Biosynthesis Of Betanin

The importance of diet in reducing the incidence of chronic and degenerative diseases such as cancer and cardiovascular disease is well recognized (1,2). Epidemiological evidence, especially regarding the Mediterranean population (3,4), pointed out on the importance of herbs, fruits, grains, and vegetables. Among other components such as fiber and micronutrients, antioxidants in these foods are thought to be active agents responsible for some of the beneficial effects. Some of the dietary antioxidants are essential nutrients, such as vitamin E, C, and A, and minerals such as Cu, Mn, Zn, and Se, which are cofactors of antioxidant enzymes. In addition, fruits, vegetables, and herbs are particularly rich sources of other nonnutrient antioxidants, including a vast array of phytochemicals (5,6), from carotenoids and bioflavonoids to phytosterols and terpenoids. Other compounds such as betalains, less common in edible species, have more recently been studied (7-10).

The prickly pear cactus, or nopal, is a member of the Cactaceae family widely distributed in Mexico, much of Latin America, South Africa, and the Mediterranean area (11). The metabolism of its crassulacean acid gives this plant a high potential of biomass with low water consumption (12), favoring its growth under semiarid conditions. About 1500 species are in the genus Opuntia, many of which produce edible fruit.

Prickly pear has long been known in traditional medicine for treating a number of pathologies from ulcer, fatigue, and dyspnea to glaucoma, liver conditions, and wounds (11,13). Studies with different models and several experimental conditions provided some scientific basis for the popular use of this plant. Various preparations from fleshy stems (cladodes) have been tested for treatment of diabetes symptomatology in animal models (14,15), or in humans (16). The mechanism for this action is still unknown; some results, however, preclude a role for dietary fiber (15). Other studies revealed beneficial effects against ethanol-induced ulcer (17), in the treatment of benign prostatic hypertrophy (18,19), and in hypercholesterolemia in humans (20) and guinea pigs (21). Diuretic activity of cladode, flower, and fruit infusions has been shown in rats (22). Obviously, other investigations are required to gain insight into the active agents in this plant and the mechanisms involved in all the observed effects.

Cladodes of prickly pear are rich in proteins, carbohydrates, minerals, and vitamins, so Mexicans eat the young pads of the plant, also known as nopalitos, cooked as vegetables. In the industrialized countries of the Mediterranean area, cladodes are not a common nutritional source for humans, but the peeled fruits are usually consumed.

The Sicilian cultivars of prickly pear [Opuntia ficus indica (L.) Mill.] are characterized by yellow, red, and white fruits, due to the combination of two betalain pigments, the purple-red betanin and the yellow-orange indica-xanthin (23,24). The composition and nutritional properties of the prickly pear fruit have long been reported (11,25-29). In contrast, though it was known that the fruit contains vitamin C (29,30), investigations on other antioxidant components, and on its antioxidant capacities, have started only recently. Antioxidant nutrients have been researched in the authors' laboratory, and antioxidant activities of the fruit extracts and of the purified betalains have been evaluated in a number of models in vitro (9,10). In addition, the effects of the regular ingestion of prickly pear fruits on redox balance and bioavailability of betalain components in healthy humans have been investigated (31). This chapter summarizes findings to date, with special emphasis on betanin and indicaxanthin, the characteristic pigments. These phytochemicals, poorly studied until recently, have appeared as important functional components whose antioxidant activity may be a matter of future development.

II. BETALAIN PIGMENTS

A. Chemistry

Betalains are vacuole pigments restricted to flowers and fruits of 10 families of Cariophyllalae plants and to a few superior fungi of the the genus Amanita of the Basidiomycetes (32). Beet (Beta vulgaris) and prickly pear cactus are the only foods containing betalains (33,34). Betalains constitute a class of cationized nitrogenous compounds, the colors of which range from the yellow betaxanthins to the violet-red betacyanins. The betaxanthins are conjugates of betalamic acid with amino acids or the corresponding amines (including dopamine), while almost all betacyanins are derivatives of betanidin, the conjugate of betalamic acid with cyclodopa. The hydroxyl groups at the C5 and C6 position of cyclodopa can be esterified with either a carbohydrate or a carbohydrate derivative to form various betacyanins (Scheme 1). Tyrosinase and Dopa dioxygenase are the only enzymes involved in the synthesis of Dopa, cyclodopa, and betalamic acid, to form the basic skeleton of betalains in plant tissues (35-38), whereas the condensation process of betalamic acid with cyclodopa or amino acids/amines appears as a nonenzymatic reaction (39,40). Glucosyl transferase is involved in the attachment of glucose to betanidine (41).

