Antioxidant Potential, Subacute Toxicity, and Beneficiary Effects of Methanolic Extract of Pomelo (Citrus grandis L. Osbeck) in Long Evan Rats.

The aim of this study was to investigate the antioxidant potentials, subacute toxicity, and beneficiary effects of methanolic extract of pomelo (Citrus grandis L. Osbeck) in rats. Long Evans rats were divided into four groups of eight animals each. The rats were orally treated with three doses of pomelo (250, 500, and 1000 mg/kg) once daily for 21 days. Pomelo extract contained high concentrations of polyphenols, flavonoids, and ascorbic acid while exhibiting high 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power values. There was no significant change in the body weight, percentage water content, and relative organ weight at any administered doses. In addition, no significant alterations in the hematological parameters were also observed. However, rats which received 1000 mg/kg dose had a significant reduction in some serum parameters, including alanine transaminase (15.29%), alkaline phosphatase (2.5%), lactate dehydrogenase (15.5%), γ-glutamyltransferase (20%), creatinine (14.47%), urea (18.50%), uric acid (27.14%), total cholesterol (5.78%), triglyceride (21.44%), low-density lipoprotein cholesterol (40.74%), glucose (2.48%), and all atherogenic indices including cardiac risk ratio (24.30%), Castelli's risk index-2 (45.71%), atherogenic coefficient (42%), and atherogenic index of plasma (25%) compared to control. In addition, the highest dose (1000 mg/kg) caused a significant increase in iron (12.07%) and high-density lipoprotein cholesterol (8.87%) levels. Histopathological findings of the vital organs did not indicate any pathological changes indicating that pomelo is nontoxic, safe, and serves as an important source of natural antioxidants. In addition, the fruit extract has the potential to ameliorate hepato- and nephrotoxicities and cardiovascular diseases as well as iron deficiency anemia.

1. Introduction

Free radicals generated in the body through complex biochemical reactions have been implicated as mediators of various chronic diseases including cancer, atherosclerosis, and heart diseases [1-3]. Free radicals can lead to oxidation of proteins, lipids or nucleic acid and initiate chronic or degenerative diseases. Antioxidants are bioactive reducing agents which trap free radicals and prevent the oxidation of other biomolecules, subsequently protecting the cell components by neutralizing the damaging effects of the free radicals [4, 5]. Antioxidants of plant origin, including polyphenols (phenolic acids and their esters), flavonoids, and ascorbic acids, are potential scavengers of free radicals (peroxide, hydroperoxide, or lipid peroxyl), therefore inhibiting the oxidative processes which lead to degenerative disease [6].

Medicinal plants are diversified source for bioactive principles, which can either be useful or detrimental to human health [7, 8] depending on the type of plants, plant parts as well as the doses used. Since ancient times, it is believed that medicinal plants and herbs are safe and are free from toxic effects in humans. Nevertheless, the recognition of these plants having useful or toxic bioactive compounds has been ignored. Some constituents may cause dysfunction of vital organs (including the kidney, liver, and stomach as well as nervous system) [9] despite the increased dependency on medicinal plants throughout the world [10]. Therefore, toxicity studies are important prerequisite for proper identification of nontoxic, safe, and pharmacologically active plants.

Pomelo (Citrus grandis L. Osbeck) is one of the most popular citrus fruit originating from warm tropical climates in Southeast Asia and is from the Rutaceae family. It is considered as the largest citrus fruit, with a diameter of more than 12 inches with yellowish or greenish skin, white, or pinkish flesh, and has a sweetish-acidic flavor. It is consumed as whole fruits, fresh juices, preserved snacks, dipped in a salt mixture, or used in salad. Since ancient times, its pulp has been used as appetizer, antitoxin, cardiac stimulant, and stomach tonic [11]. It is a rich source of vitamin C and antioxidants such as beta-carotene, terpenoids, alkaloids, beta-sitosterol, carotene, polyphenols, and flavonoids as well as flavone glycosides [12, 13]. It is also abundant in potassium, phosphorus, folic acid, vitamins B1, B2, and B12, protein, and water [14, 15].

