ACCUMULATION OF PHENYLPROPANOIDS IN GREEN AND RED PEPPER FRUITS OF SEMI-HOT CULTIVARS Capsicum annuum L

The aim of the present work was to determine differences in C and O glycosides of flavonoids, derivatives of phenolic acids, as well as capsaicinoid content in two semi-hot pepper cultivars, cv. Tornado and Tajfun. Fruits were harvested at the green and red maturity stages. Flavonoid and capsaicinoid fractions were isolated on Sep-Pak C18 cartridges with 40% and 70% methanol-water solutions, respectively. The chemical composition of both fractions was determined by HPLC method using standards of phenolic compounds obtained in the earlier work and the capsaicin standard. The flavonoid fraction contained glucose esters of phenolic acid, mainly ferulic and sinapic acid, as well as derivatives of flavonoids, quercetin, luteolin and apigenin, which occurred as Oand Cglycosides with glucose, rhamnose, and apiose. The capsaicinoid fraction contained mainly capsaicin and dihydrocapsaicin. It was found that in green fruits flavonoid O-glycosides were the predominant phenolics, while in red ones derivatives of phenolic acids. A higher loss of O-glycosides of flavonoids than of C-glycosides was observed during the maturation of the fruit of semi-hot pepper cultivars.


INTRODUCTION
In the recent decades, we can observe the great interest of scientists in the influence of food intake on human health. It has been found that diet rich in fruit and vegetables decreases the risk of cardiovascular and neoplastic diseases and delays the aging processes (R o w l a n d , 1999). This is defined by the term "functional food" applied to food that evidently has a good influence on human health, and "nutriceuticals" used for dietary compounds that promote health. Many food components are recognized as nutriceuticals, among them vitamins, carotenoids, and phenolic compounds ( Phenolic compounds are a group of plant secondary metabolites important for nutritional and therapeutic applications (S h e t t y , 2004). They exert beneficial effects in a multitude of disease states, including cancer, cardiovascular diseases, and neurodegenerative disorders (H a r b o r n e and W i l l i a m s , 2000). The chemical activity of phenolic phytochemicals has been shown to be associated with antioxidant action in biological systems; they act as scavengers of singlet oxygen and free radicals (H a r b o r n e and W i l l i a m s , 2000). The ability to quench free radicals arises from the chemical structure of phenolics.
A number of studies have demonstrated the relationships between their structure and antioxidant activity (R i c e -E v a n s et al. 1997). It has been found that the activity of these compounds generally depends on the presence of hydroxyl substituents and their position in the molecule (R i c e -E v a n s et al. 1997; B u r d a and O l e s z e k , 2001). In pepper fruit they occur as Oand C-glycosides of flavonoids and glucose esters of phenolic acids (M a r i n et al. 2004; M a t e r s k a and P e r u c k a , 2005).
The stimulation of synthesis of protective secondary metabolites results from plant adaptation to biotic and abiotic stresses (S h e t t y , 2004). Phenolics play an important role as cellular support materials and the increase in the levels of these compounds is a response to infection, acting as a precursor for synthe-sis of lignin, suberin and other polyphenolic barriers (Y a l p a n i et al. 1994; D i a z et al. 1998).
The hot taste of peppers arrises from capsaicinoids, alkaloids specific for the Capsicum genus. According to the capsaicinoid content, peppers may be classified as hot, semi-hot and sweet. Capsaicin, the main compound of capsaicinoids, has been studied extensively to exploit its potentially therapeutic value The biosynthesis of phenolic compounds consists of two central pathways: the shikimate and the common phenylpropanoid pathway from L-phenylalanine. The vanillylamine moiety of capsaicinoids is also biosynthetically derived from L-phenylalanine (S u k r a s n o and Y e o m a n , 1993). The convergense of biosynthetic ways of phenolic compounds and capsaicinoids may lead to the synthesis of one group of compounds at the cost of the second one. This situation was confirmed in the hot pepper cultivars, where the synthesis of capsaicinoids occurred at the cost of phenolic compounds (M a t e r s k a and P e r u c k a , 2005).
The aim of the present work was to determine the relationships between phenolic acid, flavonoid and capsaicinoid accumulation during fruit maturation in semi-hot pepper cultivars. Flavonoids were investigated as Oand Cglycosides. In literature there is little information about changes in the accumulation of these compounds in ripe and unripe pepper fruits. For this reason, the relation between Oand Cglycosides in green and red maturity stages of pepper fruits was taken into account.

