Bioactive Compounds in Peppers and Their ... - ACS Publications

Chapter 4

Downloaded by UNIV DE SHERBROOKE on November 18, 2012 | http://pubs.acs.org Publication Date (Web): November 15, 2012 | doi: 10.1021/bk-2012-1109.ch004

Bioactive Compounds in Peppers and Their Antioxidant Potential G. K. Jayaprakasha,*,1 Haejin Bae,1 Kevin Crosby,1 John L. Jifon,1,2 and Bhimanagouda S. Patil1 1Vegetable

and Fruit Improvement Center, Department of Horticultural Sciences, Texas A&M University, 1500 Research Parkway A120, College Station, TX 77845-2119 2Texas A&M AgriLife Research, 2415 E. Hwy 83, Weslaco, TX 78596 *E-mail: [email protected]

Substantial recent research has focused on foods containing antioxidant bioactive compounds. Peppers (Capsicum annuum) are a good source of antioxidants and nutrients, as well as bioactive compounds such as flavonoids, phenolic acids, carotenoids and vitamins C, E, A and are also rich in natural colors and aromas. The key bioactive compounds found in peppers including flavonoids, capsaicinoids and capsinoids have been linked to biochemical and pharmacological effects including anti-oxidation and anti-inflammation activities. Capsaicinoids provide the pungent sensation in hot peppers whereas capsinoids are non-pungent compounds present in sweet peppers. Capsinoids have been reported to have anti-inflammatory activity as well as to promote energy consumption and to suppress fat accumulation increase body temperature in humans. The activities of capsinoids, and their lack of pungency, make them attractive for potential applications in food and pharmacology. Other major bioactive compounds of peppers include ascorbic acid, carotenoids, and other antioxidants. The culinary properties and biological effects of bioactive compounds make them extremely important not only for nutrition, but also as pharmacological substrates.

© 2012 American Chemical Society In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction In recent years, awareness of the benefits of functional foods and interest in the discovery of natural bioactive compounds has risen substantially (1–4). Numerous plant secondary compounds, with demonstrated or proposed bioactivities have been described in many foods (5–7). For example, peppers are rich in bioactive compounds and are one of the most valuable vegetable crops; peppers are also widely consumed and consumption of peppers has increased by 18% from 2002 to 2006 (8). Peppers belong to the Solanaceae family and are grown as a perennial shrub in warm climatic zones of the world. The Solanaceae family includes about 90 genera and 2000 species. There are five recognized cultivated species of Capsicum, the dominant globally is Capsicum annum. The other cultivated commercial species are C. baccatum, C. chinense, C. frutescens, and C. pubescens (9). Although peppers are largely used as condiments, or constituents of dishes such as salads, they are also a good source of most essential nutrients such as flavonoids, phenolic acids, carotenoids (β-carotene, capxanthin, zeaxanthin) and vitamins as C, E, A and are also rich in natural colors and aromas (10). The major pungent components in Capsicum peppers are capsaicin [(E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide] and dihydrocapsaicin (11, 12). Intake of these compounds in food can be an important health-protecting factor if they are taken daily in adequate amounts (13). Peppers are also, good source of provitamin A and oxygenated carotenoids that are important for the prevention of macular degeneration and cataracts (14), and phytochemicals such as flavonoids, which may reduce the risk of degenerative disease (15). Reactive oxygen and cellular antioxidants are also important during fruit maturation and for maintenance of food quality. During maturation, pepper fruits undergo a transformation in color, aroma and texture; production of reactive oxygen species (ROS) plays an important role in maturation, including in the biosynthesis of carotenoids and in the transformation of chloroplasts to chromoplasts (16, 17). Later, during fruit senescence, ROS are also released, cellular structures and enzymes are degraded and lipid peroxidation increases (18). Lipid peroxidation in foods is one of the main causes of deterioration of quality, leading to the appearance of unpleasant flavors and disagreeable scents and the destruction of vitamins. The interaction between ROS and proteins is quite complex, and the formation of carbonyl groups is considered, as an irreversible modification, to be a valuable marker of oxidative stress (19). In this context, the function of the cellular antioxidant system is to prevent the offset of chain oxidations and removing ROS is vital. The enzymatic system of defense includes superoxide dismutase, catalase, peroxidases and the ascorbate glutathione cycle enzymes (20). In the last 10 years, several reports have provided evidence for the involvement of antioxidants in fruit physiology, including a response at the level of mitochondria, peroxisomes and chloroplasts during pepper ripening and during fruit storage at 20 °C (18, 21). Non-enzymatic antioxidants include both polyphenols and non-phenolic compounds such as ascorbic acid, and carotenoids, which are important in vegetables (22). The human organism cannot synthesize many of these protective 44 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


chemical substances, and they can only be obtained from foods. Polyphenols are widely distributed in plants and contribute to their color and flavor; two major classes of polyphenols, flavonoids and phenolic acids, protect the organism from the damage produced by oxidative agents. Levels of polyphenols are a good indication of the antioxidant capacity of peppers (9), and numerous epidemiological studies indicate a possible association between their uptake and reduction of the risk of coronary disorders and cancer (6).

