# In Vivo Antimalarial Activity of the Solvent Fractions of Fruit Rind and Root ofCarica papaya Linn (Caricaceae) againstPlasmodium berghei in Mice

Authors: Gemechu Zeleke; Dereje Kebebe; Eshetu Mulisa; Fanta Gashe
Journal: Journal of Parasitology Research
 (2017)

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## Abstract

Background. Currently, antimalarial drug resistance poses a serious challenge. This stresses the need for newer antimalarial compounds.Carica papayais used traditionally and showedin vitroantimalarial activity. This study attempted to evaluatein vivoantimalarial activity ofC. papayain mice.Methods.In vivoantimalarial activity of solvent fractions of the plant was carried out against earlyP. bergheiinfection in mice. Parasitemia, temperature, PCV, and body weight of mice were recorded. Windows SPSS version 16 (one-way ANOVA followed by Tukey’s post hoc test) was used for data analysis.Results. The pet ether and chloroform fractions ofC. papayafruit rind and root produced a significant (p<0.001) chemosuppressive effect. A maximum parasite suppression of 61.78% was produced by pet ether fraction ofC. papayafruit rind in the highest dose (400 mg/kg/day). Only 400 mg/kg/day dose of chloroform fraction ofC. papayaroot exhibited a parasite suppression effect (48.11%). But, methanol fraction of the plant parts produced less chemosuppressive effect.Conclusion. Pet ether fraction ofC. papayafruit rind had the highest antimalarial activity and could be a potential source of lead compound. Further study should be done to show the chemical and metabolomic profile of active ingredients.


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## Body

## 1. Introduction

Even though a remarkable progress has been made, malaria remains a major health problem in Sub-Saharan Africa. It is endemic in 91 countries. The number of people infected with malaria in Sub-Saharan Africa is estimated to be 114 million in 2015. Children are especially vulnerable, accounting for more than two-thirds of global malaria deaths [1]. Malaria is also the major public health problem in Ethiopia. In Ethiopia, more than 75% of people live in malaria endemic areas, putting over 50 million people at risk of malaria. According to Federal Ministry of Health of Ethiopia 2015 report, about 1,165,843 cases of malaria were reported. In addition, it is the cause for 22,784 patients’ hospital admissions [1, 2].However, safe and effective mode of treatment is needed to control malaria and its complications. The increasing antimalarial drug resistance, insecticide resistance, and behavioral changes in Anopheles vectors threaten effective antimalarial drug therapy and malaria control and elimination [3]. Artemisinin combination therapies (ACTs) are first-line treatment for uncomplicated falciparum malaria in all endemic countries, yet partial resistance to artemisinin has emerged in the Greater Mekong Subregion [4, 5].The famous and potent antimalarial compounds quinine (obtained fromCinchonaspecies) and artemisinin (obtained fromArtemisia annua) are derived from plants [6]. Medicinal plants have been reported for their significant antimalarial activity and remain the main focus for scientists and researchers in the development of new antimalarial agents. Phytochemical compounds including alkaloids [7], phenolic compounds [8], anthraquinones [9], and flavonoids [10] are commonly implicated for the antimalarial activity of many plants.Carica papaya Linn belonging to family Caricaceae is commonly known as papaya in English, “papayyaa” in local language. Traditionally,Carica papaya leaves, root, and rind are used for treatment of a wide range of ailments including malaria. Many scientific investigations have been conducted to evaluate the biological activities of various parts ofCarica papaya including their fruits, shoots, leaves, rinds, seeds, roots, or latex. The plant possessed significant biological activities such as antioxidant [11], immunomodulatory [12], anti-inflammatory [13], analgesic [14], antitumor [15], wound healing [16], and antimicrobial [17].Previous data showed that ethanol leaf extracts ofCarica papaya exhibited a promising inhibitory activity against the CQ-sensitive strain ofP. falciparum [18]. In addition, the petroleum ether extract of the rind ofC. papayahad the highestin vitro antimalarial activity with IC50 of 15.19 μg/mL [19]. But, weak antiplasmodial activity was exhibited by the leaves and seeds ofC. papaya [20]. There is no previous study showingin vivo antimalarial activity of the plant extracts.Thus, based onin vitro efficacy and traditional claims, this study was aimed at evaluatingin vivo antimalarial activity of the solvent fractions ofCarica papaya fruit rind and root in mice.


## 2. Methods

### 2.1. Collection and Authentication of Plant Materials

The freshCarica papaya fruits and roots were collected from near Jimma town, Southwest Ethiopia, in August 2016. The plant material was authenticated by a taxonomist at the Ethiopian National Herbarium, Addis Ababa University. The specimen was deposited for future reference with voucher number JU-GZ01/2016 at the National Herbarium, College of Natural Sciences, Addis Ababa University.


