Research article Open Access
Toxicity Evaluation of the Extract and Fraction of Chrysophyllum albidum Seed Cotyledons in Rats
Adedoyin Akinmayowa Shobo1, Michael Oluwatoyin Daniyan1*, Gbola Olayiwola2, Thomas Oyebode Idowu3, Abiodun Oguntuga Ogundaini3 and Saburi Adejimi Adesanya4
1Department of Pharmacology, Faculty of Pharmacy, Obafemi A wolowo University, Ile-Ife, Nigeria.
2Department of Clinical Pharmacy and Pharmacy Administration, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
4Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
*Corresponding author: Michael Oluwatoyin Daniyan, PhD, Senior Lecturer, Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University,Ile-Ife, 220005, Osun State, Nigeria. E-mail: @
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Received: January 11, 2019; Accepted: February 15, 2019; Published: February 20, 2019
Citation: Shobo AA, Daniyan MO, Olayiwola G, et al. (2019) Toxicity Evaluation of the Extract and Fraction of Chrysophyllum albidum Seed Cotyledons in Rats. SOJ Pharm Sci, 6(1) 1-12. DOI: 10.15226/2374-6866/6/1/00194
Abstract
The seed of Chrysophyllum albidum (G.Don) Sapotaceae, is widely employed for its economic and medicinal values, necessitating the need to establish its safety. Therefore, the toxicity profiles of the methanol extract and butanol fraction of C. albidum seed cotyledons following acute and 28-day repeated dosing was investigated in rats using OECD Test Guidelines 420 and 407. The median lethal dose (LD50) of the extract and fraction were 760 and 200 mg/kg respectively, and both effect varying degree of significant changes at p < 0.05 in organ weights, organ-brain weight ratio, hematological indices (WBC, RBC, Hb, HCT, MCV, MCH, MCHC), and biochemical indices (AST, ALT, creatinine, direct and total bilirubin) as well as observed pathological changes following acute and repeated dose administration. The results showed potential to cause mild to moderate toxicity, especially more with butanol fraction, and at higher doses. The recovery data showed a potential for recovery from toxic effects, but also provided a reflection of delayed toxicity. Hence, while taking advantage of the many medicinal and economic potentials of C. albidum seed cotyledons, its potential toxic effects need to be considered.

Keywords: Medicinal plants; Toxicity profiles; Acute toxicity; Repeated dose toxicity; OECD;
Abbreviations
OECD, Organization for Economic Co-operation and Development; TG, Test Guidelines; ME and BF, Methanol extract and Butanol fraction of C. albidum seed cotyledons respectively; FOB, Functional observatory batteries; FWR, Female Wistar rats; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; WBC, White blood cell; RBC, Red blood cell; Hb, Hemoglobin concentration; HCT, Hematocrit; MCV, Mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, Mean corpuscular hemoglobin concentration.
Introduction
Medicinal plants have been employed in the treatment and management of many diseases [1,2], and there are increasing global calls, especially in Africa, to promote and integrate traditional medical practices into their health system [3]. Cost effectiveness, ease of accessibility, wider cultural acceptability, source of raw materials as well as potential chemical candidates for drug discovery are some of the factors responsible for upsurge of interest in medicinal plants [1]. However, the extensive and indiscriminate uses of these plant-based medicines among other concerns, makes the evaluation of their toxicity imperative [4].

Among the medicinal plants that are widely consumed and known for therapeutic properties is the Chrysophyllum albidum (G.Don) Sapotaceae (White Star Apple) [5]. The tree plant is popularly referred to as “agbalumo” (South-western Nigeria), “udara” (South-eastern Nigeria), agwaluma (Northern Nigerian) [2,6]. It is a dominant canopy tree of lowland mixed rain forest widely distributed throughout the tropical Central, East and West Africa regions [2,6]. The oil of C. albidum seed cotyledons is employed in soap and candle production and as lubricant, while the stem-bark is used as remedy for malaria, sleeping sickness, and yellow fever [2,7]. The leaves are used as emollients and for the treatment of skin eruptions, diarrhoea and stomach ache [2,6]. In addition, the C. albidum seed cotyledons have been reported to have a number of folkloric applications, including wound healing, treatment of oligospermia, amenorrhea, certain dermatological and vaginal infections, intestinal worms and hemorrhoids [2,8]. Previous investigation of the chemical constituents of C. albidum showed that the stem-bark contains stigmasterol, epicatechin, epigallocatechin and procyanidin B5 [9]. Phytochemical studies of the crude and methanol extracts of seed cotyledons revealed the presence of saponin, alkaloid, tannin, flavonoid, sterol and anthraquinone [10]. Earlier, bioassay-guided fractionation of the methanol extract of the C. albidum seed cotyledons led to the isolation of eleagnine, tetrahydro-2-methylharman and skatole [11]. Like many herbal products containing β-carbolines, the presence of β-carboline alkaloids in C. albidum seed cotyledons suggests its potential usefulness in the treatment of cancer, neurological disorders, malaria, jaundice and asthma [12,13]. The known pharmacological activities of C. albidum seed cotyledons, attributable to the presence of eleagnine, are antioxidant, anti-inflammatory, anti-nociceptive and anti-microbial activities [11, 14, 15, 16]. Also reported are the biochemical effect and membrane stabilization potential of eleagnine [17] and anti-hyperglycemic and hypolipidemic properties of the ethanol extract of C. albidum seed cotyledons [18].

