Effects of Aqueous Extract of Psidium guajava Leaves on Lead Acetate Induced Changes on Haematological and Oxdative Stress in Adult Wistar Rats

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Iliyasu M.O., Ibegbu A.O., Musa S.A., Akpulu P.S., Animoku A.A., and Andrew I.E
Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University, Zaria,Kaduna State-Nigeria.
Sambo J.S.
Department of Veterinary Pathology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria Kaduna State-Nigeria.
All correspondences to: Iliyasu M.O, moiliyasu@gmail.com

ABSTRACT
The antioxidant and haematoprotective effects of aqueous extract of Psidium guajava leaves on lead poisoning were studied in Adult Wistar rats. 35 Adult Wistar rats were divided into seven (7) Groups of five (5) rats per Group. The rats in Groups one (1) and two (2) were administered with distilled water and 1000mg/kg body weight respectively. The rats in Groups three (3) to seven (7) were exposed to lead acetate at 120mg/kg body weight (kbw) daily for 21 days. While the rats in Groups 5, 6 and 7 were treated with 500mg, 1000 mg/kg kbw of aqueous extract of P. guajava leaves and 10mg of Succimer (Standard drugs) respectively for another 14 days, the rats in group 3 were scarified at15th day of the experiment and Group 4 was administered with distilled water for 14 days. The rats were sacrificed at the end of the experiment and blood samples were collected via cardiac puncture for the estimation of oxidative stress markers and haematological parameters. The result showed that oxidative stress was induced in rats exposed to lead acetate by a significant increase in LPO and decrease in CAT, SOD and GSH (p<0.05). The results also showed changes in haematological parameters by a significant decreased in red blood cells and a non significant decreased in haemoglobin concentration and Packed cell volume in rats exposed to lead acetate (p<0.05). There were also non significant increase in white blood cells and Platelet count in rats exposed to lead acetate. The changes in oxidative stress markers and haematological parameters were reversed in rats treated with aqueous leaves extract of P. guajava. Thus, the present study has established that aqueous P. guajava leaves extract was able to ameliorate the effects of lead acetate induced toxicity and may likely be beneficial to population in endemic areas exposed to lead poisoning.

Keywords: Oxidative stress, haematological parameters, lead acetate, Psidium guajava.

INTRODUCTION

Free radicals are produced in normal and pathological cell metabolism. Oxidation is essential to many living organisms for the production of energy to fuel biological processes (Ibegbu et al., 2012). However, uncontrolled production of oxygen derived free radicals are involved in the onset of many diseases such as cancer, rheumatoid arthritis, cirrhosis and arteriosclerosis as well as in degenerative processes associated with exposure to heavy metal such as lead and mercury (Luo et al., 2012). Exogenous chemical like lead and endogenous metabolic processes in the human body or in the food system might produce highly reactive free radicals, which are capable of oxidizing biomolecules, resulting in cell death and tissue damage (Halliwell et al., 2003).
Lead poisoning is also known as plumbism, colica Pictonum, saturnism, Devon colic, or painter’s colic, which is a medical condition caused by increased levels of the heavy metal lead, in the body (Rossi, 2008). Lead interferes with a variety of body processes and is toxic to many organs and tissues including the heart, bones, intestine, kidneys, reproductive and nervous systems. Interference with the development of the nervous system could lead to permanent learning and behavioral disorders. Symptoms of lead toxicity include abdominal pain, confusion, headache, anaemia, irritability, and in severe cases seizures, coma, and death (Barbosa et al., 2005). Study has shown that lead intoxication has destructive effects on blood (Elias et al., 2014). Histopathologically, Lead acetate induced cellular damage in the cerebellum of adult Wistar rats and it was also observed that ascorbic acid minimized the lead-induced cellular damage in the cerebellum of adult Wistar rats (Musa et al., 2012). Thus, a lot of attention has focused on dietary antioxidants which may have a potential for therapeutic use and prevention of these diseases. Investigations into the properties of plants have resulted in the identification of a wide array of bioactive compounds that include flavonoids, phenolics, limonoids, carotenoids, coumarins, phytosterols, which have medical and health benefits (Tachakittirungrod et al., 2007).