### 2.2. Preparation Solvent Fractions

The freshCarica papaya Linn fruit rind/peel and roots were air-dried at room temperature under shade and pulverized into powder using pestle and mortar. The solvent fractions ofC. papaya fruit rind and root were obtained by sequential soxhlet extraction with petroleum ether, chloroform, and then methanol in increasing polarity [19]. 40 grams of the powdered plant material was weighed and placed in the extraction thimble of the soxhlet apparatus. Then, about 200 ml of petroleum ether was added to the flask of the soxhlet apparatus set up. Then, the petroleum ether was heated with a temperature not exceeding 40°C to evaporate and condense into plant powder containing thimble. This extraction process was continued exhaustively until clear solution in the thimble was siphoned into the solvent flask. Then, the petroleum ether fraction was filtered with Whatman number 1 filter paper and the solvent was removed by placing in oven adjusted at a temperature less than 40°C. The marc of the petroleum ether based extraction was collected and dried at room temperature to remove petroleum ether. The dried left marc was extracted using absolute chloroform following the same procedure as described for petroleum ether extraction to get the chloroform fraction. Finally, the marc of chloroform fraction was collected and dried at room temperature. Then, the whole dried marc was further extracted with methanol with the same procedure indicated above. Each of the fractions was separately stored in screw capped vials in refrigerator until used for the study.


### 2.3. Experimental Animals

Healthy male Swiss Albino mice (8–12 weeks, weighing 25–33 grams) bred and maintained at Ethiopian Public Health Institute were used. The animals were kept in cages and housed in a standard animal house under natural 12/12 h light dark cycle at room temperature, the animal house of School of Veterinary Medicine, Jimma University. They were maintained on standard pelleted diet and water ad libitum. All mice were acclimatized for one week before the study. This study was approved by the ethical review board of college of health science of Jimma University with a reference number HRPGC/578/2015. All experiments were conducted in accordance with the internationally accepted guidelines on laboratory animal use, care, and handling [21].


### 2.4. Acute Toxicity Tests

The acute toxicity studies were conducted as per the OECD guidelines 425. Acute oral toxicity of each of the solvent fractions was evaluated in healthy female mice aged of 6–8 weeks. Five female mice were fasted for three hours and orally given a dose of 2000 mg/kg of the solvent fractions. The mice were observed for lacrimation, hair erection, behavioral change, reduction in their motor, feeding activities, and mortality for three hours and followed for 24 hours and/or 14 days [22].


### 2.5. Parasite Inoculation

Chloroquine sensitiveP. bergheiANKA strain obtained from Ethiopian Public Health Institute and maintained at animal house facility was used. For the parasite maintenance serial passage of blood from infected mice to noninfected ones was made. A donor mouse with a parasitemia of approximately 30% was sacrificed and blood collected in a Petri-dish containing 2% trisodium citrate as anticoagulant. The blood was then diluted with 0.9% normal saline. Each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of 1 × 107 P. Berghei infected red blood cells [23].


### 2.6.In Vivo Antimalarial Activity Study

In vivoantiplasmodial activity of the plant extract against earlyP. berghei infection was carried out according to the method described by Peter et al. (1975). Based on acute toxicity test, three doses (100, 200, and 400 mg/kg/day) were selected for thein vivo antimalarial study of the solvent fractions [24].After parasite inoculation, 30 mice were randomly assigned into five groups (three treatment groups and two controls), 6 mice per group. The negative control group was treated with the vehicle 2% Tween 80. Likewise, positive control group was treated with standard drug chloroquine 25 mg/kg/day. The remaining three groups received three different doses (100, 200, and 400 mg/kg/day) of the plant extracts. The doses were administered orally at a volume of 10 ml/kg. The mice were treated after three hours of infection and continued for three days.Weight, rectal temperature, and packed cell volume (PCV) were recorded just before infection and on day four postinfection. On day four, a thin blood film was prepared from the tail blood of each mouse. The blood films were fixed with methanol and stained with 10% Giemsa for 10 min. Blood films were examined microscopically to determine parasitemia and parasite suppression.The mean parasitemia and % parasitemia suppression were calculated and expressed as follows [23]:(1)%Parasitemia=Total  number  of  Parasitized  red  blood  cellsTotal  number  of  Red  blood  cells×100,%Parasitaemia  suppression=Parasitaemia  in  control  group-Parasitaemia  in  study  groupParasitaemia  in  control  group×100.Moreover, each mouse was observed and monitored daily for determination of their survival time. The mean survival time (MST) for each group was calculated as follows:(2)MST=Sum  of  survival  time  of  miceTotal  number  of  mice.