Neohesperidin, hesperidin, naringenin, naringin, and rutin are among the common flavonoids reported to be present in high amounts in pomelo juice [16, 17]. The fruit extract is reported to possess a variety of pharmacological benefits including antioxidant, antimicrobial antiobesity, and antidiabetic properties [18, 19]. In spite of its diversified uses, data on its safety, dosage, and toxicology profiles of many medicinal plants including pomelo are still lacking. Therefore, this study was designed to investigate the phytochemicals, the antioxidant effects, and the possible toxic effects of methanolic extract of pomelo in rat model by conducting various biochemical, hematological, and histopathological studies.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals and reagents used in this experiment were of analytical grade.

2.2. Sample Collection and Extract Preparation

Matured pomelo fruits were freshly collected from Jahangirnagar University campus, Savar, Dhaka, Bangladesh, in the middle of June 2015. After collection, the fruits were washed with clean sterile water in a laminar blower in order to remove all possible contamination. Subsequently, the clean fruit was stored in the refrigerator (4°C) for a maximum of one day before extraction.

Methanolic extract (25%) of pomelo was prepared according to the method described by S Paul. et al. [20]. Subsequently, it was stored at -20°C until further use.

2.3. Phytochemical Analysis

The total polyphenol content (TPC) of the methanolic extract of pomelo was estimated spectrometrically based on the Folin-Ciocalteu method [21]. The total flavonoid content (TFC) was estimated by using aluminum chloride colorimetric assay [22]. The ascorbic acid content (AAC) in pomelo extract was estimated by a method established by Omaye et al. [23].

2.4. Antioxidant Activity

Antioxidant potential of pomelo was determined via DPPH radical-scavenging activity and FRAP assays. The antioxidant activities of pomelo extract was investigated according to the DPPH free radical-scavenging assay based on the method as established by Braca et al. [24]. FRAP assay was performed according to the method established by Benzie and Strain [25].

2.5. Experimental Animals

In this experiment, adult Long Evan rats of both sexes weighing between 150 and 250 g ageing between 16 and 18 weeks were used. The rats were obtained from the Department of Pharmacology, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh. The animals were maintained in the animal house facilities at the Department of Biochemistry and Molecular Biology, Jahangirnagar University, in a constant temperature of 23±2°C and in an environment with humidity ranging from 44% to 56%. The rats were placed in sterile plastic cages with soft wood-chip bedding and received a natural 12 h day-night cycle. The rats were provided with a standard laboratory pellet diet and water ad libitum. All of the studies were conducted in accordance with the internationally established principles of the US guidelines (NIH publication #85-23, revised in 1985). The experimental protocol was approved by the Biosafety, Biosecurity & Ethical Committee of Jahangirnagar University, Savar, Dhaka, Bangladesh.

2.6. Experimental Design

A total of 32 rats were acclimatized one week prior to the experiment and were divided into four groups of eight animals, each group containing an equal number of sexes (50% male and 50% female).

Group A (Normal Control). Animals received normal diets and drinking water ad libitum.

Group B (Treatment 1). Animals treated with pomelo extract (250 mg/kg) dissolved in saline water for 21 days. (All animals received standard laboratory diet and drinking water ad libitum.)

Group C (Treatment 2). Animals treated with pomelo extract (500 mg/kg) dissolved in saline water for 21 days. (All animals received standard laboratory diet and drinking water ad libitum.)

Group D (Treatment 3). Animals treated with pomelo extract (1000 mg/kg) dissolved in saline water for 21 days. (All animals received standard laboratory diet and drinking water ad libitum.)

During the experimental period the animals were observed for and possible behavioral changes in their feeding and drinking habits. Any possible signs of physiological changes such as weakness with reduced activity and diarrhea were also observed.

