ANTI-ANAEMIC POTENTIAL OF METHANOLIC LEAF EXTRACT OF MUCUNA PRURIENS ON PHENYLHYDRAZINE (PHZ) INDUCED ANAEMIC ALBINO WISTAR RATS

The antianaemic potential of methanolic leaf extracts of Mucuna pruriens was investigated using phenylhydrazine (PHZ) induced anaemic albino Wistar rats. Fifteen rats used for the study were randomized into five experimental groups. To induced anaemia, the rats (except the normal control, Group E), received 60 mg/kg of the haemolytic agent Phenylhydrazine intraperitoneally (i.p) for two consecutive days. Anaemic Wistar rats in groups A and B received a daily oral dose of 500 and 250 mg/kg of the methanolic leaf extract of Mucuna pruriens. Nweze et al. (2016) had reported a median lethal dose greater than 5000 mg/kg for the methanol leaf extract of Mucuna pruriens. Groups C and D received Vitamin B12 (10 mg/kg) and normal saline (1 ml/kg), respectively. Normal control rats also received normal saline (1 ml/kg). Extract or normal saline was administered per os (p.o) while vitamin B12 was administered i.p. for a duration of 21 days. Packed cell volume (PCV) and haemoglobin concentration were determined weekly for three weeks. The result of the study indicated that both the methanolic leaf extract of Mucuna pruriens and Vitamin B12 significantly (p < 0.05) increased the packed cell volume and haemoglobin concentrations in treated rats compared to the negative control group of rats. This indicated that the methanolic leaf extract of Mucuna pruriens has anti-anaemic properties and could be utilized in the management of anaemia.


INTRODUCTION
Mucuna pruriens is a wild tropical legume native to Africa and Asia (Duke 1981 in Nweze et al., 2017), it belong to the family Fabaceae, and is among the various under-utilized wild legumes. Mucuna pruriens is commonly called 'velvet bean ', 'cowhage' or 'cowitch' in English, Eni in Hausa, Agbala or Agbaloko in Ibo and Werepe in Yoruba. The plant is known for the intense irritation and itching, it produces on contact with the skin when the fruit is mature and dries (Andersen et al., 2015). The itch is caused by mucunain and serotonin contained in the hairs lining the seed pods called trichomes (Reddy et al., 2008).
It has nutritional potential as a rich source of protein (23-35%) (Teixeira et al., 2003), which is comparable to those of other pulses such as soybean, rice bean and lima bean (Gurumoorthi et al., 2003). In South East Asia, the immature pods and leaves of mucuna beans are used as vegetables. The ethnic groups of Eastern Nigeria use the seeds of Mucuna sloanei and Mucuna puriens as a condiment or as garnishing for the main dish (Ukachukwu et al., 2000). All parts of M. pruriens has been reported to possess valuable medicinal properties and it has been investigated in various contexts. The seeds have laxative, anthelmintic, alexipharmic and tonic effects (Taylor, 2005). The L-dopa content of the seeds is responsible for its efficacy in the management of Parkinson's disease (Manyam et al., 2004). The dried crushed root is applied for relief of toothaches (Hishika et al., 1981). The leaves possess aphrodisiac and antimicrobial activities (Warrier et al., 1996) and are used in the treatment of ulcers, cephalgia and general debility. It is reported that people suffering from debilitating disease conditions, acute blood loss and blood deficiency diseases consume leaves of M. pruriens (Akindele and Busayo, 2011). Anaemia is a condition in which haemoglobin (Hb) concentration and/or red blood cell (RBC) numbers are lower than normal and insufficient to meet an individual's physiological needs (WHO, 2011). Alternately, World health organization (WHO) defined anaemia as a haemoglobin (Hb) concentration less than 130g/l in men and 120g/l in women. Anaemia develops when the rate of red blood cell production by the bone marrow fails to keep pace with loss or destruction (Reid et al., 2008). Haemoglobin is the oxygen carrying part of red blood cells. The role of Hb in carrying oxygen to the tissues explains the most common clinical symptoms of anaemia, which include fatigue, shortness of breath, bounding pulses or palpitations, and conjunctival and palmar pallor (WHO, 2011) Approximately one-third of the world's population (32.9%) was estimated to suffer from anaemia in 2010 (Kassebaum et al., 2014). The population groups most vulnerable to anaemia as of 2016 include: (42%) children under 5 years of age, particularly infants and children under 2 years of age; (39%); women of reproductive age; and (46%) pregnant women (WHO, 2016).
Anaemia is one of the most widespread public health problems, especially in developing countries due to nutritional problems (such as iron or vitamin deficiency), low socioeconomic status, inherited disorders, recurrent infections and an increased prevalence of blood parasites especially Plasmodium, Trypanosomes and helminthic infestation (Sanni et al., 2005).
It has an important health, welfare, social, and economic consequences, these include impaired cognitive development, reduced physical work capacity and in severe cases increased risk of mortality, particularly during the perinatal period (Van den Broek et al., 2000).
In the tropics, due to prevalence of malaria and other parasitic infections, between10 to 20% of the population is reported to have less than 100 g/l of haemoglobin in the blood (Diallo et al., 2008). The great loss in terms of clinical diagnosis and treatment and even depletion in human resources as a result of anemia could be prevented with adequate exploration into medicinal plant that can naturally boost blood level. Hence, this study investigated the haematological effects of Methanol leaf extract of Mucuna pruriens on Phenylhydrazine (PHZ) induced anaemic Wistar strain of albino rats.

