Elemental compositions and antianemic property of Harungana madagascari-ensis stem bark

. Abstract The concentrations of 15 elements and heavy metals in the stem bark of Harungana madagascariensis were determined using an energy-dispersive X-ray fluorescence (EDXRF) spectrometer. The antianemic activity was done using the changes in hematological parameters (PCV, RBC and hemoglobin) influenced by phenylhydrazine HCL (80 mg/kg) and malaria parasites-induced anemia. Results show Cd, Ni, Mo, Cr and Br were in the range of 0.02 – 0.9 mg/g, while Pb, Zn, Fe, Cu and Hg were in the range of 1.5 – 7.2 mg/g. The elements with very high concentration were Ca, K, Sr, Mn and Cl and were in the range of 10.5 – 774.3 mg/g. Remarkable antianemic activity was obtained with PCV of 40 – 48%, RBC count of 81-155 x 10 4 and hemoglobin value of 57-66 g/dL after treatment; compared with 30% PCV, 67 x 10 4 RBC count and 36.5 g/dL hemoglobin value obtained for the untreated control animals. Our results suggest that H. madagascariensis stem bark extract


Introduction
Plasmodium falciparum malaria remains a massive burden of disease with an estimated two million deaths worldwide each year and responsible for two major complications, namely; cerebral malaria and severe anemia (Breman, 2001). Anemia is a major cause of morbidity and mortality in malaria endemic areas of tropical regions of the world. Anemia in malaria is caused by destruction of red blood cells in the body or the depression of red blood cell production in the bone marrow. As the red blood cells become less, the patient weakens. In severe cases the patient is unable to deal with basic life essential tasks and may die. There are various causes and types of anemia, these include: sickle-cell anemia, iron deficiency anemia, vitamin B12 anemia, drug induced anemia as side effect of drug therapy.
In the treatment of diseases like malaria and anemia, the beneficial effects of analyzed drug materials especially from plants are mainly attributed to the presence of constituents (alkaloids, saponin, terpenoids, anthraquinones, essential oils, flavonoids, tannins, etc). The efficacy of these constituents could also be influenced by inorganic components known as the macro and micro (trace) elements. There are basically 17 important elements known to be required by all higher plants. Nine of them are macronutritients (C, N, O, H, K, Ca, Mg, P and S). An additional eight elements are defined as micronutritients (B, Cl, Cu, Fe, Mn, Mo, Ni and Zn). These micronutritients (Fe, Mn and Zn) are important as co-factors for enzymes activities, Cu is bound to amino acids, while some elements, such as Mn, Nd and Ce, are bound to some bio-macromolecules forming coordination compounds. Fe, Cu, Mn, Co, are important components of many antioxidant processes in human body, a deficiency of any of these essential elements may impair the function of the overall oxidant mechanisms in both plants and animals. Therefore, accurate quantitative analysis of the elemental content of plants is very important, as a contribution to increasing concern about their potential effects on human health (Slavica et al., 2005). To mention just few examples, the following medicinal plants have been employed in the management of anemia: The stems with leaves of Indigofera tinctoria, barks stem, branch, trunk of Khaya senegalensis, leaves of Sorghum vulgare, roots of Uvaria chamae, decoction were used in West Africa. Because of the prominent iron contents of 35.7 and 35.2 mg/100 g found in the root bark of Bridelia cathartica and Lannea stuhlmannii, respectively, they were useful in East Africa for anemia. Also, in China and India, Rehmannia glutinosa and Boerhavia diffusa were used for anemia (Gupta and Nandyala, 1984;Duke and Ayensu, 1985;Adjanohoun et al., 1986;Omolo et al., 1997). This study was carried out to determine the elemental compositions and examine the efficacious claim of the use of juice and sap from the Harungana madagascariensis stem bark in the management of iron deficiency anemia induced in pregnancy and child birth in folklore medicine. The study was done using two main modes of anemic induction through malaria and phenylhydrazine drug induced anemia.

Plant materials and preparation of the extracts
H. madagascariensis stem bark samples were collected near the main University campus, Ile-Ife. The plant was identified by Mr. O. A. Oladele, Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife and a voucher specimen (No. FHI 107392) was kept at the herbarium of the Forestry Research Institute of Nigeria, Ibadan. The ethanol extraction was made by soaking 325 g of the powdered stem bark in 70:30 ethanol:water for 24 hours. After maceration, the extract was evaporated in vacuo in a rotary evaporator to dryness, yielding 9 g of the brown crude extract.

