Annals of RSCB Vol. XVI, Issue 1

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1 STUDIES INVESTIGATING THE CHANGE IN TOTAL ANTHOCYANINS IN BLACK CURRANT WITH POSTHARVEST COLD STORAGE Simona Oancea 1, Alexandra Cotinghiu 2, Letiţia Oprean 3 1 DEPARTMENT OF FOOD CHEMISTRY AND ENGINEERING; 2 DEPARTMENT OF ECOLOGY AND ENVIRONMENTAL PROTECTION; 3 DEPARTMENT OF FOOD BIOTECHNOLOGY, "LUCIAN BLAGA" UNIVERSITY OF SIBIU, SIBIU, ROMANIA Summary Anthocyanins are secondary metabolites of plants, distributed in vacuoles in the form of glycosides or acyl-glycosides. These natural pigments have shown diverse physiological effects based on their strong antioxidant properties. Epidemiological studies have shown a positive link between the consumption of anthocyanin-rich foods and reduced risk of cardiovascular, cancer or other age-related diseases. The aim of the present paper was to evaluate the level of total anthocyanins in wild and cultivated black currants (Ribes nigrum) from two different growing area in Romania. Quantitative determination was performed by the ph differential spectrophotometrical method. Wild black currants showed an increased total content of anthocyanin, expressed as cyanidin-3-o-glucoside equivalents, compared to the cultivated ones. Also, the study examined the influence of long-term cold storage of plant materials (freezing) upon anthocyanins content. The results showed an increase in anthocyanin content during long-term storage at -18 o C in both investigated samples (11-52%). The results may be of interest for food manufacturers, as any processing method may influence the content and composition of these pigments and consequently their health benefits. Keywords: black currant, anthocyanins, ph differential method, cold storage simona.oancea@ulbsibiu.ro Introduction Since ancient times, plants have been considered important sources of bioactive compounds, in particular useful for traditional medicine. Research programs developed both by academic institutions and private companies of biology, biotechnology, agronomy or pharmacological profile, continue to focus on investigation of plant derived biologically active compounds, as they have great impact on human health and nutrition. Studies in vitro have shown a positive correlation between a diet rich in flavonoids from fruits and vegetables and a reduced risk of cardiovascular diseases and cancer (Babu et al., 2009). In the most important phytochemical groups of phenolics/ flavonoids, anthocyanins have received increasing attention. Considered research 359 has been done on anthocyanin pigments based mainly on their beneficial effects in human health (Motohashi et al., 2008) and their applications as potential natural alternatives of food colorants (Shipp et al., 2010). Anthocyanins are water-soluble vacuolar plant pigments responsible for the bright colors red, purple or blue of flowers, skin, seeds, fruits and leaves. They occur almost universally in plants in different concentrations and compositions depending on both internal (genetic) and environmental factors (Mohr et al., 1995). Structurally, anthocyanins are glycosides or acylglycosides of phenyl-2- benzopyrilium salts, composed of the aglycon part called anthocyanidine (see Figure 1) and carbohydrate residues (glucose, rhamnose, xylose, galactose, arabinose, rutinose) (Harborne, 1998).

2 HO 7 5 3' 4' O + 5' 3 R 2 = H = H, pelargonidin = = H, cyanidin = =, delphinidin = H, peonidin =, petunidin, malvidin Figure 1. General chemical structure of anthocyanidine. The main health promoting effects of anthocyanins are based on their antioxidant properties, which are closely related to the particular chemical structure of an anthocyanin (Rice-Evans et al., 1995). Important dietary sources of anthocyanins from edible plants are families Vitaceae (grape), Rosaceae (cherry, plum, strawberry), Saxifragaceae (red and black currants), Ericaceae (blueberries), Solanaceae (eggplant), and Cruciferae (red cabbage). Black currants are grown mainly for their fruits, which are rich in vitamins C, pro-vitamins A, minerals and bioactive compounds, in particular anthocyanins. Black currants contain mainly delphinidin- 3-O-glucoside, delphinidin-3-o-rutinoside, cyanidin-3-o-glucoside, cyanidin-3-orutinoside and in low extent glycosides of petunidin, peonidin and pelargonidin. The aim of this research study was to evaluate total anthcoyanins in wild and cultivated black currants from two growing area in Romania. As black currant fruits are consumed either as fresh or frozen products, we have also investigated the total anthocyanins content of frozen samples and anlysed in comparison with the values obtained in fresh samples. Material and methods Plant samples Two samples of fresh Ribes nigrum harvested in 2010 were used in the present investigation: (1) wild black currants from Poiana Brasov, Romania; (2) cultivated black currants from Berivoi, Romania. Fresh samples were frozen at -18 o C for six months. 360 Determination of moisture, refractive index and refractometric dry matter Moisture content of samples was determined at 105 o C using the A&D ML-50 moisture analyzer. Refractive index of anthocyanins extracts and soluble dry matter of the Ribes nigrum juices obtained by manually pressing, was determined by refractometry using an Abbe refractometer (Kruss AR2008) at a standardised temperature (21 o C). Values are expressed as refractometric dry substance RDS ( o Brix). Anthocyanins extraction Extraction of anthocyanins from selected samples of Ribes nigrum was conducted in 70% (v/v) ethanol in water, overnight at room temperature. The extracts were filtered and centrifuged at 4 o C at 8000 rpm for 10 minutes. The NF800R refrigerated centrifuge (HT) was used. Anthocyanins assay The total content of anthocyanins in fruit extracts was determined spectrophotometrically by the ph differential method (Giusti et al., 1999). Measurements were made in duplicate. The T80 UV-Visible spectrophotometer (PG Instruments Ltd) was used. Total anthocyanins were expressed as cyanidin-3- O-glucoside (Cyn-3-O-G). Results and discussion Determination of total anthocyanins concentration in different black currant fruits was performed by using the phdifferential method at ph 1.0 and ph 4.5 by difference in absorbances at 510 nm. In the present study, two samples of fresh Ribes nigrum fruits were investigated, as follow: wild black currants from Poiana