2.7. Sacrifice of Animals

At the end of the treatment period, all rats were fasted for 24 h. Subsequently, they were anaesthetized by ketamine hydrochloride injection (500 mg/kg) [26] via the intraperitoneal veins. Blood samples (5 mL) were collected from the inferior vena cava and were divided into two portions. The first portion was transferred into a tube containing ethylene diamine tetra-acetic acid (EDTA) for hematological analyses while the second portion was placed into plain tubes for serum separation and biochemical analyses. The selected organs were immediately removed, weighed, and stored in formalin (10%)-containing tubes for histopathological examination.

2.8. Measurements of Body Weight, Relative Organ Weight, and Percentage of Water Content

During the experimental study, the body weights of the rats were recorded weekly. To measure the relative organ weight profile and percentage of water content, the following tissues samples were excised and weighed immediately after sacrifice: liver, kidney, heart, lung, spleen, caecum, pancreas, brain, testes, thymus, caput, stomach, ovary, Cowper's gland, and fallopian tube. The relative organ weight was measured by dividing the individual weight of each organ with the final body weight of each rat according to following formula [27]: relative organ weight (%) = wet organ weight/body weight ×100. The percentage of water content was estimated from the respective weight of each wet organ by subtracting the dry weight of each organ [28].

2.9. Serum Preparation

Immediately after sacrifice, the blood samples were placed in dry test tubes. They were allowed to coagulate at ambient temperature for 30 min. Serum was separated by centrifugation at 2000 rpm for 10 min.

2.10. Hematological and Biochemical Parameters Analysis

Hematological parameters for both control and treatment groups were analyzed using an automated hematology analyzer (8000i, Sysmex, Japan). The biochemical parameters for liver function tests [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ-glutamyltransferase (GGT), total bilirubin (TB), total protein (TP), albumin (ALB), globulin (GLB), and albumin/globulin (A/G) ratio]; parameters for kidney function [creatinine, urea, and uric acid]; parameters for lipid profile [total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C)]; parameters for iron profile [iron (Fe); total iron binding capacity (TIBC)]; parameters for serum electrolytes [sodium (Na+), potassium (K+), chloride (Cl−), magnesium (Mg2+), phosphate (PO3+) ions, and calcium (Ca2+)]; parameters for pancreatic function [amylase and lipase]; and parameters for glycemic status [serum glucose (GLU)] levels were determined following standard procedures. An automated chemistry analyzer (Dimension EXL with LM Integrated Chemistry System, Siemens Medical Solutions Inc., USA) was used. Another lipid profile parameter, serum low-density lipoprotein cholesterol (LDL-C) level, was estimated based on the following Friedewald formula [29].

LDL-C = TC-HDL-C- TG/5

Atherogenic indices such as cardiac risk ratio (CRR), atherogenic coefficient (AC), atherogenic index of plasma (AIP), and Castelli's risk index-2 (CRI-2) were calculated based on the following formulas:

CRR = TC/HDL-C[30]

AC = (TC-HDL-C)/HDL-C[31]

AIP = Log (TG/ HDL-C)[32]

CRI-2 = LDL-C/ HDL-C[33]

2.11. Histopathological Analysis

For histopathological examinations, the tissue samples were fixed in 10% neutral formalin and processed by a paraffin embedding technique. The histopathological specimens were cut into 5-μm-thick sections using a rotary microtome and subsequently stained with hematoxylin and eosin dye [34]. Photomicrographs at 6100 X magnification were captured by a normal-spectrum fluorescence microscope (Olympus DP 72) with an attached digital camera (Olympus, Tokyo, Japan). The pathologist performing the histopathological evaluation was blinded to the treatment assignments of the different study groups.

2.12. Statistical Analysis

All results were represented as mean ± standard deviation (SD). Data were analyzed by using SPSS (Statistical Packages for Social Science, version 16.0, IBM Corporation, New York, USA) and Microsoft Excel 2007 (Redmond, Washington, USA). All the data of treatment groups were compared with the control group by using a one-way ANOVA followed by Dunnett's multiple comparison tests. A p value of < 0.05 was considered as statistically significant.