MATERIALS AND METHODS Plant Collection and Identification
The fresh leaves of velvet bean plant were collected locally from Zaria and identified by Mallam Galla at the Herbarium of Kaduna State University.

Preparation of the Methanolic Plant Extract
The leaves of M. pruriens were washed and air dried at room temperature. Then it was pulverized and macerated in 2 L of methanol for 72 hrs.The mixture was decanted through a glass funnel fitted with Whatmann No. 1 filter paper. The filtrate was then allowed to evaporate to dryness via open air evaporation. The dried extract was afterwards weighed and stored until when required.

Experimental Animals
Wistar rats of both sexes and weighing between 70g and 130 g were purchased from the animal house unit of the Nigerian Institute for Trypanosomiasis Research (NITR) Kaduna State. The animals were housed in locally fabricated cages within the Zoology Laboratory of Department of Biological Sciences Kaduna State University and were allowed to a seven days acclimatization period before the commencement of the study. The experimental animals were maintained on animal feed and given water ad libitum. Beddings were changed twice weekly.

Haematological Parameters
Before administration of phenylhydrazine in the rats, the baseline values of two heamatological parameters were recorded, namely packed cell volume and haemoglobin concentration. Packed Cell Volume (PCV) was determined by the Microhaematocrit method, while the Haemoglobin (Hb) concentration of each animal was estimated by Cyanomethaemoglobin method using a spectrophotometer.
The dose of the extract was based on the work of Nweze et al. (2016) who reported a median lethal dose (LD50) greater than 5000 mg/kg for the methanol leaf extract of Mucuna pruriens. Treatment with methanol leaf extract of M. pruriens, vitamin B12, or normal saline was given orally throughout the duration of the study.

STATISTICAL ANALYSIS
The results of the haematological estimations were presented as mean ± SD of three animals per group. Total variations present in a set of data were estimated by One Way Analysis of Variance (ANOVA) and p value less than 0.05 was considered statistically significant.

