Ascorbic acid, phenolic content, and antioxidant capacity of red, cherry, yellow and white types of pitaya cactus fruit (Stenocereus stellatus Riccobono)

Ácido ascórbico, contenido fenólico, y capacidad antioxidante de las variedades roja, cereza, amarilla y blanca del fruto del cactus de la pitaya (Stenocereus stellatus Riccobono)

M. Carmen Beltrán–Orozco*, Tzatzil G. Oliva–Coba, Tzayhri Gallardo–Velázquez, Guillermo Osorio–Revilla

Departamento de Graduados en Investigación en Alimentos. Escuela Nacional de Ciencias Biológicas. Instituto Politécnico Nacional. Prolongación de Carpio y Plan de Ayala. Colonia Santo Tomás. 11340. Delegación Miguel Hidalgo. México D. F., México. *Author for correspondence: (cbeltran@encb.ipn.mx)

Abstract

In order to sustain the growing interest in the consumption of pitaya cactus (Stenocereus stellatus Riccobono) fruit as potential neutraceutical food, the total phenolics and ascorbic acid contents were determined in four pitaya cactus fruit types (red, cherry, yellow and white). Total phenol and ascorbic acid concentrations were correlated to the antioxidant capacity (fimol Trolox eq g^–1 fresh sample). The results showed that the white and yellow types contained a higher amount of phenol compounds and ascorbic acid than the cherry and red types. A linear relationship was found between the fruit antioxidant capacity and the total phenol content (R²=0.97) and ascorbic acid content (R²=0.79), indicating that both of them contribute together to its antioxidant properties, but the contribution of ascorbic acid accounts only for 4–6 % of antioxidant capacity. The antioxidant capacity displayed by the four pitaya types are similar to those reported for some fruits of the Vaccinium genus, regarded as the fruits having the highest antioxidant capacity. The consumption of pitaya fruits could provide the same protecting effect against free radicals than berries of the Vaccinium genus, reducing risk of chronic diseases; thus pitaya fruits can be considered as potencial nutraceutical food.

Keywords: Stenocereus stellatus Riccobono, antioxidant capacity, ascorbic acid, nutraceutic, pitaya, total phenolic content.

Resumen

Con el fin de mantener el creciente interés en el consumo del fruto de la pitaya (Stenocereus stellatus Riccobono) como alimento nutracéutico potencial, se determinó el contenido fenólico y de ácido ascórbico total en cuatro variedades de frutos de pitaya (roja, cereza, amarilla y blanca). Las concentraciones fenólicas y de ácido ascórbico totales se correlacionaron con la capacidad antioxidante (mmol Trolox eq g^–1 muestra fresca). Los resultados mostraron que las variedades blanca y amarilla tuvieron mayor contenido de fenoles y de ácido ascórbico que la cereza y la roja. Se encontró una relación lineal entre la capacidad antioxidante del fruto y el contenido de fenoles totales (R²=0.97) y de ácido ascórbico (R²=0.97), lo que indica que ambos contribuyen en sus propiedades antioxidantes; sin embargo, dicha contribución representa sólo 4–6 % de la capacidad antioxidante. La capacidad antioxidante mostrada por las cuatro variedades de pitaya es similar a la de algunos frutos del género Vaccinium, considerados como los de mayor capacidad antioxidante. El consumo de frutos de pitaya podría proporcionar el mismo efecto protector contra radicales libres que las bayas del género Vaccinium, reduciendo el riesgo de enfermedades crónicas; así, los frutos de la pitaya pueden considerarse como alimento nutracéutico potencial.

Palabras clave: Stenocereus stellatus Riccobono, capacidad antioxidante, ácido ascórbico, nútraceutico, pitaya, contenido fenólico total.

