International Journal of Trend in Scientific Research and Development (IJTSRD)

Volume 5 Issue 5, July-August 2021 Available Online: www.ijtsrd.com e-ISSN: 2456 – 6470

Hepato-Protective Assessment of Pawpaw Leaves, Neem, Lemon

Grass and Acts on Plasmodium Berghei Parasitized Wistar Rats
Nnyaha Anthonia E., Igbokwe Ugochukwu V., Okonkwo Onyeka Chukwudi,
Ajeka Prisca O., Nwaissac Ikechukwu S., Okpa Precious N.
Department of Human Physiology, Faculty of Basic Medical Sciences, Nnamdi Azikiwe University, Nnewi, Nigeria

ABSTRACT KEYWORDS: Hepato-protective,

Malaria is a major concern in Nigeria, and stands as the second AzadirachtaIndica, Carica papaya
leading cause of death from all infectious disease in Africa. Several Linn, CymbopogonCitratus, ethanol
studies have reported the damaging effect of the parasite to various extract, plasmodium berghei, ACTs
body organs especially the liver. Reports over time has shown the
How to cite this paper: Nnyaha
benefits of various plants extracts in ethno-medicine. However, not Anthonia E. | Igbokwe Ugochukwu V. |
much have been done on the effects of some of these extracts in Okonkwo Onyeka Chukwudi | Ajeka
combined form on its hepato-protective assessment in comparison Prisca O. | Nwaissac
with any known ACT based anti-malaria. The focus of this study was Ikechukwu S. |
to explore the hepato-protective properties of ethanoic extract of Okpa Precious N.
Carica papaya Linn, AzadirachtaIndica, CymbopogonCitratusagainst "Hepato-Protective
ACT based antimalarial therapy on plasmodium berghei parasitized Assessment of
wistar rats. Phytochemical analysis of the extracts were done Pawpaw Leaves,
according to the method described by Treaseand Evans. Hepato- Neem, Lemon Grass IJTSRD46292
protective assessment were done using the liver function tests and and Acts on
Plasmodium Berghei Parasitized Wistar
assay of the liver histology respectively. One hundred and ten (110)
Rats" Published in International Journal
rats distributed into 11 groups, each group having 10rats were used of Trend in Scientific Research and
for the experiment. Negative control received just feed and water, Development (ijtsrd), ISSN: 2456-6470,
Positive control were induced with the malaria parasite and given Volume-5 | Issue-5, August 2021,
feed and water only. The tests groups were induced with malaria, pp.2024-2030, URL:
received feed and water and treated with 500mg/kg, 250mg/kg and www.ijtsrd.com/pap
165mg/kg doses of the extracts, both individually and in combined ers/ijtsrd46292.pdf
forms, as well as the standard ACT anti-malaria. Phytochemical
screening showed that the plant extracts possessed high concentration Copyright © 2021 by author (s) and
of Tannins, Flavonoids, Saponins and Alkaloids. Plasmodium International Journal of Trend in
berghei increased the activities of ALP, ASP and ALT when Scientific Research and Development
Journal. This is an Open Access article
compared with the positive control group. This may be attributed to
distributed under the terms of the
increase in functional capacity of the liver as a result of the presence Creative Commons Attribution License
of the infection for the tests groups. Treatment with the plant extracts (CC BY 4.0)
decreased ALP and ALT levels significantly (P<0.05), as well as (http://creativecommons.org/licenses/by/4.0)
AST levels except for the Neem extract. Histological examination of
the liver of test animals showed no extensive damage to the tissue by
the individual extracts when compared to the negative control group.