Elemental compositions analysis
The elemental analysis of the plant material was performed by an energy-dispersive X-ray flurorescence (EDXRF) spectrometer. The spectrometer consisted of a Siemens FKO-04 tube with Mo anode, a kristalloflex 710H X-ray Generator installed in the Centre for Energy Research and Development, Obafemi Awolowo University, Ile-Ife. The equipment is operated under QXAS (Quantitative X-ray Analysis System) software (QXAS, Manual 1993). The sample and standard formulated pellet was irradiated for 20 min in a fixed tube at 30 kV and 10 mA. The samples was analyzed before the standard. All the values obtained are results of an average of three measurements on the plant material.

Animals
Mice and rats of either sex, weighing between 18-22 and 180-250 g respectively were used. The animals were maintained at 25 ± 1°C under natural 12 hours daylight/night conditions for at least 5 days before the experiment. All the animals were fed with standard diet in the Department of Pharmacology Animal House and water was given ad libitum.

Parasites
The N67 chloroquine sensitive strain of Plasmodium yoelii nigeriensis was donated generously by the Malaria Research and Reference Reagent Resource Center (MR4) Manassas, VA, USA, and was obtained from Institute of Medical Research and Training courtesy of Dr. O. G. Ademowo.

Antianemia activity
Antianemic activity was assessed using a) drug induced anemia using phenylhydrazine hydrochloride and b) malaria-induced anemia. The animals were randomly distributed into the following six groups: Group 1: Non-anemic control without treatment, Group 2: Anemic control without treatment, Group 3: Anemic rats treated with vitamin B12, Group 4: Anemic rats treated with 20 mg/kg extract, Group 5: Anemic rats treated with 40 mg/kg extract, Group 6: Anemic rats treated with 80 mg/kg extract The experiment in drug-induced anemia using phenylhydrazine was performed for 3 weeks. All the rats (except the ones in the first group) were injected with a single subcutaneous dose of 80 mg/kg of phenyl hydrazine hydrochloride (Yeshoda, 1942;Berger, 1985). On day 0, before initiation of anemia, initial weight, PCV, RBC and hemoglobin were taken in all the groups. Anemia was allowed to establish in 24 hours (Nath and Prasannan, 1958). On the day of establishment of anemia, the weight, PCV, RBC and hemoglobin were taken in all the groups. Treatment with the extract (20, 40, 80 mg/kg), vitamin B12 (100 μg/ kg), commenced 24 hours after the administration of phenylhydrazine hydrochloride and continues for 21 days on daily basis. Measurement of PCV, RBC and hemoglobin were taken daily for the first 3 days then on day 5, 7, 9, 13, 17 and 21.
For the malarial-induced anemia experiment, six groups of mice were inoculated with a standard inoculum size of 1 x 10 7 (0.2 mL) infected erythrocytes of P. yoelii nigeriensis intraperitoneally on day zero. Using the 4 day schizonticidal suppressive mode of treatment, the extracts (20, 40, 80 mg/kg), vitamin B12 (100 μg/kg), artemether (25 mg/kg) were given to the mice for 4 days. Measurement of PCV, RBC and hemoglobin were taken at day 0, 3 and 6.

Packed cell volume determination
PCV of the rats was determined by obtaining blood from a slightly cut rat tail and allowing the blood to flow through a capillary tube to more than two-third of the tube. The end with blood was then sealed off using plasticine. The capillary tubes were placed in the centrifuge and centrifuged for 15 min at 3,000 rpm. The capillary tubes were placed on the microhemotocrit to determine the packed cell volume.

Red blood cell count determination
Using the red blood cell pipette, the rat's blood is sucked to 0.5 mL mark. The mouth of the pipette was wiped using a tissue paper and hayem solution was sucked to 101 mark. The blood and hayem solution were properly mixed. The mixture is placed in between the cover slide and the counting chamber of the hemocytometer. The red blood cells were counted under the microscope.

Hemoglobin level determination
The dilution tube was filled to the mark 10 with freshly prepared 0.1N HCL. Blood was sucked to 20 mL mark of the pipette from a slightly cut tail of a rat and blown gently into the acid. It was allowed to stand for 5 min for the acid hematin to form. Distilled water was added drop by drop and stirred with a rod after each addition. This was continued until the dilution tube is slightly darker than that of the standard at daylight against a white background. More distilled water was added until a slightly lighter color was obtained. The average of the value obtained for slightly darker and slightly lighter was taking as hemoglobin level.

