Evaluation of haematological changes in Entamoeba histolytica-infected rats administered with extracts of Ziziphus mauritiana leaves

 

Mansour Abdulnabi H. Mehdi^1*, Fadel Y. S. Alarabi¹, Gozif Mohammed N. Omar²,

Khaled Nasher Q. Salem³, Vidya Pradhan¹

¹Department of Zoology, Dr. Rafiq Zakaria College for Women, Aurangabad, 431001 India.

²Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431001, India.

³Department of Biology, Faculty of Science, Aden University-Yemen.

 

ABSTRACT:

This study was designed to evaluate the changes in some heamatological parameters of Entamoeba histolytica-infected rats treated with ethanolic and aqueous extracts of Ziziphus mauritiana leaves. The ethanolic and aqueous extracts of the leave at 500mg/kg-1 body weight/day, while a standard antiamoebic drug, metronidazole, at a dose of 500mg/kg-1 body weight was administered on the positive control group for 10 days. The negative control group was uninfected and untreated. The haematological parametrats in the different groups were monitored throughout the period of study which was 10 days on three stages. The results show a significant increase at P≤0.05 in red blood cell count (RBC), haemoglobin (Hb), hematocrit (HCT), mean cell volume (MCV), red cell distribution width (RDW), procalcitonin test (PCT) and total white blood cell (WBC) in groups which treated with Z. mauritiana extracts compare with metronidazole drug and the control group during treatment stages. While the results show a significant decrease at P≤0.05 in platelet (PLT),  mean platelet volume (MPV), platelet distribution width (PDW), monocyte (MO) and granulocytes (GR) when compared to the control groups of rats. The results also indicate no significant changes at P>0.05 in mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and count lymphocyte (LY) during treatment stages. Conclusion: The findings of this study, that the efficiency of Z. mauritiana extracts in improving blood standards through variations occurring in blood proportion and not adversely affect the haematological parameters.

 

 

INTRODUCTION:

Entamoeba histolytica affects millions around the world. It is the second leading cause of death in humans after malaria^1,2. Despite decades of research, amoebiasis infection remains a major global health problem with high mortality and morbidity than any other infectious disease where the World Health Organization (WHO) estimates that the E. histolytica parasite causes approximately 50 million cases of dysentery and 100,000 deaths per year³.

 

Despite decades of research, metronidazole remains a therapeutic drug of choice for the treatment of amoebiasis⁴, but is experiencing drug resistance by E. histolytica, so that resulting in the need for increased doses to overcome the infection. In addition, this drug has several untoward side effects such as headaches, metallic taste in the mouth and vomiting as well as neurotoxicity^5,6. The resistance of amoebiasis to drugs such as metronidazole has become a serious problem in developing countries⁷. Medicinal plants are popular for use especially in developing countries because their products are safe and widely available at low cost. There is also a need to provide reliable scientific data to determine whether the plants currently used to treat amoebiasis are actually effective and have no adverse effects on the system. This plant is found in most the countries including drought prone regions and has been widely investigated for its medicinal properties without any adverse effects. Z. mauritiana is a very rich source of alkaloids, ascorbic acid and other phytochemicals⁸.  

 

The study aimed to investigate the effect of Z. mauritiana leaves extracts on haematological changes in rats infected by E. histolytica parasite.

 

MATERIALS AND METHODS:

Collection of Z. mauritiana leaves:

The Z. mauritiana leaves were collected at the Aurangabad from the Himayat Bagh area-India. The leaves were washed and dried in temperature room. The dried leaves were pulverized into a fine powder using an electric grinder and stored in air tight bottles until required.

 

Preparation of plant extract:

From the dried powdered sample of Z. mauritiana leaves, 40g was weighed and dissolved in 400ml distilled water in beaker by mixing using a magnetic stirrer for 24 h. The mixture was filtrated by four layers of gauze cloth. The filtrate was centrifuged at 3000rpm for 10 minutes. The supernatant was collected and refiltered through Whatman No. 1 filter paper. After that, it was transferred to an incubator for 24 h at 50°C^6,9.

