MATERIALS AND METHODS

Consumption pattern: This was obtained by use of food-frequency questionnaire administered to 101 pregnant women attending clinic in the University of Calabar Teaching Hospital (UCTH), Calabar.

Collection and preparation of vegetables for extraction: The six DGLV were obtained from Watt market in Calabar and were properly identified. Each vegetable type was washed, handpicked and chopped into very small pieces. It was then steamed in a clean pot for three minutes using a gas cooker. About 5g of the vegetable sample was transferred into a sterile sample bottle, sealed, labeled and stored in a refrigerator at -20^oC for High Performance Liquid Chromatography (HPLC) analysis within one week.

Extraction and quantification of β-carotene from the vegetable samples: Each frozen sample was left to thaw at room temperature. The methods used in extraction and quantitation of β-carotene in the vegetable samples were similar to those developed by Hart and Scott (1995) with minor modifications: ethyl β-apo-8'-carotenoate was used as internal standard instead of echinenone; mobile phase consisted of methanol, acetonitrile, chloroform (MeOH: ACN: CHCL3; 47: 47: 6v/v/v) instead of acetonitrile, methanol, dichloromethane (ACN: MeOH: DCM; 75: 20: 5v/v/v) (Thurnham et al., 1988). All manipulations were carried out under gold fluorescent lighting. In principle, a given quantity of vegetable sample is treated with magnesium carbonate, which neutralizes any organic acids in the sample and the carotenoids are extracted from the vegetable matrix using a mixture of suitable organic solvents. An aliquot of the organic phase is dried, diluted with the mobile phase to a suitable concentration and injected onto an HPLC column. A sensitive UV/Vis detector set at 450nm detects β-carotene, which is eluted as a sharp peak within 19-22 minutes. β-carotene is then quantified by use of peak height ratios or peak area ratios relative to an internal standard and the results expressed in μg/100g edible portion (EP).

Preparation of vegetable dishes: Each soup was prepared using the customary/ traditional method. The mean portion sizes as consumed by the women were obtained by interview. The ingredient compositions of the various soups and portion sizes were computed using the procedures outlined by Olusanya (1977).

Estimation of vitamin A activity in the dishes: The vitamin A value (retinol equivalent or RE) of the soups as consumed was computed using the value obtained by direct chemical analysis in the present study and from Food Composition Table (FAO, 1968). The vitamin A value of portion sizes was obtained based on the revised operational equivalence 14 μg of dietary β-carotene yields 1μg of vitamin A activity or 1 μg of dietary β-carotene equals 0.071μg RE (Van Het Hof et al., 1999; WHO/ FAO, 2004). The amount of vitamin A in the portion sizes were expressed as percentage of total vitamin A content and daily recommended intakes.

Statistical analysis: Where appropriate, percentages (%) were used. The mean±standard deviation for the measured variables was also determined.

RESULTS

Table 1 shows the relative frequency of consumption of the six DGLV used in the study. One hundred percent (100%) of the women consumed each of the leafy vegetables. Mon-mon ikong (T. triangulare) was the most frequently consumed vegetable as 73.3% of the women consumed it at least 4 times per week. Ikong ubong (T. occidentalis) consumed by 66.3% of the women at least 4 times per week and Afang (Gnetum africanum) consumed 2-3 times per week by 91.9% of them were also extensively utilized. Inyang afia (A. hybridus), Atama (H. crinita) and Editan (L. africana) were less utilized as most of the women consumed them only once a week or occasionally.

HPLC analysis of the leafy vegetables showed that Ikong ubong had the highest β-carotene concentration (12,038±1.53 μg/100g) and vitamin A value (860±0.10 μg RE/100g). Inyang afia also recorded a comparatively high β-carotene content (11,938±2.65 μg/100g) and vitamin A value (853± 0.19 μg RE/100g). Atama leaves had the least vitamin A quality. Its β-carotene content and vitamin A value were respectively 2,213±0.58 μg/100g and 26±0.04 μg RE/100g. (Table 2). The total vitamin A value per portion size of the various dishes commonly consumed by the pregnant women covered in the study are presented in Table 3. The results showed that thedark green-leafy vegetable-based dishes which include Ikong, Afang, Editan, Ekpang nkukwo and Etighi, had the highest vitamin A values ranging from 2010.52±297 μg RE/100g in Etighi to 4759.75±672 μg RE/100g in Ikong.