### 2.7. Determination of Packed Cell Volume

For packed cell volume (PCV) determination, blood was drawn from the tail of the different group of mice using heparinized capillary tubes before infection and on day 4 after infection.The tubes were filled with blood up to (3/4)th of their volume and sealed at the dry end with sealing clay. The tubes were then placed in hematocrit centrifuge with the sealed end outwards and centrifuged for 5 min at 5,000 rpm [25].(3)Packed  Cell  Volume=Volume  of  erythrocytes  in  a  given  volume  of  bloodTotal  blood  volume×100.


### 2.8. Phytochemical Screening Test

The solvent fractions of root and rind ofCarica papaya were qualitatively screened for the presence of secondary metabolites. Thus, tests for alkaloids, flavonoids, terpenoids, phenolic compounds, tannins, saponins, anthraquinones, and cardiac glycosides were performed using standard test procedures [26, 27].


### 2.9. Data Analysis

The data was analyzed using windows software SPSS version 16 and expressed as mean ± standard error of mean (M ± SEM). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey post hoc test to compare the measured parameters (parasitemia suppression, weight, rectal temperature, and survival time) within and between groups. The analysis was performed with 95% confidence interval andp values less than 0.05 were considered to be statistically significant.


## 2.1. Collection and Authentication of Plant Materials

The freshCarica papaya fruits and roots were collected from near Jimma town, Southwest Ethiopia, in August 2016. The plant material was authenticated by a taxonomist at the Ethiopian National Herbarium, Addis Ababa University. The specimen was deposited for future reference with voucher number JU-GZ01/2016 at the National Herbarium, College of Natural Sciences, Addis Ababa University.


## 2.2. Preparation Solvent Fractions

The freshCarica papaya Linn fruit rind/peel and roots were air-dried at room temperature under shade and pulverized into powder using pestle and mortar. The solvent fractions ofC. papaya fruit rind and root were obtained by sequential soxhlet extraction with petroleum ether, chloroform, and then methanol in increasing polarity [19]. 40 grams of the powdered plant material was weighed and placed in the extraction thimble of the soxhlet apparatus. Then, about 200 ml of petroleum ether was added to the flask of the soxhlet apparatus set up. Then, the petroleum ether was heated with a temperature not exceeding 40°C to evaporate and condense into plant powder containing thimble. This extraction process was continued exhaustively until clear solution in the thimble was siphoned into the solvent flask. Then, the petroleum ether fraction was filtered with Whatman number 1 filter paper and the solvent was removed by placing in oven adjusted at a temperature less than 40°C. The marc of the petroleum ether based extraction was collected and dried at room temperature to remove petroleum ether. The dried left marc was extracted using absolute chloroform following the same procedure as described for petroleum ether extraction to get the chloroform fraction. Finally, the marc of chloroform fraction was collected and dried at room temperature. Then, the whole dried marc was further extracted with methanol with the same procedure indicated above. Each of the fractions was separately stored in screw capped vials in refrigerator until used for the study.


## 2.3. Experimental Animals

Healthy male Swiss Albino mice (8–12 weeks, weighing 25–33 grams) bred and maintained at Ethiopian Public Health Institute were used. The animals were kept in cages and housed in a standard animal house under natural 12/12 h light dark cycle at room temperature, the animal house of School of Veterinary Medicine, Jimma University. They were maintained on standard pelleted diet and water ad libitum. All mice were acclimatized for one week before the study. This study was approved by the ethical review board of college of health science of Jimma University with a reference number HRPGC/578/2015. All experiments were conducted in accordance with the internationally accepted guidelines on laboratory animal use, care, and handling [21].


## 2.4. Acute Toxicity Tests

The acute toxicity studies were conducted as per the OECD guidelines 425. Acute oral toxicity of each of the solvent fractions was evaluated in healthy female mice aged of 6–8 weeks. Five female mice were fasted for three hours and orally given a dose of 2000 mg/kg of the solvent fractions. The mice were observed for lacrimation, hair erection, behavioral change, reduction in their motor, feeding activities, and mortality for three hours and followed for 24 hours and/or 14 days [22].


## 2.5. Parasite Inoculation

Chloroquine sensitiveP. bergheiANKA strain obtained from Ethiopian Public Health Institute and maintained at animal house facility was used. For the parasite maintenance serial passage of blood from infected mice to noninfected ones was made. A donor mouse with a parasitemia of approximately 30% was sacrificed and blood collected in a Petri-dish containing 2% trisodium citrate as anticoagulant. The blood was then diluted with 0.9% normal saline. Each mouse used in the experiment was inoculated intraperitoneally with 0.2 ml of 1 × 107 P. Berghei infected red blood cells [23].


