Sickle cell disease (SCD) is a neglected tropical disease [
Various methods have been developed in an effort to find principles that prevent or reduce crises in SCD. Conventional medicines have achieved a great level of success in SCD management. In recent years, bone marrow transplantation has been found to be efficient in the treatment of SCD. However, the cost implications, unavailability of necessary expertise, and the problems of finding suitable donor constitute a major setback to this approach in developing countries [
On the understanding that herbal remedies and medicinal plant products from indigenous flora have long been used in traditional medicine for SCD management, it appears that proper and intense scientific investigations on these medicinal plants could be of remarkable help in developing effective and safer drugs for SCD treatment. Research on phytomedicine has led to the development of Nicosan (herbal based drug used to treat SCD). Other plants that have gained scientific backing as antisickling agents include
Some equipment used included Shimadzu GC-17A system, light microscope (Olympus CX 21), spectrophotometer (Jenway 6400), incubator (grant JB series), oven (Gallenkamp), and centrifuge (Labofuge 300). Chemicals such as Para-hydroxybenzoic acid (PABA) and sodium metabisulphite were procured from Sigma Chemical Company, Paderborn, Germany.
Fresh samples of
Blood (2 mL) was obtained by venipuncture from homozygous sickle cell disease (HbSS) patients in stable state at Ahmadu Bello University Teaching Hospital (ABUTH), Zaria. Approval by the Health Research Ethical Committee (HREC) and consent of participants were obtained in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
Differentiation of major genotypes method by hemoglobin electrophoresis technique was used.
The principle of hemoglobin electrophoresis (at alkaline pH 8.4–8.9) was based on charge change in hemoglobin molecules, which correlates with the rate of hemoglobin movement towards the anode.
Blood samples of different, but known, genotypes were spotted at the point of origin on the paper. It was then placed in the electrophoretic component containing a buffer system and connected to a source of current. Hemoglobin movement towards the anode (represented by hemoglobin bands) was peculiar to each genotype (HbAA, HbAS, HbSS, and HbSC). This was noted and was used as standard. Subsequent test samples were treated as mentioned above. Those that showed similar pattern of movement as HbSS (when compared with the standard) were obtained and used in this research.
The blood was placed in sodium ethylene diamine tetraacetic acid (EDTA) bottle and thoroughly mixed by gently rolling the bottle. Blood samples were used for the studies within 12 hrs of collection.
The minimum sample size in this study is determined by the formula of [
The bioassay of both methanolic and aqueous extracts of the plant materials for antisickling activity was carried out by measuring the level of reversal of sickled red blood cells (RBCs).
Evaluation of antisickling activity was carried out according to the procedure of [
Washed erythrocytes were mixed with 2% sodium metabisulphite. Sodium metabisulphite initiates a decrease in oxygen tension thereby inducing the typical sickle shape of red blood cells. This is then followed by introduction of test extract in the test tube. The degree of reversal of sickling was measured by counting the number of cells unsickled under the light microscope.
Half a milliliter (0.5 mL) of the washed erythrocytes was mixed with 0.5 mL of freshly prepared 2% sodium metabisulphite in a clean test tube. It was incubated in water bath at 37°C for 30 minutes. A drop of the mixture was then viewed under the microscope. Equal volumes (0.5 mL) each of normal saline and the extracts were added to the blood-metabisulphite mixtures in different test tube and incubated at 37°C for another 30 minutes. Aliquots were taken at 30 minutes intervals, for up to 2 hours.
The procedure described by [
Percentage cells unsickled with time by 10 mg mL−1 extracts of leaves, seeds, and stem. LEA: aqueous leaves extract, LEM: methanolic leaves extract, SEA: aqueous seed extract, SEM: methanolic seeds extract, STA: aqueous stem extract, STM: methanolic stem extract, NS: normal saline, and PABA: Para-hydroxybenzoic acid.
It was calculated by the following formula:
P-Hydroxybenzoic acid (5 mg mL−1) and normal saline were, respectively, employed as positive and negative controls.
The membrane stabilizing assay method was based on the procedure described by [
The assay mixture consisted of 2 mL of 0.25% (w/v) NaCl, 1.0 mL of 0.15 M sodium phosphate buffer (pH 7.4), 0.75 mL of various concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mg mL−1) of plant extracts, and 0.5 mL of (2% v/v) erythrocyte suspension. The control was prepared as mentioned above, but without drug or extract (blood control), while the drug control was without extract. The standard drugs used were Indomethacin and Ibuprofen. The reaction mixtures were incubated at 56°C for 30 minutes, cooled under running water, and then centrifuged at 3913 ×g.
The principle behind this assay is the spectrophotometric measurement of the amount of hemoglobin released (read at 560 nm) by sickled erythrocytes, which is dependent on the extent of stabilization of sickled red blood cells’ membrane exerted by the test extract.
The percentage membrane stability was estimated thus:
Commercially prepared TLC aluminum sheets (20 cm by 20 cm) lined with silica gel were used. The plate was cut to fitted size of 5 × 5 cm. The extract was dissolved in 95% methanol and spotted at the bottom of the TLC plate (about 0.5 cm from the base) using a micro haematocrit capillary tube. The plate was placed in a chromatographic tank and eluted with a mixture of different solvents. The gradient mixture of hexane and ethyl acetate mixture gave the best resolution. Hexane/ethyl acetate at different ratios (100% hexane 9 : 1 v/v, 8 : 2 v/v, 7 : 3 v/v, 6 : 4 v/v, 5 : 5 v/v, 4 : 6 v/v, 3 : 7 v/v, 2 : 8 v/v, 1 : 9 v/v, and 100% ethyl acetate) was used. The plate was then removed, air-dried, and developed by spraying with 10% sulphuric acid in methanol. It was viewed under UV light. Spots, where seen, hence adjudged the solvent system of choice for use in column chromatography.
