**Nutrient and Total Polyphenol Contents of Dark Green Leafy Vegetables, and Estimation of Their Iron Bioaccessibility Using the In Vitro Digestion/Caco-2 Cell Model**

**Francis Kweku Amagloh 1,\* ,† ID , Richard Atinpoore Atuna 2,†, Richard McBride 3,† , Edward Ewing Carey 4,† and Tatiana Christides 3,† ID**


Received: 8 May 2017; Accepted: 4 July 2017; Published: 22 July 2017

**Abstract:** Dark green leafy vegetables (DGLVs) are considered as important sources of iron and vitamin A. However, iron concentration may not indicate bioaccessibility. The objectives of this study were to compare the nutrient content and iron bioaccessibility of five sweet potato cultivars, including three orange-fleshed types, with other commonly consumed DGLVs in Ghana: cocoyam, corchorus, baobab, kenaf and moringa, using the in vitro digestion/Caco-2 cell model. Moringa had the highest numbers of iron absorption enhancers on an "as-would-be-eaten" basis, β-carotene (14169 µg/100 g; *p* < 0.05) and ascorbic acid (46.30 mg/100 g; *p* < 0.001), and the best iron bioaccessibility (10.28 ng ferritin/mg protein). Baobab and an orange-fleshed sweet potato with purplish young leaves had a lower iron bioaccessibility (6.51 and 6.76 ng ferritin/mg protein, respectively) compared with that of moringa, although these three greens contained similar (*p* > 0.05) iron (averaging 4.18 mg/100 g) and β-carotene levels. The ascorbic acid concentration of 25.50 mg/100 g in the cooked baobab did not enhance the iron bioaccessibility. Baobab and the orange-fleshed sweet potato with purplish young leaves contained the highest levels of total polyphenols (1646.75 and 506.95 mg Gallic Acid Equivalents/100 g, respectively; *p* < 0.001). This suggests that iron bioaccessibility in greens cannot be inferred based on the mineral concentration. Based on the similarity of the iron bioaccessibility of the sweet potato leaves and cocoyam leaf (a widely-promoted "nutritious" DGLV in Ghana), the former greens have an added advantage of increasing the dietary intake of provitamin A.

**Keywords:** β-carotene; Caco-2 cell; iron bioaccessibility; leafy vegetable; polyphenols

#### **1. Introduction**

It is generally accepted that dark green leafy vegetables (DGLVs) are important sources of micronutrients such as iron and vitamin A. For example, on the basis of compositional data, DGLVs were reported to contribute about 19–39% of iron and 42–68% of vitamin A [1] in the diets of rural South Africans. However, iron and vitamin A deficiencies are perennial malnutrition problems in developing countries where DGLVs are important food ingredients [2,3]. One of the common food ingredients,

possibly with a high concentration of micronutrients such as iron and β-carotene (provitamin A), are the greens. However, Cercamondi and co-workers [4] reported that sauce prepared from amaranth (*Amaranthus cruentus*) or Jew's mallow/corchorus (*Corchorus olitorius*) and examples of DGLVs, eaten with a thick maize paste by young Burkinabe women, did not increase the amount of iron absorbed. An inadequate dietary intake of bioavailable iron and vitamin A could be the primary cause of iron and vitamin A deficiencies. Therefore, the bioaccessibility of minerals from food may not solely depend on their concentration, but also on other constituents in the food.

Polyphenols and phytates in cereal and leguminous foods have been shown to limit the bioaccessibility, and consequently, the bioavailability of essential micronutrients including iron, calcium and zinc [5,6]; these staples are usually consumed with DGLVs that may also contain significant levels of these inhibitors. In a human feeding trial conducted by Garcia-Casal and co-workers [7], it was found that β-carotene enhances iron absorption when added to cereal-based diets. This finding was confirmed using Caco-2 cells as a model for iron availability [8]. Thus, the consumption of these greens, reported to be rich in micronutrients such as β-carotene [9,10], should have a double impact as a provitamin A dietary source, and also as an enhancer of iron absorption. However, this was contrary to the findings of Cercamondi and co-workers [4]. This calls for the need to investigate the iron bioaccessibility of commonly consumed DGLVs in Ghana, as anaemia (not categorised) prevalence has consistently been stated to be above 73% for children under 5 years, and at 35% among women of reproductive age in northern Ghana [11–13], where the consumption of greens is high. Vitamin A deficiency among Ghanaian children under 5 years was approximately 79% [14], expectedly, as micronutrient deficiencies usually occur together. In Ghana, DGLVs have been reported to be reliable sources of β-carotene for the majority of the population [10].