However, in spite of these plethoras of pharmacologic and economic potentials, the toxicity profile remains largely unexplored. In this study, we report the evaluation of the toxicity profile of the extract and fraction of C. albidum seed cotyledons.
Materials and Methods
Plant Material: Collection, extraction and fraction
The fresh fruits of C. albidum were bought from Sabo fruit market at Ile-Ife, Osun State, Nigeria. It was identified and authenticated by Mr. A.T. Oladele of the Department of Pharmacognosy, Faculty of Pharmacy, Obafemi Awolowo University (OAU), Ile-Ife, Osun State, Nigeria, where the herbarium specimen, with a voucher number FPH/S/001, was deposited. The plant name was also checked with http://www. theplantlist.org for confirmation. The seeds were separated from the fresh fruits, de-shelled to obtain the white cotyledons, and air-dried at room temperature. The air-dried cotyledons (500 g) were subsequently pulverized using mortar and pestle, and extracted with 2.5 litres of 100 % methanol three times at room temperature for 72 hours. The pooled extract was concentrated in a rotary evaporator to obtain the crude methanol extract (68 g). Forty grams (40 g) of the crude extract was dissolved in distilled water and successively partitioned with ethyl acetate and butanol. The resultant ethyl acetate, butanol and aqueous fractions were concentrated to dryness in vacuo.
Care and use of experimental Animals
Experiments were performed using nulliparous Female Wistar rats (FWRs), weighing 130 – 150 g, and bred locally in the animal holdings of the Department of Pharmacology, Faculty of Pharmacy, OAU, Ile-Ife, Nigeria. FWRs were housed in standard plastic cages, exposed to natural room temperature and lighting conditions, and allowed one week acclimatization with free access to standard laboratory pellets (Grand Cereals, United African Company Plc, Nigeria) and water ad libitum. The procedure for the care and use of animals was in strict compliance with the recommendations in the “Guide for the Care and Use of Laboratory Animals – Eighth Edition” of the National Research Council of the National Academies, USA [19]. The protocol was approved by the Committee on the care and use of laboratory animals, Obafemi Awolowo University, Ile-Ife, Nigeria (Protocol number PHP12/13/H/0601).
Median lethal dose (LD50) determination and Sighting study
Median lethal dose (LD50) values of ME and BF were determined using Lorke’s method [20] and Hodge and Sterner scale [21] was used in the categorization of the degree of toxicity. A preliminary sighting study [22] using functional observatory battery (FOB) [23] was conducted to determine the humane endpoint criteria and for the selection of the appropriate doses for the main study. Test agents were administered orally. FOB used in this study consists of twenty functional and behavioural activities, including behavioral changes, motor activity, sensory reflex, coordination, respiratory distress, vocalisation, oculonasal discharge, fear and death. Using LD50 values as guide, the starting dose for the sighting study, selected from the Organisation for Economic, Co-operation and Development (OECD) Test Guideline (TG) 420 [22] fixed dose levels of 5, 50, 300 and 2000 mg/kg, was fixed at 50 mg/kg for BF and 300 mg/kg for ME. Based on the resultant evident or lack of evident toxicity / mortality, subsequent dose levels were selected from the OECD fixed dose levels, including a control. Each dose level uses one (1) FWR with a period of 24 hours in-between dosing. Where death occurs, a confirmatory test with a second FWR was conducted. FOB were monitored continuously for the first 30 minutes, then every 30 minutes for 4 hours, and thereafter at regular interval for 24 hours and daily for a total of 14 days.
Experimental design
The experiment was divided into two test phases: Acute and repeated dose toxicity studies. FWRs were fasted overnight before the start of the studies. Test agents were administered orally.

The acute toxicity study was conducted using the OECD TG 420 [22], with 35 FWRs, randomly allotted to 7 groups of 5 rats each, namely, control group 1, ME treated groups 2, 3, 4 and BF treated groups 5, 6, 7. Group 1 receives single oral dose of normal saline (0.9 % NaCl w/v). Groups 2, 3 and 4 were administered single oral doses of 150, 300, and 600 mg/kg body weight ME respectively. Groups 5, 6 and 7 were treated orally with single doses of 40, 80, 160 mg/kg body weight BF respectively. FWR body weights were taken before dose administration, and at least twice weekly for two weeks. FOBS were monitored as described above.

The repeated dose toxicity test was conducted in accordance with the OECD TG 407 [24]. Fifty FWRs were used, and were randomly divided to 5 groups of 10 rats each for control (group 1), ME treated groups 2 and 3 and BF treated groups 4 and 5. Group 1 received normal saline (0.9 % NaCl w/v), groups 2 and 3 received 100 and 300 mg/kg body weight of ME respectively, while groups 4 and 5 were given 50 and 150 mg/kg body weight BF respectively. All doses were administered once daily for 28 days and FWRs body weights were taken periodically. On day 28, the control and surviving members of each treated groups 2, 3, 4 and 5, were randomly divided into two equal Sets: Toxicity and Recovery. Recovery set were allowed a further 21 days of nondosing recovery period. Following each daily administration, FOB were monitored continuously for the first 30 minutes, then once every 30 minutes for 4 hours.