## 2.6.In Vivo Antimalarial Activity Study

In vivoantiplasmodial activity of the plant extract against earlyP. berghei infection was carried out according to the method described by Peter et al. (1975). Based on acute toxicity test, three doses (100, 200, and 400 mg/kg/day) were selected for thein vivo antimalarial study of the solvent fractions [24].After parasite inoculation, 30 mice were randomly assigned into five groups (three treatment groups and two controls), 6 mice per group. The negative control group was treated with the vehicle 2% Tween 80. Likewise, positive control group was treated with standard drug chloroquine 25 mg/kg/day. The remaining three groups received three different doses (100, 200, and 400 mg/kg/day) of the plant extracts. The doses were administered orally at a volume of 10 ml/kg. The mice were treated after three hours of infection and continued for three days.Weight, rectal temperature, and packed cell volume (PCV) were recorded just before infection and on day four postinfection. On day four, a thin blood film was prepared from the tail blood of each mouse. The blood films were fixed with methanol and stained with 10% Giemsa for 10 min. Blood films were examined microscopically to determine parasitemia and parasite suppression.The mean parasitemia and % parasitemia suppression were calculated and expressed as follows [23]:(1)%Parasitemia=Total  number  of  Parasitized  red  blood  cellsTotal  number  of  Red  blood  cells×100,%Parasitaemia  suppression=Parasitaemia  in  control  group-Parasitaemia  in  study  groupParasitaemia  in  control  group×100.Moreover, each mouse was observed and monitored daily for determination of their survival time. The mean survival time (MST) for each group was calculated as follows:(2)MST=Sum  of  survival  time  of  miceTotal  number  of  mice.


## 2.7. Determination of Packed Cell Volume

For packed cell volume (PCV) determination, blood was drawn from the tail of the different group of mice using heparinized capillary tubes before infection and on day 4 after infection.The tubes were filled with blood up to (3/4)th of their volume and sealed at the dry end with sealing clay. The tubes were then placed in hematocrit centrifuge with the sealed end outwards and centrifuged for 5 min at 5,000 rpm [25].(3)Packed  Cell  Volume=Volume  of  erythrocytes  in  a  given  volume  of  bloodTotal  blood  volume×100.


## 2.8. Phytochemical Screening Test

The solvent fractions of root and rind ofCarica papaya were qualitatively screened for the presence of secondary metabolites. Thus, tests for alkaloids, flavonoids, terpenoids, phenolic compounds, tannins, saponins, anthraquinones, and cardiac glycosides were performed using standard test procedures [26, 27].


## 2.9. Data Analysis

The data was analyzed using windows software SPSS version 16 and expressed as mean ± standard error of mean (M ± SEM). Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey post hoc test to compare the measured parameters (parasitemia suppression, weight, rectal temperature, and survival time) within and between groups. The analysis was performed with 95% confidence interval andp values less than 0.05 were considered to be statistically significant.