INTRODUCTION

Evaluation of nutritional components of a food product is not sufficient to understand and evaluate the effects of that food on the organism. It is required as well, to consider a series of microcomponents (phytonutrients), previously known as non–nutritional or secondary components, which include antioxidant compounds (Moure et al., 2001; Wang et al., 1996). Secondary plant metabolites are found in most of the fruits, vegetables, and teas (Amakura et al., 2000). Plants are the basis of all traditional medicinal therapy (Zheng and Wang, 2001) and in 1992 the positive effect of antioxidants was found in fruit and vegetables (Ames et al., 1993; Hertog et al., 1992, 1993, 1995). Free radicals are the leading cause of degenerative diseases such as several forms of cancer, cardiovascular disease, and neurological diseases (Halliwell, 1994; Yu, 1994). Vegetables and fruits antioxidants work as singlet and triplet oxygen quenchers, free radical scavengers, peroxide decomposers, and enzyme inhibitors (Wang and Lim, 2000). Many of their protective biological effects are derived from their antioxidants functions (Velioglu et al., 1998). There is an increasing interest to know the antioxidant capacity of fruits and vegetables in order to use their potential as nutraceuticals or functional foods.

Pitayo (Stenocerus stellatus Riccobono) is a columnar cactus very resistant to drought, which produces edible fruits locally known as pitayas that represent a valuable economic alternative for farmers living in Mexico's semiarid regions. The pitayas commercially available includes the genera: Hylocereus, Pachycereus, Stenocereus, Carnegiea, Machaerocereus and Echinocereus (Bravo and Sánchez, 1991).

In México, the genus Stenocereus spp. is represented by about 19 species distributed from Baja California peninsula, along the Pacific basin to the state of Chiapas, and also along the Gulf basin from the state of Tamaulipas to Veracruz. The genus Stenocereus is most abundant in the south and southeast of the Trans–Mexican Volcanic Belt. The most important species of this genus, from an economic point of view are: S. marginatus, S. stellatus, S. treleasei, S. griseus, S. fricii, S. queretaroensis and S. quevedonis (Granados et al., 1999). The pulp of this fruit is a fresh and sweet food, the nutritional importance of which derives from its high sugar content plus considerable amounts of vitamins B, C and E (Bravo and Scheinvar, 1998).

A number of applications have been developed for the fruit pigments, mainly betalains, and mucilage present in the pulp and mesocarp of these types of fruits (Stintzing et al., 2002). There are reports about the antioxidant capacity and quantification of phenol compounds in pitahaya (Hylocereus undatus) (Wybraniec and Mizrahi, 2002). However, few reports were found for the genus Stenocereus, which is the most cultivated and popular pitaya–producing cactus in México.

The present study was carried out to quantify the total phenolic and ascorbic acid contents in the pulp of four pitaya types of the genus Stenocereus stellatus Riccobono, and to correlate them with the antioxidant capacity of these fruits, to sustain the growing interest about these fruits as potencial nutraceuticals foods.

MATERIALS AND METHODS

Fruits

Ten kilograms of fruit were collected, peeled and the pulp classified by color into yellow, red, cherry and white pulp. The fruit pulp of each color (including seeds) was homogenized and separated in portions of 150 g which were stored at –20 °C in sealed plastic bags until further analysis. Three pulp sample bags of each type were unfrozen overnight and each one analyzed in triplicate, as described below, and averaged. The results for the three sample bags were averaged again and the standard deviation calculated for each type.

Ascorbic acid determination

Ascorbic acid content was determined through xylene–extraction (Ranganna, 1986), using a Perkin Elmer UV/VIS lambda 3 spectrophotometer.

Standard curve

Test tubes were prepared with 0.0, 0.5, 0.75, 1, 1.5 and 2 mL of standard ascorbic acid solution in 3 % H₃PO₃ (0.1mg mL^–1) and made up to 2 mL with 3 % H₃PO₃ solution. Then 2 mL of acetate buffer (pH 4), 3 mL of 2, 6 dichlorophenol indophenol solution (0.0007 M) and 15 mL of xylene were added in rapid succession. Tubes were capped and stirred for 10–15 s. Phase separation was allowed. The xylene phase was extracted and absorbance was measured at 520 nm using xylene as blank.

Sample analysis

From each pitaya pulp type 100 g were ground in 3 % H₃PO₃, making up the volume to 200 mL. This solution was filtered and 2 mL aliquot was transferred to a test tube, adding 2 mL of the acetate buffer solution (pH 4), 3 mL of 2,6 dichlorophenol indophenol solution (0.0007 M) and 15 mL of xylene in a rapid succession. The tube was capped and stirred for 10–15 s. The xylene phase was extracted and absorbance was read at 520 nm. The absorbance of the sample was measured against a blank prepared as described above, but adding distilled water instead of the 2,6 dichlorophenol indophenol solution. The ascorbic acid content was expressed in mg acid 100 g^–1 sample.