Psidium guajava leaves are important part of guava tree which are useful in treating many health problems. Guava leaves have been shown to contain anti-oxidant, haemato-protective, antibacterial, anti-cancer and anti ulcer properties needed in the treatment of many diseases (Vibha et al., 2012). Antioxidant activity by scavenging of reactive oxygen species generated by lead is important in preventing potential damage to cellular components such as DNA, proteins, and lipids (Tachakittirungrod et al., 2007). The present study was aimed at evaluating the effects of aqueous extract of Psidium guajava leaves on lead acetate induced changes on haematological and oxdative stress markers in adult Wistar rats.

MATERIALS AND METHODS
Animals
Thirty five (35) adult male Wistar rats were obtained from Animal House of the Department of Human Anatomy, Ahmadu Bello University, Zaria-Nigeria. The rats were acclimatized to experimental condition for a period of two weeks and feed with rat chow and water was allowed ad libitum.

Psidium guajava leaves
Fresh leaves of P. guajava were obtained from the Faculty of Medicine premises, Ahmadu Bello University, Zaria. The leaves were identified and authenticated with a voucher number of 3253 in the Department of Biological Sciences Herbarium, Ahmadu Bello University, Zaria.

Preparation of Extract
Extraction of the guava leaves were done in the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. Aqueous extraction of the guava leaves was prepared according to the procedure of Ofodile et al., (2013). In this procedure, the fresh leaves of guava were collected, washed thoroughly, shade-dried and pulverized using a mechanical grinder. The powder obtained was macerated with distilled water for 72 hours at 30±4°C. The mixture was filtered and the filtrate was concentrated in water bath at 50°C. Finally, the concentrated crude aqueous extract was subjected to drying to solid mass. The testing samples were prepared by dissolving the solid mass with distilled water to obtain a final concentration of 500mg/ml per kg body weight (kbw) and 1000mg/ml per kbw used for the experiment.

Experimental Protocol
The LD50 of Psidium guajava leaves aqueous extract has been established to be 5000mg/kg body weight
(Shubhangi, et al., 2013) following the (OECD, 2001) guidelines. Therefore, doses of 10% (500mg/kg) and 20% (1000mg/kg) of the LD50 were used in this study.
Based on the reported oral LD50 of Lead acetate which was 600mg/kg bw for Wistar rats (Sujatha et al., 2011), 20% of the LD50 (120mg of lead acetate/kg body weight) was used in this study. 10mg/kg body weight of Meso-2,3-dimercaptosuccinic acid (DMSA), known as Succimer was used according to Alan and Miller, (1998).

Experimental Design
The thirty five (35) adult male Wistar rats were divided into seven (7) groups of five (5) animals per group. Group 1 was administered with distilled water for 35 days, Group 2 was administered with 1000mg/kg bwt of Psidium gaujava leaves extract only for 35 days, Groups 3 to 7 were administered with 120mg/kg bwt of lead acetate from 1st to 21st and animals in Group 3 were sacrificed on the 22nd day. Group 4 was administered with distilled water from the 22nd to 35th day, while Group 5 was administered with 500mg/kg of Psidium gaujava leaf extract from the 22nd to 35th day, Group 6 was administered with 1000mg/kg of Psidium gaujava leaf extract from the 22nd to 35th day, and Group 7 was administered with 10mg/kg of Succimer from 22nd to 35th day. All the administrations were through oral route within 35 days.

Animals Sacrifice
After the last administration, the animals were allowed to fast for 24 hours before they were humanly sacrificed. Blood samples were collected via cardiac puncture from each rat and the blood was divided into two. One half was put in Tripotassium salt of Ethylenediamine tetra acetic acid as anticoagulant and blood was collected for haematological estimation using Systemex XE-2100 Automated Blood Analyser according to the method of Monica, (2000), while the other half in plain bottles, was allowed to coagulate and serum was harvested for biochemical estimation.