INTRODUCTION: holo-endemic (Salako et al., 1994) with more than

Malaria is a mosquito-borne disease predominate in 75% of children aged 2-9 years infected. Malaria is
the tropics and caused by a plasmodium parasite. It is becoming more difficult to manage particularly in
a major concern in Nigeria, and stands as the second areas of multi-drug resistance.
leading cause of death from all infectious disease in Various orthodox pharmacological options has been
Africa. Malaria is one of the major killer diseases in used over time in tackling this disease. They include
Africa causing more than 1 million deaths every year. drugs like Chloroquine, Quinine, andArtemisinin
In Nigeria, the infection rate has been described as derivatives like Artesunate, Artemether, Arteether as

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 International Journal of Trend in Scientific Research and Development @ www.ijtsrd.com eISSN: 2456-6470
well as others. One major problem that arise from the dried in the oven at 60oC for a period of 45 hours.
use of these drugs has been issues of resistance, and Leaves were further dried at ambient temperature for
the fact that most anti-malaria drugs are not a period of two weeks, and then grounded. 250g each
affordable by People. This necessitates the shift to of the powdered leaves were dissolved separately in
self-medication using medicinal plants (Arese, 2001; 1000ml of 98% ethanol and allowed for 48hours at
Muregi et al., 2003). Herbs had been used by all room temperature after which it was sieved using
cultures throughout history. Plants have always been porcelain clothe. It was filtered further using a filter
a component of mankind’s healthcare system. This is paper No 1. The filtrate was concentrated using
either directly or indirectly. Directly, the plant parts digital rotary evaporator (TT-52 technel and technel
like leaves, fruits, stem, bark, roots etc. or even the USA) and was dried using thermostat oven (DHG-
whole plant are themselves used in the treatment of 9023A PEC medicals USA) into a gel like substance
illnesses. They are the first line treatment for many of and stored individually in a refrigerator (NEXUS).
the world’s population, being readily available,
LETHALITHY (LD50) TEST: The mean lethal
traditional and relatively inexpensive (Olaniyi, 1998;
dose LD50 of the extracts was determined using
Okpara et al., 2007).
Lorkes method as described in (1983) and modified
A number of traditional herbs have been tested and by Imafidon et al, (2015). A total of 17 wistar rats
used in the prevention and also treatment of malaria were used (9 in phase one and 8 in phase two). The
including Artemisia annua (Akininyi et al, 1986), old extracts were administered via oral routes and were
leaves of Carica papaya, roots and leaves of closely monitored for 24 hours for physical signs of
Guinensis, unripe fruit of Capsicum frutescence and toxicity such as gasping, palpitation, sluggishness etc.
Azadirachtaindica popularly called Dangoyaro in
PHASE 1: 3 groups with 3 rats each
Nigeria. There is high reliance on traditional medicine
in the treatment of malaria, most of which are Group one received 10mg/kg of the extract.
prepared from plants and available at affordable Group two received 100mg/kg of the extract.
prices (Alves and Rosa, 2007). Ethnobotanical studies
(Odugbemi et al., 2007; Ajibesin et al., 2008; Titanji Group three received 1000mg/kg of the extract.
et al., 2008) have been carried out on medicinal PHASE 2: 4 groups with 2 rats each..
plants useful in treating malaria in Africa, Nigeria
inclusive. Group one received 750mg/kg of the extract

Mature leaves of Carica papaya (Caricaceae; Group two received 1500mg/kg of the extract
Common name: pawpaw) is widely used to treat Group three received 3000mg/kg of the extract
malaria and splenomegaly (Adjanohoun JE,
Group four received 6000mg/kg of the extract.
Aboubakar N, Dramane K, Ebot ME, Ekpere JA,
Enoworock EG, et al 1996). The essential oils of PREPARATION OF EXTRACT AND DOSE OF
Cymbopogoncitratus were found to produced 86.6% STANDARD DRUG:
suppression in growth of Plasmodium berghei when Average weight of animals (Kg) x Dose (mg/ml)
compare to a standard drug chloroquine Stock solution (mg/ml)
(Tchoumbougnang, F; Zollo 2005) Numerous EXPERIMENTAL ANIMALS AND
biological and pharmacological activities have been TREATMENTS: A hundred and ten (110) male rats
reported of AzadirachtaIndica including antibacterial, between 100g-250g in weight were used for this
antifungal and anti-inflammatory activities(A. Kher et study. They were obtained from the animal house at
al,1997). the college of medicine, university of Nigeria and
There is inadequate information in literature on the kept in well aerated laboratory cages in the Human
hepato-protective activities associated with the physiology department animal house Nnewi, with
ethanol extract of Carica papaya Linn, dark and light cycles of 12hrs each observed, fed with
CymbopogonCitratus and AzadirachtaIndica. This water and rat feed (from vital feeds company).The
study was designed to explore the hepato-protective rats acclimatized for a period of two weeks prior to
abilities of these various plant extracts against ACTs the commencement of the study, and were handled in
treated plasmodium berghei parasitized wistar rats. adherence to the guidelines and recommendation of
the ethics committee on the use of animals for
MATERIALS AND METHODS
research of NnamdiAzikiwe University Awka,
Plant Materials and Preparation of Extracts:Fresh
Anambra, Nigeria. They were distributed into 11
leaves of pawpaw, neem and lemon grass were
groups of 10 rats each.
collected, identified (in the pharmacology department
of NnamdiAzikiwe University, Agulu), washed and Group 1 (Positive control): Feed and water only.