3 Brasov, Romania (sample Fr1) and cultivated black currants from Berivoi, Romania (sample Fr2). Anthocyanin analysis was done using Et/H 2 O extractions. Physico-chemical characteristics (moisture, RDS, refractive index) of the anthocyanin extracts and juices, and total anthocyanin level of the fresh samples extracts is presented in Table 1. RDS values is a quality control parameter useful in the fruit products industries and represents a measure of sugar content (indicator of fruit maturity and ripeness). Extract Fr1 showed a significantly higher content of total anthocyanins ( mg/100 g FW) than extract Fr2 ( mg/100 g FW), as seen in Table 1. The two plant samples were grown in regions that slighlty differ in pedoclimatic conditions and from different growing cultures, wild and plantation, respectively. The region Poiana Brasov is situated at an altitude of 1030 meters, has a mean annual rainfall of 855 litres per square meters and is mostly characterized by spontaneous vegetation. The region Berivoi is situated at an altitude of 515 m. It was shown that black currants prefer humid and relatively cold climate and fertile, permeable, acidic soils. Our results showed that growing area of black currant fruits and different cultural practices strongly influence the anthocyanin content. Table 1. Moisture, refractive index, RDS and total anthocyanin contents (milligrams per 100 g of fresh wt) of the investigated black currants fresh samples (Fr1, Fr2). Sample Fr1 Fr2 Parameters Moisture (%) n RDS (%) Total anthocyanin content (mg%) Studies regarding research on flavonoids and anthocyanins in berries as potent antioxidants reported different values of total anthocyanin content for black currants (Ribes nigrum), showing a great variability on environmental factors, genetic types, cultural practices, harvest period and not at least, different methods of extraction and quantitative analysis of pigments. For general comparison insight, values of total anthocyanins in black currants from reported results in literature are shown in Table 2. Black currants are consumed for their health-promoting properties not only as nutrients but also as antioxidants. Food processing of fruits may alter the native state of anthocyanins and the initial total amount. Table 2. Content of total anthocyanins from black currants from selected results from literature (milligrams per 100 g of fresh wt). Sample Reported total anthocyanin content Ref. (mg%) (Wu et al., 2004) black currant (Ribes nigrum) (Timberlake et al., 1988), (Wu et al., 2006), (Clifford, 2000) (Toldam-Andersen et al., 1997), (Kähkönen et al., 2001) Consequently, we have studied the influence of freezing on the total anthocyanin content of the investigated samples after a seven months period of storage at -18 o C. We have found an increase of 11% for sample Fr1 and 52% for sample Fr2 after 7 months of cold storage of the original samples (Figure 2). After 6 months of storage, anthocyanin contents of frozen samples were found quite stable. Research results on some other fruits 361