 

Experimental animals:

Twelve white albino rats (Rattus norvegicus) weighing between 200g and 220g (3-3.5 months old) were used for the study. The rats were acclimatized for two weeks to laboratory conditions and were fed food and water ad libitum. Nine rats were infected by oral administration (17×10³cell/ml) of E. histolytica obtained from the stool. Infected rats were divided into three groups in additional to, control group. Each per group containing three rats.

 

Group 1: The control group uninfected and untreated.

Group 2: Infected and administered with metronidazole (500mg/kg body weight).

Group 3: Infected and administered with ethanolic extract (500mg/kg body weight).

Group 4: Infected and administered with aqueous extract (500mg/kg body weight).

 

Collection of blood sample:

Blood was collected from all rats in this study on three stages: pre-treatment, mid-treatment and post-treatment in each group from the vein at region next to the eye using capillary tubes. Then the blood was put in sterile vials (1.0–2.0mL) containing EDTA which were used as anticoagulant for blood. After that blood was tested for complete blood count (CBC).

 

Heamatological analysis:

Heamatological analysis was carried out using an Automatic Haematology Analyser (CBC Mindray BC-3000Plus) as described by the manufacturer.

 

Statistical analysis:

The results of the present study were analyzed by GenStat 5.2 using general treatment structure (no blocking), factorial experiment, with 3 replications. Least significant different test (LSD) was used to test the difference between means (groups) at P≤0.05 and was considered significant.

 

RESULTS:

Effects of extracts of Z. mauritiana leaves on erythrocytic parameter profiles in albino rats infected with E. histolytica parasite:

The heamatological profile of rats treated with ethanolic and aqueous leaves extracts of Z. mauritiana a significant decrease in RBC, Hb, HCT, MCV, RDW and MCH decreased significantly (P≤0.05) in rats infected with E. histolytica parasite (Pre-treatment stage) in comparison with negative control group (Table 1), also, in the same table shown rats infected with E. histolytica a significant increase (P ≤0.05) in MCHC compared with negative control (Pre-treatment stage).

 

After five days of administration with extracts of Z. mauritiana (Mid-treatment stage) a significant increase was observed (P≤0.05) in RBC, Hb, HCT and RDW in groups which were treated by ethanolic and aqueous extracts of Z. mauritiana in comparison with pre-treatment stage and with negative control and metronidazole group. While the values of MCV, MCH and MCHC showed a slight increase after five days of treatment but non-significant (P>0.05) in comparison with pre-treatment stage.

 

After ten days (Post-treatment stage) of administration the results showed a significant increase in RBC, Hb, HCT, MCV and RDW in groups which were treated by ethanolic and aqueous extracts of Z. mauritiona when compared with metronidazole group. Also, the results showed that MCH and MCHC no significant differences (P>0.05) when compared with metronidazole group during the treatment period.

 

Table 1: Effects of ethanolic and aqueous leaves extracts of Z. mauritiana on erythrocytic parameter profiles in albino rats infected with E. histolytica parasite.

LSD: Least significant differences, Red blood cell (RBC), Hemoglobin (Hb), Haematocrit (HCT), Mean cell volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin (MCHC), Red cell distribution width (RDW).

 

Table 2: Effects of ethanolic and aqueous leaves extracts of Z. mauritiana on platelets and their related parameter profiles in albino rats infected with E. histolytica parasite.

LSD: Least significant differences, Platelet (PLT), Procalcitonin test (PCT), Mean platelet volume (MPV), platelet distribution width (PDW)

 

Effects of extracts of Z. mauritiana leaves on platelets and their related parameter profilesin albino rats infected with E. histolytica parasite:

The results have shown changes in platelets and their related parameters in rats infected with E. histolytica parasite (Table 2) where shown the results in the Pre-Treatment stage that PLT, PCT and PDW increased significantly (P≤0.05) compared with negative control group. On the other hand, the results showed that MPV no significant different (P>0.05) compared with negative control group. 

 

After five days (Mid-treatment stage) of administration of the ethanolic and aqueous leaves extracts of Z. mauritiana caused changes in platelets and their related parameters in rats infected with E. histolytica parasite (Table 3). Where the dose of 500mg/kg caused a significant decrease in MPV, PCT and PDW in groups which treated with extracts when compared with and pre-treatment stage.