3. Results

3.1. Phytochemicals and Antioxidant Properties

The bioactive polyphenols, flavonoids, and ascorbic acid content in pomelo are presented in Table 1. The antioxidant potentials of the methanolic extract of pomelo were investigated by estimating the free radical-scavenging activities of DPPH and ferric reducing antioxidant power (FRAP) value (Table 1).

Table 1

Polyphenol, flavonoid, and ascorbic acid contents as well as DPPH radical scavenging activity and FRAP values for pomelo methanolic extract.

Antioxidant activity	Results
Total polyphenols (mg/100 g GAEs)	515.45 ± 4.62
Total flavonoids (mg/100 g CEQs)	21.71 ± 0.22
Ascorbic acid (mg/100 g AEs)	87.25 ± 0.90
DPPH radical scavenging activity (IC50)(μg/mL)	1.70 ± 0.00
FRAP value (μM Fe[II]/100 g)	1848.2 ± 5.34

Data are presented as mean ± SD, n=3, where GAEs=gallic acid equivalent, CEQs= catechin equivalents, and AEs= ascorbic acid equivalents.

3.2. The Effects of Pomelo Extract on Body Weight, Relative Organ Weight, and Percent Water Content of Rat's Organs

There was no significant change in the body weight gain when the treatment groups were compared with the control. During the experimental period, the body weight increased gradually but the changes were not significantly different from the first to the third week (Figure 1). There was also no significant difference in both relative organ weight and percentage of water content of different rat's organs (Tables 2 and 3).

Figure 1

The effects of pomelo extract on body weight (g) gain. Results are expressed as mean ± SD. (n=8). Nonsignificant changes were found when all treatment groups were compared against the control group (p< 0.05) as determined using one-way ANOVA followed by Dunnett's multiple comparison tests.

Table 2

The effects of different concentrations of methanolic extract of pomelo on the relative organ weight.

Organs	Control	250 mg/kg	500 mg/kg	1000 mg/kg
Heart	0.280 ± 0.01	0.278 ± 0.03	0.282 ± 0.03	0.300 ± 0.01
Kidney	0.589 ± 0.03	0.617 ± 0.01	0.645 ± 0.01	0.657 ± 0.06
Lung	0.548 ± 0.08	0.552 ± 0.10	0.549 ± 0.06∗	0.551 ± 0.13
Liver	3.060 ± 0.29	3.031 ± 0.18	3.309 ± 0.11	3.00 ± 1.43
Spleen	0.352 ± 0.05	0.368 ± 0.04	0.348 ± 0.03	0.360 ± 0.22
Caecum	0.352 ± 0.05	0.325 ± 0.45	0.347 ± 0.03	0.355 ± 0.07
Pancreas	0.165 ± 0.03	0.181 ± 0.10	0.162 ± 0.10	0.151 ± 0.04
Brain	0.811 ± 0.08	0.775 ± 0.00	0.804 ± 0.09	0.857 ± 0.15
Testes	0.072 ± 0.01	0.078 ± 0.02	0.077 ± 0.01	0.080 ± 0.00
Thymus	0.183 ± 0.01	0.139 ± 0.14	0.179 ± 0.03	0.167 ± 0.05
Caput	0.356 ± 0.09	0.408 ± 0.04	0.354 ± 0.03	0.346 ± 0.02
CPG	0.131 ± 0.01	0.129 ± 0.01	0.126 ± 0.00	0.122 ± 0.01
Stomach	0.620 ± 0.02	0.573 ± 0.22	0.692 ± 0.05	0.675 ± 0.14
Ovary	0.065 ± 0.02	0.067 ± 0.02	0.070 ± 0.01	0.062 ± 0.00
FT	0.263 ± 0.00	0.290 ± 0.05	0.270 ± 0.05	0.286 ± 0.05

Results are expressed as mean ± SD (n=8).