MATERIALS AND METHODS
The fruits of two semi-hot pepper cultivars: Tornado and Tajfun, were obtained from the experimental field of the Department of Vegetables of the University of Life Sciences in Lublin (Poland). For analysis, the pericarp of pepper fruits at the two maturity stages: mature green (fully developed but not colored) and red ripe (completely red skin), was taken. Immediately after harvest, the pericarps were cut, freeze-dried and stored at -20 o C. Dry matter of fresh pericarps was assayed using the dryer method.
The freeze-dried pericarps of pepper were homogenized with 80% ethanol and after solvent evaporation two fractions of phenylpropanoids were isolated by solid phase extraction (SPE) on a Sep-Pak C18 (Waters) column. The procedure used for the isola-tion of phenolic compounds is described in detail in M a t e r s k a and P e r u c k a (2005). Both fractions were evaporated until dryness at 40 o C and equilibrated to 1 cm 3 and analyzed by HPLC method.
The qualitative and quantitative analysis of particular components of the fractions was made by highperformance liquid chromatography (HPLC) on the WellChrom chromatograph (Knauer) with the UV detector. Separation was done on a column filled with modified silica gel RP-18 (Vertex Eurosil Bioselect 300 Å, 5 m, 4x30 mm, endcapped). Particular derivatives of flavonoids and of phenolic acids were quantitatively determined with the use of standards of phenolic compounds isolated from pepper fruit by means of preparative liquid chromatography and identified by spectral methods NMR and MS, according to the method of S t o c h m a l et al. (2001) and M a t e r s k a et al. (2003a; 2003b). The quantitative determination of the capsaicinoid fraction was conducted by the HPLC method in the isocratic system with 55% CH 3 CN (Merck), with the flow rate of 1ml/min and detection at 280 nm. The capsaicin and dihydrocapsaicin content was determined on the basis of the standard capsaicin solution also containing dihydrocapsaicin (Merck).
The statistical analysis compared the results obtained over the three years of the experiments done in three replications. For each set of data, the standard deviation was calculated and Tukey's multiple test was applied to evaluate the significance of the differences between the means, assuming a 5% error of probability. To define correlations between sets of data, the correlation coefficient was calculated. The calculations were done using Statgraphic v 3.0 for Windows software.

RESULTS
The chemical composition of flavonoids with phenolic acid fractions was determined by HPLC method (Fig. 1). The analyzed derivatives of phenolic acids were as follows: trans-p-feruloyl--D-glucopyranoside and trans-p-sinapoyl--D-glucopyranoside. The Tornado cultivar was characterized by higher levels of phenolic derivatives than Tajfun (Tables 1, 2). It was found that luteolin derivatives were the main flavonoid in pepper fruit of both semi-hot cultivars (Table 1). During fruit maturation, considerable Table 1 The content of flavonoid derivatives in semi-hot pepper cultivars at two maturity stages ( g x g -1 fw.)

Cultivar
Dry matter   changes in phenolic content were found. There was noted a higher level of flavonoid derivatives and total phenolics at green maturity stage than at red ripe stage, while the changes of phenolic acid derivatives during pepper ripening were inverse. The concentration of these compounds increased four times in red fruits of cv. Tornado and 60% in cv. Tajfun when compared to the green fruits (Table 2). It was also found that the Oand Cglycosylated flavonoid content was higher in green fruits than in red ones in the two analyzed pepper cultivars (Table  2). Additionally, it was observed that the decomposition of O-glycosylated derivatives of flavonoids was more dynamic than that of C-glycosylated derivatives. During fruit maturation, O-glycosylated flavonoid content decreased by about 60% in the two cultivars, when compared to green fruits. The highest concentrations of O-glycosylated flavonoids were noted in green fruits of cv. Tornado. The content of C-glycosylated flavonoids was lower than of O-glycosylated flavonoids, and during fruit ripening the levels of these compounds decreased by about 40% and 37% in cv. Tornado and Tajfun, respectively ( Table 2). The capsaicinoid content increased about 47% during maturation of cv. Tajfun. In cv. Tornado a decrease in the concentration of these compounds was observed during ripening, but changes were not significant statistically.