Total phenolic compounds

This determination was carried out according to Singleton and Rossi (1965), using a Perkin Elmer UV/VIS lambda 3 spectrophotometer.

Solutions containing 100, 200, 300, 400 and 500 mg L^–1 galic acid were prepared. From each solution 100 µL were transferred to a test tubes adding 100 µL of deionized water, 1mL of the Folin Ciocalteu reagent and 0.8 mL of sodium carbonate solution (7.5 %). Tubes were stirred and allowed to stand in the dark for 30 min. Absorbance at 765 nm was measured against a blank prepared in the same way substituting the galic acid solution by distilled water.

Sample analysis

From each pitaya type 20 g pulp were ground and extracted in 2 % HCl in methanol for 24 h in the dark at room temperature. The extracts were diluted with the same solvent used for extraction in 100 mL and filtered; l00 µL aliquots were transferred to a test tube, adding 100 µL of deionized water, 1 mL of the Folin Ciocalteu reagent and 0.8 mL of sodium carbonate solution (7.5 %). Tubes were stirred and then left to stand for 30 min in the dark. The absorbance was determined at 765 nm against a blank prepared as described above, but changing the Folin Ciocalteu reagent by distilled water. Since the ascorbic acid also shows reaction with the Folin Ciocalteu reagent, the absorbance obtained as explained before, was corrected by the ascorbic acid content as follows: solutions of ascorbic acid with the same concentration found in 20 g of each type of fruit were prepared and the total phenols technique was applied to them. The absorbance obtained corresponding to the ascorbic acid was subtracted from that obtained for the fruit, an the total phenolic content was then calculated and expressed as galic acid equivalent (GAE) per 100 g of dry sample.

Antioxidant capacity

The antioxidant capacity (AC) of the four types of pitaya, was determined as Trolox (6–Hydroxy–2,5,7,8–tetramethylchroman–2–carboxylic acid) equivalent AC (TEAC) and ascorbic acid equivalent AC (AEAC), following the method described by Fogliano et al. (1999) which is based on the formation of a colored cationic DMPD⁺ (N–N–dimethyl–phenylenediamine) radical (purple) in the presence of an oxidizing solution (Fe³⁺). This free radical becomes colorless as a result of the transfer of an hydrogen atom from an antioxidant compound (pitaya sample) or antioxidant standard as Trolox.

DMPD solution (100 mM), was prepared dissolving 209 mg of DMPD in 10 mL of deionized water; 1 mL of this solution was added to 100 mL of 0.1 acetate buffer (pH 5.25), and the colored radical cation (DMPD⁺) was obtained by adding 0.2 mL of ferric chloride solution (0.05 M).

From this solution 1 mL was directly placed in 1 cm cuvette and its absorbance at 505 nm was measured, corresponding to the uninhibited signal (Ao). Solutions of the antioxidant standard Trolox were prepared diluting with methanol a solution of 1 mg mL^–1 of Trolox (0.1 g in 100 mL of methanol) to suitable concentrations. 50 mL of the Trolox standard solutions were added to 950 mL of the colored radical DMPD⁺solution. The mixture was stirred for 10 min, and the absorbance at 505 nm was measured (A_f). A dose–response curve was derived for Trolox, by plotting the absorbance at 505 nm as percentage of the absorbance of the uninhibited radical cation solution (blank) using the following equation: Inhibition of A₅₀₅ (%) = (1–A_f/ Ao) X 100, where Ao is the absorbance of the uninhibited radical cation and A_f is the absorbance measured 10 min after the addition of the antioxidant standard solutions of Trolox or the antioxidant in the extract of pitaya samples.

For the AEAC, ascorbic acid standard solutions were prepared in deionized water and used in the same way as described for Trolox standard solutions.

Sample analysis

The results obtained for the TEAC were correlated with both, galic acid equivalent (GAE) as an indicator for the total phenolic content and ascorbic acid content to obtain the contribution of each one to the total AC.