4 (strawberries, cherries, blueberries, oranges, grapes) reported by different groups of workers (Kalt et al., 1999), (Connor et al., 2002), (Goncalves et al., 2005), (Sanchez- Ballesta et al., 2007) have also shown an increase in anthocyanin content by longterm cold storage, but some other results showed no significant different amounts after storage at low temperatures (Lohachoompo et al., 2004). anthocyanins, mg% Fr1/Fz1 Fr2/Fz2 0 month 3 months 7 months fresh Fr and frozen Fz samples Ribes nigrum Figure 2. Anthocyanin content of Ribes nigrum samples after freezing storage (3-7 months). Anthocyanin contents of frozen samples were found higher than those of the original fresh samples of black currants fruits. Nevertheless, comparison of levels of anthocyanins from different reported data should be conducted with caution as reported values were dependent on the extraction strategies and methods used. Conclusions Anthocyanins are widely distributed water-soluble plant pigments that have proved diverse physiological effects based on their strong antioxidant properties. Epidemiological studies have shown a positive link between the consumption of anthocyanin-rich foods (fruits and vegetables) and reduced risk of cardiovascular, cancer or other age-related diseases. These effects are based on the strong antioxidant properties of the bioactive compounds contained in such foods. As black currants (Ribes nigrum) are fruits consumed in different forms (fresh, juices, products) we have evaluated 362 the total anthocyanin amount of wild and cultivated varieties from two different growing area in Romania. Wild black currants showed an increased total content of anthocyanin, expressed as cyanidin-3-o-glucoside equivalents, compared to the cultivated ones. Both investigated samples proved an increase in anthocyanin content during long-term storage at -18 o C. The results may be of interest for food manufacturers, as any processing method may influence the content and composition of these pigments and consequently their health benefits. Acknowledgements This work was cofinanced from the European Social Fund through Sectoral Operational Programme-Human Resources Development , project POSDRU 89/1.5/S/63258 Postdoctoral school for zootechnical biodiversity and food biotechnology based on the eco-economy and the bio-economy required by ecosanogenesis".

5 References Babu, P.V.A., Liu, D., In: Complementary and alternative therapies and the aging population, pp , Edited by R.R. Watson, Academic Press, San Diego. Clifford, M.N.: Anthocyanins nature, occurrence and dietary burden, J. Sci. Food Agric., 80, , Connor, A.M., Luby, J.J., Hancock, J.F.: Changes in fruit antioxidant activity among blueberry cultivars during cold-temperature storage, J. Agric. Food Chem., 50, , Giusti, M.M., Rodriguez-Saona, Wrolstad, R.E.: Molar absorptivity and color characteristics of acylated and non-acylated pelargonidin-based anthocyanins: J. Agric. Food Chem., 47, No. 11, , Goncalves, B., Landbo, A.K., Knudsen, D.: Effect of ripeness and postharvest storage on the phenolic profiles of cherries (Prunus avium L.), J. Agric. Food Chem., 52, , Harborne, J.B., Phytochemical methods, Chapman&Hall, Thomson Science, London, Kalt, W., Forney, C.F., Martin, A.: Antioxidant capacity, vitamin C, phenolic, and anthocyanins after fresh storage of small fruits, J. Agric. Food Chem., 47, , Kähkönen, M.P., Hopia, A.I., Heinonen, M.: Berry phenolics and their antioxidant activity, J. Agric. Food Chem. 49, , Lohachoompo, V., Srzednicki, G., Craske, J.: The change of total anthocyanins in blueberries and their antioxidant effect after drying and freezing, J. Biomed. Biotechnol., 5, , Mohr, H., Schopfer, P., Biosynthetic Metabolism, In: Plant Physiology, pp , 1995, 4th Ed. Springer, New York. Motohashi, N., Sakagami, H.: Functionality of anthocyanins as alternative medicine. Top Heterocycl. Chem., 15, 1, Rice-Evans, C.A., Miller, N.J., Bolwell, P.G., Bramley, P.M., Pridham, J.B.: Free Radic. Res., 22, 375, Sanchez-Ballesta, M.T., Romero, I., Jimenez, J.B.: Involvement of the phenylpropanoid pathway in the response of table grapes to low temperature and high CO 2 levels, Postharvest Biol. Technol., 46, 29 35, Shipp, J., El-Sayed M. Abdel-Aal: Food applications and physiological effects of anthocyanins as functional food ingredients: The Open Food Science Journal, 4, 7, Timberlake, C.F.: The biological properties of anthocyanin compounds, NATCOL Q Bull., 1, 4 15, Toldam-Andersen, T.B., Hansen, P.: Growth and development in black currant (Ribes nigrum). III. Seasonal changes in sugars, organic acids, chlorophyll and anthocyanins and their possible metabolic background, J. Hortic. Sci., 72, , Wu, X., et al.: Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity, J. Agric. Food Chem., 52, 7846, Wu, X.L., Beecher, G.R., Holden, J.M., et al.: Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption, J. Agric. Food Chem., 54, ,

Dr. Pran Kishore Deb Dr. Balakumar Chandrasekaran Assistant Professor Pharmaceutical Medicinal Chemistry Faculty of Pharmacy, Philadelphia

Dr. Pran Kishore Deb Dr. Balakumar Chandrasekaran Assistant Professor Pharmaceutical Medicinal Chemistry Faculty of Pharmacy, Philadelphia University-Jordan Email: pdeb@philadelphia.edu.jo 1 Learning Outcomes