In all the experiments, doses were prepared using physiological saline (0.9 % NaCl w/v) and volume of administered doses was not more than 5 ml / kg body weight. Furthermore, on day 14 for acute toxicity test, and days 29 and 49 for repeated dose Toxicity and Recovery sets respectively, the surviving rats were euthanized using cervical dislocation, and all efforts were made to minimize suffering. Blood samples were collected by cardiac puncture into EDTA sample bottles and processed for hematological and biochemical assays, while organs samples (brain, liver and kidney) were isolated and weighed. Liver and kidney were then processed for histopathological analysis.
Biochemical assays
Blood samples in EDTA tubes were centrifuged at 3000 rpm for 5 minutes to obtain the plasma for biochemical assays. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (total and direct) and creatinine, were assayed using standard biochemical kits as per manufacturer’s instructions (Randox Laboratories Limited, Crumlin, County Antrim, BT294QY, United Kingdom).
Hematological assays
The whole blood samples were subjected to hematological analysis using Mindray BC 2800 Haematology Auto-Analyzer [25]. WBC, RBC, Hb, HCT, MCV, MCH, MCHC were analyzed.
Histopathological examination
The liver and the two kidneys were fixed in 10% buffered formalin and dehydrated using increasing concentrations of isopropyl alcohol (80 - 100%). The organs were embedded in paraffin, and sectioned at 5 μm thickness using a Leica rotary microtome (Bright B5143 Huntington, England). The sections were subjected to routine hematoxylin–eosin (HE) staining, involving deparaffinization, hydration, staining, rinsing and clearing in xylene in line with standard procedures [26,27]. Slides were viewed under light microscope with photomicrographs taken with a Leica DM750 Camera Microscope (X 400).
Statistical analysis
Data were expressed as mean ± standard error of mean (SEM), and analyzed using Student’s t – test and / or one – way analysis of variance (ANOVA) followed by Dunnett’s post hoc test using Graph Pad Prism version 5.01 (Graph Pad software, San Diego, California, U.S.A). The level of significance was set at p < 0.05.
Results and Discussion
Medicinal plants remain an indispensable alternative and complementary pharmacotherapy in the management of many diseases, necessitating the need to establish their safety [1]. In this report, we have evaluated the toxicity of C. albidum seed cotyledons methanol extract and butanol fraction using acute and repeated dose toxicity models with a view to ascertain their safety and the possibility of reversibility or persistence of their toxic effects. Butanol fraction was selected based on our earlier work that found eleaginine, its essential active phytochemicals, in butanol fraction following TLC derivatization of the three fractions using Draggendorff’s reagent [11,17].
Median lethal dose (LD50) and humane endpoint criteria
The estimated oral LD50 of the ME (760 mg/kg) and BF (200 mg/kg) provided an indication that they could be slightly and moderately toxic respectively [21]. Also, the sighting studies revealed that loss of righting reflex and respiratory distress are the obvious FOB observed in all rats that died (Table 1), and are therefore used as humane endpoint criteria in subsequent acute and repeated dose toxicity studies.
Table 1:Summary of cage side observations following single and repeated oral dose administrations of Methanol extract and Butanol fraction of C.albidum cotyledon

Signs of toxicity

Single dose oral administration (x/5)

Repeated dose oral administration (x/10)

Control

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

Control

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

150

300

600

40

80

160

100

300

50

150

Piloerection*

0/5

0/5

0/5

2/5

0/5

1/5

1/5

0/10

0/10

2/10

2/10

1/10

Reaction to handling

0/5

0/5

0/5

0/5

0/5

1/5

0/5

0/10

0/10

3/10

0/10

2/10

Palpebral closure

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

5/10

0/10

0/10

Eye colour

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Lacrimation

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Gait

0/5

0/5

0/5

0/5

0/5

1/5

0/5

0/10

0/10

3/10

0/10

4/10

Sedation

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Skin colour

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Respiration distress#

0/5

0/5

0/5

2/5

0/5

0/5

1/5

0/10

0/10

2/10

1/10

4/10

Tremor

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Convulsion

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Defecation

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Vocalisation*

0/5

0/5

0/5

2/5

0/5

1/5

2/5

0/10

0/10

4/10

4/10

5/10

Loss of righting reflex#

0/5

0/5

0/5

2/5

0/5

0/5

1/5

0/10

0/10

2/10

1/10

4/10

Oculonasal discharge*

0/5

0/5

0/5

2/5

1/5

1/5

2/5

0/10

8/10

10/10

6/10

8/10

Hypokinesia*

0/5

0/5

1/5

2/5

0/5

2/5

3/5

0/10

0/10

9/10

3/10

6/10

Tail elevation

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Inappetence*

0/5

0/5

1/5

2/5

0/5

2/5

4/5

0/10

0/10

3/10

1/10

4/10

Fear

0/5

0/5

0/5

0/5

0/5

0/5

0/5

0/10

0/10

0/10

0/10

0/10

Death

0/5

0/5

0/5

2/5

0/5

0/5

1/5

0/10

0/10

2/10

1/10

4/10

Functional observational battery (FOB) in toxicity studies
The acute toxicity study shows no clear signs of intoxication (including mortality) aside general malaise such as lethargy, transient hypoactivity and inappetence in the FWR administered, except at 600 mg/kg methanol extract (ME) where 40% mortality was observed and 20% mortality at 160 mg/kg butanol fraction (BF) (Table 1). On the other hand, repeated dose toxicity study showed 20% mortality at 300 mg/kg ME, and 10% and 40 % mortalities at 50 mg/kg and 150 mg/kg BF respectively (Table 1). It should be noted that FOB [23], revealed that while the behavioural alterations during the course of the acute and 28 days repeated administration are similar, they also correlated well with the rate of mortality, indicating that BF could be more toxic than ME (Table 1). It is proposed that the observed toxicity may be connected with the reported depressant or inhibitory effect of the test agents on the central nervous system (CNS) [17].