Data Analysis
Data obtained were expressed as Mean ± SEM (Standard error of mean). One-way analysis of variance (ANOVA) was used to compare the mean differences followed by Turkey’s post-hoc test. P-value less than to 0.05 was considered to be statistically significant. All the results were analyzed using the Statistical Package for Social Scientist (SPSS version 16).

RESULTS
Analysis of oxidative stress parameters
The study revealed a significant increase in lipid peroxidation among rats in Groups 3, 4 and 5 (Table 1) when compared with those in Group 1 (P < 0.05). On the other hand, there was a significant decrease of catalase levels in rats of Groups 3 and 4 (P < 0.05) compared to the value obtained in Group 1. While the superoxide dismutase levels decrease insignificantly in all the Groups and the mean glutathione levels were significantly lower in Groups 3 (P < 0.05) when Haematological Analysis
The results showed that the mean Red blood cell (RBC) count, Haemoglobin concentration (Hb) and Packed cell volume (PCV) values were reduced in Groups 3 and 4, though the differences were not statistically significant when compared with Group 1 (P < 0.05). However, the mean RBC and Hb values increased insignificantly in Groups 2, 5, 6 and 7 compared to Group 1. It was observed that the mean RBC was significantly decreased in Groups 3 and 4 when compared with Group 2 (p <
0.05). The mean Platelet count (PLT) increased insignificantly in Groups 3, 4, 5, 6 and 7 when compared with Group 1 (P < 0.05). The mean total White blood cells (WBC) counts were found to have increased in Groups 3 and 4. However, these increases observed were not statistically significant (P < 0.05). The mean values of differential leucocytes counts with respect to lymphocytes, neutrophils, Eosinophil and monocytes also varied insignificantly among the groups (Table 2).