## 3. Result

The findings from the four-day suppressive test showed that petroleum ether ofCarica papaya fruit rind has a considerable antiplasmodial activityin vivoagainstP. berghei on early infections. In this study, the petroleum ether fraction ofC. papaya fruit rind produced a dose dependent chemosuppressive effect at three doses evaluated (100, 200, and 400 mg/kg/day), with a chemosuppression of 23.03%, 34.38%, and 61.78%, respectively (Table 1). At all dose levels evaluated, the three fractions produced a statistically significant (p<0.001) parasite suppression as compared to negative control. The highest chemosuppressive effect (61.78%) was exhibited by petroleum ether fraction ofC. papaya fruit rind at 400 mg/kg/day dose.Table 1
Effect of the solvent fractions ofC. papaya fruit rind on parasitemia and survival of P. berghei infected mice on 4-day Peter’s suppression test.
Treatment
Dose in mg/kg/day
% parasitemia
% chemosuppression
Survival date
Petroleum ether fraction
100
40.45 ± 2.1
23.03a3,b3,d2,e3
8.67 ± 0.52b3,e2
200
34.48 ± 4.9
34.38a3,b3,c2,e3
8.83 ± 0.75b3,e2
400
20.08 ± 2.4
61.78a3,b3,c3,d3
10.33 ± 1.03a3,c2,d2
Chloroform fraction
100
46.92 ± 4.7
10.72a1,b3,d2,e3
8.33 ± 0.52b3
200
39.83 ± 3.3
24.20a3,b3,c2,e2
8.67 ± 0.82b3
400
32.77 ± 1.61
37.65a3,b3,c3,d2
9.5 ± 1.05a2,b3
Methanol fraction
100
48.25 ± 4.38
8.18b3
7.67 ± 0.52b3
200
43.53 ± 3.25
17.16a2,b3
8.50 ± 0.55b3
400
42.88 ± 4.88
18.39a3,b3
8.17 ± 0.75b3
Vehicle
1 ml
52.55 ± 5.6
-
7.83 ± 0.75
Chloroquine
25
0
100a3,c3
30.00 ± 0.00a3
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80), comparedb to chloroquine 25 mg/kg, comparedc to 100 mg/kg/day of the fraction, comparedd to 200 mg/kg/day of the fraction, and comparede to 400 mg/kg/day of the fraction; p1<0.05; p2<0.01; p3<0.001.However, a mild chemosuppression was produced by the methanol fraction ofC. papaya fruit rind. On the other hand, the standard drug, chloroquine, caused chemosuppression of 100%. Compared to negative control, only the highest dose (400 mg/kg/day) of petroleum ether (p<0.001) and chloroform (p<0.05) fractions caused a statistically significant prolongation of survival time. However, the methanol fraction was not associated with significant prolongation of survival time when compared with the negative control.As shown in Table2,C. papayaroot fractions produced a dose dependent and statistically significant (p<0.001) chemosuppressive effect at the three doses evaluated (100, 200, and 400 mg/kg/day). The parasite suppression by petroleum ether fraction was 21.85%, 31.53%, and 43.77% for 100, 200, and 400 mg/kg/day doses, respectively. Likewise, the parasite suppression by chloroform fraction was 9.77%, 25.25%, and 48.11% for 100, 200, and 400 mg/kg/day doses. The 400 mg/kg/day dose of the ether and chloroform fractions produced the highest parasite suppression relative to the other doses.Table 2
Effect of the solvent fractions ofC. papaya root on parasitemia and survival of infected mice with P. bergheion 4-day Peter’s suppression test.
Treatment
Dose mg/kg/day
% parasitemia
% chemosuppression
Survival date
Petroleum ether fraction
100
41.07 ± 2.55
21.85a3,b3,d1,e3
8.00 ± 0.63b3,e2
200
35.98 ± 3.67
31.53a3,b3,c1,e2
9.17 ± 0.41a1,b3
400
29.55 ± 4.42
43.77a3,b3,c3,d2
9.83 ± 1.33a3,b3,c2
Chloroform fraction
100
47.42 ± 1.94
9.77a2,b3,d3,e3
7.83 ± 0.41b3,e3
200
39.28 ± 1.53
25.25a3,b3,c3,e3
8.83 ± 0.75b3,e2
400
27.27 ± 3.36
48.11a3,b3,c3,d3
10.17 ± 0.75a3,b3,c3,d2
Methanol fraction
100
47.13 ± 4.39
10.31b3,e2
7.83 ± 0.75b3
200
47.22 ± 4.97
10.15b3,e2
8.33 ± 1.03b3
400
39.08 ± 2.76
25.63a3,b3,c2,d2
9.00 ± 0.89b3
Vehicle
1 ml
52.55 ± 5.6
-
7.83 ± 0.75
Chloroquine
25
0.00 ± 0.00
100
30.00 ± 0.00
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80), comparedb to chloroquine 25 mg/kg, comparedc to 100 mg/kg/day of the fraction, comparedd to 200 mg/kg/day of the fraction, and comparede to 400 mg/kg/day of the fraction; p1<0.05; p2<0.01; p3<0.001.Both 200 and 400 mg/kg/day doses of the ether fraction caused statistically significant (p<0.05) survival time prolongation effect, with the mean survival time of 9.83 and 10.17 days, respectively. Similarly, 400 mg/kg/day dose of the chloroform fraction exhibited statistically significant (p<0.01) survival time (10.17±0.75 days) prolongation effect compared to the negative control group. This effect was still by much lower (p<0.001) than that attained by chloroquine (30±0.00).Analysis of PCV change (between day 0 and day 4 after infection) shows that the higher two doses (200 and 400 mg/kg/day) of petroleum ether fraction ofC. papayafruit rind significantly (p<0.01) prevented reduction in PCV as compared to the negative control. Likewise, only the highest administered dose (400 mg/kg/day) of chloroform fraction ofC. papayafruit rind significantly (p<0.01) prevented reduction in PCV (Table 3, Figure 1).Table 3
Effect of the solvent fractions ofCarica papaya rind on packed cell volume (PCV) of P. berghei infected mice on 4-day Peter’s suppression test.
Treatment group
Dose mg/kg/day
Packed cell volume
Day 0
Day 4
% change
Pet ether fraction
100
57.7 ± 2.34
56.0 ± 2.83
−3.02
200
58.2 ± 2.04
57.0 ± 1.67
−2.09a1
400
57.3 ± 2.42
57.2 ± 2.40
−0.29a3
Chloroform fraction
100
56.3 ± 1.97
53.7 ± 2.94
−5.09
200
56.3 ± 1.50
54.3 ± 2.94
−3.85a1
400
55.0 ± 1.09
54.7 ± 1.21
−0.62a3
Methanol fraction
100
57.0 ± 1.09
55.7 ± 2.34
−2.49
200
56.0 ± 1.79
56.0 ± 2.19
−0.04
400
56.3 ± 1.50
56.0 ± 2.83
−0.70
Vehicle
1 ml
56.0 ± 1.79
50.67 ± 3.26
−10.79
Chloroquine
25
57.0 ± 1.55
58.00 ± 2.10
1.69
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80); p1<0.05; p3<0.001. Day 0 = pretreatment value on day 0. Day 4 = posttreatment value on day four.Figure 1
