Abstract

The nutritional and phytochemical benefits that can be obtained from packaging corn-based foods using Marantochloa species are poorly documented scientifically. This paper therefore aims to determine the edibility and nutritional potential of M. cuspidata, M. purpurea, and M. mannii in the packaging of these foods in Greater Lomé. The properties of the leaves were analyzed using aqueous extraction. Their toxicity was assessed using human blood mononuclear cells (PBMC). The antioxidant potential of the leaves was assessed using DPPH (2,2-diphenyl-1-picrylhydrazyl). Total phenols were determined by spectrophotometry using the Folin-Ciocalteu colorimetric method. The toxicity of the three species is identical to that of the average tolerated negative control standard and significantly lower than that of the 100% DMSO positive control. All the aqueous extracts showed with DPPH inhibition percentages of 58.50 ± 3.31 for M. purpurea, 40.94 ± 28 for M. mannii, and 14.90 ± 8.17 for M. cuspidata, respectively. In addition to the insignificant presence of phenol, bioactive metabolites such as tannins, terpenes, phytosterols, and cardiac glycosides were identified. These results therefore show that the leaves studied have antimicrobial and anti-inflammatory properties. They help preserve the texture, color, and smell of packaged food, as well as its vitamin and lipid content. It is therefore necessary to promote the domestication of these species for the health, economic, and ecological well-being of the Togolese capital.

1. Introduction

Corn is an essential grain that is integral to the cuisine, diet, and even the basic nutritional intake of the inhabitants of Lomé and other major urban centers in West Africa ^{1, 2}. It is rich in carbohydrates (starch), fiber, and vitamins, and contributes to the production of minerals. The pantothenic acid (vitamin B5) in this food is part of a key coenzyme that enables the body to properly use synthesized food energy ³. It also participates in the synthesis of steroid hormones, neurotransmitters, and hemoglobin. Folate (vitamin B9) is used to make all the cells in the body, including red blood cells. It plays an essential role in the production of DNA and RNA, in the functioning of the nervous and immune systems, and in the healing of injuries and wounds ^{4, 5}. The cultural and gastronomic significance of this food is reflected in the daily diet of the inhabitants of Lomé, where it is processed and consumed in various forms: several varieties of porridge, paste, and flour ⁶. Whether for breakfast, lunch, dinner, or even snacks, corn-based dishes are very popular and consumed in Greater Lomé by adults, children, the elderly, and pregnant women alike. However, for packaging used as a medium for processing or direct handling, especially when hot, it has been found that synthetic materials, mainly plastics, are more widely used ⁷. In addition, this toxic form of packaging for corn-based foods is gradually replacing plant-based packaging such as M. cuspidata, M. purpurea, and M. mannii. In fact, apart from their containment and transport functions, the interesting properties of packaging leaves can enhance the nutritional quality of corn-based foods, provided that exchanges between packaging and products are effective ⁸. Depending on the variety, M. cuspidata, M. purpurea, and M. mannii can contribute to the transfer of magnesium, potassium, and iron to the foods they wrap. These plant species can preserve the nutritional value of the wrapped food by stabilizing vitamins and extending their shelf life ⁹. In the Togolese capital, as in other West African cities, the invasion of modern food practices is increasingly taking the place of the important role played by M. cuspidata, M. purpurea, and M. mannii in the packaging of corn-based foods due to their physical and phytochemical properties. As these plants are becoming more difficult to access, even in rural areas, their availability and high cost are increasingly steering users towards the use of plastic packaging. In this context, the research aims to determine the edibility and nutritional potential of M. cuspidata, M. purpurea, and M. mannii in the packaging of corn-based foods in Greater Lomé. These data could contribute to plan actions to promote the consumption of foods whose nutritional value could be significantly enhanced using these leaves. The focus will be on antioxidant activity, total phenol content, flavonoids, and then on identifying other compounds.