DISCUSSION
Oxidative stress markers
The present study provided evidence that lead has induced oxidative stress in Wistar rats by increasing LPO and decreasing CAT, SOD and GSH. Reduced glutathione (GSH) concentrations, as well as modifications in CAT and superoxide dismutase (SOD) activities are the most frequently used markers in tissues or in blood (Khaki et al.,
2010). Lead-induced oxidative stress in blood and other soft tissues has been postulated to be among the possible mechanisms of toxicity (Pande et al., 2002; Auman et al., 2007; Waters et al., 2008). Disruption of pro-oxidant and antioxidant balance might lead to tissue injury. It was reported that lead increased the level of lipid peroxidation (Upasani et al., 2001) and brain thiobarbituric acid-reactive substances and altered the antioxidant defense system (Adanaylo et al., 1999).
This study further established that treatment with P. guava leaves extract could lead to recovery from oxidative stress caused by lead acetate. There were no significant differences in serum LPO and GSH concentrations, CAT and SOD activities of the Control and those of the animals treated with P. guava after administration of lead acetate. On the other hand, rats treated with lead acetate only showed a significant increase in serum LPO level and significant decrease in GSH level, CAT and SOD activities. This observation supports the report of Neeraj et al., (2012), that aqueous extract of Psidium guajava leaves was protective against sodium arsenite-induced toxicity in experimental rats. In that earlier study, treatment with aqueous extract of P. guajava significantly restored the level of oxidative stress markers. It was concluded that prophylactic co-administration of aqueous extract of the leaves could provide specific protection from oxidative injury.
Psidium guajava crude leaf aqueous extract, contains many flavonoids, especially quercetin (Witness et al., 2008), which is a potent antioxidant has been shown to protect nitric oxide from the scavenging actions of superoxide anion (Ajay et al., 2006). Quercetin chelates lead by forming a coordination bond with the lead ions through its orthophenolic groups located on the quercetin B ring (Bravo and Anacona, 2001). The hydroxyl groups of quercetin along with the carbonyl group easily donate electrons by undergoing resonance and stabilize free radicals that can initiate lipid peroxidation (Beecher, 2003). Study has demonstrated that T. brucei infection resulted in high amounts of MDA and low concentrations of GSH in the T. brucei-infected animals, however the aqueous extract of P. guajava leaves was able to reduce the trypanosomosis associated lipid peroxidation as well as raise the level of GSH in the treatment group (Akanji et al., 2009). Therefore, it could be pertinent to speculate that the ability of the leaf extract of P. guajava to lower the MDA concentrations in the treatment group may be attributed to similar antioxidant properties.
Haematological indexes
The present study showed a significant reduction of Red Blood Cell (RBC) count in rats administered with lead acetate only but RBC, Hb and PCV increased significantly in rats administered with lead acetate and then treated with aqueous extract of P. guajava leaves. The observation in the present study was in agreement with other studies such as Suradkar et al. (2009), who observed that the reduction could be as a result of the effect of lead acetate on the activity of aminolevulinic acid dehydratase (ALAD), a key enzyme in heme synthesis. Moreover lead also inhibit the conversion of coproporphyrinogen III to protoporphyrin IX leading to reduction in haemoglobin production and shortened life span of erythrocytes (Klassen, 2001). Progressive destruction of RBCs due to binding of lead with RBCs, leading to increased fragility and destruction could be the main reason for decrease haematological values (Rous, 2000). Significant decrease in Hb and PCV were observed following exposure of rats to lead acetate according to the work of Helmy et al. (2000).
The increase in WBC and platelet counts were not significant and were variance with the study of Nwokocha et al. (2011), who showed that a significant increase in
platelet and WBC count were observed, and the speculation the increase could have been as a result of marrow infiltration by toxic substance and with a reactionary increase in some parameters. This was also contrary to the decrease in WBC which may directly be related with either decrease in their production from germinal centers of the lymphoid organs or increased lysis due to the presence of lead in the body (Avdheshumar et al., 1998).
Significant improvement in RBC, Hb and PCV values and reduction in the WBC and PLT values in Psidium guajava and Succimer treated groups caused amelioration when compared to lead administered groups. This might be due to hemoprotective immune stimulation and anti-inflammatory effects and antioxidative property of Psidium guajava. The findings were in line with the results of Friday et al., (2010) which showed that the red blood cell counts, hematocrit and hemoglobin concentration increased significantly on the administration of guajava extract to both male and female rats but has no significant effect on the white blood cell counts. Extract of P. guajava leaves may possibly serve as an acceptable blood booster in an anemic condition or for prophylactic purposes. Although the specific mechanism through which the extract facilitated the increase in these hematological indices was not ascertained. It is possible that the extract contains constituent(s) that can interact and stimulate the formation and secretion of erythropoietin, hematopoietic growth factors or committed stem cells (Friday et al., 2010). Specifically, stimulations of hematopoietic growth factors and erythropoietin systems have been reported to enhance rapid synthesis of blood cells (Murray, 2000). Moreover, the hematopoietic potential of the leaf extract of P. guajava may be related to its antioxidative activity (Friday et al., 2010).

CONCLUSION
From the observations in the present study, there were alterations in value of oxidative stress markers in rats exposed to lead acetate. The LPO increased while CAT, SOD and GSH increased significantly. The effects were reversed in lead exposed rats and treated with aqueous extract of Psidium guajava leaves. The present study also revealed the deleterious effects of lead acetate on haematological indices of rats exposed to lead acetate were ameliorated when treated with aqueous extract of Psidium guajava leaves.
The study has shown that aqueous extract of Psidium guajava leaves ameliorated the changes induced by lead acetate in oxidative stress markers and haematological indices in adult Wistar rats. Thus, aqueous extract of Psidium guajava leaves may be useful therapy in lead exposed patients, especially in lead poisoning endemic area.

 

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