Effect of the solvent fractions ofC. papaya fruit rind on packed cell volume (PCV) ofP. berghei infected mice on 4-day Peter’s suppression test.The solvent fractions ofC. papaya fruit rind exhibited a significant protection against body temperature reduction on day 4. Analysis of percent of body temperature change, between days 0 and 4, indicated thatP. bergheiinfected mice treated with the three doses of the pet ether and chloroform fractions showed a statistically significant (p<0.01) difference when compared to negative control (Table 4). The attenuation of the reduction in body temperature produced by 200 and 400 mg/kg/day doses of pet ether fraction ofC. papaya fruit rind had a comparable effect to chloroquine. In chloroquine treated group, no significant change in both body temperature and PCV was observed.Table 4
Effect of the solvent fractions ofCarica papaya rind on body temperature and weight of P. berghei infected mice on 4-day Peter’s suppression test.
Treatment
Dose mg/kg
Temperature
Weight
Day 0
Day 4
% change
Day 0
Day 4
% change
Pet ether fraction
100
37.1 ± 0.07
36.6 ± 0.33
−1.37a3
27.3 ± 1.03
26.3 ± 1.97
−4.15
200
37.1 ± 0.13
36.9 ± 0.43
−0.64a3
22.0 ± 1.90
23.5 ± 1.22
6.41a3
400
37.1 ± 0.07
36.9 ± 0.25
−0.41a3
26.0 ± 0.89
25.8 ± 1.17
−0.72a2
Chloroform fraction
100
37.2 ± 0.14
35.7 ± 0.29
−1.27a3
24.2 ± 2.14
23.5 ± 2.17
−3.01
200
37.1 ± 0.12
37.0 ± 0.32
−0.36a3
26.5 ± 2.51
26.7 ± 3.20
0.30a2
400
37.1 ± 0.08
36.9 ± 0.13
−0.41a3
24.7 ± 1.97
25.0 ± 1.55
1.23a2
Methanol fraction
100
37.1 ± 0.13
35.5 ± 0.34
−1.65a3
25.5 ± 1.05
24.2 ± 1.33
−5.66a1
200
37.1 ± 0.15
35.7 ± 0.29
−1.09a3
23.8 ± 0.98
24.3 ± 1.21
1.91a3
400
37.0 ± 0.08
35.6 ± 0.55
−1.29a3
24.5 ± 1.22
24.7 ± 1.21
0.62a3
Vehicle
1 ml
37.1 ± 0.14
34.05 ± 0.78
−5.89
25.33 ± 2.16
22.3 ± 2.25
−13.66
Chloroquine
25
37.1 ± 0.39
37.30 ± 0.32
0.41a3
33.3 ± 5.05
34.3 ± 5.35
3.16
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80); p1<0.05; p2<0.01; p3<0.001. Day 0 = pretreatment value on day 0. Day 4 = posttreatment value on day four.On the other hand, the chloroform fraction (at all doses evaluated) ofC. papaya fruit rind averted loss of body weight associated with infection compared to negative control. In contrast, body weight reduction caused by inoculation of the parasite was not significantly prevented by the ether fraction.Percent PCV change analysis, between days 0 and 4, revealed that the three doses (100 mg/kg withp<0.05, 200 mg/kg with p<0.01, and 400 mg/kg with p<0.001) of petroleum ether fraction ofC. papaya root significantly attenuated the PCV reduction compared with the negative control. Similarly, the 200 mg/kg (p<0.05) and 400 mg/kg (p<0.001) doses of chloroform fraction showed a statistically significant PCV protection effect unlike methanol fraction (Table 5, Figure 2).Table 5
Effect of the solvent fractions ofCarica papaya root on PCV, body temperature, and weight of P. berghei infected mice on 4-day Peter’s suppression test.
Treatment group
Dose mg/kg/day
Packed cell volume
Day 0
Day 4
% change
Petroleum ether fraction
100
55.3 ± 2.94
53.5 ± 1.22
−3.45
200
55.5 ± 1.52
54.3 ± 3.44
−2.34a1
400
56.3 ± 1.97
55.5 ± 1.52
−1.51a3
Chloroform fraction
100
55.7 ± 1.50
52.17 ± 2.23
−6.81
200
55.0 ± 1.09
52.7 ± 1.63
−4.47
400
54.7 ± 1.03
53.5 ± 2.17
−2.28a2
Methanol fraction
100
56.0 ± 1.26
51.7 ± 1.50
−8.41
200
55.0 ± 1.09
49.8 ± 2.23
−10.50
400
55.7 ± 1.50
54 ± 1.79
−3.11
Vehicle
1 ml
56.0 ± 1.79
50.7 ± 3.27
−10.79
Chloroquine
25
56.0 ± 1.55
56.0 ± 2.10
1.69
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80); p1<0.05; p2<0.01; p3<0.001. Day 0 = pretreatment value on day 0. Day 4 = posttreatment value on day four.Figure 2
Effect of the solvent fractions ofC. papaya root on packed cell volume (PCV) ofP. berghei infected mice on 4-day Peter’s suppression test.Analysis of percent of body temperature change, between days 0 and 4, revealed that at the evaluated doses the three fractionsC. papaya root showed a statistically significant (p<0.05) difference when compared to negative control. This difference was not significant when compared with chloroquine treated group. On the other hand, the effect of petroleum ether and chloroform fractions on body weight was not dose dependent and consistent. As indicated in Table 6, the lower doses ofC. papaya root solvent fractions produced a significant (p<0.05) parasite induced body weight reduction.Table 6
Effect of the solvent fractions ofC. papaya root on body temperature and body weight of P. berghei infected mice on 4-day Peter’s suppression test.
Treatment group
Dose mg/kg/day
Temperature
Weight
Day 0
Day 4
% change
Day 0
Day 4
% change
Petroleum ether fraction
100
37.2 ± 0.15
35.9 ± 0.23
−0.68a3
24.7 ± 3.26
24.2 ± 3.12
−2.20a1
200
37.1 ± 0.12
37.0 ± 0.13
−0.18a3
28.3 ± 2.25
27.5 ± 1.76
−3.04a1
400
37.1 ± 0.09
37.0 ± 0.19
−0.22a3
22.2 ± 1.94
20.8 ± 2.99
−7.36
Chloroform fraction
100
37.1 ± 0.10
35.5 ± 0.38
−1.70a3
20.2 ± 1.17
19.5 ± 1.05
−3.52a1
200
37.2 ± 0.07
36.9 ± 0.10
−0.63a3
20.5 ± 1.38
19.8 ± 1.72
−3.75a1
400
37.1 ± 0.12
36.9 ± 0.16
−0.50a3
22.7 ± 1.37
21.5 ± 1.52
−5.62
Methanol fraction
100
37.1 ± 0.12
34.5 ± 0.35
−1.60a3
21.2 ± 1.60
22.5 ± 1.64
1.39
200
37.1 ± 0.18
34.6 ± 0.27
−1.37a3
21.0 ± 0.89
18.8 ± 1.94
−12.32
400
37.1 ± 0.12
35.7 ± 0.29
−1.09a3
20.3 ± 1.63
19.8 ± 1.72
−2.60a1
Vehicle
1 ml
37.1 ± 0.13
34.0 ± 0.78
−5.89
25.3 ± 2.16
22.3 ± 2.25
−13.66
Chloroquine
25
37.1 ± 0.39
37.3 ± 0.32
0.41a3
33.3 ± 5.05
34.3 ± 5.35
3.16a1
Data are expressed as mean ± SEM;n = 6; compareda to negative control (vehicle; 2% Tween 80); p1<0.05; p3<0.001. Day 0 = pretreatment value on day 0. Day 4 = posttreatment value on day four.


