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Sickle cell disease or drepanocytosis is caused by the polymerisation of abnormal haemoglobin S when oxygen tension decreases. This lead to the changes in the shape of red blood cells and anaemia. It has also been postulated that the red cells of patients with sickle cell disease contain a higher than normal concentration of calcium ions. These ions are bound to membrane proteins resulting in dehydration and loss of red blood cell deformability and cell-to-cell adherence. Anthocyanins extracted from some Congolese plants used in traditional medicine against sickle cell disease have recently been shown to have anti-sickling activity in vitro. Justicia secunda is a plant used in Congo by Jehovah’s Witnesses, well known for their refusal of blood transfusions, against anaemia.
Emmel, Itano and osmotic fragility tests were used to test the effect of anthocyanin extracts from Justicia secunda leaves on haemoglobin S solubility and sickle cell membrane stability.
Anthocyanins from Justicia secunda were found to possess anti-sickling activity. Treated SS red blood cells recovered a normal, classical biconcave form with a radius of 3.3±0.3 μm, similar to that of normal erythrocytes. The solubility of deoxyhaemoglobin S increased and the osmotic fragility of drepanocytes decreased upon treatment with anthocyanin extracts.
These findings suggest that anthocyanin extracts play a role in both stabilising the red blood cell membrane and inhibiting polymerisation of haemoglobin S. This provides a possible molecular basis for earlier reports on the anti-sickling properties of anthocyanins from some Congolese plants and their use in the management of sickle cell disease by Congolese traditional healers.
Each year approximately 100,000 children in the world are born with sickle cell disease (SCD) which is a genetic disorder considered as a public health problem in many countries, particularly in west and central Africa1,2.
The molecular and cellular basis of the pathophysiology of SCD is attributed to both sickle cell deoxyhaemoglobin (deoxy HbS) and the behaviour of the red blood cell (RBC) membrane3,4. Under certain physiological stresses, such as a decrease in oxygen tension, the deoxy HbS protein polymerises, leading to a rigid chain that can distort the RBC into the shape of a sickle5,6. The structure of the resulting HbS polymer has been studied by both X-ray diffraction and electron microscopy and has been found to take a helical from with ten outer filaments of haemoglobin molecules and four inner filaments. These filaments are paired as double strands. The transition from a fibre to a crystal structure has been observed within RBC over prolonged periods. Certain regions of the crystal structure of deoxy HbS have been recognised to be involved in the up-down and side-to-side contacts between haemoglobin molecules in the double strands7–11.
There is substantial evidence that the polymerisation of deoxy HbS, which requires a high concentration of haemoglobin, substantially lowers the protein’s oxygen affinity. It was postulated that, in the sickled state, the RBC of patients with SCD contain 3–4 times more than the normal concentration of calcium ions as a result of ATP depletion. Since the cells contain few or no endocytic vesicles for the storage of calcium ions, most of these ions are bound to membrane proteins, thereby resulting in dehydration and haemochrome formation with a resulting loss of RBC deformability and cell-to-cell adherence12,13.
However, several studies have indicated that some experimental anti-sickling agents may directly inhibit polymerisation of haemoglobin or modify membrane stability, as evaluated by measuring RBC haemolysis, as a result of red cell hydration, in graded hypotonic saline solutions.
A number of plant products that could serve as anti-sickling agents have been described 1,14–17. Our research group recently investigated the effects of crude aqueous and/or ethanol extracts of some Congolese medicinal plants in the control of the sickling process in vitro2. We demonstrated that the anti-sickling activity of these plants is due to the anthocyanins they contain18–26. However, the molecular mechanism for the effect of the anthocyanin extracts has not yet been determined.
The present study was performed with the aim of providing further cellular and molecular information by investigating the effects of anthocyanin extracts on both the inhibition of polymerisation of deoxy HbS and erythrocyte membrane stability as determined by Itano and osmotic fragility tests, respectively. The plant studied, Justicia secunda, is used as a treatment of anaemia in Congo by Jehovah’s Witnesses, well known for their refusal of blood transfusions.
Leaves of Justicia secunda Vahl were collected from plants growing in Kinshasa, DRC and were authenticated by Mr. B.L. Nlandu of the National Institute of Agronomic Research (INERA). A specimen is deposited at the INERA Herbarium of the Faculty of Science (University of Kinshasa).
The dried and powdered plant material (leaves, 10 g) underwent repeated extraction procedures by cold percolation with 95% ethanol and water (100 mL x 1) for 48 h. The aqueous and organic extracts were chemically screened using a well known protocol27. Fractions were filtered and concentrated to dryness under reduced pressure using a rotary evaporator. Anthocyanins were extracted from 100 g of dried powdered plant material with distilled water and diethyl ether following an established protocol, as previously reported18,19.