RESULTS AND DISCUSSION

Results of ascorbic acid content in the pulp of the four pitaya types studied are shown in Table 1. The ascorbic acid content in the pitaya types, per 100 g edible pulp, accounts for 21 % of the recommended daily intake of ascorbic acid in adults (60 mg d^–1) (Muñoz et al., 2002).

Ascorbic acid content for the four pitaya types varied between 8 and 14 mg 100 g^–1 with a mean of 13 mg 100 g^–1, which is similar to the following fruits: capulin (Prunus serotina Cav.) 13 mg 100 g^–1; chicozapote (Manilkara sapota L.) 12 mg 100 g^–1; banano (Musa paradisiaca L.) 13 mg 100 g^–1; and yellow plum (Prunus domestica L.) 12 mg 100 g^–1. The vegetables with a similar ascorbic acid supply include: zucchini (Cucurbita pepo L. var melopepo) 13 mg 100 g^–1; chayote (Sechium edule L.) 12 mg 100 g^–1; cucumber (Cucumis sativus L.) 13 mg 100 g *; white onion (Allium cepa L.) 12 mg 100 g^–1; and avocado (Persea americana L.) 14 mg 100 g^–1 (Muñoz et al., 2002). Besides, the cherry and yellow types of pitaya show the lowest and highest ascorbic acid content.

The total phenolic content corrected by ascorbic acid showed considerably variation between the four fruit types (Table 2). Opposed to what it was expected, the red and cherry types showed a lower total phenolic compounds than the yellow and white types. Thus the red and cherry types have similar content of total phenols (1384–1552 GAE) are at the lower end; the yellow and white types also have similar total phenols content (2129–2395 GAE) but considerably higher.

The total phenolic content in the red and cherry types (1384 to 1552 GAE) is similar to that of fruits with a high antioxidant activity derived from their content of phenol compounds: apple (Malus pumila) 1300–1310 GAE, and strawberry (Fragaria ananassa) 1600 to 1800 GAE. The yellow and white types (2129 and 2395 GAE) are similar to cranberry (Vaccinium oxycoccus) 2200 GAE, raspberry (Rubus idaeus) 2700–2900 GAE, being lower than bilberry (Vaccinium myrtillus) 3003–3800 GAE, which is regarded as one of the fruits with the highest antioxidant power due to its high phenolic content (Kähkönen et al., 2001).

The AC for the four pitaya types studied, expressed as TEAC, was considerably higher (16.8–17.3 µmol g^–1) for the yellow and white types than that of the red and cherry types (11–12.2 µmol g^–1) in agreement with the total phenolic content in (Table 3). For the four pitaya types TEAC varied between 11 and 17.3 µmol g^–1 which is comparable to: cabbage (Brassica oleracea) 17.7 jumol Trolox eq g^–1 of edible sample; strawberry (Fragaria ananassa) 15.4 µmol Trolox eq g^–1 edible sample; and spinach (Spinacia oleracea) 12.6 µmol Trolox eq g^–1 edible sample (Pellegrini et al., 2003). Compared to the Vaccinium genus berry, considered the fruit with the highest AC, the yellow and white pitaya types showed an AC (16.8–17.3 µmol g^–1) equivalent to the lowest value reported for Vaccinium corymbosum (17.0 µmol g^–1 ±1.0 µmol Trolox eq g^–1), whereas the AC of the red and cherry pitaya types (11–12.2 µmol g^–1) was similar to the Climax type of Vaccinium ashei (13.9±4.1) (Prior et al., 1998).

Based on the results obtained for ascorbic acid, total phenols and AC, pitaya fruits can be regarded as functional food, since besides their nutrients, they have an excellent AC. Therefore, consumption of these fruits could provide protecting effects against free radicals, reducing risk of chronic diseases. According to Bickford et al. (1997), fruits rich in antioxidant phytochemicals, like strawberry and Vaccinium berries, have a beneficial effect delaying the deficiencies in the central nervous system that occur when these cells age, besides protecting the organism from oxidizing stress (Bickford et al., 1997). Thus pitaya consumption, with an AC within the range reported for Vaccinium berries, may have the potential of producing the same effect. However, further in vivo studies on the pitaya AC are required. Prior et al. (1998) also reported that an increase in the blood plasma AC occurs with an intake of 3–4 mmol Trolox eq d^–1, equivalent to a daily intake of 4 to 5 cherry or red pitaya fruits, or 3 to 4 white or yellow pitaya fruits.

