Abstract
Objective: Anemia is a pathological condition characterized by reduced oxygen bioavailability and/or changes in hematological parameters. This study investigated the anti-anemic activities of Carica papaya (CP) phytoconstituents in aluminium-chloride-induced anemic rats. Method: Twenty-seven rats were randomized into nine groups of three rats as follows; group 1 was the normal (non-induced) group, 2-9 were anemic rats administered 1 mL distilled water, standard drug (3 mg/kg body weight (bw) ferrous sulphate), 100, 300 and 500 mg/kg bw of crude methanolic extract of CP (CMECP) of the leaf and 100, 300 and 500 mg/kg bw of CMECP of the seed respectively in the first stage of the study. In the second stage, thirty-three rats were randomized into eleven groups of three rats as follows; group 1 was the normal group, 2-11 were anemic rats treated with 1 mL distilled water, standard drug, 75 mg/kg bw, 150 mg/kg of alkaloid fraction of CP seed, 75 mg/kg bw, 150 mg/kg bw of flavonoid fraction of CP seed, 75 mg/kg bw and 150 mg/kg of alkaloid fraction of CP leaf, 75 mg/kg bw and 150 mg/kg bw of flavonoid fraction of CP leaf respectively. Results: Treatment of anemic rats with CP extracts and fractions of the seed and leaf significantly reversed the hematological parameters and body weight of anemic rats in a dose independent fashion. The CMECP leaf at 100 and 500 mg/kg gave PCV of 42.50±0.50 and 47.00±0.50, while the seed gave 49.50±0.50 and 42.50±0.50 respectively after 2 weeks of treatment. However, the alkaloid and flavonoid fraction of CP presented better anti-anemic properties probably due to constituents’ synergism. Conclusion: This study concluded that CP possesses phytoconstituents which potentiates it as a safe anti-anemic drug candidate.
Keywords: Anti-anemic, aluminium-chloride, Carica papaya, phytoconstituents, hematological parameters
Introduction
Anemia is a pathological and hematological condition resulting in reduction in blood parameters [1]. This condition may arise from lack of oxygen supply to epithelial tissues for various physiological body functions. Pathological conditions such as morphological or mechanical defects lead to reduction in the oxygen-carrying capacity of hemoglobin (Hb) with signs and symptoms including weakness, irregular heartbeat, pale skin, and nail bed and headache [2,3]. At the early stage, mild symptoms might be observed, however as the anemia progresses, its symptoms become severe leading to conditions such as insomnia, hemolysis, renal impairments, and hypertension [4].
Anemic conditions can be induced externally with chemicals like phenyl-hydrazine, dapsone, hydroxylamine, divicine, and aluminum chloride [5]. Aluminum (Al) is a trivalent metallic cation that contributes to hemolytic free radical production causing damage to various proteins, DNA, membrane lipids, and prevents the binding of iron to transferrin. Aluminum also produces peroxidative changes in the erythrocyte’s membrane causing hemolysis [6].
In 2021, 1.92 billion (24.3%) global cases of anemia were reported. However, the age, sex, and geographical distribution vary. For example, children below 5 years, females, sub-Saharan Africa, and south Africa are more affected by anemia [7]. Although, anemia is not listed as a disease of global concern, in Africa it causes significant morbidity and mortality; moreover, it decreases the already limited resources in less developed countries, contributing to social effects such as reduced quality of life and depression [8]. In Nigeria, more than 40% of the population is anemic with children and women being the most impacted [9].
The strategies used in the management and treatment of anemia, especially iron deficiency induced anemia are oral or intravenous administration and supplementation with iron (ferrous) [10]. Blood transfusion and bone marrow transplantation can also be used to treat anemia; however, they are cost intensive. Anti-anemic drug such as fersolates presents risk of iron overdose and mutagenicity [4]. Haemochromatosis (iron overload disorder) can also result from the use of anti-anemic drugs which can lead to cancer and heart diseases if left untreated. Excess iron can also get stored in the liver, heart, and pancreas, resulting in other pathological conditions [11]. Therefore, it becomes pertinent to explore other safer treatment options.
With the growing exploration of medicinal plants as alternative drug candidates, there is high demand for probes into their constituents to generate information for safer and effective drug development purposes. Moringa oleifera, Ipomoea batata, Mormodica charantia are among plants of reported anti-anemic properties [12], however, a large number of plants are yet to be reported for their anti-anemic potential. Carica papaya (family: Caricaceae; common name ‘pawpaw’) is widely cultivated for its nutritional and medicinal values [13]. This plant has several traditional uses such as treatment of fibroid, kidney, dental, and heart diseases, it is also used to boost memory and low sperm count [14]. It leaves have been reported to possess anticancer, anti-plamodial, anti-inflammatory, antioxidant, antimicrobial, and hepatoprotective effects [15]. Due to well documented reports implicating the role of oxygen-based free radicals Al-induced anemia and presence of a large array of phytoconstituents in C. papaya, this study investigated the anti-anemic potential of selected phytochemicals of C. papaya leaf and seed extracts in aluminium chloride-induced (AlCl3)-induced anemia in an animal model, with a view to formulating new cost-effective drugs that are easily accessible, non-toxic, and anti-anemic.
Materials and methods
Fresh leaves of C. papaya were collected from the biological garden of Federal University of Technology (FUT), Minna, Niger State in November 2018, while the seeds were obtained from fruit sellers in Bosso Local Government Minna, Niger State in November 2018. The plant materials were identified by Mr. Dangana, M. C. of Biological Science Department, FUT, Minna, Nigeria.
Reagents, chemicals, and equipment
All the chemicals and reagents used in this study were products of Sigma chemical Co, USA while the equipment used was provided by the Department of Biochemistry, FUT Minna, Nigeria.
Experimental animals
A total of sixty rats (weighing 125-160 g) were obtained from the animal breeding unit of the National Veterinary Research Institute, Vom, Nigeria. The animals were housed in standard plastic cages (temperature maintained at 22°C±3°C, relative humidity of 50%±5%, and a 12-hour dark and 12-hour light cycle) and fed a commercial rat diet with free access to water.
Ethical approval
This study was approved by the Animal Use and Research Committee of Federal University of Technology, Minna, Nigeria (BCHFUTMINNA-012019). All the procedures performed in the animal experiments were in accordance with the ethical standards of this institution.
Preparation of extracts
The plant materials were rinsed, air-dried, and pulverized using an electronic blending machine. The powdered samples were extracted with methanol in the ratio 1:8, refluxed for 2 hours in a distillation flask mounted on a heating mantle. The mixture was filtered and concentrated to give the crude methanolic extracts of C. papaya (CMECP). The extracts obtained were weighed for percentage yield estimation using the equation:
Percentage yield = Weight (g) of extract/Weight (g) of pulverized sample × 100
Extraction of total flavonoid
The extraction of total flavonoid was carried out as described by Jouad et al. [16]. Briefly, 350 g each of powdered C. papaya seed and leaf were extracted with methanol in a Soxhlet extractor. The extracts were concentrated under reduced pressure using rotary evaporator. Subsequently, 41 g of the methanol extracts were dissolved in 400 mL of distilled water and extracted with n-butanol saturated with distilled water. This was then concentrated, and the butanol extract was subjected to column chromatography on silica gel eluted with n-hexane and methanol.