The blood samples used in the evaluation of the anti-sickling activity of the plant extracts in this study were taken from adolescent patients known to have sickle cell disease attending the “Centre de Médecine Mixte et d’Anémie SS” and “Centre Hospitalier Monkole”, both located in the Kinshasa area, DRC. None of the patients had been transfused recently with Hb AA blood. All anti-sickling experiments were carried out using a sodium citrate suspension of freshly collected blood. In order to confirm their SS nature, the above-mentioned blood samples were first characterised by haemoglobin electrophoresis on cellulose acetate gel at pH 8.5. They were confirmed to contain SS red blood cells and were then stored at ± 4° C in a refrigerator.
Sickle cell blood was diluted with 150 mM phosphate-buffered saline (NaH2PO4 30mM, Na2HPO4 120mM, NaCl 150 mM) and mixed with an equivalent volume of 2% sodium metabisulfite. A drop from the mixture was spotted on a microscope slide in the presence or absence of an anthocyanin extracts and covered with a cover slip. Paraffin was applied to seal the edges of the cover slip completely to exclude air. The RBC were analysed by measuring various parameters including the area, perimeter and radius of each RBC using a computer assisted image analysis system (Motic Images 2000, version 1.3). The data were processed using Microcal Origin 6.1 package software.
RBC were washed twice in physiological saline solution (NaCl 0.9%) by centrifugation at 3000 rpm for 10 min, and then re-suspended in hypotonic medium. The haemolysate of the RBC was centrifuged and an equivalent volume of 2% metabisulfite was added to supernatant, which was then incubated at room temperature for 45 min.
At fixed time points aliquots (50 μL) of the 2% sodium metabisulfite pre-treated haemolysate were diluted with 500 μL of phosphate buffer (pH 7.5) containing (NH2)2 SO4 30%, saponin 1% and K2HPO4 1.2%. Next, 50 μL of crude anthocyanin extract were added to the test sample, mixed and incubated for 10 min. An equivalent volume of phosphate-buffered saline was added to the control sample instead of the extract. At predetermined time intervals aliquots of test or control samples were removed and centrifuged at 3500 rpm at room temperature for 5 min. The absorbance of the supernatant was measured at 540 nm. The solubility of deoxygenated sickle cell haemoglobin was expressed as the increase of the optical density at 540 nm.
The fragility of RBC was determined by placing the cells in graded series of hypotonic saline solutions buffered at pH 7.4 with 150 mM phosphate. Concentrations ranging from 0.2% to 0.9% NaCl were made up in a final volume of 10 mL. A 10 μL sample of washed SS RBC was added to 1990 μL of each hypotonic saline solution and immediately mixed by inverting several times. The tubes were allowed to stand for 150 min at room temperature. To determine the effect of the anthocyanin extracts, 10 μL of extract (30 mg/mL) were added to 1980 μL of each hypotonic saline solution, then 10 μL of RBC added and the mixture treated as described earlier. The number of RBC not lysed at each saline concentration was determined using a photonic microscope (OLYMPUS×21) and a haemacytometer (Neubauer’s cell). Haemolysis was calculated using the following equation: number of RBC after 150 min×100/number of RBC inoculated (0 min).
The mean corpuscular fragility (determined from the concentration of saline causing 50% haemolysis of the RBC) was obtained from a plot of lysis (%) versus NaCl concentration.
pH values were determined using a Metrohm E 604 pH-meter equipped with a glass electrode. This electrode was kept soaked in 3 mol/L KCl solution and calibrated with aqueous standard buffers. A GENESYS UV-Visible spectrophotometer was used.
The in vitro bioassays were performed in triplicate and the number of observed erythrocytes was determined using Neubauer’s cell. All results presented in this study are mean ±S.D.
The phytochemical screening of leaves of Justicia secunda Vahl revealed the presence of alkaloids and polyphenols such as flavonoids, tannins, leuco-anthocyanins, quinones and anthocyanins.
The calculated average values of the radius, perimeter and surface of drepanocytes before and after treatment with anthocyanins extracted from Justicia secunda are presented in Table I.
The effect of anthocyanins on the polymerisation of deoxy-HbS can be determined by studying the solubility of deoxy-HbS in the absence and in the presence of anthocyanin extracts. This was done by monitoring the optical density of free HbS at 540 nm and of polymerised HbS at 700 nm at different times. Table II shows the optical density at 540 nm for untreated HbS samples and HbS samples treated with anthocyanin extracts (HbS+ACE).
Effect of anthocyanin extracts on membrane stability
The effect of anthocyanins on the membrane stability of RBC can be evaluated by comparing the percentage of haemolysis of untreated and treated SS RBCs using the osmotic fragility test30. Table III shows the percentage lysis of untreated and treated SS RBC at different saline concentrations.