Partial purification of active crude extracts using a modified method of [
The purified fraction that showed peak antisickling activity was characterized using Gas Chromatography linked Mass Spectroscopy (GC-MS). The fraction was dissolved in methanol and applied on a Gas Chromatography (GC). It was separated at 60°C at a flow rate of 1.83 mL/min, 109.6 Kpa pressure for 4.15 min. Eluents were bombarded with a stream of protons from mass spectra (MS) and analyzed.
Results were presented as mean ± standard deviation (SD). Within and between the groups, comparisons were performed by analysis of variance (ANOVA) (using SPSS 17.0 computer software package). Significant differences were compared using Duncan Multiple Range Test (DMRT), and a probability level of less than 5% (
Data on
Peak antisickling effect of crude aqueous and methanolic extracts of
Crude extract | % of cells unsickled at 120 min | ||
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10 mg | 1 mg | 0.1 mg | |
LEA | |
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LEM | |
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STA | |
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STM | |
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SEA | |
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SEM | — | — | |
Values in the same row with different superscripts (a–c) are significantly different at
Values in the same column with different superscripts (1–5) are significantly different at
LEA: leaves aqueous extract, LEM: leaves methanolic extract, STA: stem aqueous extract, STM: stem methanolic extract, SEA: seeds aqueous extract, and SEM: seed methanolic extract.
Note: “—” represents complete lysis of hemoglobin.
Aqueous and methanolic crude extracts of the stem demonstrated a dose- and time-dependent increase in reverting sickled cells back to normal. However, the methanolic extract of seeds triggered partial lysis of erythrocytes at the maximum concentration of 10 mg mL−1, after 30 minutes of incubation. Complete lysis was seen between 60 and 120 minutes, by the effect of 10 mg mL−1 of methanolic seeds extract. This may imply that, at high concentration and over time, seeds extract of
Table
Membrane stabilizing activity of
Extracts | Concentration (mg/mL) | ||||
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0.5 | 1.0 | 1.5 | 2.0 | 2.5 | |
LEA |
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LEM |
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STA |
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STM |
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SEA |
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SEM |
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Indomethacin |
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Ibuprofen |
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LEA: leaves aqueous extract, LEM: leaves methanolic extract, STA: stem aqueous extract, STM: stem methanolic extract, SEA: seeds aqueous extract, and SEM: seed methanolic extract.
Sixty (60) fractions were obtained from column chromatography of the most active crude extract (aqueous leaves extract). A gradient mixture of hexane and ethyl acetate was used as eluent. Fractions with the same number of spots,
The GC-MS analysis of purified fraction that showed peak antisickling activity (Fraction C) revealed some associated bioactive components. Their molecular weight, chemical formula and retention index are shown in Table
Results obtained from this research indicated that
Peak antisickling effect of partially purified fractions of crude aqueous leaves extracts of
Partially purified fractions | Percentage (%) of cells unsickled at 120 min | ||
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10 mg | 1 mg | 0.1 mg | |
Fraction B | |
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Fraction C | |
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Fraction D | |
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Fraction E | |
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PABA (5 mg mL−1) | |
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Normal saline | |
GC-MS analysis of fraction C.
S/no | Name of compound | Chemical formula | M. wt. | RI |
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Pentadecanoic acid, 14-methyl ester | C17H34O2 | 270 | 619 |
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Octadecanoic acid | C18H36O2 | 283 | 749 |
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Hexadecanoic acid | C16H32O2 | 256 | 648 |
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9-Octadecadienoic acid (Z)-, 2,3-dihydroxypropyl ester | C21H40O4 | 356 | 756 |
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C19H16O | 152 | 1123 |
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Cyclohexanespiro-5′,4′-methyl-2′-phenyl-2′-oxazoline | C15H19NO | 229 | 947 |
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C10H16 | 136 | 1243 |
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16-Octadecenoic acid | C19H36O2 | 296 | 718 |
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Borneol | C10H18O | 154 | 1164 |
Bioactive compounds such as
Alpha-Phellandrene (
Depending on its half-life, it would be expected that its periodic administration would reduce both frequency and duration of crises.
Pathophysiology of sickle cell disease has been attributed to both sickle hemoglobin and erythrocyte-membrane behavior [
On the basis of these results, it could be inferred that the aqueous and methanolic extracts of leaves, stem, and seeds of
The results of this study scientifically validated the
Sickle cell disease
Thin Layer Chromatography
Retention factor
Analysis of variance
Duncan Multiple Range Test
Homozygous sickle cell disease
Health Research Ethical Committee
Red blood cells
Aqueous leaves extract
Methanolic leaves extract
Aqueous seed extract
Methanolic seed extract
Aqueous stem extract
Methanolic stem extract and Para-hydroxybenzoic acid (PABA).
Present address of Samuel Atabo: Department of Biological Science, University of Agriculture, Makurdi, Benue State, Nigeria.
The authors declare that there are no competing interests regarding the publication of this paper.
This work was self-funded with support from family.