2. Materials and Methods

Leaves material used for food wrapping was collected in May 2025 in forest galleries located on Akposso plateau (Wawa prefecture, Plateaux region in Togo).

Leaves aqueous extracts were prepared and analyzed in two research units in University of Lome: “Food Microbiology and Quality Control” Laboratory (for toxicity assay) and "Physiology, horticulture and biotechnology” research team (for bioactive components characterization and measurement).

In order to analyze the properties of the above mentionned leaves, an aqueous extraction was carried out. The aqueous extracts were obtained by a method adapted from that described by Azwanida (2015). The collected leaves samples of Marantochloa cuspidata, Marantochloa purpurea and Marantochloa mannii were washed with clean water and then cut into small pieces. The extraction was carried out by infusing 100 g of fresh leaves in 500 ml of boiling water for 15 minutes. The extract was then filtered through cotton and then through Whatman paper. Less diluted extract solutions ranging from 100 to 3.125 mg/ml was then obtained. These extracts were stored at - 19°C.

Human Blood Cell Isolation

Human blood cells were used to assess the toxicity of these packaging leaves, according to the NF EN ISO 10993-5 standard. These are peripheral blood mononuclear cells (PBMCs). They were isolated by Ficoll density gradient centrifugation from venous blood samples of three (03) healthy volunteers, then cultured in RPMI 1640 media and incubated for 22 hours at 37°C under 5% CO2 in a humid atmosphere (Katawa et al., 2015).

Toxicity assessment

These assays were performed on peripheral blood mononuclear cells (PBMCs) following the method of Kamiloglu et al. (2020) based on trypan blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) staining techniques. Peripheral blood mononuclear cells from three voluntary adults consenting donors were isolated by Ficoll density gradient centrifugation using the method of Katawa et al. (2015). After counting, the isolated cells were cultured in the presence of the M. cuspidata, M. purpurea and M. mannii extracts in 96-well culture plates containing the same culture medium. 100 µl of cell suspension containing 2×10⁵ cells was therefore, distributed in each well. Then 100 µl of each concentration of leaves extracts were added to the corresponding wells, each concentration constituting a separate treatment. Dimethyl sulfoxide (DMSO) at 10 and 100 and the culture medium (RMPI 1640 medium supplemented) were used as positive and negative controls, respectively. After incubation for 22 hours at 37 °C under 5% CO₂ in a humid atmosphere, the different stainings were performed. The coloring with trypan blue allowed to determine the mortality rate (dead cells are colored blue). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) staining is useful to assess cell viability which depends on the intensity of insoluble purple formazan produced by active dehydrogenase in living cells.

Antioxidant activity

The antioxidant potential of the extracts of Marantochloa cuspidata, Marantochloa purpurea and Marantochloa mannii was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) stable free radical scavenging test according to the method implemented by Merhoudj and Zaid (2023). The DPPH test is based on the ability of antioxidants to reduce the DPPH free radical, resulting in a decrease in violet absorbance at 515 nm and a yellow discoloration towards yellow, proportional to the antioxidant activity. A 0.00478% (w/v) DPPH solution was prepared in ethanol and stored in the dark. 100 µL of each extract was then added to 3 mL of this DPPH solution. A control was prepared by replacing the extract with distilled water. After incubation for 30 minutes in the dark, the absorbance (OD) of the mixtures was measured at 523 nm using a spectrophotometer, with the blank consisting of ethanol supplemented with the extract, but without DPPH. Antioxidant activity was expressed as percentage inhibition, according to the formula proposed by Matkowski et al. (2008).

Determination of Total Phenol Concentration

Total phenols were determined spectrophotometrically using the Folin-Ciocalteu reagent colorimetric method, as described by Merhoudj and Zaid (2023). In hemolysis tubes, 400 μL the 3 extracts were mixed with 2 mL of 10% (v/v) Folin-Ciocalteu reagent. After standing for 5 minutes, 1600 μL of 7.5% (w/v) sodium carbonate (Na₂CO₃) solution was added to the mixture. The tubes were shaken and left in the dark for 30 minutes to allow the colored complex to form. The absorbance was then measured at 765 nm using a spectrophotometer whose blank was prepared under the same conditions by replacing the leaves extracts with distilled water. The concentration of total phenol is expressed in mg of gallic acid equivalent.