Figure 1 shows that the majority of the control, untreated red blood cells (Figure 1) were sickle-shaped, confirming the SS nature of the blood. However, when the SS red blood cells were mixed with an anthocyanin extract (Figure 2), the majority of the erythrocytes recovered a normal shape. These morphological changes were observed in hypoxic conditions, i.e. after deoxygenation of haemoglobin. This morphological normalisation of SS erythrocytes following treatment with anthocyanins extracted from Justicia secunda indicates the influence of the extract on the propensity to sickle of the RBC. It was recently reported that anthocyanins from some others Congolese plants used in traditional medicine for the management of sickle cell disease give the same results18–26.
Statistical analysis using Student’s t-test31, applied with probability thresholds of both 0.01 and 0.05 for 20 degrees of freedom revealed statistically significant differences between the average values of both the perimeter and the surface of the untreated and treated erythrocytes (Table I), thus confirming the modification of RBC shape in the presence of the anthocyanin extract. Indeed, the RBC were observed to change from a sickled shape to normal biconcave cells. These values were in agreement with previously reported values18,19. The blood values of the treated SS RBC were remarkably similar to those of normal RBC.
The pathophysiology of SCD has been attributed to both the sickle haemoglobin and erythrocyte membrane behaviour. In this regard, two mechanisms of action could be suggested to explain the anti-sickling activity of anthocyanins. Given their property of binding to proteins, anthocyanins could inhibit the polymerisation of deoxy HbS into tactoids inside the RBC. This would lead to an increased concentration of haemoglobin in solution. Experimentally, this can be evaluated by the Itano solubility test using an UV/visible light spectrophotometer.
Another possible mechanism of action of anthocyanins lies in their effect on RBC membrane stability. Indeed, pharmacological agents that enhance SS RBC re-hydration rate may be promising drugs for the effective management of SCD. Cell re-hydration can be evaluated by the osmotic fragility test.
Table II shows that the solubility of deoxy-HbS (expessed as the increase of optical density of haemoglobin at 540 nm) increases with time after treatment with anthocyanin extracts (HbS+ACE), while it decreases in untreated HbS samples. In fact, after 90 min the absorbance of untreated HbS cells decreased from 1.360 to 0.255 (an approximately 80% decrease). When treated with anthocyanins, the absorbance of HbS solution did not decrease, but actually increased over the same period from 0.470 to 0.506 (an increase of almost 8%). These results indicate that anthocyanin extracts from Justcia secunda enhance the solubility of deoxy HbS, thereby substantially inhibiting gelification. The anti-sickling effect of anthocyanins would be achieved through direct interaction with HbS molecules. It has been postulated that the polymerisation of deoxy-HbS reduces its oxygen affinity13.
Haemolysis of SS RBC decreases with exposure to increasing concentrations of hypotonic saline. The mean corpuscular fragility values for untreated and treated SS RBC were 0.59 and 0.63, respectively (Table III). This indicates that the anthocyanin extract improved the ability of SS RBC to take up water without lysis occurring. This stabilisation effect could be explained by noting that anthocyanins rendered the SS RBC capable of withstanding higher concentrations of NaCl by increasing the volume of the RBC, reverting the sickling to produce biconcave cells, and, thereby, maintaining membrane integrity. Such effects have been reported for aqueous extracts of Zanthoxylum macrophylla roots Garcinia kola seeds and homoserine32–34.
Collectively, the above results suggest that anthocyanins could be used as an anti-sickling treatment for patients with SCD. The effect of these plant extracts on Fe3+/Fe2+ ratio in SS RBC is still being testing.
We recently demonstrated the in vitro anti-sickling activity of anthocyanin extracts from some Congolese medicinal plants, although how these extract exert their anti-sickling effect is not well understood. Here, we have shown that the mechanisms of the anti-sickling effect of anthocyanin extracts may involve both direct binding of the extract with deoxy-HbS molecules and stabilisation of the SS RBC membrane. Indeed, the anthocyanin extract decreased intracellular haemoglobin concentration by inhibiting cell dehydration.
The results of the present research have paved the way to envisaging in vivo studies of anthocyanin extracts as a therapeutic agent in patients with SCD and may provide a rational explanation for the use of Justicia secunda Vahl in managing SCD by Congolese traditional practitioners.
X-ray diffraction and electron microscopy studies will be helpful in elucidating the precise mechanism of action of anthocyanins.
The authors are indebted to the Third World Academy of Science (TWAS) (Grant No. 08-030 LDC/CHE/AF/AC-UNESCO FR-3240204448) for providing financial assistance for this study.