Statistical analysis
Data are expressed as mean ± S.E.M and analyzed using Students' t-test. The significant level was set at p<0.05. The IC50 values were calculated using the Microsoft Excel program.

Results
The elemental analysis of H. madagascariensis stem bark sample revealed the presence of 15 elements and heavy metals, Ca, K, Mn, Sr, Cl, Br, Cr, Cd, Cu, Fe, Ni, Pb, Zn, Mo and Hg in three categories of various concentrations. For example, Cd, Ni, Mo, Cr and Br were in the range of 0.02-0.9 mg/g, while Pb, Zn, Fe, Cu and Hg were in the range of 1.5-7.2 mg/g. The elements with very high concentration were Ca, K, Sr, Mn and Cl and were in the range of 10.5-774.3 mg/g (Table I). Table II showed the effects of the extract on the anemia induced by malaria parasites on selected hematological parameters of albino mice. Administrations of the extract to the animals at various doses (20, 40 and 80 mg/kg body weight) produced significant increase (p <0.05) in the hemoglobin content compared to the control mice; however, the values are not in dosedependent manner. Remarkable antianemic activity was obtained with PCV of 40-48%, RBC count of 81-55 x 10 4 and hemoglobin value of 57-66 g/dL after treatment compared with 30% PCV, 67 x 10 4 RBC count and 36.5 g/dL hemoglobin value obtained for the untreated animals. Figures 1, 2 and 3, showed the effects of the extract on the anemia induced by pheylhydrazine hydrochloride in rats. The three selected hematological parameters (PCV, RBC and hemoglobin) significant decreased (p<0.05). However, vitamin B12 and the extract increased significantly (p<0.05) these hemoglobin contents (Figure 1, 2, 3). This similar pattern of increase in PCV, hemoglobin and RBC was also observed from day 5 to day 21. These increases were significant (p<0.05), however not dosedependent.