Toxicological assessment of C. albidum seed cotyledons extract and fraction

Effect on body weight, organ weights and organ-brain weight ratio

Available evidence has shown that body weight gain, organ weights and organ - brain weight ratio are important and sensitive indices of toxic effects [28]. Essentially, organ – brain weight ratio is a more relevant index for toxicity in cases where significant variations in body weight is inevitable, as test materials that alter body weight generally do not alter brain weight [28]. In the acute toxicity study, though significant differences (p < 0.05) in relative body weight were observed in all treated FWRs at all tested doses when compared to control (Table 2), lack of significant changes in relative organ weights and organ–brain weight ratio (Table 4) suggest that any potential toxicity of the test agents may be temporary. On the other hand, following repeated oral administration, significant increase in body weights between sample days (p > 0.05) and during recovery were observed (Table 3). However, 50 mg/kg BF produced a significant decrease in organs weights, and at higher doses of 300 mg/kg ME and 150 mg/kg BF, there were significant decrease in organs weights and significant increase in organ-brain weight ratio (P < 0.05) (Table 4). Therefore, the significant reduction in relative organs weights, as well as significant increases in organ - brain weights ratio at higher doses, suggest that at higher doses, which is often the case with indiscriminate use of medicinal plants, there may be an increased risk of organs toxicity [28]. Also, the general increase in organ - brain weight ratio in recovery group compared to toxicity group in all tested doses (Table 5) may suggest a form of delayed toxicity [29].
Effect on hematological indices
Blood plays important roles in maintaining body functions and homeostasis [30]. Generally, following single and repeated dose administrations of either the extract or fraction (Table 6), the results showed varying degree of significant changes (p < 0.05) in the hematological indices (WBC, RBC, Hb, HCT, MCV, MCH, MCHC) and appear to be dose dependent and more pronounced with BF (Table 6). In acute toxicity test, apart from ME at 150 mg/kg, where RBC was significantly higher, other doses showed significant decreases in RBC. Also, there were consistent significant decreases in hemoglobin concentration, HCT, MCH and MCHC in all tested doses of ME and BF. And while all the doses of BF showed significant reduction in MCV, only at 150 mg/kg ME was there such an effect observed. Meanwhile, upon repeated administration of the test substances (ME and BF), there were significant changes in RBC, MCV and MCH in all the tested doses and only at 50 mg/kg BF was changes in MCHC not significant (Table 6). However, while the pattern of changes were similar for hemoglobin concentration, hematocrit and MCHC, the effect of repeated doses on RBC, MCV, and MCH showed opposite pattern between ME and BF, when comparing lower doses with higher doses. For instance, RBC was significantly higher at lower dose of 100 mg/kg ME, and lower at higher dose of 300 mg/kg ME, but the reverse was the case with BF, suggesting a different pattern or mechanism of toxic action of ME and BF (Table 6). It should be noted that significant changes in critical hematological indices following acute and repeated doses, especially with BF and at higher doses, is suggestive of potential toxicity [30,31]. Generally, the differential dose dependent effects on various hematological indices, may provide insights into the potential toxic effects resulting from the cumulative doses of both the extract and fraction. In addition, though the Recovery set showed significant improvement in the hematological indices when compared with Toxicity set, suggesting potential for recovery from toxic effects, such recovery may take longer at higher doses, especially with the butanol fraction (Table 7).
Table 2:Changes in body weights following single and repeated dose oral administration of extract and fraction of C albidum seed cotyledon

Dose (mg/kg)

Acute Toxicity**

Dose / Groups

Repeated Dose Toxicity#

Day 7 (%)

Day 14 (%)

Day 7 (%)

Day 14 (%)

Day 21 (%)

Day 28 (%)

 

 

Methanol Extract

Control (n=5)

121.80 ± 1.36

131.20 ± 1.96

Control (n=5)

102.46 ± 0.54

104.76 ± 0.50

106.73 ± 1.08

109.27 ± 1.32

150 (n=5)

103.00 ± 0.45*

116.00 ± 3.28*

100 (n=5)

103.21 ± 0.72

105.30 ± 0.65

108.03  ± 1.07

109.72  ± 0.99

300 (n=5)

107.20 ± 0.29*

110.40 ± 1.08*

300 (n=4)

103.03 ± 0.91

105.23 ± 0.73

107.01 ± 0.96

110.47 ± 1.28

600 (n=3)

104.40 ± 1.69*

118.40 ± 3.03*

 

 

Butanol Fraction

Control (n=5)

121.80 ± 1.36

131.20 ± 1.96

Control (n=5)

102.46 ± 0.54

104.76 ± 0.50

106.73 ± 1.08

109.27 ± 1.32

40 (n=5)

106.00 ± 1.23*

112.80 ± 1.24*

50 (n=5)

102.74 ± 0.56

104.83 ± 0.78

106.44 ± 1.24

110.14 ± 1.49

80 (n=5)

105.20 ± 2.08*

109.40 ± 2.29*

150 (n=3)

103.36 ± 1.43

103.96 ± 1.01

105.22 ± 0.82

107.85 ± 2.12

160 (n=4)

94.00 ± 2.11*

94.80 ± 3.12*

*Significant difference at p < 0.05 when compared to Control. **Significant difference at p < 0.05 when comparing Day 7 to Day 14. #Significant difference between Sampling Days using One Way ANOVA at p < 0.05.
Table 3:Changes in body weights of Recovery set of the repeated dose toxicity studies of extract and fraction of C albidum cotyledon

Dose / Groups

Last Read         (Day 28)

Recovery (Post Day 28)#

Day 7 (%)

Day 14 (%)

Day 21 (%)

Methanol Extract

Control (n=5)

106.73 ± 1.08

116.07 ± 0.97

123.50 ± 0.86

131.35 ± 2.08

100 (n=5)

108.03  ± 1.07

94.45  ± 0.87*

101.25  ± 1.03*

109.05  ± 1.13*

300 (n=4)

107.01 ± 0.96

93.02 ± 0.96*

100.15 ± 0.96*

108.15 ± 0.96*

Butanol Fraction

Control (n=5)

106.73 ± 1.08

116.07 ± 0.97

123.50 ± 0.86

131.35 ± 2.08

50 (n=4)

106.44 ± 1.24

87.36 ± 0.82*

93.44 ± 1.24*

102.54 ± 1.26*

150 (n=3)

105.22 ± 0.82

78.83 ± 0.78*

84.56 ± 1.01*

93.56 ± 1.01*

*Significant difference at p < 0.05 when compared to Control. #Significant difference at p < 0.05 Read. using One Way ANOVA followed by Dunnett’s posthoc test comparing with Last
Table 4:Organ weights and Organ - brain weight ratio following single and repeated dose oral administration of extract and fraction of C. albidum seed cotyledon