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Group 2 (Negative control): Feed, water and 0.2ml PHYTOCHEMICAL SCREENING: The ethanoic
malaria parasite via intraperitoneal route. extracts of the plants were subjected to preliminary
Group 3 (Lemon grass extract): Feed, water, 0.2ml phytochemical analysis by methods described by
malaria parasite and 500mg/kg lemon grass extract. Trease and Evans (1983) to test for alkaloids,
flavonoids,and glycosides, reducing sugars, steroids,
Group 4 (Pawpaw leaf extract): Feed, water, 0.2ml saponins and tannins.
malaria parasite and 500mg/kg pawpaw leaf extract.
LIVER ENZYMES ASSAY:The activities of
Group 5 (Neem extract): Feed, water, 0.2ml malaria Alkaline phosphatase (ALP), Aspartate amino
parasite and 500mg/kg neem extract. transferase(AST) and Alanine aminotransferase
Group 6 (Neem and pawpaw leaf extract): Feed, (ALP) were evaluated using the method as described
water, 0.2ml malaria parasite and 250mg/kg of neem by WHO (2004).
and pawpaw leaf extract each. LIVER SACRIFICE AND HISTOLOGY: Liver
Group 7 (Neem and Lemon grass extract): Feed, tissue were harvested from test animals seven days
water, 0.2ml malaria parasite and 250mg/kg of neem after treatment. Tissue sections were brought to
and lemon grass extract each. distilled water, stained with Erhlich’shaematoxylin
for 30 minutes and rinsed in running water. After
Group 8 (Neem, Pawpaw leaf and Lemon grass drying out, the tissue were differentiated with 1%
extract): Feed, water, 0.2ml malaria parasite and acid alcohol until only nuclei were stained.
165mg/kg of neem, pawpaw leaf and lemon grass Thereafter, it was rinsed in running water and ‘blued’
extract each. in scott’s tap water substitute for 3minutes. The
Group 9 (Pawpaw leaf and lemon grass extract): sections were further rinsed in tap water, stained with
Feed, water, 0.2ml malaria parasite and 250mg/kg of Eosin for 2 minutes, dehydrated, cleared and viewed
pawpaw leaf and lemon grass extract each. under a microscope.
Group 10 (Artemether/Lumefantrine): Feed, water. DATA ANALYSIS: Results obtained were
0.2ml malaria parasite and 4mg/kg expressed as mean ± SEM as described by (Duncan et
Artemether/lumefantrine. al., 1997). The data were statistically analyzed using
one-way analysis of variance (ANOVA) with
Group 11 (Dihydoartemisinine/piperaquine Turkey’s multiple comparison post hoc test to
phosphate): Feed, water, 0.2ml malaria parasite and compare the level of significance between the test
4mg/kg dihydroartemisinine/piperaquine phosphate. groups. The values of p<0.05 were considered as
All treatments were done for 7days with the extracts significant for differences in means.
and ACT administered orally.
RESULTS:
Table 1: Phytochemical analysis of the ethanoic extract of pawpaw leaf, lemon grass and neem
PHYTOCHEMICALS LEMON GRASS PAWPAW LEAF NEEM
Tannins + + +
Saponins + + +
Flavonoids + + +
Glycosides - + -
Alkaloids + + +
Steroids + - +
Key: + shows presence of phytochemicals, - Shows absence of phytochemicals
Table 2: ACUTE LETHAL DOSE OF PLANT EXTRACTS.
DOSE NEEM LEMON GRASS PAWPAW OBSERVATION
10mg/kg 0/3 0/3 0/3 0/3 No death
100mg/kg 0/3 0/3 0/3 0/3 No death
1000mg/kg 0/3 0/3 0/3 0/3 No death
750mg/kg 0/2 0/2 0/2 No death
1500mg/kg 0/2 0/2 0/2 No death
3000mg/kg 0/2 0/2 0/2 No death
6000mg/kg ½ ½ 0/2 Two deaths
LD50 =√A×B