% Yield = Weight of the flavonoid/Weight of the powdered C. papaya × 100
Extraction of total alkaloids
The Soxhlet method as described by Gonzales et al. [17] was used for the alkaloid extraction. Briefly, 300 g of powdered C. papaya leaf and seed was weighed separately and packed in a cheese cloth bag which served as an extraction thimble. 95% ethanol was added to each sample in the thimble to moisten it before the addition of ammonia test solution (T.S) The extraction was done in a Soxhlet extractor for 24 hours after which 95% ethanol was then placed in the solvent flask and further extracted for another 3-4 hours. The mixture obtained was filtered and concentrated at 60°C. The resulting crude alkaloid extract obtained was treated with 1.0 N HCl and filtered. Ammonia T.S was then added to the filtrate. Afterwards, chloroform was added to the mixture and thoroughly shaken in a separating funnel to form two layers where the lower chloroform layer contained the alkaloid, and the aqueous portion formed the upper layer. The chloroform layer was carefully collected and concentrated using a rotary evaporator. The percentage alkaloid yield was calculated as:
% Yield = Weight of the alkaloidal residue/Weight of the powdered C. papaya × 100
Acute toxicity test of crude extract of C. papaya
This test was conducted using Lorke’s method [18]. Three groups comprising three animals were administered 10, 100 and 1000 mg/kg of the extract per group in the first phase. Behavioral changes and mortality were observed for 24 hours. In the second phase, three animals were administered 1600, 2900 and 5000 mg/kg of the extract respectively and observed for behavioral changes and mortality for 24 hours. Then the lethal dose (LD50) was calculated using the formula:
LD50 = √(D0 × D100)
D0 = Mortality at the highest dose; D100 = Mortality at the lowest dose.
Experimental model for anti-anemic study
The protective potential effect of crude methanolic extracts and crude alkaloids and flavonoid fractions of C. papaya were evaluated in aluminium-chloride (AlCl3)-induced anemic rats. The anemic condition was induced with 0.5 mg/kg for ten minutes followed by a 14-day oral treatment period. The study was carried out in two stages as follows:
The first stage involved treatment with CMECP leaf and seed. Nine groups comprising 3 rats each were treated as follows: group 1 was non-anemic (normal control), groups 2 and 3 were anemic rats administered distilled water (1 mL) and standard drug [(ferrous sulphate (3 mg/kg)] respectively, groups 4-6 were anemic rats treated with 100, 300 and 500 mg/kg extract of C. papaya leaf respectively while groups 7-9 were anemic rats treated with 100, 300 and 500 mg/kg of C. papaya seed respectively.
The second stage involved the treatment of anemic rats with crude alkaloid fraction of Carica papaya (CAFCP) and crude flavonoid fractions of Carica papaya (CFFCP) leaf and seed. Nine groups comprising 3 rats each were treated as follows: group 1 was non-anemic (normal control), groups 2 and 3 were anemic rats administered distilled water (1 mL) and ferrous sulphate (3 mg/kg) respectively, groups 4 and 5 were anemic rats treated with 75, and 150 mg/kg of the CAFCP seed. Similarly, groups 6 and 7 were treated with 75, and 150 mg/kg of CFFCP seed. Groups 8 and 9 were treated with 75, and 150 mg/kg of CAFCP leaf, while groups 10 and 11 were treated with 75, and 150 mg/kg of CFFCP leaf.
Blood sample collection and determination of hematological parameters
During the 2 weeks treatment period, the animals’ body weight was recorded within an interval of 3 days before they were finally sacrificed on the 15th day of treatment [19]. Blood sample (3 mL) was collected by cardiac puncture into a well-labeled heparinized sample bottle and analyzed immediately for hematological parameters using the SYSMEX KX21 (SYSMEX, Co, Japan) automated analyzer [20].
Data analysis
Data generated were analyzed using Statistical Package for Social Science (SPSS v20). All the data in the results are expressed as mean ± standard error of three replicate determinations. One Way ANOVA (analysis of variance) was used for comparison between groups where a P-value <0.05 was considered significant. Values or bars with different letters of the alphabet are significantly (P<0.05) different.
Results
Phytochemicals compositions of C. papaya leaf and seed
The percentage yield of crude methanolic extract, alkaloid, and flavonoid fractions of C. papaya leaf and seed are presented in Table 1. The percentage yields of crude C. papaya leaf extract (17.51±1.93%) was significantly (P<0.05) higher than the that of the crude seed extract (11.59±0.78%) (Table 1A). Upon crude alkaloid and flavonoid fractionation of the leaf and seed, a higher flavonoid yield was recorded in the leaf and seed fractions when compared to their alkaloid yields (Table 1A). The CMECP possessed different secondary metabolites in different concentrations (Table 1B). For the leaf and seed respectively, saponins had the highest concentrations; 1249.83±13.05 and 723.65±0.39 mg/g, followed by phenols (261.34±1.07 and 171.45±0.91 mg/g), alkaloids (67.75±1.06 and 35.42±0.50 mg/g), tannins (45.25±0.46 and 40.67±0.50 mg/g) and flavonoids (34.89±0.66 and 25.80±0.99 mg/g).
Table 1A.
Percentage yield of crude methanol extract, alkaloid and flavonoid fractions of C. papaya leaf and seed
| Plant Samples | % Yield | ||
|---|---|---|---|
|
| |||
| Methanolic extract | Leaf | Seed | |
| C. papaya leaf | 17.51±1.93* | - | - |
| C. papaya seed | 11.59±0.78 | - | - |
| Crude C. papaya alkaloid fraction | - | 2.65±0.03a | 2.97±0.04a |
| Crude C. papaya flavonoid fraction | - | 7.05±0.65b | 7.96±0.29b |
Data are presented in Mean ± standard error of one paired determination.
Significantly higher as compared to crude (seed) extract at P<0.05.
Values with superscript alphabet b is significantly higher than value with superscript alphabet a.
Table 1B.
Quantitative phytochemicals composition of crude methanolic extract of C. papaya leaf and seed
| Parameters | Leaf (mg/100 g) | Seed (mg/100 g) |
|---|---|---|
| Phenols | 261.34±1.07a | 171.45±0.91b |
| Flavonoid | 34.89±0.66a | 25.80±0.99b |
| Tannins | 45.25±0.46a | 40.67±0.50b |
| Alkaloids | 67.75±1.06a | 35.42±0.50b |
| Saponins | 1249.83±13.05a | 723.65±0.39b |
Data are presented in Mean ± standard error of triplicate determinations. Values with different superscript alphabets (a and b) are significantly different across a row at P<0.05.
Acute oral toxicity test for CMECP leaf and seed
The result of the oral toxicity of CMECP is presented in Table 2. The lethal dose (LD50) of CMECP seed and leaf in rats was >5000 mg/kg while the safe doses were 1600 and 2900 mg/kg bw, respectively. However, animals administered 2900 mg/kg and 5000 mg/kg bw demonstrated behavioral signs including rubbing of mouth on the surface of the cage, hyperactivity/restlessness but no death was recorded.