Identification of other bioactive components

Tannins, terpenes, phytosterols, terpenoids, cardiac glycoside and saponins were detected in the aqueous extracts using the method described by Melila et al. (2021) presented in Table 1. To do this, the appropriate reagent is added to hemolysis tubes containing a volume of extract and the coloration is observed. If the latter is characteristic, then the desired metabolite is present in the extract.

The various data collected were processed using Excel 2013 spreadsheet software. Histograms of the means were plotted using GraphPad software as well as data analysis. The latter were subjected to the Shapiro-Wilk normality test at the 5% threshold. Statistical comparisons between the different treatments were carried out using ANOVA analysis of variance for distributed and mixed data and the Kruskal-Walis test for non-distributed data.

3. Results

Mortality rate

Figure 1 shows the results of toxicity assessment from the cell mortality point of view. Indeed, the PBMCs’ mortality rate when treated with all the three species aqueous extracts varies from 0 to 18%. Analysis of the results using the Kruskal-Walis test reveals, on the one hand, that these rates are statistically identical to that of the medium (negative control), whose mortality rate was 5%, and on the other hand, these rates are significantly lower compared to that of the positive control DMSO 100% (78.5%). Furthermore, no dose-dependent effect was observed.

Viability rate

Cell viability was assessed by measuring cellular metabolic activity as a function of absorbance as illustrated in Figure 2. The results indicate a decrease in cell viability compared to the medium for all the three species aqueous extract tested with variability depending on the species and the concentration of the extract. For each species, the analysis of variance of the different concentrations compared to the medium reveals significant differences with p-values varying between 0.05 and 0.001, with the exception of a few concentrations

The aqueous extracts of the leaves extract studied, at some concentrations, could contain substances that inhibit cell multiplication.

All the aqueous extracts showed antioxidant activity with a percentage of DPPH inhibition of respectively 58,50 ± 3,31 for M. purpurea, 40,94 ± 28 for M. mannii and 14,90 ± 8,17 for M. cuspidata. Aqueous extract from dried M. mannii leaves inhibited 2.55% of DPPH while Marantochloa purpurea and Marantochloa cuspidata recorded 0% inhibition. This difference was not significant at the 5% level (Table 2). Although the last value is very different from the previous ones, statistical analysis reveals that these three percentages are not significantly different.

As illustrated by Table 3, all the leaves extracts reveal similar and non-significantly different values for total phenols concentration.

Fresh leaves prepared extracts were also tested for the presence or not of some other bioactive metabolites.

Table 4 indicates that all the samples assessed for the purpose contain general tannins, phytosterol + terpenes, cardiac glycosides and saponins.