### 3.1. Phytochemical Screening

In the qualitative phytochemical analysis, the solvent fractions of fruit rind and root ofCarica papaya showed the presence of secondary metabolites such as alkaloids, flavonoids, polyphenols, tannins, and terpenoids as indicated in Table 7.Table 7
Qualitative phytochemical screening of the solvent fractions of fruit rind and root ofCarica papaya.
Secondary metabolites
C. papaya fruit rind fractions
Carica papaya root fractions
Pet ether
Chloroform
Methanol
Pet ether
Chloroform
Methanol
Alkaloids
+
+
+
+
+
+
Flavonoids
+
+
−
+
+
−
Polyphenols
+
+
+
+
+
+
Tannins
+
+
+
+
+
+
Terpenoids
+
+
+
+
+
Cardiac glycosides
−
−
−
−
−
−
Saponin
−
−
+
−
−
−
Note. + indicates the presence and − indicates absence of particular metabolites.


## 3.1. Phytochemical Screening

In the qualitative phytochemical analysis, the solvent fractions of fruit rind and root ofCarica papaya showed the presence of secondary metabolites such as alkaloids, flavonoids, polyphenols, tannins, and terpenoids as indicated in Table 7.Table 7
Qualitative phytochemical screening of the solvent fractions of fruit rind and root ofCarica papaya.
Secondary metabolites
C. papaya fruit rind fractions
Carica papaya root fractions
Pet ether
Chloroform
Methanol
Pet ether
Chloroform
Methanol
Alkaloids
+
+
+
+
+
+
Flavonoids
+
+
−
+
+
−
Polyphenols
+
+
+
+
+
+
Tannins
+
+
+
+
+
+
Terpenoids
+
+
+
+
+
Cardiac glycosides
−
−
−
−
−
−
Saponin
−
−
+
−
−
−
Note. + indicates the presence and − indicates absence of particular metabolites.