Table 2.
Safe dose of methanol seed and leaf extract of C. papaya
| Phases | Plant part/groups | Dose (mg/kg) | Number of rats | Number of deaths after 24 hours | % Mortality | Sign of toxicity |
|
| ||||||
| PHASE 1 | Seed | |||||
|
| ||||||
| Group 1 | 10 | 3 | 0 | 0 | Nil | |
| Group 2 | 100 | 3 | 0 | 0 | Nil | |
| Group 3 | 1000 | 3 | 0 | 0 | Nil | |
|
|
||||||
| Leaf | ||||||
|
| ||||||
| 10 | 3 | 0 | 0 | Nil | ||
| 100 | 3 | 0 | 0 | Nil | ||
| 1000 | 3 | 0 | 0 | Nil | ||
|
| ||||||
| PHASE 2 | Seed | |||||
|
| ||||||
| Group 1 | 1600 | 3 | 0 | 0 | Nil | |
| Group 2 | 2900 | 3 | 0 | 0 | Rubbing of the mouth on the surface of the cage | |
| Group 3 | 5000 | 3 | 0 | 0 | Hyperactivity/restlessness | |
|
|
||||||
| Leaf | ||||||
|
|
||||||
| Group 1 | 1600 | 3 | 0 | 0 | Nil | |
| Group 2 | 2900 | 3 | 0 | 0 | Nil | |
| Group 3 | 5000 | 3 | 0 | 0 | Hyperactivity/restlessness | |
Body weight and PCV changes in AlCl3-induced anemic rats treated with CMECP seed and leaf
The changes in weight of AlCl3-induced anemic rats treated with CMECP seed and leaf over a period of 2 weeks are presented in Table 3. A decrease in the weight of untreated anemic rats relative to the normal and treatment groups was observed. However, treatment with CMECP led to an increase in the body weights of the animals.
Table 3.
Effect of crude methanol extract of C. papaya leaf and seed on body weight of AlCl3-induced anemic rats
| Group | Day 0 | Day 3 | Day 7 | Day 10 | Day 14 |
|---|---|---|---|---|---|
| Normal Control | 191.50±10.50a,b | 196.50±23.50a | 199.25±36.45b | 203.00±39.00a | 209.75±37.25b |
| Untreated anemic rats | 107.00±33.00b | 102.00±30.00a | 94.05±11.35a,b | 81.50±9.50a | 71.00±11.00a,b |
| 3 mg/kg bw FeSO4 | 133.50±19.50a | 137.75±12.75a | 142.00±17.00a | 148.00±26.00a | 152.50±11.50a |
| 100 mg/kg bw CMECP leaf | 163.25±2.75a,b | 154.75±0.25a | 162.50±1.50a,b | 171.50±6.50a | 185.00±5.50a,b |
| 300 mg/kg bw CMECP leaf | 196.25±23.75a,b | 180.50±30.20a | 201.50±22.50a,b | 190.00±37.00a | 206.75±39.75a,b |
| 500 mg/kg bw CMECP leaf | 161.50±10.50a,b | 168.50±9.50a | 175.75±7.25a,b | 169.50±6.50a | 177.80±4.20a,b |
| 100 mg/kg bw CMECP seed | 202.50±10.50a,b | 209.50±14.50a | 222.00±19.00a,b | 226.00±33.00a | 230.25±12.75a,b |
| 300 mg/kg bw CMECP seed | 156.25±0.25a,b | 173.50±10.50a | 186.350±3.50a,b | 186.00±12.00a | 191.50±15.00a,b |
| 500 mg/kg bw CMECP seed | 134.00±38.00a | 143.00±36.00a | 151.35±34.35a | 151.50±37.50a | 168.25±28.25a |
Data are presented in Mean ± standard error of triplicate determinations. Values with different superscript alphabets (a and b) are significantly different between groups at P<0.05.
The effect of 2 weeks CMECP treatment on the packed cell volume (PCV) of AlCl3-induced anemic rats is presented in Table 4. The reduced PCV count of the untreated group relative to other groups was observed. However, the extract groups had higher PCV counts than the normal control.
Table 4.
Changes in Packed Cell Volume (PCV) in aluminium chloride-induced anemic rats treated with methanol extracts of C. papaya for two weeks
| GROUPS | DAY 0 | DAY 4 | DAY 8 | DAY 12 | DAY 15 |
|---|---|---|---|---|---|
| Normal Control | 38.37±0.43a,b | 40.51±0.45b | 41.03±0.09c | 41.63±0.36d | 43.00±2.00a,b |
| Untreated anemic rats | 40.50±1.81d | 36.43±0.97c | 31.36±2.19a | 28.70±1.30a | 26.50±3.50a |
| 3 mg/kg bw FeSO4 | 38.21±0.39a,b | 37.75±0.38a | 38.43±0.25b | 38.69±0.42b | 40.00±0.00b |
| 100 mg/kg bw CMECP leaf | 37.87±0.14a | 37.81±0.41a | 39.10±0.32b | 40.48±0.33c | 42.50±0.50b |
| 300 mg/kg bw CMECP leaf | 37.43±0.43a | 37.30±0.85a | 36.92±0.45b | 39.16±0.22b,c | 40.50±0.50b |
| 500 mg/kg bw CMECP leaf | 40.43±0.27b | 42.58±0.29c | 43.94±0.04d | 45.78±0.09e | 47.00±0.50c,d |
| 100 mg/kg bw CMECP seed | 42.72±0.43c | 43.95±0.04c | 46.22±0.40d | 47.72±0.26f | 49.50±0.00d |
| 300 mg/kg bw CMECP seed | 36.87±0.46a | 37.52±0.37a | 38.56±0.29b | 38.00±0.58b | 40.00±0.00b |
| 500 mg/kg bw CMECP seed | 38.00±0.57a | 38.43±0.58a | 38.22±0.39b | 39.66±0.33b,c | 42.50±2.50a,b |
Data are presented in Mean ± standard error of triplicate determinations. Values with different superscript alphabets (a, b, c, d, e, and f) are significantly different between groups at P<0.05.
Changes in body weight and PCV of AlCl3-induced anemic rats treated with alkaloids and flavonoid fractions of C. papaya seed and leaf
The changes in body weight of AlCl3-induced anemic rats treated with alkaloids and flavonoid fractions of C. papaya seed and leaf are presented in Table 5. Aluminium-chloride caused a decrease in the body weight of the rats (Table 5). However, treatment with the fractions brought about improvement in the body weights of the animals at the end of the 2 weeks treatment except in the 150 mg/kg bw CAFCP leaf group.
Table 5.