4. Discussion

The mortality rates obtained indicate that aqueous extracts of the leaves of the species studied are not toxic to human peripheral blood mononuclear cells. A similar study carried out in Benin using the toxicity assessment method based on the lethal concentration (LC50), the value of which was determined using the review of S. Hu et al. ¹⁰ revealed the non-toxicity of many plant species used as packaging ¹¹. Indeed, these authors report that no toxic effect of crude aqueous extracts obtained by centrifugation of the leaves of Tectona grandis, Musa sapientum, Manihot esculenta and Thalia geniculata, used as food packaging in Benin, was observed on brine shrimp larvae (Artemia salina LEACH). Similarly, the technique using MTT staining, Y. Kanga et al. ¹² showed that the ethanolic extract of Hunteria eburnea, a medicinal plant, had no toxic effects on fetal human fibroblast cells. Regarding cell viability, the results show that aqueous extracts of the species studied lead to a decrease in cell proliferation, revealing a cytostatic effect of the extracts at certain concentrations. This result, although contradictory to the previous conclusion, is similar to that of Y. Kanga et al. ¹² who observed, after MTT staining, a slight decrease in the viability of dividing cells in the presence of ethanolic extract of Hunteria eburnea compared to confluent cells. Indeed, the authors, studying 15 Mexican plants, noted that methanolic extracts of H. inuloides, J. dioica, J. spicigera, P. peltatum and P. obtusifolium induced the proliferation of Vero cells while the other extracts induced a reduction in cell viability between concentrations of 250 and 500 µg/ml on these same cells. On the other hand, extracts of K. daigremontiana, A. adstringens, S. mexicanum, J. spicigera, L. tridentata and M. tenuiflora induced the proliferation of PBMC cells while the other extracts induced a reduction in viability at concentrations of 250 and 500 µg/ml. It therefore appears that aqueous extracts of M. mannii, M. leucantha, M. purpurea, M. cuspidata, M. alnifolia and S. prionogonium are not toxic to peripheral blood mononuclear cells ¹³. However, they may contain substances that inhibit the division and/or multiplication of the same cells.

In terms of nutritional benefits, the phenols present in M. cuspidata, M. purpurea, and M. mannii are natural antioxidants that protect the vitamins in the corn-based foods they package from oxidation ¹⁴. The presence of phenols also shows that these three plant species enhance the safety and preservation of the corn foods they package by acting as antimicrobials ¹⁵. This reduces the risk of toxicity and infection for consumers ¹⁶. In addition, the migration of phenols to corn-based foods in Lomé transfers anti-inflammatory and cell-protective properties to the food ¹⁷. For example, packaging a dough made from corn with M. cuspidata, M. purpurea, and M. mannii reduces the consumer's chances of exposure to chronic inflammatory diseases.

In general, not only can M. cuspidata, M. purpurea, and M. mannii indirectly strengthen the protection of natural ecosystems through the significant reduction of the ecological effects of plastic waste, but they also constitute an important lever for economic development ²². Indeed, the more these species are valued and promoted in terms of use, the less public health problems will affect the populations of Greater Lomé due to the regular consumption of corn-based foods in this city. Furthermore, it should be noted that this health benefit may, in the long term, contribute to boosting the country's economic growth, which will be able to fully reap its economic dividends due to its young population ²³. However, for the research results to be widely applicable, animal testing is needed to study acute and subchronic toxicity. Here, particular emphasis should be placed on food intake, the animals' reaction before and after intake, the appearance of their feces, their physiological condition (weight of certain organs such as the liver and kidneys), and even their lifespan.

5. Conclusion

Corn-based foods come in many varieties and are deeply rooted in the daily food culture of the inhabitants of Greater Lomé, but their packaging made from plant species for transport or consumption is tending to disappear as a result of the use of plastics. However, these leaves have nutritional and phytochemical properties that can protect and preserve food and improve consumer health. These properties have been documented for M. cuspidata, M. purpurea, and M. mannii, and the research aimed to determine them in order to consider promoting corn-based foods packaged in these leaves. The results of the analyses showed that the leaves contain antioxidants, phenols, tannins, terpenes, phytosterols, and cardiac glycosides. These compounds generally protect the vitamin content of corn-based foods, preserve their freshness, and give them a pleasant smell. They also contain antioxidants that can act against microbial attacks, fungi, and yeasts. They therefore contribute to a significant reduction in the risk of food poisoning and inflammation. In addition, they can improve the fixation of certain minerals in food in small doses, thus benefiting consumer health. It is therefore important to promote the widespread cultivation of these plants in order to improve the quality of food packaging in Greater Lomé and, in turn, protect natural ecosystems from degradation, reduce public health expenditure, and increase the benefits of the demographic dividend.

ACKNOWLEDGEMENT

The authors would like to thank Ms. Fékandine Victoire Douti supervised by Dr. Gnatoulma Katawa, for their great support in toxicity assessment in collected leaves’ extracts.

References