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A = Maximum dose with 0% mortality
B = Minimum dose with 100% mortality.
Table 3: Comparison of the various enzymes levels between the negative control group and the test
groups after 7days of treatment with the various plant extracts and ACT combined malaria therapy.
EXPERINMENT AST ALT ALP P-
P-VALUE P-VALUE
GROUP Mean±SEM Mean±SEM Mean±SEM VALUE
GROUP 2 172.00±0.95 42.80±0.86 452.80±2.24
GROUP 3 (LG) 94.20±0.80 0.000* 28.80±0.49 0.000* 194.80±0.37 0.000*
GROUP 4 (PL) 141.6±7.10 0.000* 26.80±2.31 0.000* 204.40±1.12 0.000*
GROUP 5 (N) 173.20±1.62 0.850 24.20±1.28 0.000* 208.40±1.89 0.000*
GROUP 6 (N/PL) 160.80±9.72 0.082 23.40±0.93 0.000* 178.00±8.04 0.000*
GROUP 7 (N/LG)) 127.00±2.17 0.000* 21.20±0.86 0.000* 167.40±2.38 0.000*
GROUP 8 (N/LG/PL) 124.80±5.63 0.000* 26.80±0.86 0.000* 157.20±1.39 0.000*
GROUP 9(PL/LG) 115.00±5.30 0.000* 24.40±0.81 0.000* 145.80±6.36 0.000*
GROUP 10(ATL) 129.60±1.44 0.000* 25.20±1.02 0.000* 156.00±5.63 0.000*
GTROUP 11(DAP) 137.20±0.86 0.000* 29.80±1.71 0.000* 160.80±2.94 0.00*

* The values of p<0.05 is considered as significant for differences in means when compared with group 2. LG=
Lemon grass, PL= Pawpaw leaf, N= Neem, ATL= artemetger/lumefantrine, DAP=
Dihydroartemisine/piperaquine phosphate.
From the table above, it is observed that the test groups showed significant decrease in the ALT and ALP levels
when compared with the negative control group.There was also significant decrease in the AST level of all the
test groups when compared with the negative control group except group 5
Table 4: Comparison of the various enzymes levels between negative control group and the test
groups 14 days after treatment with the various plant extracts and ACT combined malaria therapy.
AST ALT ALP
EXPERIMENT GROUP P-VALUE P-VALUE P-VALUE
Mean±SEM Mean±SEM Mean±SEM
GROUP 2 100.00±6.32 68.80±1.16 465.20±4.53
GROUP 3 (LG) 93.60±0.51 0.000 19.80±1.59 0.000 152.00±2.43 0.000
GROUP 4 (PL) 58.80±0.37 0.000 15.60±0.24 0.000 138.00±0.55 0.000
GROUP 5 (N) 58.00±2.43 0.850 15.40±0.93 0.000 132.80±1.93 0.000
GROUP 6 (N/PL) 71.40±1.69 0.082 19.80±0.86 0.000 142.60±2.56 0.000
GROUP 7 (N/LG) 67.60±3.19 0.000 18.80±1.07 0.000 143.80±1.69 0.000
GROUP 8 (N/LG/PL) 76.40±3.88 0.000 19.80±0.86 0.000 142.60±2.29 0.000
GROUP 9 (PL/LG) 60.40±1.33 0.000 18.40±0.68 0.000 132.00±1.55 0.000
GROUP 10 (ATL) 55.60±0.51 0.000 22.60±0.51 0.000 148.20±2.62 0.000
GROUP 11 (DAP) 58.40±0.68 0.000 24.60±0.87 0.000 152.80±0.97 0.000
* The values of p<0.05 is considered as significant for differences in means when compared with group 2. LG=
Lemon grass, PL= Pawpaw leaf, N= Neem, ATL= artemetger/lumefantrine, DAP=
Dihydroartemisine/piperaquine phosphate
All the test groups showed significant decrease in the AST, ALT and ALP levels except the AST level of groups
5 and 6 which showed no significant difference when compared with group 2.
The histological assessment of the liver tissue gave an in-depth information on the protective activities of liver
enzymes in ensuring organ recovery and maintaining the functionality of the liver. There were no visible lesions
in the positive control group as well as the negative control, lemon grass, pawpaw leaf, neem, neem and lemon
grass and dihydroartemisinine/piperaquine groups respectively and their hepatocytes were uncompromised. The
group treated with neem and pawpaw leaf (6) showed an area of mild lymphocytic infiltration. The group with all
three extracts (8) andthe artemether/lumefantrine group (10) showed moderately hypertrophied central vein with
mild fluid exudation. Group 9 (pawpaw leaf and lemon grass) showed early signs of lipid infiltration on the liver
tissue.

