HESPERIDIN IN CITRUS SPECIES, QUANTITATIVE DISTRIBUTION DURING FRUIT MATURATION AND OPTIMAL HARVESTING TIME

The experiment was done in the Citrus Research Organisation located in Ramsar (North of Iran) and was conducted through two successive seasons of 1997 and 1998. We have studied the effect of harvest time on fruit growth and hesperidin content of 4 Citrus species. Weight, diameter and peel thickness were characteristic to the species. The highest hesperidin content in different Citrus species, used in those investigations, was obtained 50 to 60 days after full bloom. Citrus species had been differentiated according to their hesperidin content. As the local sweet orange is cultivated on large scale in the North of Iran, therefore it is a suitable source to produce hesperidin. INTRODUCTION A well-documented characteristics of the Citrus genus is the accumulation of high amounts of flavonone glycosides in the fruits (Ortuno et al., 1997, Maier and Hasegawa, 1970). Some of these flavonone glycosides are of commercial interest because they are used in the pharmaceutical and food industries. Hesperidin is indicated as oedema protective as well as in the prevention of vein blood stagnation and the regeneration of the normal wall capillary permeability (Gabor, 1998, Ortuno et al., 1997). Several studies have shown that the accumulation of flavonoids in various Citrus species is related to certain stages of fruit growth, the highest levels of these secondary metabolites being detected in very young tissues of the fruit (Castillo et al., 1992, Del Rio and Ortuno, 1994, Ortuno et al., 1995). Therefore, this study was oriented to determine the optimal time of harvest of the immature fruits, to obtain the maximum yield of hesperidin. MATERIALS AND METHODS Plant Material The flavonone glycoside hesperidin was isolated and quantitatively evaluated in local sweet orange (Citrus sinensis), Thomson navel orange (Citrus sinensis), clementine mandarin (Citrus reticulata) and unshiu (satsuma) mandarin (Citrus unshiu). The statistical design used was a factorial examination on the basis of complete randomised design. The experiment was done in the Citrus Research Organisation located in Ramsar (North of Iran) and was conducted through two successive seasons of 1997 and 1998. The main characteristics of the chosen samples were: immature fruits of local sweet orange from 25 years old trees with fruit diameter varying from 12 up to 42 mm. , immature fruits of Thomson navel orange from 17 years old trees with fruit diameter varying from 12 up to 47 mm, immature fruits of clementine mandarin from 25 years old trees with fruit diameter varying from 10 up to 29 mm and immature fruits of unshiu (satsuma) mandarin from 17 years old trees with fruit diameter varying from 10 up to 38 mm. The fruits were harvested at 44, 60, 72, 88 days and 30, 40, 53, 63, 74, 90 days after full bloom in 1997 and 1998, respectively. Crude Hesperidin Extraction After the collection the immature fruits were immediately dried at 50 °C then Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 576, ISHS 2002 92 grounded. After homogenisation, 100 g from each powder sample were exhaustively extracted in a Soxhlet apparatus in two steps, first with petroleum ether (60°C for 24h) to remove the lipophilic fraction, then with methanol (70°C for 48h) to obtain the total extract. Crude hesperidin was completely precipitated by cooling the methanolic extract in the refrigerator. After filtration and washing with cold methanol, the crude hesperidin was dried in an oven at 50°C. Analysis of Hesperidin Content The purity of the crude hesperidin was determined by HPLC. A μ Bondapak C18 (300×3.9 mm) reverse phase column with an average particle size of 10 μm protected by a C18 (10 μm) guard column cartridge was used (Fischer, 1978). HPLC grade solvents were used for the mobile phase in an isocratic mode with acetonitrile 21.5% and water 78.5% at 2ml/min flow rate. The absorbency was monitored at 258 nm with a U.V. detector. All samples were prepared as follows: 5 mg crude hesperidin powder were dissolved in 6 ml dimethyl sulfoxide (DMSO), then the volume was adjusted to 100 ml with methanol. The obtained solutions were filtered through a 0.45 μm single use filter. From each sample, 5μl was injected into the chromatograph. Pure hesperidin standard (97 %) was obtained from Aldrich Co.