Anthocyanins Extracts from
Morinda lucida Benth (Rubiaceae)
P.T. Mpiana1*, V. Mudogo1, K.N. Ngbolua2,
D.S.T. Tshibangu1 and E.K. Atibu1
1Département de Chimie, 2Département de Biologie,
Faculté des Sciences B.P. 190, Université de Kinshasa, Kinshasa XI, R.D. Congo
ABSTRACT
In the present work, anthocyanins extracts from a Congolese plant Morinda lucida were evaluated for their antisickling activity using microscopic technique. The red blood cells (RBCs) were observed to change from the sickled shape to normal biconcave cells in the presence of 2 per cent sodium metabisulfite. The treated SS RBCs demonstrated a remarkable similarity to normal blood values (Radius =3.3±0.3mm).The minimal concentration of normalization (MCN) of sickle cell erythrocytes was 0.195 µg/mL. The antisickling activity was found to be dose dependent. Anthocyanins extracts was found to be responsible of the inhibition of the sickling process, thus, justifying the claims of the traditional medical practitioners and suggesting a possible correlation between the chemical composition of this plant and its use in traditional medicine.
Keywords:Morinda lucida, Sodium metabisulfite, Antisickling activity, Anthocyanins extracts.
Introduction
Several reports indicate that pharmacological agents that inhibit haemoglobin S (HbS) gelation could be used in the control of the sickling process of red blood cells (RBCs), which is a major pathological event of the sickle cell disease (SCD) (Iwu et al., 1988; Nwaoguikpe et al., 2005).
Sickling of RBCs occur as a result of polymerization of deoxygenated HbS molecules, so that, they become stacked linearly. Clinical symptoms occur in homozygote individuals and develop from at about 6 months old. There is a chronic haemolytic anaemia and recurrent painful vasocclusive crises because of the sickled RBCs blocking small vessels. This leads to tissue ischemia and infarction, mostly affecting the liver, spleen, lungs, brain and retina. These crises may be precipitated by infection, cold, exercise, dehydration and pregnancy (Parveen and Michael, 1999).
Many investigations have been carried out on the role of phenolics such as benzoic acid derivatives and divanilloyl quinic acids in the management of SCD (Elujoba and Sofowora, 1977b, Adesanya and Sofowora, 1983; Osoba et al., 1989; Akojie and Fung, 1992; Ouatara et al., 2004) and the importance of the plant materials in the maintenance of health is well established (Neuwinger, 2000; Cordeiro et al., 2004; Elujoba et al., 2005a, Ekeke et al., 1997).
These phytochemical compounds have been found to reduce the in vitro sickling of RBCs.
Different species of plants from tropical Africa region are very rich sources of phenolics. Because of the antisickling effects of certain phenolics such as anthocyanins (Mpiana et al., 2007a, Mpiana et al., 2007b), we were prompted to investigate the antisickling potency of a Congolese plant Morinda lucida Benth., which is a medical plant widely used by traditional medical practitioner in Democratic Republic of Congo (DRC) to cure the SCD.
The plant Morinda lucida Benth. belongs to the family of Rubiaceae. It is commonly known as Nsiki (Bas Congo), Mukakadi or Mutebenolozi (Bandundu), Endombe or Kolomboka (Equateur), Mundundama (Kasai-Occidental), Kakate (Kasai-Oriental), isuku (Katanga) in the specified Congolese languages.
It is a small tree, approximately 15m (50ft) high, widely distributed in Tropical Africa. It has been found in DRC, Republic of Congo, Benin, Ivory Coast, Nigeria, Central African Republic, Senegal, Togo, Sao Tomé et Principé.
The plant has been reported to have varying traditional medicinal uses (Kerharo, 1974). It has been shown to have antibacterial (Bokolo et al., 2006), antitrypanocidal, and antimalarial (Azuzu and Chineme, 1990) activities. Anthocyanins are powerful free radical scavengers (Kahkonen et al., 2003a). They also show antioxidant activity in lipid environments (Satué-Gracia et al., 1997).
The antisickling activity of anthocyanins extracted from this plant is evaluated in vitro on SS blood using Emmel’s test in the presence of Sodium metabisulfite (Courtejoie and Hartaing, 1992). This activity is expressed as the normalization of sickled cells. The minimal concentration of normalization (MCN) of sickle cell erythrocytes will be determined.
Materials and Methods
Plant Material
The leaves of Morinda lucida Benth were collected from plants growing in Kinshasa, DRC and were authenticated by Mr. B.L. Nlandu of the INERA (Institut National d’Etudes et Recherches Agronomiques). Voucher specimen is on deposit at the INERA Herbarium of the Faculty of Science (Université de Kinshasa).
Extraction
The dried and powdered plant material (leaves, 10 g) was repeatedly extracted by cold percolation with 95 per cent ethanol (EtOH) and water (100 ml x 1) for 48 hrs. Fractions were filtered and concentrated to dryness under reduced pressure using a rotary evaporator. Extraction of anthocyanins was then done using 100 g of dried powdered plant material with distillated water and diethyl ether following an established protocol as previously reported (Mpiana et al., 2007a, Mpiana et al., 2007c).
Biological Material
The sodium citrate suspension of blood samples used to evaluate the antisickling activity of the plant extracts in this study were taken from known sickle cell adolescent patients attending the “Centre de Médecine Mixte et d’Anémie SS” and “Centre Hospitalier Monkole”, both located in Kinshasa area, DRC. None of the patients had been transfused recently with Hb AA blood. All antisickling experiments were carried out with freshly collected blood. In order to confirm their SS nature, the above-mentioned blood samples were first characterized by haemoglobin electrophoresis on cellulose acetate gel at pH 8.5. They were found to be SS blood and were then stored at±4° C in a refrigerator.
Antisickling Assay
In order to evaluate the antisickling activity of our plant extract samples, an in vitro antisickling assay was performed, in which the 2 per cent sodium metabisulfite pre-treated blood sample is put in contact with plants extracts at different concentrations, according to Emmel’s test procedure as previously reported (Mpiana et al., 2007a; Mpiana et al.,
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