Effect of alkaloids and flavonoid fractions of C. papaya seed and leaf on body weight changes in AlCl3-induced anemic rats
| Group | Day 0 | Day 3 | Day 7 | Day 10 | Day 14 |
|---|---|---|---|---|---|
| Normal Control | 105.67±5.78a,b | 109.33±6.64a,b | 115.00±4.93a,b,c | 117.67±3.48a,b,c | 119.33±3.18a,b,c |
| Untreated anemic | 99.66±34.48a | 96.67±9.24a,b | 93.33±9.52a,b | 92.00±10.12a | 87.00±7.81a |
| AlCl3 + 3 mg/kg bw FeSO4 | 116.33±2.91a,b | 120.00±2.08a,b,c | 123.33±2.40a,b,c | 128.00±2.65a,b,c | 129.33±1.86a,b,c |
| AlCl3 + 75 mg/kg bw CAFCP seed | 95.33±16.15a | 104.67±19.38a,b | 108.00±19.86a,b | 112.00±16.46a,b | 114.00±16.16a,b |
| AlCl3 + 150 mg/kg bw CAFCP seed | 98.66±10.68a | 110.67±14.89a,b | 106.67±12.45a,b | 108.67±16.46a,b | 109.33±11.72a,b |
| AlCl3 + 75 mg/kg bw CFFCP seed | 81.00±7.21a | 87.67±5.78a,b | 90.67±6.33a | 95.33±6.57a | 98.00±6.43a |
| AlCl3 + 150 mg/kg bw CFFCP seed | 72.66±4.67a | 76.67±4.91a | 81.33±4.25a | 87.67±6.01a | 89.33±5.93a |
| AlCl3 + 75 mg/kg bw CAFCP leaf | 95.33±16.15a | 104.67±19.38a,b | 108.00±19.86a,b | 112.00±16.46a,b | 114.00±16.16a,b |
| AlCl3 + 150 mg/kg bw CAFCP leaf | 98.66±10.68a | 110.67±14.89a,b | 106.67±12.45a,b | 108.67±16.46a,b | 109.33±11.72a,b |
| AlCl3 + 75 mg/kg bw CFFCP leaf | 81.00±7.21a | 87.67±5.78a,b | 90.67±6.33a | 95.33±6.57a | 98.00±6.43a |
| AlCl3 + 150 mg/kg bw CFFCP leaf | 72.66±4.67a | 76.67±4.91a | 81.33±4.25a | 87.67±6.01a | 89.33±5.93a |
Data are presented in Mean ± standard error of triplicate determinations. Values with different superscript alphabets (a, b, and c) are significantly different between groups at P<0.05.
The changes in PCV of AlCl3-induced anemic rats treated with alkaloids and flavonoid fractions of C. papaya seed and leaf are presented in Table 6. The consistent reduction observed in the PCV of the untreated anemic rat was reversed in the alkaloid and flavonoid fractions of C. papaya seed and leaf treated groups over the treatment period (Table 6).
Table 6.
Effect of alkaloids and flavonoid fractions of C. papaya seed and leaf on the changes in packed cell volume (PCV) in aluminium chloride-induced anemic rats
| Group | Day 0 | Day 4 | Day 8 | Day 12 | Day 15 |
|---|---|---|---|---|---|
| Normal Control | 36.56±9.85a | 34.56±0.54a | 37.46±0.49b | 38.13±0.35b | 40.00±2.00b |
| Untreated anemic | 40.90±0.35b | 38.20±0.42b | 35.06±0.33a | 27.56±0.34c | 26.50±3.50c |
| AlCl3 + 3 mg/kg bw FeSO4 | 35.66±0.35a | 35.96±0.27a | 29.06±0.42c | 33.50±0.50a | 37.00±0.00b |
| AlCl3 + 75 mg/kg bw CAFCP seed | 36.36±11.68a | 28.30±0.37c | 30.90±0.32a,b | 33.86±0.38a,b | 36.00±0.58a,b |
| AlCl3 + 150 mg/kg bw CAFCP seed | 40.90±0.06a,b | 38.13±0.44b | 32.63±0.50a,b | 34.86±0.15a,b | 36.00±3.06b |
| AlCl3 + 75 mg/kg bw CFFCP seed | 34.16±0.33d | 34.53±0.29d | 27.80±0.55b,c | 25.06±0.22b,c | 23.67±3.28b,c |
| AlCl3 + 150 mg/kg bw CFFCP seed | 36.96±0.09a | 27.90±0.05c | 31.36±0.37b,c | 26.13±0.33b,c | 23.67±1.86b,c |
| AlCl3 + 75 mg/kg bw CAFCP leaf | 42.10±0.55b | 38.86±1.00a,b | 41.36±0.72b | 37.23±1.07a | 39.33±2.40a,b |
| AlCl3 + 150 mg/kg bw CAFCP leaf | 40.73±0.13b | 37.33±0.48a | 35.10±0.38a | 36.53±0.29a | 37.66±0.88a |
| AlCl3 + 75 mg/kg bw CFFCP leaf | 35.20±0.61a | 37.03±0.28a | 32.00±0.58a,b | 28.20±0.42d | 29.66±2.40d |
| AlCl3 + 150 mg/kg bw CFFCP leaf | 38.26±0.37a | 37.830.12a | 31.46±0.12a | 33.50±0.55b,c | 34.33±5.17b,c |
Data are presented in Mean ± standard error of triplicate determinations. Values with different superscript alphabets (a, b, c, and d) are significantly different between groups at P<0.05.
Anti-anemic activity of CMECP leaf and seed in AlCl3-induced anemic rats
The effect of CMECP leaf and seed in AlCl3-induced anemic rats are presented in Figure 1. The untreated anemic rats showed reduced PCV, Hgb and RBC counts relative to the normal and extract treated groups (Figure 1A). The CMECP treated groups showed dose dependent increase in RBC relative to the normal and reference drug treated groups. However, PCV and Hgb in the extract treated groups at all tested concentrations were different from the non-induced group. Figure 1B shows the white blood cell (WBC), lymphocytes (L), neutrophils (N) and red blood cell wide counts (RDWC) in AlCl3-induced anemic rats. A decrease in the WBC and lymphocytes of the untreated group was improved upon treatment with the extracts to levels comparable to the normal. Interestingly, the neutrophil counts in the CMECP treated groups were higher than the normal group. The mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin counts (MCHC) in AlCl3-induced anemic rats are presented in Figure 1C. Except in the 300 mg/kg bw CMECP treated group, no appreciable difference was recorded in MCH and MCHC levels of the other CMECP treated groups relative to the untreated and normal groups. Similar trend was observed difference in the MCV counts of all the groups. There was an increase in the platelet counts of the groups treated with the standard drug, 100 and 300 mg/kg of CP leaf extracts when compared to the untreated. However, other treatment groups showed no appreciable difference in platelet counts from the anemic group (Figure 1D).
Figure 1.
Effect of leaf and seed extracts of C. papaya on (A) packed cell volume, hemoglobin, and red blood cells (B) white blood cell, lymphocytes, neutrophils, and red blood cell wide counts in AlCl3-induced anemic rats (C) mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin (D) platelet counts in AlCl3-induced anemic rats. Bars are presented in Mean ± standard error of triplicate determinations. Bars with different superscript alphabets (a, b, c, and d) are significantly different between groups at P<0.05.