CPG: Cowper gland and FT: fallopian tube. Nonsignificant changes were found when all treatment groups were compared against the control group (p< 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests. Table 2 is reproduced from Sudip Paul et al. (2017) [under the Creative Commons Attribution License/public domain].

Table 3

The effects of different concentrations of methanolic extract of pomelo on the percentage of water content of different organs.

Organs	Control	250 mg/kg	500 mg/kg	1000 mg/kg
Heart	73.00 ± 2.30	72.05 ± 2.08	76.68 ± 2.07	76.08 ± 1.64
Kidney	73.47 ± 2.07	74.18 ± 3.05	72.34 ± 2.50	73.03 ± 1.99
Lung	71.84 ± 3.92	74.88 ± 4.30	72.55 ± 2.67	72.12 ± 3.81
Liver	65.12 ± 4.00	65.13 ± 2.75	64.48 ± 3.07	64.24 ± 2.66
Spleen	74.61 ± 3.11	75.36 ± 2.79	76.92 ± 2.55	76.92 ± 1.60
Caecum	74.60 ± 2.02	73.72 ± 2.97	74.50 ± 2.92	73.80 ± 3.73
Pancreas	66.23 ± 1.00	65.96 ± 1.32	66.07 ± 3.53	63.83 ± 2.07
Brain	51.70 ± 3.38	54.50 ± 2.85	55.23 ± 2.64	58.00 ± 2.13
Testes	76.33 ± 2.62	77.84 ± 2.32	76.40 ± 1.99	75.57 ± 2.16
Thymus	69.68 ± 2.12	74.80 ± 3.01	68.88 ± 2.12	70.80 ± 1.52
Caput	72.73 ± 2.53	75.84 ± 3.06	75.07 ± 1.89	74.94 ± 1.54
CPG	73.60 ± 3.17	72.93 ± 2.61	76.58 ± 1.66	75.76 ± 1.84
Stomach	76.44 ± 1.38	76.14 ± 0.82	76.30 ± 1.78	76.22 ± 1.63
Ovary	56.85 ± 2.56	55.34 ± 1.53	55.23 ± 2.35	55.93 ± 1.50
FT	76.95 ± 1.68	77.69 ± 2.58	77.13 ± 0.86	75.92 ± 1.89

Results are expressed as mean ± SD (n=8).

CPG: Cowper gland and FT: fallopian tube. Nonsignificant changes were found when all treatment groups were compared against the control group (p< 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests.

3.3. The Effects of Pomelo Extract on Hematological Parameters

Pomelo extract did not significantly affect the hematological parameters of the treatments groups as compared to the control (Table 4).

Table 4

The effects of different concentrations methanolic extract of pomelo on the hematological parameters of whole blood.

Hematological parameters	Control	250 mg/kg	500 mg/kg	1000 mg/kg
RBC (10∧6/μL)	7.41 ± 0.97	7.24 ± 0.48	6.56 ± 0.43	7.26 ± 1.32
HGB (g/dL)	14.10 ± 1.83	13.86 ± 0.90	12.64 ± 0.73	13.81 ± 2.52
HCT (%)	38.98 ± 4.82	38.26 ± 2.26	35.10 ± 1.82	38.90 ± 6.53
MCV (fL)	52.78 ± 1.19	52.88 ± 0.88	53.54 ± 1.03	53.75 ± 3.32
MCH (pg)	19.08 ± 0.42	19.16 ± 0.47	19.28 ± 0.37	19.02 ± 0.60
MCHC (g/dL)	36.13 ± 0.70	36.22 ± 0.45	36.00 ± 0.35	35.45 ± 1.24
RDW-SD (fL)	32.41 ± 2.81	33.14 ± 1.82	31.10 ± 1.25	34.50 ± 3.92
RDW-CV (%)	20.98 ± 2.48	21.02 ± 1.02	19.06 ± 1.23	21.40 ± 1.55
WBC (10∧3/μL)	4.55 ± 1.32	4.48 ± 0.90	4.94 ± 1.14	4.27 ± 1.70
LYMPH (%)	2.79 ± 1.08	2.83 ± 0.66	3.34 ± 0.83	2.68 ± 1.20
MONO (%)	0.05 ± 0.01	0.05 ± 0.01	0.05 ± 0.02	0.04 ± 0.01
GRANULOCYTES (%)	0.01 ± 0.01	0.15 ± 0.06	0.04 ± 0.06	0.03 ± 0.03
PLT (10∧3/μL)	4.55 ± 1.32	4.48 ± 0.90	4.94 ± 1.14	4.27 ± 1.70
MPV (fL)	9.48 ± 0.31	9.48 ± 0.34	9.40 ± 0.18	9.16 ± 0.26
PDW (fL)	10.85 ± 0.71	10.94 ± 0.62	10.40 ± 0.31	10.30 ± 0.54
PCT (%)	0.77 ± 0.11	0.79 ± 0.04	0.78 ± 0.04	0.76 ± 0.11
P-LCR (%)	21.21 ± 2.68	21.44 ± 3.11	20.18 ± 1.55	19.01 ± 2.19