B. Betanin and Indicaxanthin from Prickly Pear Fruit

The structure of the major betalains occurring in the fruits of O.ficus indica is shown in Figure 1. They are the betacyanin betanin (5-O-glucose betanidine) and the betaxanthin indicaxanthin, the adduct of betalamic acid with proline (9,23,24,42-44). Minor amounts of a few other betacyanins and betaxanthins have been found (44).

Betalains possess high molar absorption coefficients in visible light (33,45), which allows their detection in extracts from various vegetal sources. Indicaxanthin (MW 309) has a clear absorbance peak at 482 nm (A482 = 42,600). Betanin (MW 565) shows an absorbance peak at a wavelength of 536 nm (A536 = 65,000); however, it also absorbs at 482 nm (Fig. 2). Owing to the overlapping of betanin absorbance on the absorbance of indicaxanthin (calculated betanin A482 = 30,900) (9), the indicaxanthin concentration in crude extracts containing both pigments should be measured according to the following equation:

This was applied to investigate the amounts of both betalains in methanolic extracts from fruit of the white, yellow, and red Sicilian cultivars of prickly pear (9).

Scheme 1 Biosynthetic pathway of betaxanthins and betacyanins.

N COOH

N COOH

Figure 1 Structure of betanin and indicaxanthin.

r cooH

r cooH

Betanin Indicaxanthin

Figure 1 Structure of betanin and indicaxanthin.

Simple chromatographic methods have been described to isolate and purify the two major pigments from the fruit of prickly pear (9,42-44).

C. Redox Potential

The oxidation potentials of betanin and indicaxanthin have been evaluated by cyclic voltammetry (9). The cyclic voltammogram showed two and three anodic waves for indicaxanthin and betanin, respectively, indicating that both

Wavelength (nm)

Figure 2 Visible-light absorption spectra of indicaxanthin and betanin.

Wavelength (nm)

Figure 2 Visible-light absorption spectra of indicaxanthin and betanin.

are able to donate their electrons. Three peak potentials of 404, 616, and 998 mV, and two peak potentials of 611 and 895 mV have been calculated for betanin and indicaxanthin, respectively, from the differential pulse voltam-mogram (Fig. 3).

D. Safety

Betalains are important natural pigments for industry. They have been exploited as colorants in processed food (46-48), cosmetics, and pharmaceutical products. To this end, the safety of these compounds has been tested. Studies carried out to determine decomposition and stability (49,50), mutagenicity (48,51,52), and toxicological and toxicokinetic effects (53) showed that these pigments are not harmful. Some in vivo studies in rats indicated that betalains did not have toxic effects with any of the doses tested, up to 5 g/kg body weight (53).

In the authors' laboratory the potential prooxidant activity of the pure betanin and indicaxanthin was tested in a model of copper-stimulated oxidation of human LDL (10). Neither betalain showed adverse effects when assayed in a concentration range of 0.05-50 aM. Rather, both compounds were able to decrease dose-dependently the conjugated diene lipid hydro-

REDPBSDP YEDP50

REDPBSDP YEDP50

Figure 3 Differential pulse voltammetry of betanin and indicaxanthin. The arrows represent the Ep(a) of the anodic waves. (Modified from Ref. 9.)

Potential, V

Figure 3 Differential pulse voltammetry of betanin and indicaxanthin. The arrows represent the Ep(a) of the anodic waves. (Modified from Ref. 9.)

peroxides formed in 120 min, with indicaxanthin more effective than betanin in the range 0.05-1.0 ||M. Above 1.0 ||M both betalains completely inhibited LDL lipid oxidation for the period of observation.

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