Figure 1. Structures of flavonoids and phenolic acids found in peppers. 45 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Polyphenols, Flavonoids, and Their Antioxidant Potential Flavonoids and other polyphenols (Figure 1) are ubiquitous phytochemicals and some of the most abundant and important bioactive compounds present in green, sweet, and hot peppers. Many studies have focused on identifying and quantifying flavonoid levels in peppers (23–27). Glycosides and aglycones of myricetin, quercetin, luteolin, kaempferol, and apigenin are also found in peppers (23). Flavonoids show high antioxidant and anticancer activities, and this is related to the presence of the numbers of hydroxyl groups at certain position and a double bond at the C2-C3 position. Based on the relationship of structure and antioxidant activity, myricetin seems to be one of the most powerful flavonoid (28–30). The structures of C- and O-glycosides of flavonoids in peppers have been identified based on MS–MS fragmentation and UV spectra (31). The four reported quercetin glycosides are: quercetin 3-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-glucoside, quercetin 3-O-glucoside-7-O-rhamnoside and quercetin glycosylated with rhamnoside-glucoside attached either at the C-3 or C-7 position (32). Two luteolin O-glycosides, luteolin (apiosyl-acetyl)-glucoside and luteolin 7-O-(2-apiosyl)-glucoside and five luteolin C-glycosides, luteolin 6-C-hexoside, luteolin 8-C-hexoside, luteolin 6-C-pentoside-8-C-hexoside, luteolin 6-C-hexoside-8-C-pentoside and luteolin 6,8-di-C-hexoside, were found in the pericarp of pepper fruits. In addition, two apigenin C-glycosides were also identified as apigenin 6-C-pentoside-8-C-hexoside and apigenin 6, 8-di-C-hexoside (33). Flavonoid levels change depending on genetic, developmental and environmental conditions. For example, the highest amounts of flavonoids are found in the red fruits of most cultivars, except cv. Cyklon (34). By contrast, quercetin 3-O-R-L-rhamnopyranoside was the main compound in the flavonoid fraction from green fruits, and the highest amounts were reported in cv. Cyklon. The levels of quercetin glycoside were high in green fruit and decreased during ripening (34, 35). Measurement of bioactive compounds is both crucial for unraveling their effects, and difficult because of their different chemical structures. For example, the antioxidant capacity of extracts made with a specific solvent does not represent all the bioactive compounds present in the pepper, because peppers have various polar and non-polar antioxidants, such as ascorbic acid, carotenoids, capsaicinoids and flavonoids. All compounds cannot be extracted using one solvent. To reveal the full antioxidant capacity of peppers, we have studied a range of extraction conditions and solvents of different polarities, to extract both polar and non-polar bioactive compounds (36). Our group recently measured the levels of flavonoids including quercetin, myricetin, luteolin and kaemperol and apigenin in Paprika and Habanero peppers (Figure 1) (23). Ethanol and methanol extraction showed the highest total phenolics and maximum radical scavenging activity (23). Furthermore, eight pepper cultivars include cayenne (C. annuum L. cv. ‘CA408’ and ‘Mesilla’), jalapeño (C. annuum L. cv. ‘Ixtapa’), and serrano (C. annuum L. cv. ‘Tuxtlas’) were studied to examine the antioxidant potential and active chemical constituents in various polar fractions. Five of these cultivars were commercial types (TMH, Mesilla, Ixtapa, Tuxtlas, and TMJ) and three 46 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


were advanced breeding lines (PA137, B58, and CA408). Different levels of flavonoids such as quercetin, luteolin, kaempferol, and apigenin were found in these pepper extracts (10, 36). Flavonoids were not detected in hexane extracts and the remaining four solvents extracted differential levels of flavonoids in all pepper cultivars. Figure 2 lists the levels of flavonoids in eight different pepper cultivars, as determined using extraction with five different solvents. Hydrophilic flavonoids were extracted to the maximum using methanol. The highest levels (152.2 µg/g) of flavonoids were found in methanol extract of paprika (B58). In contrast to peppers, root vegetables such as carrot, radish, burdock, and potato contain simple polyphenolic acids and their glucosides (Figure 1). Leaf vegetables such as cabbage, chive, lettuce, and spinach has flavones and flavonols mainly in the glycoside form. Among leafy vegetables, celery and parsley are classified as herb vegetables along with peppermint, sage, oregano, and thyme (32, 37). They contain aglycon forms of flavones and flavonols at relatively high levels. Interestingly, peppers found to have polyphenolics, flavones and flavonols (Figure 1).

Figure 2. The levels of total flavonoids found in eight cultivars of hot and mild peppers (µg/g) determined by reversed phase HPLC (10, 37). Values are means ± standard error of triplicate analysis. The same letter within a column and a pepper cultivar is not significantly different using Tukey’s test.