Dose (mg/kg)

Organ weights (g)

Organ – brain weight ratio

Liver

Kidney#

Brain

Liver

Left kidney

Right kidney

 

 

 

Acute Toxicity

 

 

Methanol Extract

Control (n=5)

4.02 ± 0.28

0.70 ± 0.02

0.91 ± 0.18

4.49 ± 0.44

0.39 ± 0.04

0.37 ± 0.03

150
(n=5)

3.57 ± 0.11

0.64 ± 0.03

0.88 ± 0.10

4.46 ± 0.88

0.39 ± 0.07

0.34 ± 0.02

300
(n=5)

3.63 ± 0.07

0.69 ± 0.02

0.94 ± 0.04

3.88 ± 0.22

0.35 ± 0.03

0.36 ± 0.01

600
(n=3)

3.45 ± 0.24

0.70 ± 0.02

0.98 ± 0.05

3.54 ± 0.20

0.37 ± 0.01

0.35 ± 0.02

 

Butanol Fraction

Control (n=5)

4.02 ± 0.28

0.70 ± 0.02

0.91 ± 0.18

4.49 ± 0.44

0.39 ± 0.04

0.37 ± 0.03

40
(n=5)

3.81 ± 0.13

0.69 ± 0.05

0.95 ± 0.04

4.08 ± 0.29

0.36 ± 0.04

0.39 ± 0.04

80
(n=5)

3.83 ± 0.10

0.66 ± 0.02

0.92 ± 0.04

4.21 ± 0.26

0.37 ± 0.02

0.36 ± 0.02

160
(n=4)

4.22 ± 0.16

0.71 ± 0.05

0.94 ± 0.08

4.62 ± 0.47

0.37 ± 0.05

0.38 ± 0.05

 

 

 

Repeated Dose Toxicity

 

 

Methanol Extract

Control (n=5)

4.00 ± 0.22

0.66 ± 0.02

1.04 ± 0.02

3.89 ± 0.26

0.32 ± 0.01

0.31 ± 0.01

100
(n=5)

3.47 ± 0.08

0.66 ± 0.03

1.04 ± 0.05

3.39 ± 0.20

0.31 ± 0.03

0.33 ± 0.02

300
(n=4)

2.22 ± 0.26*

0.38 ± 0.05*

0.47 ± 0.03*

4.80 ± 0.10*

0.43 ± 0.03*

0.39 ± 0.03*

 

 

Butanol Fraction

Control (n=5)

4.00 ± 0.22

0.66 ± 0.02

1.04 ± 0.02

3.89 ± 0.26

0.32 ± 0.01

0.31 ± 0.01

50
(n=5)

2.85 ± 0.21*

0.49 ± 0.02*

0.83 ± 0.02*

3.86 ± 0.28

0.31 ± 0.07

0.29 ± 0.07

150
(n=3)

1.60 ± 0.08*

0.28 ± 0.07*

0.35 ± 0.02*

4.84 ± 0.13*

0.41 ± 0.03*

0.38 ± 0.02*

*Significant at p < 0.05 when compared to control group. Data are expressed as mean ± SEM. #indicate combined Relative weights of the two kidneys
Table 5:Comparison of the Organs weights and Organ - Brain weight ratio of animals in repeated dose toxicity and recovery Sets

Organ Weights (g)

Organ  - Brain Weight Ratio

Methanol Extract

100 mg/kg (n=5)

300 mg/kg (n=4)

100 mg/kg (n=5)

300 mg/kg (n=4)

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Liver

4.69 ± 0.11

6.90 ± 0.47*

3.28 ± 1.45

4.25 ± 1.75

Liver

3.39 ± 0.20

4.68 ± 1.22

4.80 ± 0.10

5.82 ± 1.50

Kidneys#

0.89 ± 0.03

1.08 ± 0.08

0.53 ± 0.22

0.62 ± 0.26

Left kidney

0.31 ± 0.03

0.35 ± 0.09

0.43 ± 0.03

0.46 ± 0.11

Brain

1.40 ± 0.07

0.99 ± 0.18

0.83 ± 0.34

0.73 ± 0.31

Right kidney

0.33 ± 0.02

0.37 ± 0.10

0.39 ± 0.03

0.40 ± 0.11

Butanol Fraction

50 mg/kg (n=4)

150 mg/kg (n=3)

50 mg/kg (n=4)

150 mg/kg (n=3)

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Liver

4.02 ± 1.03

7.13 ± 0.43*

2.36 ± 1.45

4.47 ± 1.90

Liver

3.56 ± 0.28

5.05 ± 0.34*

4.84 ± 0.13

5.26 ± 1.28

Kidneys#

0.49 ± 0.12

0.78 ± 0.04

0.28 ± 0.17

0.69 ± 0.29

Left Kidney

0.31 ± 0.07

0.35 ± 0.03

0.41 ± 0.03

0.44 ± 0.10

Brain

1.14 ± 0.29

1.41 ± 0.06

0.51 ± 0.32

0.85 ± 0.35

Right Kidney

0.29 ± 0.07

0.30 ± 0.03

0.38 ± 0.02

0.42 ± 0.10

*Significant at P < 0.05 when compared to Test group. Data are expressed as mean ± SEM
Table 6:Hematological parameters of animals administered single and repeated doses of extract and fraction of C. albidum seed cotyledon

Acute Toxicity

 

Hematological Parameters

Control
(n = 5)

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

150 (n=5)

300 (n=5)

600 (n=3)

40 (n=5)

80 (n=5)

160 (n=4)

White Cell count (103 / µl)