A linear relationship (R²=0.97) was found between the total phenolic content of the four pitaya types and the AC of the fruit (Figure 1). This high coefficient suggests that phenol components represent a significant contribution to the pitaya. This regression equation (Figure 1) can be used to calculate the antioxidant capacity of this fruit from its phenol content. In Figure 1, the cherry and red types lie at the lower end of the total phenol content, and hence of TEAC, whereas the yellow and white types show the highest TEAC concentrations corresponding to the higher AC.

There was a linear relationship (R²=0.79) between ascorbic acid content and the AC for the four pitaya types studied (Figure 2). This coefficient suggests that pitaya's ascorbic acid contents makes a lower contribution to the fruit AC as compared that of the total phenolic content. This assumption can be corroborated if the ascorbic acid content of each pitaya type (Table 2) is subtracted from the total AC expressed as AEAC (Table 4).

The ascorbic acid content in each fruit type accounts only for about 4 to 6 % of the total AC (Table 4), corroborating the assumption that the main contribution to the total AC is due to phenols and other compounds. Based on this, those fruit types with larger concentrations of phenols will show a higher AC as is the case of the yellow and white fruit types.

In Figure 2 it is shown that the cherry and red types, which contain less concentration of ascorbic acid, lie at the lower end of correlation line. The yellow and white types, with the highest concentrations of ascorbic acid, show the highest TEAC corresponding to the higher antioxidant capacities.

CONCLUSIONS

The Stenocereus stellatus Riccobono types studied (red, cherry, yellow and white) showed an antioxidant capacity similar to that of the Vaccinium genus, considered the fruits with the highest antioxidant capacity. Therefore, the consumption of these fruits could provide a similar protection against free radicals than berries of the Vaccinium genus. Thus, they could be considered as nutraceutical food.

White and yellow pitaya types showed higher phenolic content than cherry and red types, showing a higher antioxidant capacity. Likewise, the ascorbic acid content of white and yellow pitaya types was higher than the other two pitaya types.

ACKNOWLEDGEMENTS

Financial support is acknowledged to the Secretaria de Investigación y Posgrado del Instituto Politécnico Nacional, México, and grants (EDD, COFAA) given to the authors that made this workpossible.

LITERATURE CITED

Amakura, Y., Y. Umino, S. Tsuji, and Y. Tonogai. 2000. Influence of jam processing on the radical scavenging activity and phenolic content in berries. J. Agric. Food Chem. 48: 6292–6297.        [ Links ]

Ames, B. M., M. K. Shigenaga, and T. M. Hagen. 1993. Oxidants, antioxidants and the degenerative diseases of aging. Proc. of the National Academy of Sci. U.S.A. 90: 7915–7922.        [ Links ]

Bickford, P. C., K. Chadman, G. Taglialatea, B. Shukitt–Hale, R. L. Prior, G. Cao, and J. A. Joseph. 1997. Dietary strawberry supplementation protects against the age–accelerated CNS effects of oxidative stress. Fed. Am. Soc. Exp. Biol. J. 11: A176.        [ Links ]

Bravo, H. H., y M. H. Sánchez. 1991. Las Cactáceas de México, 2nd. ed. UNAM. México, D. F. México. pp: 501–518.        [ Links ]

Bravo, H. H., y L. Scheinvar. 1998. Donde crecen las cactáceas. In: Retif, A. (ed). El Interesante Mundo de las Cactáceas. CONACYT y Fondo de Cultura Económica, México. pp: 9–22.        [ Links ]

Fogliano V., V. Verde, G. Randazzo, and A. Ritieni. 1999. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem. 47: 1035–1040.        [ Links ]

Granados, S. D., B. A. Mercado, y R. G. López. 1999. Las Pitayas de México. Ciencia y Desarrollo 145 (2): 58–67.        [ Links ]

Halliwell, B. 1994. Free radicals and antioxidants. Nutrition Rev. 52: 253–265.        [ Links ]