Anti-anemic activity of AlCl3-induced anemic rats treated with alkaloid and flavonoid fractions of C. papaya seed
The anti-anemic activity alkaloid and flavonoid fractions of C. papaya seed in AlCl3-induced anemic rats is displayed in Figure 2. The effects of crude alkaloids and flavonoid fractions of CP seed on PCV, Hgb and RBC on AlCl3-induced anemic rats is presented in Figure 2A. The reduced PCV, Hgb and RBC counts in the anemic untreated rats were increased upon fractions treatment. Figure 2B shows the MCV, MCH and MCHC levels of anemic rats. Except in the untreated rats, the MCH level in the groups compared well with the normal whereas the anemic untreated rats showed decreasing MCHC and MCV levels compared to the other groups.
Figure 2.
Effect of alkaloids and flavonoid of C. papaya seed fractions on (A) packed cell volume, hemoglobin, and red blood cells (B) mean corpuscular volume (MCV) mean cell hemoglobin (MCH), and mean cell hemoglobin counts (MCHC) (C) white blood cell, neutrophils, Lymphocytes, and red blood cell wide (D) platelet counts in AlCl3-induced anemic rats. Bars are presented in Mean ± standard error of triplicate determinations. Bars with different superscript alphabets (a, b, c, and d) are significantly different between groups at P<0.05.
The effect of alkaloids and flavonoid fractions of C. papaya seed extracts on WBC, N, L, and RDWC counts in AlCl3-induced anemic rats are presented in Figure 2C. These counts were higher in both the alkaloid and flavonoid fractions of C. papaya seed relative to the untreated group. These fractions also compared well with the normal and reference drug groups. Except in the 150 mg/kg bw of CFFCP seed treated group, an increased WBC in the flavonoid fraction treatment group was recorded compared to the normal and untreated groups. Differences in N, L, and RDWC counts were seen in the flavonoid fraction treated rats relative to the untreated and normal groups. The platelet count of the anemic rats is presented in Figure 2D. A lowered platelet count in the 75 mg/kg body weight CFFCP seed compared with all other groups was observed. However, all the other treatment groups had higher platelet counts than the anemic rats.
Anti-anemic activity of AlCl3-induced anemic rats treated with alkaloids and flavonoid fractions of C. papaya leaf
The anti-anemic activity alkaloid and flavonoid fractions of C. papaya leaf in AlCl3-induced anemic rats is displayed in Figure 3. The effects of crude alkaloids and flavonoid fractions of CP leaf on PCV, Hb, and RBC in AlCl3-induced anemic rats are presented in Figure 3A. The lowered PCV, Hb and RBC counts in the anemic untreated group were increased upon fractions treatments which compared well to the normal and standard groups.
Figure 3.
Effect of alkaloids and flavonoid of C. papaya leaf fractions on (A) packed cell volume, hemoglobin, and red blood cells (B) white blood cell, lymphocytes, neutrophils, and red blood cell wide (C) packed cell volume, hemoglobin, and red blood cells (D) platelet counts of AlCl3-induced anemic rats. Bars are presented in Mean ± standard error of triplicate determinations. Bars with different superscript alphabets (a, b, c, and d) are significantly different between groups at P<0.05.
The WBC, L. N and RDWC in AlCl3-induced anemic rats are presented in Figure 3B. Higher levels of WBC, L, N, and RDWC were observed when the rats were treated with CAFCP and CFFCP leaf at all the concentrations tested when compared to untreated anemic rats and normal group. The MCV, MCH and MCHC counts are presented in Figure 3C. MCV was improved upon treatment with alkaloids and flavonoid fractions of C. papaya leaf relative to normal control, standard drug, and untreated groups. MCH was not significantly (P>0.05) different in all extract treated groups relative to the normal group. However, MCHC level was higher in the normal group when compared to the extract treated and untreated groups.
The platelet counts of AlCl3-induced anemic rats are presented in Figure 3D. Higher platelet counts were recorded in the normal and standard groups relative to the extract treated groups and untreated groups. However, decreased platelet counts were observed in the treatment groups when compared with the untreated except the 150 mg/kg bw alkaloids fraction of C. papaya leaf.
Discussion
Anemia is a condition of reduced hematological parameters constituting health, economic and social concerns particularly in the developing and less developed countries [21]. Due to the cost and safety issues associated with conventional medicine, plants are being explored as better alternatives especially since they are rich in phytochemicals of medicinal values. Therefore, our study investigated the protective effect of C. papaya phytochemicals in AlCl3-induced anemic rats.
Phytochemicals
Plant phytochemicals serve protective, hormonal, growth, and developmental purposes to plant [22]. These metabolites are abundant in all plants in different quantities including flavonoids, saponins, alkaloids, dietary fibers, carotenoids, polyphenols, phytoseterols, tannins among others. Several studies have demonstrated the antimicrobial, antidiabetic, antioxidant, anti-dyslipidemia, antiallergic, anti-anemic, anti-obesity, anti-inflammatory, immune boosting, anticancer, gene, neurotransmitter and enzyme modulatory effects and lots more, these functions are attributable to the presence of these chemicals in plants [23-26]. Furthermore, quality and quantity of phytochemicals obtained in a sample are dependent on factors including season, climate, soil type. The choice of solvent and/or heat is also considered for their application in the fields of medicinal and nutraceuticals [27]. The use of compatible solvent medium and suitable method of extraction helps in the retainment of functional properties [28,29].
Carica papaya methanolic extract contains varying quantities of alkaloids, flavonoid, saponins, phenols and tannins in both the leaf and seed. This observation is in line with several studies [14,30] where they reported several phytochemicals in the plant. Saponins are glycosides with distinctive foaming characteristics and had higher contents in the CMECP leaf and seed than the other phytochemicals analyzed. Alkaloids are bioactive compounds of medicinal properties with the ability to intercalate with DNA [31]. Flavonoid and phenols are essential biological antioxidants having the ability to mediate against free radicals’ deleterious effects. Low antioxidants to reactive oxygen species ratio may result in reactions that cause damage to the cells. Cellular damage results in various diseases. The flavonoid content of C. papaya in this study suggests its prevention and management against diseases resulting from oxidative stress such as cardiovascular diseases, atherosclerosis, chronic ulcer, arthritis, ischemic injury, and neuro-degenerative diseases [32].
With the knowledge that each phytochemical has its own function, we proceeded to fractionate alkaloid and flavonoid from the leaf and seed of C. papaya. The findings that the crude methanolic extract outperformed the fractions in terms of anti-anemic and weight increment are suggestive of the possible synergistic effects of the phytochemicals in the plant while the fractions displayed an individual effect. Improved pharmacological effects are reportedly obtained with combinations of compounds [33].
Toxicity test for CMECP
In testing for the safety of potential drug candidates, any substance that does not result to mortality at 5000 mg/kg bw is considered safe and non-lethal [18]. In this study, crude extract of C. papaya leaves, and seed showed high safety margin since no mortality was recorded within 24 hours post extract treatments except for rubbing of the mouth on the surface of the cage and hyperactivity suggesting the relative safety of the extracts. This finding agrees with the findings of [34-36] where they reported that C. papaya did not show any toxic effects at 5000 mg/kg bw and at higher doses used traditionally. However, LD50 may vary due to biological, environmental, and social differences in organisms and may hence not be used to make a general assumption [37].