Discussion
Practical approach to malaria is essential if the global objectives of malaria control are to be achieved. Anemia is a universal feature of malaria. It is common among the general population in the tropics, particularly in Africa and especially in children and pregnant women (Beales, 1997). In plants and animals development, their metabolic reactions require some essential elements. These elements are also important for diseases and disorders control in man. However, they could produce harmful effects in excessive concentrations; therefore, their different roles in the physiological processes strongly depend on their concentrations in a particular plant tissue. The plant analyzed in this work is known all over the world for their different pharmacological activities and, thus, have a very important place in phytotherapy (EMEA 1999;Gurib-Fakim 2007). Toxic heavy metals such as Cd, Pb, and Hg were detected in the sample of H. madagascariensis stem bark, but they were of minimal concentrations. Trace elements (e.g., B, Cu, Mn, Ni, Zn) that are phytotoxic at concentrations not harmful to human are of much less concern than elements that are easily concentrated in plants at toxic levels to humans and animals (e.g., Cd, Co, Se, Mo  According to Dhawan and Srimal (1997), the normal values for PCV, RBC count and hemoglobin in normal control rats are 40-52%, 6.5-9.0 x 10 4 and 60.4-76.8 g/dL respectively qualities of blood, bones, teeth and blood formation and also of cardiac function. They also play a predominant role in oxygen and electron transport. The large amounts of Fe and Ca in H. madagascariensis stem bark shows that it could be specifically useful in the treatment of diseases and disorders like: malaria, anemia in pregnant mothers at a very low dose, as its main medicinal activities are as an anti-infective (against malaria, trichomonads, bacterial, fungi) (Okoli et al., 2002;Iwalewa et al., 2008a), antianemia, (Erah et al., 2003), bleeding and amenorrhea (Duke and Ayensu, 2002). For example, the red juice from the plant leaves and stem bark are reputed for arresting post-partum or post-abortal bleedings in Sierra Leone, while the unopened buds are equally reputed for treating puerperal infection in Liberia (Olagunju et al., 2000). Jubi formula, a herbal preparation made from three medicinal herbs (Parquetina nigrescens, Sorghum bicolor and H. madagasca -riensis) has been successfully used in the treatment of anemia in humans and in the treatment of Trypanosoma brucei brucei-induced anemia in rabbits (Erah et al., 2003). The presence of these elements could also serve as ingredients that favor its antioxidant effect, antimalarial, antitrichomonal and anti-amoebic activities of H. madagascariensis stem bark (Tona et al., 2000;Iwalewa et al., 2008a,b). The beneficial effects of copper to plants and animals are numerous. Copper as a trace element has an inhibitory effect on edema and inflammation. The effect of copper on resistance to bacterial and viral infection in ruminants has been highlighted by Suttle and Jones (1989). Likewise, several investigators have explained the effect of iron overload on microbial infections. Van Asbeck et al. (1982) demonstrated that iron has a deleterious effect on the phagocytic capacity of both monocytes and granulocytes in vitro. Mencacci et al. (1997) indicated that iron overload may negatively affect CD4 Th1 development in mice with candidiasis, a function efficiently restored by therapy with deferoxa- mine, an iron chelator. This report of H. madagascariensis stem bark is a milestone as it shows that the elemental constituents of this plant is a source of dietary and food supplement for both animals and man especially when taking in very small doses. It could also be a pointer to how the plant produces its biological activities.
Several workers like Adams, 1953;Beutler and Blaisdell, (1958) have used biochemical indices like hemoglobin concentration, RBC count and PCV to evaluate the state of iron deficiency anemia, hence, the use of these indices in this study. According to Dhawan and Srimal (1997), the normal values for PCV, RBC count and hemoglobin in normal control rats are 40-52%, 6.5-9.0 x 10 4 and 60.4-76.8 g/dL respectively. From the results of malaria induced anemia groups, it is glaringly clear that the constituents of H. madagascariensis stem bark have elevated and contributed to the depleted constituents of the blood. The result of our study also indicated that anemia induced by malaria parasites in this experiment is not due to vitamin B12 and artemether as antimalarial agent has little effect on anemia induced by rodent malaria parasites. Artemether and vitamin B12 could not effectively reverse the anemic condition by increa-sing the number of RBC. The inabilities of artemether and vitamin B12 to reduce anemia could be possibly attributed to the various pathophysiological effects produced during malaria infection in mice. It has been established that the degree of anemia always correlates well with increase in parasitemia and levels of hemotocrit (Iwalewa, 1995), therefore as indicated in this study, the effect of extract on anemia induced by malaria shows that anemia was reversible. There was a continuous reduction in the value of PCV, RBCs and hemoglobin contents in the control group between D0 and D6, however in all the treated groups between D3 and D6 of infection, there was increase in the value of PCV, RBCs and hemoglobin. This is an indication that the elemental constituents in the extract has the ability to effectively inhibit the growth of the parasite in the RBC as previously reported in our earlier study (Iwalewa et al., 2008a) and couple with the fact that the extract contains Fe, the PCV, RBCs and hemoglobin contents could have increased.
Oxidative stress on erythrocytes is considered an important mechanism of hemolysis. Disruption of membrane integrity arises from fragility, dehydration as well as increased production of reactive oxygen species. Chronic hemolysis leads to loss of hemoglobin. These metabolic changes lead to depletion of essential nutrients and micronutrients which are required for proper cell function. Since non-parasitized erythrocytes are also destroyed, and in acute malaria, the presence of pro-oxidants is well established, the released constituents at the extracellular environment of the erythro-cyte may be a contributor to the oxidative stress.
Micronutrient deficiencies and infectious disease often coexist and show complex interactions leading to mutually reinforced detrimental clinical effects especially in underprivileged people of developing countries, particularly in rural regions. Several micronutrients such as trace elements (Zn, Fe, Se) modulate immune function and influence the susceptibility of the host to infection. Some trace elements have been found beneficial in the control of anemia under this condition. These include Fe, Cu, Zn and folate (Prasad, 1999). There have been a linkage or a direct correlation between antioxidants effect of trace elements, malaria and anemia. Deficiencies of trace elements do have negative impacts on malaria and anemia (Okochi and Okpuzor, 2005). Therefore, in this study, the presence of these trace elements in Harungana could serve as anti -oxidants, hence contributed to the plant antianemic and anti-malarial properties. This however, corroborates our earlier findings (Iwalewa et al., 2008a,b).
In conclusion, this study shows that H. madascariensis stem bark extract constituents' exhibit antianemic activity.
The effects in both malaria and phenylhydrazineinduced anemia are suggested to be influenced with the presence of the macro (K, Ca, Mg, Cr) and micro (trace) elements (Cu, Fe, Mn, Mo, Ni, Zn) detected in different concentrations that could produce toxicity if administered in large quantity.

Financial Support
Self-funded

Ethical Issue
The study was approved by the Ethical Committee of the Obafemi Awolowo University. The Principle of laboratory animal care (National Institute of Health-NIH publication No. 85-23) guidelines and procedures were followed in this study (NIH publication revised, 1985).