The samples studied are plants harvested in the South Benin region and brought back to the laboratory the same day. The samples were spread out in a cold drying room (22˚C) for about 14 days, after which time they are practically anhydrous and brittle. They are then ground, sieved with a sieve with 710 µm diameter pores and then stored in airtight containers. These are the leafy stems of Gossypium barbadense (Gb) and Justicia secunda (Js), the leafy panicums of Sorghum bicolor (Sb) and the calyxes of Hibiscus sabdariffa (Hs).

The rats used for the anti-anaemic activity are from the wistar line of weight varying between 180 and 220 g. They are acclimatized and fed with water and appropriate pellets, one week before the start and then during the experiment.

The larvae used for the toxicity test come from Artemia salina shrimp eggs purchased on the market.

The extractions carried out by decoction and by maceration produced variable yields depending on the solvents and the plant species studied (Table 1).

The highest yield, 39.20% ± 1.54%, is obtained with the aqueous extract of the Hibiscus sabbariffa plant and the lowest 5.85% ± 0.03% with the aqueous extract of the Sorghum bicolor plant. This difference in yields could be explained by the presence of polar chemical compounds sometimes highly, moderately or weakly concentrated in one species than others. The yields of the hydroethanolic extracts of Justicia secunda (14.08% ± 0.23%) and Sorghum bicolor (13.58% ± 1.18%) are 2.5 times higher than those of their decoctions. We deduce that these plants could contain moderately polar compounds having more affinity with ethanol than water. This disparity in yields could also be explained by the thermosensitivity of certain compounds during the decoction.

Phytochemical screening of the species studied revealed that they are rich in secondary metabolites and all contain gallic tannins, anthocyanins, reducing compounds, saponosides, terpenes and steroids. On the other hand, the cyanogenic and cardiotonic derivatives which are plant phytotoxins have not been characterized in the species studied (Table 2).

Apart from Justicia secunda, all the species studied contain flavonoids, while only S. bicolor does not contain an alkaloid. It should therefore be noted that the most common chemical groups in coloring plants traditionally used for their anti-anaemic effects are polyphenols (tannins, anthocyanins, flavonoids), alkaloids, reducing compounds and saponosides. These results are comparable to those obtained by Kambou et al. [25] to those of Yamoah et al. [29], and Gbénou et al. [30] who also noted the presence of these chemical groups in J. secunda plant and spirulina algae extracts tested for their anti-anaemic activity.

The absence of alkaloids was observed only in Sorghm bicolor as revealed by the work of Sènou et al. [31]. Similarly, the absence of flavonoids noted in Justicia secunda was confirmed by the work of Akibou et al. [32], however, the work of Abiodun et al. [33], revealed the presence of flavonoids in the extracts methanolic, ethanolic and aqueous of J. secunda studied for these antibacterial activities in Nigeria.

The Artemia larvae lethality test, used in the interest of predicting the cytotoxicity

Note: *: number of species containing a given chemical group, #: number of chemical groups characterized in a given species; +: presence of chemical group, −: absence of chemical group.

of the extracts studied, showed that the lethal doses causing the death of 50% of the larvae vary very little according to the plant and control extracts applied (Figure 1). They are between 3.74 ± 0.16 mg/mL and 4.64 ± 0.62 mg/mL provided respectively by the positive control Ranferon-12 and the aqueous extract of S. bicolor applied.

Analysis of these results reveals that it was at very high doses of substances applied that it was possible to achieve 50% mortality in the row of shrimp larvae. In comparison with the toxicity reference table of the brine shrimp test established by Mousseux [34], the LC₅₀ values from the extracts tested are on average thirty times higher than the limit set (0.1 mg/mL) to declare that a substance is toxic. Consequently, these results reflect a priori the safety of the extracts and that of the Ranferon-12 control tested against these organisms.

It should be noted that the hydroethanolic extract provided a moderately acceptable extraction yield at the level of all the species and that there is no significant difference between their chemical composition and their toxicity, only the hydroethanolic extract is used for carrying out anti-anaemic activities.

Three hematological parameters were characterized during the induction of anemia and its treatment with hydroethanolic extracts of the plants studied and the reference anti-anaemic molecule used. These are the hemoglobin level, the hematocrit level and the red blood cell count.

Administration of phenylhydrazine caused a moderate and significant drop in hemoglobin, red blood cell count and hematocrit. The results obtained are similar to those of Ryu and Yook [35] and Yamoah et al. [29] who observed a decrease in these hematological parameters of 50% and 55% respectively, with an administration of 40 mg/kg/d of phenylhydrazine in Sprague Drawley rats.

