydrazyl (DPPH^?); Polyoxyethylene sorbitan monopalmitate (Tween 80); Ethanol; lead acetate-water (1:10,v/v), methanol-water (1:1, v/v), butanol, acetic acid, Folin-Ciocalteu reagent, acetic acid, acetonitrile, vanillic, caffeic acid, p-coumaric acid, protocatechuic acid, ferulic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, (Sigma H 5882: Sigma Co., St Louis, 2004) All the other solvents/chemicals used were of analytical grade and purchased from Sigma Aldrich.

Extraction for Total Phenolics

Total phenolic compounds were extracted from the peel and pulp as described by Makkar (1999). The peel, or pulp sample (2 g) was extracted with 50 per cent aqueous methanol (10 ml). The cell walls were broken by sornicating for 10 min followed by centrifugation at 3, 000 rpm for 10 min. Supernatant was transferred into sample bottles for analysis.

Determination of Total Phenolic Content using Folin-Ciocalteu Method

Total phenolic compounds were determined following the method by Makkar (1999). To a sample (20 ml), distilled water (2.98 ml) was added to make up to 3 ml followed by 1N Folin C. reagent (500 ml) and sodium carbonate (500 ml). After 40 min at room temperature absorbance at 725 nm was read on a Spectronic 20 Genesys Tm spectrophotometer against a blank that contained methanol instead of sample. Total phenolics were expressed in terms of Gallic Acid Equivalent (GAE).

Extraction and hydrolysis for HPLC

Total phenolic compounds were extracted from the peel, and pulp. The peel and pulp sample (2 g) was sornicated for 10 min and hydrolysed with 2 M HCL in boiling water bath for 30 min followed by extraction with 50 per cent aqueous methanol (5 ml). The hydrolysates were centrifuged at 3 000 rpm for 10 min. The supernatant was filtered through and transferred into sample bottles for analysis.

Chromatographic Conditions in HPLC

A Shimadzu HPLC with a SCL-6B Shimadzu systems controller, C-R AX Shimadzu chromatopac, Shimadzu SPD-10 AV UV-Vis detector fitted with a Dynamax 60 A C18 column was used for analysis of phenolic compounds. The amount of sample injected was 5 µl and the flow rate was 1 ml/min. Two mobile phases were employed for elution, water-acetic acid (98:2, v/v, A) and water-acetonitrile-acetic acid (78:20:2, v/v/v, B). The gradient profile used is shown in Table 18.2. Detection was carried out at 280 nm.

Table 18.2: Time Programme for HPLC Analysis of Phenolic Compounds

Free Radical Scavenging Activity with 2, 2–Diphenyl–1–picrylhydrazyl (DPPH•

DPPH• (0.001 g) was dissolved into absolute alcohol (100 ml). To DPPH• (3 ml) the extract (20 ml) was added and mixed. The discoloring of DPPH^? was monitored by taking absorbance readings at 517 nm at 30°C in a spectrophotometer at an interval of 15 min. Alcohol was used as a blank while ascorbic acid (50 mg/ml) was used as control. Scavenging activity was calculated as: [Absorbance at time n (t_n)/absorbance at time zero (t_o)]

Antioxidant Activity Using β-carotene-linoleic Acid Model System

β-Carotene (2 mg) was dissolved in 10 ml chloroform. One ml of the b-carotene solution was pipetted into a 100 ml round-bottom flask and evaporated using a rotavapor at 40°C for 10 min. Tween 80 (400 ml), linoleic acid (40 ml) as well as distilled water (100 ml) were added to the 100 ml flask containing β-carotene. The contents were vigorously shaken to form an emulsion. A blank devoid of

β-carotene was used to zero the machine. Absorbance was read at 470 nm at an interval of 15 min for 120 min.

Results

Determination of Total Phenolic Compounds in Pyrus communis L. (Common Pear)

Secondary metabolites, for example phenolic compounds, are important in determining the nutritional value of a fruit as well as the adaptability of the plant to environmental factors. These factors assist the breeders in identifying the write germplasm in their programs. It is invaluable then to quantify and qualify these compounds for proper reference of the germplasm.

The reaction of phenolic compounds with the Folin and Ciocalteu’s phenol reagent was measured at 725 nm to give the amount of phenolic compounds in 50 per cent methanol extracts. The phenolic content was determined by reference to a standard gallic acid curve.

The green coloured Clapp’s favourite peel had the highest total phenolics (1.541 mg GAE/g); while Starkrimson peel had the least. The amount of total phenolics in Clapp’s favourite peel extracts (Table 18.3) is more than double that of wild type Starkrimson peel. It can be observed that green coloured fruits have peel extracts containing higher total phenolics compared to the red coloured Starkrimson fruit peels. The pulp extracts of Starkrimson (0.065 mg GAE/g) had the highest total phenolics, while those of Clapp’s favourite (0.014 mg GAE/g) had the least.

Stay updated, free articles. Join our Telegram channel

Join