Amaranth and jute are widely consumed DGLV in northern Ghana, in addition to others such as baobab (*Adansonia digitata*), and moringa (*Moringa oleifera)* [15]. Sweet potato (*Ipomoea batatas*) is available in northern Ghana [16], but is mainly cultivated for the roots. Sweet potato leaf has been reported to contain appreciable levels of vitamin A, iron and other essential nutrients, including water-soluble vitamins [17,18], and the crop can be cultivated with low agricultural inputs [19]. Also, it has been reported that the sweet potato leaves have higher caffeoylquinic acid derivatives (polyphenols) than commercial vegetables with physiological functions, due to their enhanced antimutagenic and antioxidative properties [20]. Although the polyphenols have health benefits, they may compromise the iron bioaccessibility from the DGLVs. Different polyphenols exist, and have differing effects on the iron bioaccessibility [21–24]. Based on the nutrient superiority of the sweet potato leaf [17], it could serve as an alternative source of leafy vegetables to the populace in tropical regions of the world, particularly in Africa, where vitamin A and iron deficiencies often co-exist and remain public health problems [2,3]. The compositional data suggest that sweet potato and moringa leaves might be better sources of bioavailable iron, compared with other leafy green vegetables, as both have high levels of iron and β-carotene—a dietary factor that has been reported to improve iron bioaccessibility. However, the use of the greens of sweet potato as a leafy vegetable in Ghana is limited.

There is a need to do a comparative study of leaves commonly consumed, and sweet potato leaf before the latter could be suggested as an alternative green in Ghana, as a source of bioavailable iron or β-carotene. The in vitro digestion/Caco-2 cell model has been suggested to be less expensive than human trials [25,26], a more physiological tool for screening iron availability in comparison with solubility and dialysability methods, and an effective approach for predicting the iron bioaccessibility from food for humans [27,28]. Therefore, the in vitro digestion/Caco-2 cell model, with ferritin formation as a marker for iron absorption, was used to measure the iron bioaccessibility of selected greens available in Ghana, in comparison with sweet potato leaves.

The objectives of this study were to compare the nutrient contents and iron bioaccessibility using the in vitro digestion/Caco-2 cell model of five different cultivars of sweet potato, with five other commonly consumed DGLVs in Ghana: cocoyam (*Xanthosoma sagittifolium*), corchorus, baobab, kenaf (*Hibiscus cannabinus*) and moringa.

#### **2. Materials and Methods**

#### *2.1. Sample Cultivation and Collection*

Five cultivars of sweet potato—three orange-fleshed (Coded OFSP1, OFSP2 and OFSP3), one purple-fleshed (PFSP), and one white-fleshed (WFSP)—and three other DGLVs, namely moringa, corchorus and kenaf (Figures 1 and 2), were nursed in a screen house up to maturity (8 weeks). Each DGLV was cultivated in three replicates, and each replicate contained five pots of the particular green. Baobab and cocoyam were purposively sampled from three different geographical locations. Baobab leaves were collected from trees near settlements from the Upper East, Upper West and North regions, while cocoyam leaves were harvested from farmlands from the Ashanti, East and Brong-Ahafo regions of Ghana. The baobab was not nursed due to a relatively long time for the initiation of vegetative growth. Cocoyam is normally cultivated in the rainforest regions in Ghana and not in northern Ghana. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

‐ **Figure 1.** Cultivars of sweet potato ( ‐ ‐ *Ipomoea batatas*) leaves used in the study. OFSP: orange-fleshed sweet potato; PFSP: purple-fleshed sweet potato; WFSP: white-fleshed sweet potato. ‐

**Figure 2.** Commonly consumed dark green leafy vegetables (DGLVs) used in the study.

#### *2.2. Sample Preparation*

The replicates of the DGLVs were separately washed twice under running tap water and rinsed in distilled water; about two handfuls of DGLVs put into a stainless steel cup with 100 mL of distilled water added were covered with aluminium foil and boiled until soft, for between approximately 15 and 20 min. The cooked DGLVs were allowed to cool, and all the contents of the cup were transferred into coded, transparent, low-density polyethylene zip-lock bags, and stored in a freezer at −18 ◦C for 2 weeks. Prior to storage in the freezer, about 5 g aliquot portions were taken for moisture determination. The frozen samples were then freeze-dried (TK-118 Vacuum Freeze-Dryer, True Ten Industrial Company Limited, Taichung, Taiwan) for 72 h. The samples were then milled (Thomas Scientific, Dayton Electric Manufacturing Company Limited, Niles, IL, USA) and sieved into fine powder using a 60 mm sieve.

Triplicate aliquots of three-letter-coded powdered samples were couriered to the University of Greenwich at Medway, Chatham-Maritime United Kingdom, and Massey University, Palmerston North, New Zealand, from Ghana. The moisture determination of fresh leaves was performed in Ghana.

#### *2.3. Compositional Analysis*

#### 2.3.1. Moisture and Protein

The moisture contents of freshly harvested leaves and cooked leaves were gravimetrically determined using the forced air oven method (AOAC 925.10). For the milled freeze-dried samples, the vacuum oven protocol (AOAC 926.12), as published in the official methods of analysis of AOAC International [29], was used for the moisture determination.

The concentration of nitrogen in the freeze-dried greens was performed by the Dumas method (AOAC 968.06), and a nitrogen-to-protein conversion factor of 6.25 was used to quantify the amount of protein in the leaves on a fee-for-service basis by Massey University Nutrition Laboratory, Palmerston North, New Zealand.

