Plant samples used in this work (whole plants of Euphobiahirta L., leaves of Diclipteracolorata C. B. Clarke, seeds of Sorghum bicolor (L.) Moench) were harvested in the vicinity of Bukavu city on April 2012. The collected materials were identified and deposited at Herbarium of the Faculty of Science, University of Kinshasa.

Five grams of dried and powdered plant materials were repeatedly extracted by cold percolation with water (50 ml × 1) for 24 h. Fractions were filtered and the solvent was evaporated under reduced pressure using a rotary evaporator. Chemical screening was done on the three plants using aqueous fractions as previously reported [22]. Extraction of anthocyanins was then done using 100 g of dried powdered plant material with distillated water and diethyl ether according to the universal procedures [22].The aqueous and anthocyanin solid crude extracts were conserved for the preparation of different solutions used for biological tests.

The blood samples used for the bioassays in this study were taken from adolescent SCD patients attending the “Centre HospitalierMalkiawaAmani” and “HôpitalGé- néral de RéférenceProvinciale de Bukavu”, both located in the Bukavuarea, DR Congo (2˚30'55"S and 28˚50'42" E.). A written consent for each patient was obtained before the experiment. The protocol was approved by the national ethic committee (N˚BE117). Ethical clearance on the use of SS blood was strictly observed according to international rules [23].

In order to confirm their SS nature, the above-mentioned blood samples were first characterized by hemoglobin electrophoresis on cellulose acetate gel at pH 8.5 and then stored at ±4˚C in a refrigerator.

Emmel’s test and Hemolysis test were performed as previously reported [14,15]. The RBCs digitize micrographs were treated with a computer assisted image analysis system (Motic Images 2000, version 1.3), statistical data analysis and curves were processed using Microcal Origin 8.6 package software. All anti-sickling experiments were carried out in triplicate using a sodium citrate suspension of freshly collected blood.

Figure 1 illustrates the morphology of SS blood erythrocytes (Control) and that of SS blood erythrocytes in the presence of plantsaqueous extracts. Figure 1(a) shows that nearly all RBCs adopt a sickle-shape in hypoxic conditions which, additionally, confirms the SS nature of the used blood samples. As shown in Figure 1(b), in the same experimental conditions, these sickle erythrocytes present a different morphology: they almost recover the biconcave normal form. This is unambiguously due to the presence of aqueous extracts of E. hirta L. which is the unique different input of the two microscopic preparations. The aqueous crude extracts of D. colorata C. B. Clarkeand S. bicolor (L.) Moenchshowed the same morphology, indicating the antisickling activity of these threeplants (Figures 1(c) and (d)). It should be noted that in the antisickling bioassays there is not yet established standard molecule that can be used as a positive control.

Perimeter, surface and radius were calculated for untreated and treated sickle RBCs with plant extracts in order to confirm the modification showed by micrographies (Table 1).

Table 1 shows that the average radius for the RBCs of the sickle blood could not be calculated; because sickled RBCs of untreated blood are not circular. The average radius appeared after treatment of sickle RBCs with plant aqueous extracts indicating the re-appearance of the normal form of RBCs.

Statistical treatment (Student test applied with a probability threshold of 0.05) [15] enabled the determination of a significant difference between the average values of both the perimeter and the surface of blood cells on the micrographies, thus confirming the modification RBCs morphology in the presence of plant extracts. These findings confirm previous results obtained for aqueous extracts from other plants used in Congolese traditional

Table 1. Average values of the perimeters, surfaces and the radius of untreated and treated sickle RBCs.

medicine against sickle cell anaemia [21].

The maximal normalization rate or minimal concentration of normalization (MCN) of aqueous crude extract of D. colorata C. B. Clarke, E. hirta L. and S. bicolor (L.) Moench was determined. Figure 2 shows the evolution of normalization rate of the extracts of these three plants on drepanocytes.

These curves show that the normalization rate of drepanocytes increases with the concentration of crude extracts, reaching maximum and constant value. The minimal concentration corresponding to the maximal normalization rate is called minimal concentration of normalization. A low value of the MCN is a good indication of the antisickling activity [17-19,21].

These results show that the aqueous extract of E. hirta L. is more active of with the normalization rate evaluated to be >70% as compared to D. colorata C. B. Clarkeand S. bicolor (L. Moench) for which this rate is >50%.

As it was noticed in the conducted survey the mode of traditional receipts preparation were decoction for S. bicolor (L.) Moench, infusion for D. colorata C. B. Clarkeand maceration for E. hirta L., the phytochemical screening was done on aqueous extracts obtained from the three modes of preparation.

The macerated aqueous extracts revealed the presence of anthocyanins in all three species while alkaloids and saponins were found only in D. colorata C. B. Clarkeand flavonoids and tannins only in S. bicolor (L.) Moench. Quinones were present both in D. colorata C. B. Clarkeand E. hirta and leucoanthocyanins in D. colorata C. B. Clarkeand S. bicolor (L.) Moench.

In the decoction extracts, alkaloids and saponins have been found only in D. colorata C. B. Clarkeandleucoanthocyanins in D. colorata C. B. Clarkeand S. bicolor (L.) Moenchas in case of the macerated aqueous extracts. Anthocyanins were present only in D. colorata C. B. Clarke, and flavonoids were present in all the three extracts while tannins were not found in any extracts.

In extracts obtained from infusion, flavonoids were present in D. colorata C. B. Clarkeand E. hirta L., quinones and anthocyanins were present in all the three extracts while alkaloids, saponins and leucoanthocyanins were found only in D. colorata C. B. Clarke.

The presence of various secondary metabolites in these plants would justify their medical use. E. hirta L. for example, is reported to treat some bronchial and respiretory diseases (including asthma, bronchitis, hay fever) and gastrointestinal diseases (including diarrhea, dysentery, intestinal parasitosis) [24-28]. Polyphenols com-

pounds, which are significantly present in all these plants, are well known for their large spectrum of pharmacological properties, including antimicrobial, antioxidant, antifungal, antiprotozoal, antiviral activities [22].

There is, therefore, more evidence that these extracts contain some metabolites which inhibit the sickling process of erythrocytes. More promising candidates responsible of this biological activity are polyphenols, particularly anthocyanins since besides their remarkable well-known antioxidant properties they have shown in vitro antisickling activity [15,21]. This is even supported by their simultaneous presence in all the three investigated species as revealed by the phytochemical screening on macerated aqueous extracts. Anthocyanins were then extracted and tested. Extraction yields of anthocyanins for D. colorata C. B. Clarke, S. bicolor (L.) Moench and E. hirta L. are presented in Figure 3.

According to these results, it is clearly shown that D. colorata C. B. Clarke, for which the calculated values of the output in anthocyanins is 7.58%, presents the highest content of anthocyanins, followed by S. bicolor (L.) Moench (7.04%) and E. hirtha L. (2.54%). In comparison to some other results, e.g. Maesopsiseminii (2.36%) and Alchorneacordifolia (1.28%), these three species are richer in anthocyanins [14].

Figure 4 typifiesmicrographies of the drepanocytes in the presence of anthocyanins extracted from D. colorata C. B. Clarke, E. hirta L. and S. bicolor (L.) Moench. As it can be seen from this micrography, the majority of drepanocytes reversed their sickle shapes to the normal biconcave as compared to the negative control Figure 1(a); confirming thus the antisickling activity of anthocyanins.