RESULTS
Prior to the administration of phenylhydrazine (PHZ), the Packed cell volumes (PCV) of rats were between 49 and 55 % (Table 1), with no significant difference (p > 0.05) in the mean PCV values of rats in the various experimental groups. Upon administration of phenylhydrazine (60 mg/kg body weight) there was an acute decline in mean PCV with values ranging from 31-35 percent in all the experimental groups except in the normal control. Between the PHZ treated groups, there was no statistically significant difference in the packed cell volumes in all the anaemic rats (p > 0.05). However, the mean packed cell volumes of anaemic rats were significantly lower (p < 0.05) than that of the normal control group (Table 1).
On day 21, the pattern of changes in the haematological parameters under investigation amongst group 3 (PHZ + 10mg/kg vitamin B12: positive control) rats were observed to be essentially similar to the pattern obtained amongst group 1 (PHZ + low dose extract) rats and group 2 (PHZ + high dose extract). Furthermore, when the extract treated groups were compared with the Normal control (Group 5), there was no significant difference (p > 0.05) with PCV values of the extract treated group approaching those of normal control. However, when compared with group 4 (PHZ + 1ml/kg normal saline: negative control) there was significant difference (p < 0.05) with elevations in PCV among the extract treated groups.  The effect of treatment with methanolic leaf extract of Mucuna pruriens on Haemoglobin Concentration (Hb) of rats is shown in Table 2. Haemoglobin Concentration of anaemic rats treated with 250 and 500 mg/kg of the methanolic leaf extract recorded steady increase in mean Hb concentration compared to group 4 (PHZ + 1ml/kg normal saline: negative control). At Day 21, the Hb values of rats that received the extracts and Vitamins B12 had return to normal values, resulting to no significant difference in the mean values when compared with the normal control group (p > 0.05). But when compared with group 4 (PHZ + 1ml/kg normal saline: negative control) there was high significant difference (p < 0.05).

DISCUSSION
The results of this study indicate that phenylhydrazine induced anaemia in rats. These findings agree with those of Dholi et al. (2016), Droucoula et al. (2017) and Umaru et al. (2018) who also reported that administration of PHZ induces acute anaemia in rats. Phenylhydrazine induces haemolytic anaemia though oxidative alternations to red cell membrane proteins resulting in red cell injury and lysis (Berger, 2007).
The elevations in haematological parameters recorded in anaemic rats treated with either Vitamin B12 or the leaf extract of Mucuna pruriens are indicative of their haematopoietic properties. Vitamin B12 (cobalamin) is an essential nutrient required in blood cell formation and maturation. When in insufficient quantity, vitamin B12 deficiency results in Megaloblastic anaemia, a condition characterized by proliferation of immature red cell with reduced lifespan (Castellanos-Sinco et al., 2015;Koury and Ponka, 2004). There anti-anaemic activity demonstrated with the doses of the methanol leaf extract of Mucuna pruriens agrees with those of Obioma et al. (2014) who investigated the anti-anaemic potentials of the aqueous extract and raw leaves of Mucuna pruriens. Similarly, Madukwe et al. (2014) also reported that the fresh and shade-dried leaf extracts of Mucuna pruriens significantly increased haemoglobin, packed cell volume and white cell counts in anaemic rats. In a review article, Ravindra and Ashvini (2019) also mentioned Mucuna pruriens (Agbala) as a plant having anti-anaemic potentials. Furthermore, Nweze et al. (2017) reported that the fresh leaves of M. pruriens are washed and macerated in water to make a decoction which is drunk to boost blood supply. The anti-anaemic activity as demonstrated by Mucuna pruriens may be due to the presence of blood forming moieties in the leaf extract. Proximate analysis of the leaves showed the presence of iron, vitamin C and pro-vitamin A (Akomas et al., 2014;Madukwe et al., 2014). Iron is an essential haematopoietic factor required in the formation of the haem component of haemoglobin found in red cells. Vitamin C has also been reported to participate in haematopoiesis (Musyoka et al., 2016).
Phytochemical screening of the leaf extracts has also revealed the presence of saponins, flavonoids and alkaloids (Nweze et al., 2016). Certain class flavonoids and alkaloids have been reported to possessing antioxidant activity. Thus, the presence of these antioxidants in the leaf extract of M. pruriens may facilitate the reversal of the damaging effect of phenyl hydrazine on red cells. Phenyl hydrazine is reported to induce anaemia through oxidative damage to red cell membrane by increasing the formation of reactive oxygen species (Lavanya et al., 2018). However, alkaloids and flavonoids protect cells as powerful antioxidants which prevent or repair damage done to red cells by free radicals or highly reactive oxygen species (Beack et al., 2020).
In conclusion, the results of the analysis carried out on Phenylhydrazine (PHZ) induced anaemia in Wistar strain of albino rats orally treated with methanolic leaf extract of M. pruriens revealed that the extracts boosted blood production, thereby suggesting its antianaemic potentials which can be used in folklore medicine for the management of anaemia.