Levels of Capsaicinoids and Their Beneficial Properties for Human Health In 1816, P. A. Bucholz isolated the capsaicin molecule for the first time (38), but the spicy properties of hot peppers have intrigued the human palate for thousands of years. Capsaicinoids are responsible for the pungent sensation in fruits of the genus Capsicum and the two major capsaicinoids, capsaicin and dihydrocapsaicin, and are responsible up to 90% of the total 47 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


pungency of pepper fruits. Other minor capsaicinoids are nordihydrocapsaicin, homodihydrocapsaicin, homocapsaicin and nonivamide (Figure 3). The degree of pungency depends on the cultivar and environmental conditions during plant growth and fruit development/maturation Also, the levels of capsaicinoids are affected by different factors such as the developmental stage of the fruit (39) and the environmental growth conditions (40–42). Moreover, measurement of capsaicinoids also depends on extraction conditions, since peppers have a wide variety of compounds (e.g. polar and non-polar compounds), the observed functional properties such as antioxidant capacity will also depend on the assay method. Different solvents have different capacities for extracting compounds of interest from different media (43). The importance of optimized analytical methods for characterizing the levels and diversity of bioactive constituents in foods can, therefore, not be overemphasized. Few studies have critically assessed the importance of solvent properties in the accurate isolation and quantification of bioactive compounds in foods (23, 43). The complexity of the bioactive constituents in peppers makes it difficult to isolate each compound with precision. For instance, the prevalence of pigment-fatty acid complexes in peppers makes it difficult to efficiently extract certain bioactive compounds (44). Thus maximum extraction requires use of a saponification step to hydrolyze these pigment-fatty acid complexes, and hence optimize extraction and simplify the quantification of bioactive compounds in pepper. Table 1 shows the total capsaicinoid concentrations in samples from four different cultivars of hot pepper extracted with different solvents. The levels of capsaicinoid extraction were observed in the following order: hexane > EtOAC > acetone. Capsaicinoids were not found in MeOH and MeOH:water (80:20) extracts. The maximum amounts of capsaicinoids were extracted in hexane. The highest level (5072 µg/g) of capsaicinoids was found in the Ixtapa, and the lowest amount (71.5 µg/g) was found in Tuxtlas pepper (36). Multiple studies indicate that consuming capsaicinoids may provide health benefits; for example, these compounds are reported to inhibit iron-mediated lipid peroxidation and copper-dependent oxidation of low-density lipoprotein (38), an effect ascribed to their capacity to form complexes with reduced metals and act as hydrogen donors. Capsaicin also demonstrated for anti-obesity activity in an animal model (45). Moreover there is epidemiological evidence for an association between consumption of capsaicinoid-containing foods and lower incidence of obesity. It is widely accepted that increasing energy expenditure and reducing energy intake form the basis for management of weight (46, 47). Consuming capsaicin one hour before low intensity exercise improved lipolysis and it may be a valuable supplement in the treatment of individuals with hyperlipidemia and obesity (48). According to animal and human studies, dietary capsaicin may be considered a functional agent that helps to prevent obesity. However, it is not ideal for controlling obesity in humans because the long-term consumption of capsaicin may be limited by its pungency. Capsaicin can also prevent the oxidation of oleic acid at cooking temperatures (49) as well as the formation of lipid hydroperoxides from the autoxidation of linoleic acid (37). Despite these intriguing findings, the use of capsaicinoids as food antioxidants is obviously limited by their strong pungency and potentially noxious properties. 48 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Chemical structures of capsaicinoids from hot peppers.

49 In Hispanic Foods: Chemistry and Bioactive Compounds; Tunick, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Table 1. Levels of capsaicinoids (µg/g) found in four hot pepper cultivars from different individual solvent and total content. Dried peppers were extracted with various solvents and quantified by HPLC (10, 36) Solvents used for extraction

CA408

Mesilla

Ixtapa

Tuxtlas

Hexane

83.8 ± 0.3

549.3 ± 12.9a

3511.4 ± 10.1a

51.9 ± 0.4a

EtOAC

0

18.0 ± 0.5b

1035.3 ± 26.8b

11.4 ± 1.5b

Acetone

0

7.4 ± 3.2c

525.6 ± 42.1c

8.2 ± 1.0c

83.8

574.8

5072.4

71.5


Total

Values are means ± standard error of triplicate analysis; nd: not detected. The same letter within a column and a pepper cultivar is not significantly different using Tukey’s test.

In addition to dietary benefits, capsaicin is also applied topically for chronic pain, to reduce aches and burning feelings, the frequently reported symptoms of painful neuropathy (50). It is currently used in topical creams and high-dose dermal patches to relieve the pain of peripheral neuropathies such as post-herpetic neuralgia caused by shingles, and can be directly applied to abdominal skin with no side effects in animal models and humans (51).

Capsinoids from Sweet Peppers Capsinoids are non-offensive and devoid of pungency, having an ester group instead of the amide moiety in capsaicinoids. Capsinoids (Figure 4) are found in the fruits of non-pungent cultivars of Capsicum annuum L. (CH-10 sweet) peppers at

Bioactive Compounds in Peppers and Their ... - ACS Publications

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