6.3 ± 0.26

6.36 ± 0.25

5.88 ± 0.23

8.02 ± 0.26*

6.42 ± 0.23

8.1 ± 0.16*

4.78 ± 0.30*

Red Cell count  (106 / µl)

4.52 ± 0.04

4.92 ± 0.06*

3.86 ± 0.05*

3.63 ± 0.06*

4.22 ± 0.04*

4.35± 0.02*

3.78 ± 0.06*

 

Hemoglobin (g/dL)

18.74 ± 1.44

13.22 ± 1.04*

13.7 ± 0.44*

13.48 ± 0.62*

13.46 ± 0.86*

13.68 ± 1.63*

12.33 ± 1.36*

 

Hemocrit (%)

50.2 ± 3.12

39.6 ± 3.15*

41 ± 1.34*

40.5 ± 1.87*

40.4 ± 2.56*

41 ± 2.45*

37 ± 2.03*

 

MCV (fl)

111.06 ± 3.2

80.49 ± 2.52*

106.22 ± 2.7

111.57 ± 3.8

95.73 ± 3.8*

94.25 ± 3.14*

97.88 ± 3.96*

 

MCH (pg)

41.46 ± 1.10

26.87 ± 1.05*

35.49 ± 1.13*

37.13 ± 1.56

31.90 ± 1.57*

31.45 ± 1.06*

32.62 ± 4.12*

 

MCHC (g/L)

37.33 ± 0.33

33.38 ± 0.22*

33.41 ± 0.12*

33.28 ± 0.21*

33.32 ± 0.23*

33.37 ± 0.41*

33.32 ± 0.13*

 

Repeated Dose Toxicity

Hematological Parameters

Control
(n = 5)

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

100 (n=5)

300 (n=4)

50 (n=5)

150 (n=3)

White Cell count (103 / µl)

6.08 ± 0.31

5.42 ± 0.43

6.2 ± 0.20

6.43 ± 0.11

6.3 ± 0.11

Red Cell count  (106 / µl)

3.02 ± 0.09

8.78 ± 0.60*#

1.77 ± 0.14*

2.55 ± 0.02*#

5.77 ± 0.23*

Hemoglobin (g/dL)

13.66 ± 1.49

16.12 ± 0.52

13.57 ± 1.30

14.9 ± 0.43#

13.47 ± 0.43

Hemocrit (%)

41.2 ± 2.42

42.2 ± 2.55#

30 ± 1.64*

45 ± 2.02#

34 ± 1.08*

MCV (fl)

136.42 ± 3.14

48.06 ± 1.76*#

169.49 ± 6.1*

176.47 ± 2.36*#

58.93 ± 2.92*

MCH (pg)

45.23 ± 2.16

18.36 ± 0.93*#

76.6 ± 4.3*

58.43 ± 2.00*#

23.34 ± 1.45*

MCHC (g/L)

33.16 ± 0.48

38.20 ± 1.08*#

45.23 ± 0.67*

33.10 ± 0.13#

39.62 ± 0.52*

 

*Significant at P < 0.05 when compared to control. #Significant at P < 0.05, when comparing lower dose vs higher dose in the repeated dose Toxicity study. Data are expressed as mean ± SEM
Table 7:Comparison of the hematological indices of animals in the repeated dose toxicity and recovery Sets (methanol extract and Butanol Fraction)

Hematological Parameters

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

100 (n=5)

300 (n=4)

50 (n=4)

150 (n=3)

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

White Cell count (x 103 / µl)

5.42 ± 0.43*

3.62 ± 0.06

6.2 ± 0.20*

1.8 ± 0.05

6.43 ± 0.11*

2.12 ± 0.07

6.3 ± 0.11*

4.5 ± 0.10

Red Cell count  (x 106 / µl)

8.78 ± 0.60

7.9 ± 0.84

1.77 ± 0.14*

3.1 ± 0.16

2.55 ± 0.02*

5.48 ± 0.04

5.77 ± 0.23*

4.83 ± 0.12

Hemoglobin (g/dL)

16.12 ± 0.52

14.88 ± 0.42

13.57 ± 1.30

13.63 ± 1.13

14.9 ± 0.43*

13.46 ± 0.33

13.47 ± 0.43

14.37 ± 0.23

Hemocrit (%)

42.2 ± 2.55*

51.4 ± 2.15

30 ± 1.64*

56.67 ± 1.20

45 ± 2.02

50.4 ± 1.20

34 ± 1.08*

47.67 ± 2.16

MCV (fl)

50.44 ± 1.76*

70.06 ± 2.34

174.8 ± 6.1

192.5 ± 6.68

176.45 ± 2.36*

93.9 ± 1.49

65.2 ± 2.92*

113.03 ± 6.12

MCH (pg)

18.62 ± 0.93

20.2 ± 1.68

79.13 ± 4.3*

45.7 ± 2.35

58.5 ± 2.00*

25.16 ± 1.13

28.8 ± 1.45

33.63 ± 2.25

MCHC (g/L)

39.64 ± 1.08*

29.34 ± 2.23

45.17 ± 0.67*

24.17 ± 2.42

33.525 ± 0.13*

26.7 ± 0.39

40.83 ± 0.52*

31.63 ± 2.34

*Significant at P < 0.05 for Toxicity group vs Recovery group. Data are expressed as mean ± SEM
Effect on biochemical indices
We also examined the effects of the extract and fraction on plasma levels of AST, ALT, creatinine and bilirubin. Elevated levels of AST, ALT, and bilirubin have been reported to be indicators of underlying cellular injuries [29,32,33]. The single and repeated dose administrations of the extract or fraction showed varying degree of significant changes (p < 0.05) in the biochemical indices (Table 8). Interestingly, following both acute and repeated doses, there were consistent dose dependent significant changes in ALT and AST activities, and creatinine and direct bilirubin level, at all tested doses of ME and BF when compared with control (Table 8). These changes were more pronounced with repeated doses, suggesting more toxic responses to cumulative doses of the test agents.

