Ascorbic Acid Content in Five Yellow-Flesh Kiwifruit Genotypes

In this study, the content of ascorbic acid (AsA) in five kinds of yellow-flesh kiwifruit genotypes was determined by high performance liquid chromatography. Content of AsA and GSH involved in AsA-GSH cycle were compared in different yellow flesh kiwifruit genotypes. The results indicated that the AsA levels changed vary remarkably in different yellow flesh kiwifruit genotypes, and the AsA levels in ‘Fengyue’ and ‘Guihai 4’ were higher.


Introduction
Kiwifruit belongs to Actinidiaceae Actinidia Lindl, originated in China. Kiwifruit riches in Vc and has high nutritional value, known as "the king of fruits" [1]. Ascorbic acid (AsA) is a high-abundance small molecule antioxidant that is commonly found in plant tissues [2][3][4]. AsA is not only an essential substance for maintaining human health, but also has important physiological functions for the plant itself. Ascorbic acid is an important antioxidant and a cofactor of many enzymes in organisms and plays an important role in plant growth and development and its resistance to stress [3]. The AsA content of plants is highly regulated by its own biosynthetic capacity, and it has been confirmed that L-galactose pathway is the main pathway for AsA synthesis [5][6][7]. In this study, we determined the AsA content of five yellow flesh kiwifruit genotypes using high performance liquid chromatography (HPLC).

Plant material
Five yellow kiwifruit genotypes used in this study were harvested from Kiwifruit Resource Orchard in Shifang (104°16′N, 31°13′E), Chengdu, China. Guihai 4, Jinshi 2, Fengyue, Jinnong and Hort 16A kiwifruit all belong to A.chinensis. Fruits were selected according to the uniformity of the shape when samples have reached physiological maturity (total soluble solid content was 7-8%). At least 10 fruits were harvested for every sample. Prior to preparation of the test samples, the fruit samples were exposed to room temperature to reach easting maturity (total soluble solid content was 10-11%). These fruits were chopped and homogenised under liquid nitrogen in a high-speed blender for 1 min, then immediately frozen in liquid nitrogen and stored at -80°C until use.

Assays for AsA
Frozen tissue (0.5g) was added to 3ml of 0.2% metaphosphoric acid and ground. The homogenate was centrifuged and the supernatant was diluted with 0.2% metaphosphoric acid to 10ml and used for AsA determination. To determine the total AsA (T-AsA) level, method as described by Li et al was used. [8][9]. Thus, a 1000µl aliquot of supernatant was incubated for 4h in the dark with10µl of 200mM dithiothreitol (DTT). AsA was determined as described by Huang et al. [10] and Zhang et al. [11] via a high performance liquid chromatography (HPLC) with system with a photodiode array detector, Chromeleon software (Dinex), and a reverse C18 column. The mobile phase was composed of 15% methanol and 85% metaphosphoric acid aqueous solution, pH2.5. The column temperature was set at 35℃. Spectra were acquired at wavelengths between 200 and 400nm and AsA quantification was performed at 243nm.

T-AsA and AsA levels
As is shown in Figure 1, T-AsA and AsA levels showed great differences in flesh of 5 kiwifruit genotypes. Values for T-AsA and AsA ranged from 14.23 (Hort 16A) to 31.51 μmol/g FW (Guihai 4) and 6.49 (Hort 16A) to 15.28 μmol/g FW (Guihai 4). The T-AsA and AsA content of 'Guihai 4' was significant higher than that of other genotypes, while the lowest value for this parameter was found in 'Hort 16A'. Meanwhile, the results indicated that T-AsA and AsA levels of 'Guihai 4' kiwifruit were higher than 'Hort 16A' kiwifruit. The ratio of AsA/DHA in 'Fengyue' kiwifruit was more than one, but that in other genotypes was less than one. The ratio of AsA/DHA results showed that the AsA in 'Fengyue' kiwifruit flesh was mainly in the reduced state, while that in other kiwifruit genotypes was chiefly in the oxidation state (Figure 1).

T-GSH and GSH levels
Values for T-GSH and GSH differed significantly among kiwifruit genotypes, ranging from 0.41 (Jinshi 2) to 0.92 μmol/g FW (Fengyue) and 0.21 (Jinshi 2) to 0.54 μmol/g FW (Fengyue) in flesh (Figure 2). 'Fengyue' contained the highest contents of T-GSH and GSH in flesh whereas the lowest values were measured from 'Jinshi 2'. The ratios of GSH/GSSG of 'Fengyue', 'Jinnong' and 'Hort 16A' kiwifruit were all more than one, while that of 'Jinshi 2' kiwifruit was less than one, and that of 'Guihai 4' kiwifruit was 1.0015. The ratio of GSH/GSSG results showed that the GSH in 'Fengyue', 'Jinnong' and 'Hort 16A' kiwifruit flesh were mainly in the reduced state, while that in 'Jinshi 2' kiwifruit was chiefly in the oxidation state (Figure 2). There was no correlation between the contents of T-AsA, AsA, T-GSH and GSH, as discovery in apple [12] and persimmon [13].These results indicated that GSH content would not be a key factor of controlling AsA content.

Conclusion
The contentof AsA and GSH involved in AsA-GSH cycle were compared in different yellow flesh kiwifruit genotypes. The results indicated that the AsA levels changed vary remarkably in different yellow flesh kiwifruit genotypes, and the AsA levels of 'Fengyue' and 'Guihai 4' kiwifruit were higher. The AsA content of different kiwifruit genotypes is also different.