Hematopoietic effect of CMECP
The administration of CMECP improved the effect of aluminium induced toxicity. The observed body weight increases in the treated groups relative to the anemic untreated group might be adduced to the nutrients present in C. papaya [38]. This agrees with the findings of other researchers [39]. The significant loss in weight could also be linked to aluminium-induced oxidative stress which may result in free radical mediated cytotoxicity and reduced cholinergic function, decrease in disaccharidases (sucrase and lactase enzymes that catalyzed the last stage of carbohydrate digestion), or inhibition of enzymes such as hexokinase, acid and alkaline phosphatase, phosphodiesterase and phosphooxidase activities and consequently results in tissue wasting [40,41].
The pathological, nutritional, and physiological status of organisms can be examined via the blood [42]. Examination of hematological parameters could also indicate the effect of xenobiotics in animals [43]. The significant decrease in PCV, Hb and RBC levels in the anemic group could be due to toxicity caused by AlCl3. Aluminium chloride toxicity reportedly inhibits heme synthesis, inhibition of iron utilization, destruction of matured RBC and enzyme activity resulting in a decrease in PCV and RBC [44,45]. However, aluminum-induced hematological alterations in MCV, MCH and MCHC might be adduced to microcytic hypochromic anemia.
The reduction observed in RBC, Hgb and PCV levels of the anemic rats was probably due to the oxidation of Fe2+ to form Fe3+ by aluminium, thus causing alterations in RBC, Hgb and PCV. However, the reversal of this effect upon extract administration might be due to the haemopoietic promoting factors such as iron, potassium, calcium, vitamins, and fiber content of the plant [46,47]. The detoxifying function of saponins which allows for the lysing of RBC [48] could have been circumvented by the extract availing iron for the synthesis of new RBCs thus improving the Hb, PCV and RBC of the extract treated groups. The increase in treated groups lymphocyte counts may suggest the inhibition of possible lymphocytosis that could have occurred in the anemic rats [49]. Again, AlCl3 probably caused a decrease in the immune system predisposing animals to infection. The immune boosting potential of the extracts could have led to elevation in the WBC levels [50]. Interestingly, the high platelet counts in the extract treated groups could be due to their ability to produce stimulating effects on platelet production, suggesting their role as a clot-formation agent [51].
According to the study of Osman et al. [52] reduced MCV, MCH and MCHC counts are reported in cases of aluminium toxicity. However, crude extracted groups could not reverse the reduced levels observed in the anemic group while the increase in the fraction treated groups could imply that the induction of anemia was reversed, and the fraction might probably prevent other pathological conditions like liver cirrhosis and hemolytic anemia [53]. The increase in RBC, WBC, Hb, and PCV levels of the extract treated groups might be due to the high flavonoid and alkaloid levels present in the plant.
Hematopoietic effect of alkaloids and flavonoid seed and leaf fractions of C. papaya
The reduction in weight of anemic rats was reversed in the treatment groups. The significant weight loss could arise from the deleterious effect of the reactive oxides induced by administration of aluminium chloride [54]. The destruction of matured RBC by aluminium could have resulted in the reduced hematological parameters such as RBC, PCV, and Hb observed in the untreated anemic rats. This fact is also supported by the findings of Muhammad and Oloyede [45] and Ogbonnia et al. [55] that heme biosynthesis is impaired in aluminium induced anemia. The reduction in RBC count and PCV may also be due to the hemolytic action of aluminium resulting from the replacement of Fe3+ by Al3+ during hemoglobin formation [56]. Treatment with alkaloids and flavonoid fractions of C. papaya leaf prevented the occurrence of anemia in the experimental groups as significantly higher RBC count, hemoglobin and PCV were obtained as compared to the non-treated group (AlCl3 only administered rats). Flavonoid and alkaloids are antioxidants that have direct influence on blood formation with the ability to inhibit free radical-induced blood cell damage [46].
The flavonoid fraction of C. papaya seed showed no positive effect on hematopoietic system of anemic rats suggesting that flavonoid fractions of C. papaya seed are not a major hematopoietic stimulatory bioactive metabolite. However, the ability of the alkaloid fraction to improve the hematological status to levels comparable to the normal rats is suggestive of alkaloids as a hematopoietic stimulatory bioactive metabolite in C. papaya seed. The fractions also brought about improvement in immune response as observed in the elevated WBC levels whereas, the anemic rats could have compromised immune system that have overwhelmed the WBC defense mechanism thus the reduced WBC level in the anemic untreated rat [57,58].
Conclusion
Carica papaya leaves and seeds are rich in phytocompounds, this makes it a promising in phytotherapy and could be used in the protective and management of diseases resulting from oxidative stress such as anemia. Due to its phytoconstituents, C. papaya had shown the ability to protect and restore the integrity of hemoglobin, PCV and RBC and other hematological parameters that were compromised by AlCl3-induction, therefore it may possess haemato-protective and hematinic potentials. The general observation of improved anti-anemic capability and weight gain effect of the crude methanolic extract is suggestive of the combinatory effect of the phytoconstituents. However, further studies should be carried out to investigate the mode of anti-anemic action of C. papaya, its effects iron deficiency anemia as well as metabolomic profiling of C. papaya constituents.
Acknowledgements
The authors would like to extend their gratitude to King Saud University (Riyadh, Saudi Arabia) for supporting this research through Researchers Supporting Project number (RSP-2024-R406).
Disclosure of conflict of interest
None.