Generally, observed changes in assayed biochemical indices indicate that BF could be more toxic and that there is a potential for increased cumulative toxic effects at higher doses (Table 8). While these changes are more pronounced with BF, the observed significant increase in ALT activities and direct bilirubin, (Table 4) may imply a potential to cause hepatocellular injury, possibly due to increased workload on the liver [32,33], although, it is unclear if this injury is sufficient to cause leakage in the mitochondrial AST enzyme [29]. Also, the significant increase in creatinine levels at all doses of ME and 80 mg/kg BF (Table 8), may be a reflection of an impaired kidney function [29,32,33]. Additionally, the sustained elevated level of DBIL at all tested doses of ME and BF following period of recovery (Table 9), may be a reflection of delayed toxicity and an indication of a potential for irreversibility of toxic effects.
Table 8:Changes in plasma biochemical indices following single and repeated doses administration of the extract and fraction of C. albidum seedcotyledon

Acute Toxicity

Parameters

Control
(n = 5)

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

150 (n=5)

300 (n=5)

600 (n=3)

40 (n=5)

80 (n=5)

160 (n=4)

ALT (IU/L)

24.40 ± 2.16

29.60 ± 2.02

32.80 ± 1.86*

33.20 ± 2.09*

29.80 ± 2.42

31.80 ± 1.52*

69.40 ± 3.21*

AST (IU/L)

58.40 ± 2.90

51.00 ± 3.32

48.20 ± 2.50*

40.20 ± 2.20*

53.00 ± 2.45

46.00 ± 1.73*

38.20 ± 1.56*

Creatinine (g/dl)

1.36 ± 0.09

2.02 ± 0.13*

3.54 ± 0.15*

2.82 ± 0.12*

1.27 ± 0.06

1.68 ± 0.07*

0.95 ± 0.03*

Direct Bilirubin (g/dl)

0.03 ± 0.01

0.10 ± 0.06

0.41 ± 0.07*

0.09 ± 0.01*

0.04 ± 0.01

0.16 ± 0.04*

0.30 ± 0.02*

Indirect Bilirubin (g/dl)

0.66 ± 0.04

0.55 ± 0.08

0.28 ± 0.03*

0.53 ± 0.08

0.70 ± 0.06

0.60 ± 0.06

0.24 ± 0.01*

Total Bilirubin (g/dl)

0.69 ± 0.03

0.59 ± 0.09

0.51 ± 0.07*

0.62 ± 0.07

0.74 ± 0.05

0.75 ± 0.03

0.53 ± 0.04*

 

Repeated Dose Toxicity

Parameters

Control    (n = 5)

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

100 (n=5)

300 (n=4)

50 (n=5)

150 (n=3)

ALT (IU/L)

40.20 ± 0.49

49.00 ± 2.83*#

26.60 ± 1.88*

28.80 ± 1.26*#

19.40 ± 1.88*

AST (IU/L)

34.60 ± 2.98

60.80 ± 2.43*#

22.00 ± 2.33*

26.40 ± 2.24*#

13.40 ± 1.24*

Creatinine (g/dl)

0.75 ± 0.08

1.18 ± 0.06*

1.05 ± 0.04*

0.63 ± 0.02*#

0.31 ± 0.02*

Direct Bilirubin (g/dl)

0.05 ± 0.01

0.06 ± 0.01

0.03 ± 0.01

0.08 ± 0.02

0.03 ± 0.01

Indirect Bilirubin(g/dl)

0.55 ± 0.05

0.44 ± 0.03#

0.17 ± 0.05*

0.39 ± 0.03*#

0.24 ± 0.03*

Total Bilirubin (g/dl)

0.60 ± 0.04

0.50 ± 0.02#

0.20 ± 0.03*

0.47 ± 0.02*#

0.26 ± 0.06*

 

*P < 0.05; Dose (s) vs. Control group; #P < 0.05, lower dose vs. higher dose. Data are expressed as mean ± SEM
Table 9:Comparison of the plasma biomarkers of animals in the repeated dose toxicity and recovery Sets (methanol extract and butanol fractions)

Biomakers

Methanol Extract (mg/kg)

Butanol Fraction (mg/kg)

100 (n=5)

300 (n=4)

50 (n=4)

150 (n=3)

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

Toxicity

Recovery

ALT (IU/L)

49.00 ± 2.83

42.00 ± 1.52

26.60 ± 1.88

29.00 ± 1.85

28.80 ± 1.26*

42.60 ± 1.25

19.40 ± 1.88*

27.80 ± 1.37

AST (IU/L)

60.80 ± 2.43*

45.80 ± 2.00

22.00 ± 2.33

18.20 ± 1.77

36.40 ± 2.24

40.00 ± 2.80

13.40 ± 1.24*

21.00 ± 1.87

Creatinine (g/dl)

1.18 ± 0.06

1.30 ± 0.06

1.05 ± 0.04*

0.81 ± 0.04

0.79 ± 0.02

0.82 ± 0.04

0.31 ± 0.02*

0.57 ± 0.04

Direct Bilirubin (g/dl)

0.16 ± 0.01

0.15 ± 0.02

0.23 ± 0.01

0.20 ± 0.02

0.18 ± 0.02

0.19 ± 0.02

0.43 ± 0.01

0.46 ±  0.03

Indirect Bilirubin (g/dl)

0.44 ± 0.03*

0.31 ± 0.01

0.17 ± 0.05*

0.45 ± 0.02

0.39 ± 0.03*

0.51 ± 0.04

0.24 ± 0.03

0.19 ± 0.01

Total Bilirubin (g/dl)