DISCUSSION
Similar changes in the composition of phenolic compounds in pepper fruit during the maturation process were observed by other authors (S u k r a s n o and Y e o m a n ; 1993; H o w a r d et al. 2000). Interesting conclusions may be drawn when our results are compared with those obtained by M a r i n et al. (2004), who analyzed hydroxycinnamic acid derivatives and flavonoid content of sweet pepper at different maturity stages. They found that during fruit maturation both phenolic acid derivatives and Oand Cglycosylated flavonoids decreased. This is in disagreement with our results. It may be explained by the composition of these groups of compounds. In their work, M a r i n et al. (2004) found a compound called "caffeic acid derivative"; it was found in green fruits but was not detected in red ones. Up till now, we have not identified these compounds in the earlier analyzed fractions of hot peppers (M a t e r s k a and P e r u c k a , 2005; M a t e r s k a et al. 2003a, 2003b) and, in this study, we did not show them in the fractions derived from the semi-hot pepper cultivars. On this basis we conclude that these compounds occur mainly in sweet cultivars of pepper and determine the total of hydroxycinnamic acid derivatives in unripe fruits. The second difference between phenolic compound content in sweet and semihot pepper cultivars is the fact that in sweet pepper C-glycosylated flavonoids predominated (M a r i n et al. 2004), contrary to the results obtained in the present work where it was shown that O-glycosylated flavonoids occurred in a higher concentration in semi-hot pepper cultivars (Table 2). Studying the changes in the accumulation of Oand Cglycosidic flavonoid derivatives during maturation of the fruits, we noted higher losses of O-glycosides than of C-glycosides (Table 2). M a r i n et al. (2008) showed that changes in O-and C-glycosidic derivatives of flavonoids in sweet peppers were comparable during maturation of the fruits. It may be explained by the fact that in sweet pepper cultivars changes in the content of phenolic compounds (hydoxycinnamic acids, Oand Cflavonoid derivatives) are less diverse than in semi-hot cultivars which also contain capsaicinoids.
The changes in capsaicinoid content during pepper fruit maturation were not sufficiently statistically significant at P 0.05 in cv. Tornado, but in cv. Tajfun we noticed an increase in the level of these compounds ( Table 2). The results obtained earlier for hot peper cultivars and by other authors indicate that during fruit maturation the content of capsaicinoids decreased (S u k r a s n o and Y e o m a n , 1993; P er u c k a , 1996; C o n t r e r a s -P a d i l l a and Y ah i a , 1998; M i n a m i et al. 1998; P e r u c k a and M a t e r s k a , 2001). The same tendency was noticed in cv. Tornado (Table 2). The decline in capsaicinoid concentration was a result of chemical disintegration due to photooxidation (I w a i et al. 1979) or inhibition of capsaicinoid synthesis (H a l l and Y e o m a n , 1991). During maturation, the biosynthesis of glycosylated derivatives of phenolic acids increased, possibly instead of capsaicinoids and flavonoids.
Additionally, we observed similar changes in total phenolics in the present study (a 23% decrease during maturation), likewise M a r i n et al. (2008) (a decrease of approx. 25% during sweet pepper maturation). On this basis we may conclude that the accumulation of phenolic compound derivatives during the maturation process of pepper fruits changes in different ways -depending on the pungency of the fruit, but total phenolics decrease at a comparable level in the fruit of semi-hot and sweet pepper cultivars.

CONCLUSIONS
1. The accumulation of phenylpropanoids in pepper fruit of semi-hot cultivars related to the stage of fruit maturity. During maturation, total phenolics and the level of flavonoid derivatives decreased, but at the same time the accumulation of phenolic acid derivatives increased.
2. In the fruit of semi-hot pepper cultivars, O-glycosides of flavonoids dominated in both maturity stages.