## 4. Discussion

Antimalarial drug resistance remains a major challenge and continued to emerge creating an obstacle in malaria control and elimination [5, 28]. At present, developing novel approaches and new alternative antimalarial drugs is pivotal to combat the disease [29]. From history, medicinal plants are endowed with active antimalarial compounds as artemisinin is obtained fromArtemisia annua and quinine fromCinchona bark [30].In vivoevaluations of antimalarial activity begin with the use of the rodent malaria parasite. In addition,in vivostudies take into account any prodrug effect and the role of immune system in controlling malaria infection unlikein vitroones [23]. Accordingly, this study evaluated thein vivo antimalarial activity of solvent fractions ofCarica papaya root and fruit rind using the 4-day suppressive test, which mainly evaluates the antimalarial activity of candidates on early infections, againstP. bergheiin mice. In acute toxicity studies, the observation of no death or sign of toxicity with an oral dose of 2000 mg/kg of the fractions indicated the solvent fractions are safe for use.In this study, all the solvent fractionsof Carica papaya root and fruit rind exhibited a statistically significant (p<0.001) and dose dependent parasite suppression effect on early infections (Tables 1 and 2). A maximum parasite suppression of 61.78% was produced by petroleum ether fraction ofCarica papaya fruit rind in the highest dose (400 mg/kg/day), with the longest survival time compared to other fractions treated mice and negative control. Likewise, chloroform fraction ofCarica papayaroot exhibited a higher chemosuppression effect of 48.11% at 400 mg/kg/day dose. The parasite suppression exhibited by these solvent fractions was comparable with similar studies done onD. angustifolia [25],Lophira alata [31], andParkia biglobosa [32]. But, a weak parasite suppression effect was exhibited by methanol fraction of both root and fruit rind of this plant.Malaria infected mice suffer from anemia because of erythrocyte destruction, either by malaria multiplication or by spleen reticuloendothelial cell action [33]. An ideal antimalarial candidate should prevent anemia secondary to preventing hemolysis, body weight loss, and body temperature reduction in mice. In this study, a significant attenuation of PCV and body temperature reduction effect was observed by petroleum ether and chloroform fractions at 200 and 400 mg/kg doses. This was also comparable to standard drug chloroquine. The effect of the solvent fractions on body weight was variable and produced inconsistent protection. This might be due to the nature of the ingredients present in the fractions causing apatite suppression.In vivo antiplasmodial activity can be classified as moderate, good, and very good if an extract displayed percent of parasite suppression equal to or greater than 50% at a dose of 500 mg, 250 mg, and 100 mg/kg body weight per day, respectively [34, 35]. Based on this classification, the petroleum ether fraction ofCarica papayafruit rind and chloroform fraction ofCarica papaya root exhibited a moderate antiplasmodial activity.In this study, pet ether fraction ofCarica papayafruit rind produced a promising in vivo antiplasmodial activity. This result was in agreement within vitro antiplasmodial activity of pet ether (IC50 = 15.19 μg/mL) and methanol (IC50 > 100 μg/mL) fractions ofC. papayafruit rind in previous study [19]. In another study, the leaf extract ofC. papaya produced antiplasmodial activity with IC50 of 46.23 μg/mL [20]. This signifies that the pet ether fraction ofC. papayafruit rind had the highest antimalarial activity relative to the other fractions and the plant parts. This is attributed to the possible presence of the active metabolites. On phytochemical screening, terpenoids, flavonoids, alkaloids, and phenols are present in both petroleum ether and chloroform fractions that might be responsible for the observed antimalarial activity. These phytochemicals might also exert a synergistic antiplasmodial effect. But, weak antimalarial activity by the methanol fraction may be due to the presence of trace active constituents in the administered dose.This finding may be an indicator for the presence of potential compounds with higher antimalarial activity in the petroleum ether fraction ofC. papayafruit rind. From this study, the active antimalarial compound found inC. papayafruit rind is possibly nonpolar or semipolar in nature. Moreover, there may be a commercial potential in extracting the active compound from this plant, which grows abundantly throughout the tropics, the rind of which is discarded as waste.


## 5. Conclusion

The higher dose of pet ether and chloroform fraction ofC. papayafruit rind and root exhibited a moderate antiplasmodial effect with the longest survival time compared to negative control. From this finding, the petroleum ether fraction ofC. papayafruit rind had the highest antimalarial activity and could be targeted as potential source of lead compound in the development of new antimalarial agent. Therefore, further study should be done on pet ether fraction ofC. papayafruit rind to show the chemical and metabolomic profile of active ingredients from this plant.



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*Source: 3121050-2017-12-17.xml*