On the 18th day of treatment, the rats having received the control anti-anaemic molecule as well as those treated with the plant extracts studied had almost completely recovered, which was not the case in the negative control rats treated with the saline solution. Indeed, this reversibility of the anemia due to the discontinuation of the administration of phenylhydrazine was described by Ryu and Yook [35] and Criswell et al. [36].

Treatment of anaemic rats with Ranferon-12 resulted in increased red blood cell count and hemoglobin concentration compared to rats treated with normal saline. Ranferon-12 like Feroglobulin mainly contains vitamin B12, Zinc, folic acid and iron. Iron, a component of heme, forms the core of the iron-porphyrin heme ring and together with globin chains forms hemoglobin, so its availability may contribute to the regeneration and structural consolidation of hemoglobin. The B-complex vitamins, by acting as cofactors in hematopoiesis, would promote the synthesis of blood proteins [37]. This would explain the increase in the number of red blood cells and the concentration of hemoglobins in rats fed with Ranferon-12.

Similarly, the treatment of anaemic rats with hydroethanolic extracts of the plants studied showed a significant increase in hemoglobin concentration from 9.35 g/dL to 18.40 g/dL, with the number of red blood cells rising from 3.20 × 10⁶/µL to 6.37 × 10⁶/µL over the experimental period.

These results show that the anaemic rats treated with the extracts of G barbadense, S. bicolor, J. secunda and H. sabdariffa almost completely recovered with regard to the number of red blood cells and the concentration of hemoglobin at the 18th day of treatment. This increase in the number of red blood cells could be attributed to the groups of chemical compounds identified in the plant extracts, which were thus able to reverse the effects of anemia induced by phenylhydrazine hydrochloride, as described by the work of Koffuor et al. [27] and Diallo et al. [38] on extracts of Solanum torvum and Tectona grandis administered following the induction of anemia in rats by phenylhydrazine.

The phytochemical analysis carried out on the extracts of tinctorial plants studied, revealed that these species contain polyphenols in particular tannins, anthocyanins and flavonoids as well as reducing compounds. These compounds possess a wide range of biological activities related to their reducing character and their affinity for proteins and metal ions. They have well-established antioxidant properties linked to the inhibition of oxidative stress [18] as well as they promote the regeneration of blood tissue by numerous young resistant cells [39]. The presence of these major chemical groups, due to their properties, in particular those of iron chelators, justifies the high rate of regeneration of red blood cells recorded in rats treated with these extracts. The lowest regenerative power 84.61% was recorded at the level of treatment with H. sabdariffa. Although effective, this last position of H. sabdariffa compared to other species could be justified by the fact that it contains fewer secondary metabolites. On the other hand, the strong capacity for regeneration of red blood cells induced by extracts of S. bicolor (96.80%) compared to those of G. barbadense (87.50%) and H. sabdariffa, would be related to the many chemical groups identified in this species. In addition to the polyphenols, coumarins, terpenes, reducing compounds characterized in these plants, S. bicolor also contains quinone derivatives and glycosides.

Although less rich in chemical groups, compared to the extract of S. bicolor, J secunda displays a very high potential for cell regeneration (99.06%). This capacity for resorption of red blood cells by J. secunda compared to the other species tested, would be linked to its high iron content. Indeed, according to the work of Koné et al. [40] on the iron content of anti-anaemic plants used in traditional medicine, J. secunda would contain 26.6 mg of iron per 100 g of plant matrix, compared to 3.8 mg of iron for 100g of H. sabdariffa calyces and 4.2 mg of iron for 100 g of S. bicolor panicum revealed by work carried out on the chemical composition of these species respectively by Leung et al. [41] and Sawadogo [42].

This could justify a priori the order of regeneration of red blood cells observed in this study by the plant extracts used: (H. sabdariffa < G. barbadense < S. bicolor < J. secunda).

Iron plays an important role in hematopoiesis. However, the therapeutic potential of plants cannot be established on the basis of available iron content alone because other compounds play a role in iron absorption in the body [43]. These compounds are alkaloids, flavonoids, saponins, tannins, calcium, zinc, vitamins C and potassium [44]. For example, vitamin C contributes to the bioavailability of iron in the body [45]. Phytochemical compounds, in particular polyphenols, characterized in the extracts tested, due to their metal-chelating properties and their affinity for proteins, could also explain their anti-anaemic effects. Tannins form by complexation, iron tannates, and regulate by inhibition the intestinal absorption of iron by transforming soluble iron into ferric iron.

This study has shown that the dye plants studied can play a role in part in improving the health conditions of people with anemia, especially those suffering from iron deficiency anemia without the risk of probable side effects. Indeed, this work testifies to the safety of the extracts tested in view of the very high LC₅₀ values compared to the reference concentration accepted by the applied test.