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Group 3: Photomicrograph of liver tissue

showing morphology consistent with healthy
liver histology. (H&E, X100). Group 7: Photomicrograph of liver tissue
showing morphology consistent with healthy
liver histology. (H&E, X100).

Group 4: Photomicrograph of liver tissue

showing morphology consistent with healthy
liver histology. (H&E, X100) Group 8: Photomicrograph of liver tissue
showing moderately hypertrophied central vein
with mild fluid exudation and normal
hepatocytes (H&E, X100)

Group 5: Photomicrograph of liver tissue

showing morphology consistent with healthy
liver histology. (H&E, X100) Group 9: Photomicrograph of liver tissue
showing normal liver cells but with early sign of
lipid infiltration (H&E, X100).

Group 6: Photomicrograph of liver tissue

showing normal hepatocytes, with a focal area of
mild lymphocytic infiltration (H&E, X100). Group 10: Photomicrograph of liver tissue
showing moderately hypertrophied central vein
with mild fluid exudation and normal
hepatocytes (H&E, X100).

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test groups showed significant increase in AST and
ALP level when compared to the control group, this
indicates that fourteen days after treatment the effect
of the various extract is still evident on the
experimental rats. The non-significant difference
between the ARTHEMETER+LUMENFANTRINE
and DIHYDROARTEMISINE+ PIPERAQUINE
group might be indicating that the effects of the both
drugs elapsed at the same time.
Group 11: Photomicrograph of liver tissue
showing morphology consistent with healthy Fourteen days after administration significant
liver histology. The sinusoid and hepatocytes are increase occurred in the AST level of the group that
normal with no obvious sign of injury (H&E, was treated with NEEM+PAWPAW LEAF,NEEM
X100). and NEEM+LEMON GRASS+PAWPAW LEAF
extracts when compared with the group that was
DISCUSSION treated with ARTHEMETER+LUMENFANTRINE,
The enhanced activities of these serum marker this is suggesting that the detoxification activities of
enzymes observed in all the test groups in the present ARTHEMETER+LUMENFANTRINE to the liver
study correspond to the extensive liver damage stops before that of the plants.
induced by the parasite. The present study observed
that the ALT levels in all the test groups increase High levels of ALP in serum indicate liver damage
significantly except in the group treated with which is similar to this present study where, the
neem+lemon grass. The ALP levels of the groups that increase in ALP activity in rats treated with LEMON
was treated with NEEM+LEMON GRASS, GRASS extract and DIHYDROARTEMISINE+
NEEM+LEMON GRASS+PAWPAW LEAF and PIPERAQUINE when compared with the negative
PAWPAW LEAF+LEMON GRASS respectively control group, shows the liver damage, as a result of
increased when compared to the single extract group. metabolic changes such as administration of toxin,
This observation indicates that combination of the liver cirrhosis, hepatitis, and cancer of the liver
various extracts were not as toxic to the liver than the (Trichopoulos and Willett, 1996). Thus, it can be used
individual extracts, this suggests that in the treatment as markers to estimate the extent of liver damage by
of malaria it is better to use the plants combination the parasite.
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