References
- 1.Pingali PS, Srinivas P, Reddy M. Study of anti-anaemic effect of Schrebera swietenioides roxb. In rat models. Asian J Pharm Clin Res. 2015:260–263. [Google Scholar]
- 2.Qaseem A, Humphrey LL, Fitterman N, Starkey M, Shekelle P Clinical Guidelines Committee of the American College of Physicians. Treatment of anemia in patients with heart disease: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2013;159:770–779. doi: 10.7326/0003-4819-159-11-201312030-00009. [DOI] [PubMed] [Google Scholar]
- 3.Lippi G, Schena F, Salvagno GL, Aloe R, Banfi G, Guidi GC. Foot-strike haemolysis after a 60-km ultramarathon. Blood Transfus. 2012;10:377–383. doi: 10.2450/2012.0167-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Sharda S, Shukla A, Singh C, Bigoniya P. A review on herbal anti-anaemia plants RGI. Int J Appl Sci Technol. 2011;3:31–42. [Google Scholar]
- 5.Tata PI, Afari-Sefa V, Ntsomboh NG, Ngome AF, Okolle NJ, Billa SF. Policy and institutional frameworks impacting on vegetable seed production and distribution systems in Cameroon. J Crop Improv. 2016;30:196–216. [Google Scholar]
- 6.Vota DM, Crisp RL, Nesse AB, Vittori DC. Oxidative stress due to aluminum exposure induces eryptosis which is prevented by erythropoietin. J Cell Biochem. 2012;113:1581–1589. doi: 10.1002/jcb.24026. [DOI] [PubMed] [Google Scholar]
- 7.GBD 2021 Anaemia Collaborators. Prevalence, years lived with disability, and trends in anaemia burden by severity and cause, 1990-2021: findings from the Global Burden of Disease Study 2021. Lancet Haematol. 2023;10:e713–e734. doi: 10.1016/S2352-3026(23)00160-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Zegeye B, Anyiam FE, Ahinkorah BO, Ameyaw EK, Budu E, Seidu AA, Yaya S. Prevalence of anemia and its associated factors among married women in 19 sub-Saharan African countries. Arch Public Health. 2021;79:214. doi: 10.1186/s13690-021-00733-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Azinge IE, Ogunyemi A, Ogamba CF, Jimoh RO. Prevalence of anemia and associated factors among adults in a select population in Lagos, Southwest Nigeria. J Public Health Afr. 2023;14:2224. doi: 10.4081/jphia.2023.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Iwalewa EO, Adewunmi CO, Omisore NO, Adebanji OA, Azike CK, Adigun AO, Adesina OA, Olowoyo OG. Pro- and antioxidant effects and cytoprotective potentials of nine edible vegetables in southwest Nigeria. J Med Food. 2005;8:539–544. doi: 10.1089/jmf.2005.8.539. [DOI] [PubMed] [Google Scholar]
- 11.Kimetu JM, Lehmann J. Stability and stabilisation of biochar and green manure in soil with different organic carbon contents. Soil Res. 2010;48:577–585. [Google Scholar]
- 12.Magtalas MC, Balbin PT, Cruz EC, Clemente RF, Buan AKG, Garcia JP, Lee KY, Tantengco OAG. Ethnomedicinal plants used for the prevention and treatment of anemia in the Philippines: a systematic review. Trop Med Health. 2023;51:27. doi: 10.1186/s41182-023-00515-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Oloyede HO, Lukman HY, Salawu MO. Protective potentials of ethyl acetate-ethanolic fraction of Carica papaya leaves against acetaminophen-induced liver damage in rats. Not Sci Biol. 2020;12:556–567. [Google Scholar]
- 14.Umana U, Timbuak J, Musa S, Asala S, Hambolu J, Anuka A. Acute and chronic hepatotoxicity and nephrotoxicity study of orally administered chloroform extract of Carica papaya seeds in adult Wistar rats. Int J Sci Res Publ. 2013;3:1–8. [Google Scholar]
- 15.Nugroho A, Heryani H, Choi JS, Park HJ. Identification and quantification of flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity. Asian Pac J Trop Biomed. 2017;7:208–213. [Google Scholar]
- 16.Jouad H, Haloui M, Rhiouani H, El Hilaly J, Eddouks M. Ethnobotanical survey of medicinal plants used for the treatment of diabetes, cardiac and renal diseases in the North centre region of Morocco (Fez-Boulemane) J Ethnopharmacol. 2001;77:175–182. doi: 10.1016/s0378-8741(01)00289-6. [DOI] [PubMed] [Google Scholar]
- 17.Gonzales MVM, Tolentino AG. Extraction and isolation of the alkaloids from the Samanea saman (Acacia) bark: its antiseptic potential. Int J Sci Technol Res. 2014;3:119–124. [Google Scholar]
- 18.Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol. 1983;54:275–287. doi: 10.1007/BF01234480. [DOI] [PubMed] [Google Scholar]
- 19.Yusuf Z, Zaifada A, Haruna A, Adam M, Umar M, Mohammed A. Effects of medetomidine and thiopentone anaesthesia on the haematological and some serum biochemical changes in donkeys (Equus asinus) in Maiduguri, Nigeria. Niger Vet J. 2018;39:51–56. [Google Scholar]
- 20.Dacie JV, Lewis SM. Determination of Haematological Parameters. Practical Haematology. Edinburgh, UK: Churchill Livingstone; 2002. p. 48. [Google Scholar]
- 21.Kumar SB, Arnipalli SR, Mehta P, Carrau S, Ziouzenkova O. Iron deficiency anemia: efficacy and limitations of nutritional and comprehensive mitigation strategies. Nutrients. 2022;14:2976. doi: 10.3390/nu14142976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Sharma BR, Kumar V, Gat Y, Kumar N, Parashar A, Pinakin DJ. Microbial maceration: a sustainable approach for phytochemical extraction. 3 Biotech. 2018;8:401. doi: 10.1007/s13205-018-1423-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Rowles JL 3rd, Erdman JW Jr. Carotenoids and their role in cancer prevention. Biochim Biophys Acta Mol Cell Biol Lipids. 2020;1865:158613. doi: 10.1016/j.bbalip.2020.158613. [DOI] [PubMed] [Google Scholar]
- 24.Jiang Y, Chen L, Taylor RN, Li C, Zhou X. Physiological and pathological implications of retinoid action in the endometrium. J Endocrinol. 2018;236:R169–R188. doi: 10.1530/JOE-17-0544. [DOI] [PubMed] [Google Scholar]
- 25.Jaeger R, Cuny E. Terpenoids with special pharmacological significance: a review. Nat Prod Commun. 2016;11:1373–1390. [PubMed] [Google Scholar]
- 26.Vallverdú-Coll N, Ortiz-Santaliestra ME, Mougeot F, Vidal D, Mateo R. Sublethal Pb exposure produces season-dependent effects on immune response, oxidative balance and investment in carotenoid-based coloration in red-legged partridges. Environ Sci Technol. 2015;49:3839–3850. doi: 10.1021/es505148d. [DOI] [PubMed] [Google Scholar]
- 27.Dike I, Ibojo OO, Daramola F, Omonhinmin A. Phytochemical and proximate analysis of foliage and seed of Bixa orellana linn. Int J Pharm Sci Rev Res. 2016;36:247–251. [Google Scholar]