 

After ten days (Post-treatment stage) of administration the results showed a significant decrease in PLT, PCT, MPV and PDW in groups which were treated by ethanolic and aqueous extracts of Z. mauritiana when compared with negative control group and metronidazole group.

 

Effects of extracts of Z. mauritiana leaves on total WBC and differential WBC counts in albino rats infected with E. histolytica parasite:

The total white blood cells (WBC) and differential leucocytic counts values are presented in (Table 3). The results showed significant increase (P≤0.05) in WBC and MO in rats infected with E. histolytica parasite (Pre-treatment stage) in comparison with negative control group (Table 3). On the other hand, the levels of GR decreased in rats infected with E. histolytica parasite. While the values of LY PLT showed a slight increase but non-significant (P>0.05) in comparison with negative control.

 

After five days (Mid-treatment stage) of administration of the extracts of Z. mauritiana a significant decrease was observed in WBC and GR in all groups treated with extracts when compared with pre-treatment stage but observed that values of mixed cells GR non-significant (P>0.05) in comparison with metronidazole group. While the MO showed increase in compared with pre-treatment stage but observed that value LY non-significant different (P>0.05) when with pre-treatment.

 

In the post-treatment, the results showed significant increase (P≤0.05) in WBC, MO and LY in all groups treated compared with metronidazole group but the GR was showing significant decrease (P≤0.05) compared with metronidazole group but non-significant when compared with negative control group.

 

Table 3: Effects of ethanolic and aqueous leaves extracts of Z. mauritiana on white blood cells and their differentials parameter profiles in albino rats infected with E. histolytica parasite.

Parameters	Type-Treatment	Pre-Treatment	Mid-Treatment	Post-Treatment	Means	LSD 5%
WBC (103)	Control	8.07	7.27	9.20	8.18	0.546
	Metronidazole	10.57	10.67	11.90	11.05
	Ethanolic extract	14.10	13.83	14.43	14.12
	Aqueous extract	13.77	12.23	15.90	13.97
Means		11.63	11.00	12.86	11.83
LY (%)	Control	93.07	97.00	95.77	95.28	0.601
	Metronidazole	88.97	89.93	92.00	90.30
	Ethanolic extract	93.30	92.40	93.93	93.21
	Aqueous extract	93.60	92.40	94.87	93.62
Means		92.24	92.93	94.14	93.10
MO (%)	Control	3.97	2.00	3.30	3.09	0.531
	Metronidazole	8.40	7.97	5.80	7.39
	Ethanolic extract	5.07	5.73	4.67	5.16
	Aqueous extract	5.00	5.57	4.03	4.87
Means		5.61	5.32	4.45	5.13
GR (%)	Control	1.80	1.00	1.10	1.30	0.3272
	Metronidazole	2.63	2.10	2.20	2.31
	Ethanolicn extract	1.63	2.03	1.40	1.69
	Aqueous extract	1.40	2.03	1.10	1.51
Means		1.87	1.79	1.45	1.70

LSD: Least significant differences, Total white blood cell (WBC), Lymphocyte (LY), Monocyte (MO), Granulocytes (GR).

 

DISCUSSION:

Infection with E. histolytica and nutritional deficiencies caused by them is a major health problem in many tropical and subtropical areas of the world, especially in developing countries¹⁰. Heamatological parameters are very important in useful indices that can be employed to assess the treatment potentials of plant extracts for treatment of many diseases¹¹, where that examination of heamatological parameters to be reliable in disease diagnosis, prevention and treatment¹². So this study was aimed to evaluate the effect of aqueous and alcoholic extracts of Z. mauritiana leaves on heamatological changes in E. histolytica-infected rats at different stages have been assessed.