#### 2.3.2. Mineral Analysis: Calcium, Iron, and Zinc

Approximately 0.50 g of the freeze-dried DGLV samples was microwave-digested using an accelerated reaction system (CEM MARS 5H with XP-1500 vessels) for 20 min at 400 psi and 1200 W. Subsequently, calcium, iron and zinc were quantified using an Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Perkin–Elmer Optima 4300 DV, Perkin–Elmer, Coventry, UK) using protocols as previously described [30]. A certified reference material (ERMCD281, Sigma-Aldrich, UK) was included and run in parallel with the DGLV samples. The data obtained for all three minerals in the reference material were within 5% of the expected values.

#### 2.3.3. β-Carotene

Other researchers have described the extraction and quantification methods used in this study [31]. Averagely, 0.50 g of the freeze-dried samples of the leaves was used for the extraction. A certified reference material (BCR—485, Sigma-Aldrich now Merck, provided to Sigma-Aldrich from the European Commission Joint Research Centre, Institute for Certified Reference Materials and Measurements, Geel, Belgium) was included in three out of the five batches of extraction carried out on DGLV samples. A mean recovery of 128% was obtained for the β-carotene level for the reference material. Therefore, the values obtained for DGLVs were adjusted for a systematic error of 28%.

#### 2.3.4. Ascorbic Acid

The method for vitamin C determination as published by Lee and Coates [32] was carried out by the Massey University Nutrition Laboratory, Palmerston North, New Zealand, on a fee-for-service basis.

#### 2.3.5. Polyphenols

The Folin–Ciocalteu method described by Isabelle and co-workers [33] was used to quantify the total polyphenols in the samples, as gallic acid equivalents. The Nutrition Laboratory, Massey University, New Zealand Palmerston North, New Zealand, carried out the analysis on a fee-for-service basis.

#### *2.4. In Vitro Digestion/Caco-2 Cell Model for Iron Availability*

The iron availability from the freeze-dried DGLVs as received from Ghana was assessed using the TC7 Caco-2 cell clone (INSERM U505, Paris, France) from cell passages 42–45 in the in vitro digestion/Caco-2 cell model, as previously described [34], with slight modification. Averagely, 0.5 g rather than 1 g of the sample was weighed for the assessment, as 1 g of the starting material led to a matrix that was too viscous for the multiple mixing and pH adjustments required in this method. Cells were grown in six-well tissue culture plates for the experiments and maintained in DMEM supplemented with 10% *v*/*v* foetal bovine serum (FBS). On days 12 and 13, cell media were changed to MEM without FBS, as in the method developed by Glahn [35,36], to ensure low iron media, but optimal expression of Caco-2 cell iron transport proteins [37]. On day 14, foods were subjected to in vitro digestion with a sequential addition of digestive enzymes to mimic exposure to the stomach and small intestine (pepsin at pH 2, followed by bile/pancreatin at pH 7). Digested foods (digestates) and controls, including a blank "No food/added iron" digestate, were then applied to Caco-2 cells through an upper chamber suspended over the plate wells, created using a 15 kD dialysis membrane fitted over a Transwell insert and held in place with a silicon ring. The membrane protected the cells from the digestive enzymes, and also mimicked the gut mucous layer by only allowing soluble iron of a selected size to be available for enterocyte absorption. Cells were treated for 2 h, the digestates were removed, and the cells were returned to the incubator. The cells were harvested for ferritin 24 h after the initiation of the digestive process. Ferritin was measured using a commercial enzyme-linked immunosorbent assay (Spectro ferritin, RAMCO Laboratories Inc., Stafford, TX, USA), and corrected for differing numbers of cell per tissue culture well by measurement of cell protein as an indicator of cell numbers; the cell protein was measured using the Pierce protein bicinchoninic acid assay. Ferritin values were expressed as ng ferritin/mg cell protein.

#### *2.5. Statistical Analysis*

The compositional data were converted to an "as-would-be-eaten" basis prior to statistical analysis, using the dry matter content obtained for the cooked samples prior to storage in the freezer. The univariate analysis, followed by Tukey's studentised range test with the significance set at *p* < 0.05, was used for the compositional data. For the in vitro digestion/Caco-2 cell model for iron availability, the data generated were normalised prior to using the general linear model procedure for one-factor analysis, and the results were presented as interval plots of the means with 95% confidence intervals. The Minitab 16.2.2 (Minitab Inc., State College, PA, USA) statistical package was employed for the data analysis.

#### **3. Results**

#### *3.1. Compositional Profile*

The data in Table 1 is expressed on the as-would-be-eaten basis, with the exception of the moisture value of the freshly harvested leaves. The moisture content of the sweet potato cultivars ranged from 83 to 87 g/100 g, and it was similar to other cultivars cultivated in China [38]. The greens of the sweet potato cultivars were generally not significantly different (*p* > 0.05) from each other for all the components analysed, with the exception of the total polyphenols.


**Table 1.**Moisture, micronutrient and total polyphenol levels per 100 g in DGLVs on an "as-would-be-eaten" basis#.