Table 1:	Frequency of consumption of the DGLV by the pregnant women

Table 2:	b-carotene content and vitamin A value of various boiled green leafy vegetables consumed by respondents
*EP = Edible Portion

Table 3:	Total vitamin A content of portion sizes of various dishes commonly consumed by the pregnant women
aAtama, APP, fish, crayfish, bAfang, Mon-mon ikong, RPO, fish, crayfish, cPounded yam, fish, crayfish, dRice, RPO, fish, crayfish eRice, REO, fish, crayfish, fEditan, Mon-mon ikong, RPO, fish, crayfish, gGrated cocoyam, Inyang afia, RPO, fish, crayfish, hOkro, Ikong, RPO, fish, crayfish, iMelon, Ikong, RPO, fish, crayfish, jIkong, Mon-mon ikong, RPO, fish, crayfish, kBeans, RPO, lPounded bush mango seeds, Ikong, RPO, fish, crayfish, mFresh tomatoes, REO, Abbreviations: APP = African-oil palm puree, RPO = Red palm oil, REO = Refined edible oil

Consequently, they made the highest contribution (108% and 255% respectively) to the vitamin A requirement in the women. Dishes prepared with little or no green leafy vegetables such as Abak Atama, Afia efere, Iwuk edesi, and stew or tomato sauce had much lower vitamin A values. The only exception to this is Okoti (bean pottage) which had a high vitamin A value (2138.61±425μg RE/100g) and also made a significant (115%) contribution to the vitamin A requirement in the women.

DISCUSSION

Over half of the women sampled consumed Mon-mon ikong (T. triangulare) and Ikong ubong (T. occidentalis) at least four times per week. According to Hart et al. (2005), the consumption of a food item more than four times per week shows that the food is consumed almost every day in a week. Therefore it could be said that the women in this study consumed these two vegetables daily. Overall, Mon-mon ikong had the highest frequency of consumption. This is understandable since traditionally, the vegetable is cooked and consumed together with ikong ubong (T. occidentalis) in ikong soup, afang (G. africanum) in afang soup and editan (L. africana) in editan soup and with porridges. Regarding the high frequency of Ikong ubong consumption recorded in this study, it is possible that the women were affected by the health benefits usually attributed to this vegetable. Use of this vegetable in the prevention and treatment of many life-threatening ailments have been reported including constipation, appendicitis, atherosclerotic cardiovascular disease and stroke, infection, liver problems, anaemia, convulsion, high blood pressure, hypercholesterolemia, diabetes mellitus, arthritis, impotency and loss of libido in men (Ajayi et al., 2000; Alada, 2000; Eseyin et al., 2006; Nworgu et al., 2007). In fact, it is common to observe people from the Efik/ Ibibio tribes of Southeastern Nigeria drinking the raw extract of this vegetable as preventive measure against anaemia and other diseases.

The vitamin A value of the DGLV ranged from 26±0.04μgRE/100g in atama (H. crinita) to 860±0.10μgRE/100g in ikong ubong (T. occidentalis). These values are generally lower than the values reported in earlier studies (Polacchi et al., 1982; IVACG, 1989; Rahman et al., 1990). Such variations could be due to differences in handling. A number of studies have shown that boiling results in reduction of vitamin A activity of leafy vegetables (Sweeney and Marsh, 1971; Leung et al., 1972). Another potential source of variation is the recent changes in the vitamin A equivalency of provitamin A carotenoids because of the lower bioavailability of carotenoids than earlier assumed (Wardlaw and Kessel, 2002; WHO/FAO, 2004). However, the vitamin A value obtained in the present study for inyang afia (A. hybridus) (853±0.19μgRE/100g) is slightly higher than that reported in earlier studies (Polacchi et al., 1982; Rahman et al., 1990).

In conclusion, this study reveals that all things being equal, exception of atama (abak soup) which had low values, all other DGLV dishes usually consumed by pregnant women in Calabar are adequate to meet their daily vitamin A needs. They are in the order ikong, editan, ekpang nkukwo, afang and ogbono soups. Exception of atama, the consumption of these green leafy vegetables should be encouraged.

ACKNOWLEDGEMENT

We are grateful to the Government of United States of America for the Fulbright Fellowship during which, part of this research was conducted. We also appreciate the Faculty and staff of Division of Nutrition Sciences, Cornell University, Ithaca-New York for allowing us the use of a laboratory specially conditioned for carotenoid and retinoid research. The expertise, encouragement and cooperation of Dr. R.S. Parker is also acknowledged and appreciated.