- 28.Carreira-Casais A, Otero P, Garcia-Perez P, Garcia-Oliveira P, Pereira AG, Carpena M, Soria-Lopez A, Simal-Gandara J, Prieto MA. Benefits and drawbacks of ultrasound-assisted extraction for the recovery of bioactive compounds from marine algae. Int J Environ Res Public Health. 2021;18:9153. doi: 10.3390/ijerph18179153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Quitério E, Grosso C, Ferraz R, Delerue-Matos C, Soares C. A critical comparison of the advanced extraction techniques applied to obtain health-promoting compounds from seaweeds. Mar Drugs. 2022;20:677. doi: 10.3390/md20110677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Shikov AN, Mikhailovskaya IY, Narkevich IA, Flisyuk EV, Pozharitskaya ON. Methods of extraction of medicinal plants. Evidence-Based Validation of Herbal Medicine. Elsevier; 2022. pp. 771–796. [Google Scholar]
- 31.Kasolo JN, Bimenya GS, Ojok L, Ochieng J, Ogwal-Okeng JW. Phytochemicals and uses of Moringa oleifera leaves in Ugandan rural communities. J Med Plant Res. 2010;4:753–757. [Google Scholar]
- 32.Burlon G, Ingold K. B-Carotene: an unusual type of lipid antioxidant. J Sci. 1984;224:573. doi: 10.1126/science.6710156. [DOI] [PubMed] [Google Scholar]
- 33.Ayaz M, Ullah F, Sadiq A, Ullah F, Ovais M, Ahmed J, Devkota HP. Synergistic interactions of phytochemicals with antimicrobial agents: potential strategy to counteract drug resistance. Chem Biol Interact. 2019;308:294–303. doi: 10.1016/j.cbi.2019.05.050. [DOI] [PubMed] [Google Scholar]
- 34.Adeneye AA. Subchronic and chronic toxicities of African medicinal plants. Toxicological survey of african medicinal plants. Elsevier; 2014. pp. 99–133. [Google Scholar]
- 35.Ismail Z, Halim SZ, Abdullah NR, Afzan A, Abdul Rashid BA, Jantan I. Safety evaluation of oral toxicity of Carica papaya Linn. leaves: a subchronic toxicity study in sprague dawley rats. Evid Based Complement Alternat Med. 2014;2014:741470. doi: 10.1155/2014/741470. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Halim S, Abdullah N, Afzan A, Rashid BA, Jantan I, Ismail Z. Acute toxicity study of Carica papaya leaf extract in Sprague Dawley rats. J Med Plant Res. 2011;5:1867–1872. [Google Scholar]
- 37.Devaki K, Beulah U, Akila G, Gopalakrishnan VK. Effect of aqueous extract of Passiflora edulis on biochemical and hematological parameters of Wistar albino rats. Toxicol Int. 2012;19:63–7. doi: 10.4103/0971-6580.94508. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Bhushan K. Studies on biological efficacy of various leaf extracts of Carica papaya L. ICGTETM. 2016;2:510–516. [Google Scholar]
- 39.Opasich C, Cazzola M, Scelsi L, De Feo S, Bosimini E, Lagioia R, Febo O, Ferrari R, Fucili A, Moratti R, Tramarin R, Tavazzi L. Blunted erythropoietin production and defective iron supply for erythropoiesis as major causes of anaemia in patients with chronic heart failure. Eur Heart J. 2005;26:2232–2237. doi: 10.1093/eurheartj/ehi388. [DOI] [PubMed] [Google Scholar]
- 40.Gebauer J, El-Siddig K, Ebert G. Baobab (Adansonia digitata L.): a review on a multipurpose tree with promising future in the Sudan. Gartenbauwissenschaft. 2002;67:155–160. [Google Scholar]
- 41.Al-Hashem FH. Camel’s milk alleviates oxidative stress and lipid peroxidation induced by chronic aluminum chloride exposure in rat’s testes. Am J of App Sci. 2009;6:1868. [Google Scholar]
- 42.Ahaotu E, Mbaegbu I. Effects of water leaf (Talinum triangulare) shoot meal on the performance of weaner New Zealand white rabbits. Greener Journal of Animal Breeding and Genetics. 2017;3:018–024. [Google Scholar]
- 43.Berinyuy E, Lawal B, Olalekan L, Olalekan I, Yusuf A, Sakpe S, Ossai P. Hematological status and organs/body-weight parameters in Wister rats during chronic administration of Cassia occidentalis. Int Blood Res Rev. 2015;4:1–7. [Google Scholar]
- 44.Han J, Han J, Dunn MA. Effect of dietary aluminum on tissue nonheme iron and ferritin levels in the chick. Toxicology. 2000;142:97–109. doi: 10.1016/s0300-483x(99)00119-5. [DOI] [PubMed] [Google Scholar]
- 45.Muhammad N, Oloyede O. Haematological parameters of broiler chicks fed Aspergillus niger-fermented Terminalia catappa seed meal-based diet. Global J Biotechnol Biochem. 2009;4:179–183. [Google Scholar]
- 46.Beltowski J, Wojcicka G, Gorny D, Marciniak A. The effect of dietary-induced obesity on lipid peroxidation, antioxidant enzymes and total plasma antioxidant capacity. J Physiol Pharmacol. 2000;51:883–96. [PubMed] [Google Scholar]
- 47.Ayoola P, Adeyeye A. Phytochemical and nutrient evaluation of Carica papaya (pawpaw) leaves. Int J Appl Sci. 2010;5:325–328. [Google Scholar]
- 48.Bissinger R, Modicano P, Alzoubi K, Honisch S, Faggio C, Abed M, Lang F. Effect of saponin on erythrocytes. Int J Hematol. 2014;100:51–59. doi: 10.1007/s12185-014-1605-z. [DOI] [PubMed] [Google Scholar]
- 49.Zhuang C, Huo H, Fu W, Huang W, Han L, Song M, Li Y. Aluminum chloride induced splenic lymphocytes apoptosis through NF-κB inhibition. Chem Biol Interact. 2016;257:94–100. doi: 10.1016/j.cbi.2016.07.033. [DOI] [PubMed] [Google Scholar]
- 50.Anacletus F, Onyegeme-Okerenta B. Induction of hematological and lipid profile changes by aluminium induced toxicity and ameliorative effects of selected antioxidants on Wistar rats. Health Sci Res. 2016;3:30–34. [Google Scholar]
- 51.Hall JE. Guyton and Hall Textbook of Medical Physiology. Jordanian Edition E-Book. Elsevier Health Sciences. 2016 [Google Scholar]
- 52.Osman HM, Shayoub ME, Babiker EM. Effect of ethanolic leaf extract of Moringa oleifera on aluminum-induced anemia in white albino rats. Jordan J Biol Sci. 2012;147:1–5. [Google Scholar]
- 53.Sembulingam K, Sembulingam P. Essentials of medical physiology. 5th ed. Jaypee Brothers Medical Limited; 2010. pp. 85–89. [Google Scholar]
- 54.Shackley S, Sohi S, Brownsort P, Carter S, Cook J, Cunningham C, Gaunt J, Hammond J, Ibarrola R, Mašek O. An assessment of the benefits and issues associated with the application of biochar to soil. Department for Environment, Food and Rural Affairs, UK Government, London. 2010 [Google Scholar]
- 55.Steve O, Ogbonnia E, Florence N, Anyika E. Evaluation of acute and subchronic toxicity of Stachytarpheta angustifolia extract in animals. Afr J Biotech. 2009;8:1793–1799. [Google Scholar]
- 56.Uhunmwangho ES, Omoregie ES. Antioxidant properties associated with the biochemical changes in the development of African walnut (Tetracarpidium conophorum) fruit. Saudi J Life Sci. 2017;2:217–229. [Google Scholar]
- 57.el-Demerdash FM, Yousef MI, Kedwany FS, Baghdadi HH. Role of α-tocopherol and β-carotene in ameliorating the fenvalerate-induced changes in oxidative stress, hemato-biochemical parameters, and semen quality of male rats. J Environ Sci Health B. 2004;39:443–459. doi: 10.1081/pfc-120035929. [DOI] [PubMed] [Google Scholar]
- 58.Ganong WF. Review of medical physiology. Dynamics of Blood and Lymph Flow. 1995;30:525–541. [Google Scholar]