Hertog, M. G. L., P. C. H. Hollman, and M. B. Katan. 1992. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in The Netherlands. J. Agric. Food Chem. 40: 2379–2383.        [ Links ]

Hertog, M. G. L., E. J. Feskens, and P. C. H. Hollman. 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen eldery study. Lancet 342: 1007–1011.        [ Links ]

Hertog, M. G. L., D. Kromhout, and C. Aravanis. 1995. Flavonoid intake and long–term risk of coronary–heart–disease and cancer in the 7 countries study. Arch. Internal Medicine 155: 381–386.         [ Links ]

Kähkönen, M., A. I. Hopia, and M. Heinonen. 2001. Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 49: 4076– 4082.         [ Links ]

Moure, A., M. Cruz, D. Franco, M. J. Domínguez, J. Siniero, H. Domínguez, J. M. Núñez, and C. J. Pájaro. 2001. Natural antioxidant from residual sources. Food Chem. 72: 145–171.         [ Links ]

Muñoz, D. C., S. J. Ledesma, V. A. Chávez, R. F. Pérez–Gil, M. E. Mendoza, L. J. Castañeda, C. Calvo, G. I. Castro, C. C. Sánchez,y C. A. Ávila. 2002. Los Alimentos y sus Nutrientes. Tablas de Valor Nutritivo de Alimentos. Mc Graw Hill Interamericana, México. 203 p.         [ Links ]

Pellegrini, N., M. Serafín, B. Colombi, D. Del Rio, S. Salvatore, M. Bianchi, and F. Brighenti. 2003. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. The J. Nutr. 133: 2812–2819.         [ Links ]

Prior, R. L., G. Cao, A. Martin, E. Sofic, J. McEwen, C. O'Brien, N. Lischner, M. Ehlenfeldt, W. Kalt, G. Krewer, and M. C. Mainland. 1998. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity and variety of Vaccinum species. J. Agric. Food Chem. 46: 2686–2693.         [ Links ]

Ranganna, S. 1986. Handbook of Analysis and Quality Control for Fruit and Vegetable Products. 2^nd ed. Tata Mc Graw Hill Publishing Company Ltd. India. pp: 105–107.         [ Links ]

Singleton, V. L., and J. A. Rossi. 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am. J. Enol. Viticulture 16: 144–158.         [ Links ]

Stintzing, F. C., A. Schieber, and R. Carle. 2002. Betacyanins in fruits from red–purple pitaya, Hylocereus polyrhizus (Weber) Britton & Rose. Food Chem. 77: 101–106.         [ Links ]

Velioglu, Y. S., G. Mazza, L. Gao, and B. D. Oomah. 1998. Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. J. Agric. Food Chem. 46: 413– 417.         [ Links ]

Wang, H., G. Cao, and R. L. Prior. 1996. Total antioxidant capacity of fruits. J. Agric. Food Chem. 44: 701–705.         [ Links ]

Wang, S. Y., and H. S. Lim. 2000. Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and development stage. J. Agric. Food Chem. 48: 140– 146.         [ Links ]

Wybraniec, S., and Y. Mizrahi. 2002. Fruit flesh betacyanin pigments in hylocereus cacti. J. Agric. Food Chem. 9: 6086–6089.         [ Links ]

Yu, B. P. 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 76: 139–162.         [ Links ]

Zheng, W., and S. Y. Wang. 2001. Antioxidant activity and phenolic compounds in selected herbs. J. Agric. Food Chem. 49: 5165–5170.        [ Links ] ]]> 2000 48 6292-6297 1993 90 7915-7922 1997 11 A176 1991 2nd 501-518 1998 9-22 1999 47 1035-1040 1999 145 2 2 58-67 1994 52 253-265 1992 40 2379-2383 1993 342 1007-1011 1995 155 381-386 2001 49 4076- 4082 2001 72 145-171 2002 203 2003 133 2812-2819 1998 46 2686-2693 1986 2 105-107 1965 16 144-158 2002 77 101-106 1998 46 413- 417 1996 44 701-705 2000 48 140- 146 2002 9 6086-6089 1994 76 139-162 2001 49 5165-5170