0.60 ± 0.02*

0.46 ± 0.02

0.40 ± 0.03*

0.65 ± 0.02

0.57 ± 0.02*

0.69 ±  0.05

0.67 ± 0.02

0.65 ± 0.02

*P < 0.05; Toxicity group vs Recovery group. Data were expressed as mean ± SEM
Effect on organ histology
The inclusion of pathological findings as components of pathology data in assessing oral toxicity is necessary to provide a more holistic toxicological information [22,24]. Histological findings following acute and repeated doses of the ME revealed presence of pathological lesions in the FWRs kidneys and liver, especially at higher doses (Figure 1 and Figure 2). Following acute administration of 150, 300, 600 mg/kg ME, the kidney showed presence of renal lesions, including hydropic degeneration (Figure. 1). The liver showed an enhanced depiction of the centrilobular vein, hepatocytes and sinusoids at 150 mg/kg ME, and slight presence of hydropic changes at 300 mg/kg ME. However, at 600 mg/kg ME, the presence of pathological lesions including hydropic degeneration, microvesicular steatosis, marked by small or large vacuoles can be observed (Figure. 1). On the other hand, following 28-day repeated administration of ME and 21-day recovery period, histology of the kidney revealed the presence of hydropic degeneration and mild vacuolar degeneration at 100 mg/kg and 300 mg/kg ME respectively in both Toxicity and Recovery sets. Also, while mild traces of hepatocellular lesions was seen only in Toxicity set at 100 mg/ kg ME, at 300 mg/kg ME, both Toxicity and Recovery revealed a cross section of hepatic parenchyma with hepatocellular lesions (Figure. 2). Furthermore, except at 160 mg/kg, where presence of pathological lesions including hydropic degeneration and fatty changes were observed (Figure. 3), no other pathological lesions was observed in kidney and liver following acute doses of 40, 80 and 160 mg/kg BF. In addition, following repeated administration of BF, the Toxicity set showed pathological lesion such as hydropic degeneration at 50 mg/kg BF, and presence of tubular and glomerular structure degeneration at 150 mg/kg BF in kidney architecture (Figure. 4).
Figure 1:Effects of Histology of acute doses of methanol extract of C. albidum seed cotyledon on rat Kidney (K) and Liver (L). DCT, distal convoluted tubule; PCT, proximal convoluted tubule; G, glomerulus; H, hepatocyte; V, central vein. Red arrows were used to identify pathological changes and vascular degeneration. Staining was done using H&E and magnification was x400.
Figure 2:Effects of 28-day repeated administration of methanol extract of C. albidum seed cotyledons (Toxicity - T), followed by 21 days recovery period (Recovery - R) on rat Kidney (K) and Liver (L). DCT, distal convoluted tubule; PCT, proximal convoluted tubule; G, glomerulus; US, urinary space; H, hepatocyte; V, central vein; A, hepatic artery; D, bile duct. Red arrows were used to identify pathological changes and vasculardegeneration All staining were done with H&E and Magnification was x400.
Figure 3:Photomicrographs of rat Kidney (K) and Liver (L) following administration of the acute doses of butanol fraction of C. albidum seed cotyledons. DCT, distal convoluted tubule; PCT, proximal convoluted tubule; G, glomerulus; H, hepatocyte; V, central vein; D, bile duct. Red arrows were used to identify pathological changes and vascular degeneration. All were stained using H&E and Magnification was x400.
Figure 4:Photomicrograph of rat Kidney (K) and Liver (L) following 28-day repeated administration of butanol fraction of C. albidum seed cotyledons (toxicity - T), followed by 21 days recovery period (recovery - R). DCT, distal convoluted tubule; PCT, proximal convoluted tubule; G, glomerulus; US, urinary space; H, hepatocyte; V, central vein; A, hepatic artery; D, bile duct. Red arrows were used to identify pathological changes and vascular degeneration All staining were done with H&E and Magnification was x400.
In general, the presence of pathological lesions (hydropic degeneration and microvesicular steatosis) in the histology of the FWR kidney and liver following acute and repeated doses of the ME and BF may be representative of the potential negative effect of C. albidum seed cotyledons on these organs. The observed histologic changes could be morphologic correlates of reversible cell injury [34]. Meanwhile, though observed pathological changes were maintained in the Recovery set of ME at 300 mg/ kg, suggesting persistent form of toxicity, the gradual reduction and/or complete eradication of pathological changes following recovery at other tested doses of both ME and BF are indicative of a pattern of reversible form of toxicity, and may be a reflection of the non-persistence of the negative effect on the organs [34].
Conclusions
The many useful properties of Chrysophyllum albidum [14–17] is a pointer to its potential as a natural source of drug, but also makes it a subject of potential abuse, especially among the local consumers. Toxicological findings from this study revealed that the test materials (ME and BF of C. albidum seed cotyledons) showed potential to induced toxicity with some level of persistent signs of intoxication following acute and repeated dose administration. This is consistent with their relatively low LD50 values, observed increase mortality with increasing doses and significant, though relatively reversible, changes in most hematological, biochemical and histological data. Hence, while taking advantage of its many economic and medicinal properties, there is the need to protect against indiscriminate uses and to apply caution in the use of the seed cotyledon of C. albidum.
Acknowledgement
We acknowledge the assistance of the following on histopathology aspects of the work and its analysis: Dr. O. Oladele of the Department of Morbid Anatomy and Forensic Medicine, Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile – Ife, Nigeria, Dr. A. Onaolapo and Mrs. M.O Cyril of Department of Anatomy, Ladoke Akintola University, Ogbomoso, Nigeria.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declarations
Ethical Approval: The protocol was approved by the Committee on the care and use of laboratory animals, Obafemi Awolowo University, Ile-Ife, Nigeria (Protocol number PHP12/13/H/0601).

Clinical trial registration: N/A
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