 

The present results have revealed a significant decrease haematological parameter such as RBC, Hb, HCT, MCV, RDW and MCH in rats infected with E. histolytica parasite. This can be due to lowered erythropoesis and its usually associated with mild-acute enteritis which hampers the absorption of essential nutritis for blood cell formation also may be due to the ingestion of red blood cells by E. histolytica this agree with report Hussein and Shaker¹³ who stated that E. histolytica causes decreased in RBC, Hb, PCV and MCV. Also, that E. histolytica causes digestive disturbance, which affects Hb, MCV and MCH values which could be an indication of the presence of anaemia caused by the body's inability to absorb iron which can be caused by digestive disturbance due to parasite E. histolytica. This result agrees to that found by Mehdi, et al.,¹² who stated that E. histolytica infection in the rats it has an effect on Hb, MCV and MCH values. In addition, the MCHC values were higher in rats infected with E. histolytica when compared with negative control. This increase may be occur when RBC are fragile or destroyed, also, maybe the reason why the rate of RBC production leads to the release of immature RBC into blood circulation, which may cause an increase in MCHC values. This agrees with Kotepui, et al.,¹⁴.

 

The present study showed that extracts of Z. mauritiana demonstrated of changes in erythrocytic parameter profiles in albino rats infected with E. histolytica parasite at the dose of 500mg/kgbw. The significant increase in RBC, Hb, HCT, MCV and RDW after oral administration of extracts of Z. mauritiana suggests that the extracts may contain phytochemicals and compounds that stimulate the secretion or formation of erythropoietin in the stem cells of infected rats. The increased in RBC caused to increase in HCT value. This result agrees to that found by Kabiru, et al.,¹⁵ which found that crude extracts of Phyllantus amarus have effects on HCT in Plasmodium-berghei-infected mice. Moreover, the active components found in plant extracts might not be sufficiently concentrated to significantly effect on MCH and MCHC values as was observed in this study.

 

In the present study, effects of extracts of Z. mauritiana on platelets and their related parameter profiles in rats infected with E. histolytica parasite were showed significant decrease in all platelets and their related parameter profiles during stages of treatment.

 

In the present study, platelets and their related parameter profiles in rats infected with E. histolytica parasite were showed significant increase in PLT counts in pre-treatment stage. The increase in PLT might be due to haemolytic anaemia. The finding of high platelet counts in E. histolytica-infected rats is consistent with earlier studies^16,17.

 

During of treatment stages were observed significant decrease in platelets and their related parameter profiles in rats infected with E. histolytica parasite until it reached within the expected range signifies where that this plant understudy could importantly influence the replenishment of lost blood thereby curbing anaemia that may be caused by  E. histolytica parasite. This result agrees with that found by Mwale et al.,¹⁸  who stated that plants under study increase platelets count in village chickens naturally infected with Heterakis gallinarum.

 

In the present study, the WBC, MO and GR in E. histolytica-infected rats were showed significant increase as compared with control group in pretreatment stage. While in the same stage was observed no significant change in LY. The increase in WBC might be due to the increase in GR because they reform first immune defense against the infection¹⁹. This result agrees with the result found by Mehdi et al.,¹² who observed an increase in WBC, MO and GR in E. histolytica-infected rats.

In rats treated by Z. mauritiana extracts a significant increase was observed in WBC and GR compared with control group, this increase continued during the treatment stages. This increased the in WBC count caused by extracts of Z. mauritiana maybe lead possible immune modulatory effects of the extract which augmented the production of more WBC²⁰. This will increase the body capability of generating antibodies in the process of phagocytosis and have high degree of resistance to diseases²¹. This result agrees with that found by Mehdi et al.,¹² who stated that increases the differential white blood cell WBC count in E. histolytica-infected rats which treated by T. indica extracts.

 

CONCLUSION:

It can be concluded from the results obtained in this study that the Z. mauritiana extracts do not adversely affect the hematological parameters determined when used to treat E. histolytica-infected rats, but have some beneficial potential in improving blood standards through variations occurring in blood proportion.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr. Mazahar Farooqui Principal of Maulana Azad College of Arts, Science and Commerce for his constant support and cooperation.

 

ETHICS APPROVAL:

Institutional guidelines for the care and use of animals were followed. All procedures performed in the study involving animals were in accordance with the ethical standards of the institution or practice at which the study was conducted date 16/08/2018.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

 

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Accepted on 23.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4105-4110.