Results are expressed as mean ± SD (n=8).

HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, PLT: platelets, RDW-SD: red cell distribution width-standard deviation, RDW-CV: red cell distribution width-coefficient of variation, PDW-CV: platelets distribution width, MPV: mean platelets volume, P-LCR: platelets larger cell ratio, and PCT: procalcitonin. No significant changes were found when all treatment groups were compared against the control based on a one-way ANOVA followed by Dunnett's multiple comparison tests.

3.4. The Effects of Pomelo Extract on Serum Biochemical Parameters

The effects of oral administration of pomelo on liver function markers are presented in Figure 2 and Table 5. The fruit extract exerted no adverse effect on parameters for liver function, such as ALT, AST, ALP, GGT, LDH activities, and TB level. Moreover, the levels of the investigated parameters including ALT, ALP, and LDH were significantly decreased at the highest dose (1000 mg/kg). TP, ALB, GLB, and A/G which are important biomarkers for liver function remained unchanged at the three dose levels (Table 5) indicating that the extract is safe at the administered doses.

Figure 2

The effects of different concentrations of pomelo methanolic extracts on serum hepatic marker enzymes. Results are expressed as mean ± SD (n=8). ∗ denotes the level of significant difference when compared to the control group as determined by a one-way ANOVA followed by Dunnett's multiple comparison tests.

Table 5

The effects of different concentrations of pomelo methanolic extracts on serum liver markers.

Biochemical parameters	Control	250 mg/kg	500 mg/kg	1000 mg/kg
TB (mg/dl)	0.15 ± 0.05	0.12 ± 0.08	0.12 ± 0.05	0.17 ± 0.05
TP (g/L)	55.00 ± 5.2	51.00 ± 3.5	48.75 ± 5.0	52.00 ± 3.6
ALB (g/L)	31.75 ± 3.5	29.25 ± 2.06	28.25 ± 2.21	29.00 ± 1.82
A/G	1.36 ± 0.10	1.34 ± 0.45	1.39 ± 0.13	1.34 ± 0.096

Results are expressed as mean ± SD (n=8). No significant changes were established when all treatment groups were compared against the control group using a one-way ANOVA followed by Dunnett's multiple comparison tests.

The effects of methanolic extract of pomelo on renal function were analyzed by measuring the serum urea, uric acid, and creatinine levels and major electrolytes, such as Na+, K+, Cl−, Mg2+, PO3+, and Ca2+ levels (Table 6). Pomelo extracts in all three doses ameliorated serum creatinine, uric acid, and urea levels indicating that it is renal-protective. The extract did not alter body homeostasis or affect electrolyte levels.

Table 6

The effects of different concentrations of pomelo methanolic extract on serum renal markers.

Biochemical parameters	Control	250 mg/kg	500 mg/kg	1000 mg/kg
Creatinine (mmol/L)	39.75 ± 3.09	35.00 ± 5.59∗	37.25 ± 6.34∗	34.00 ± 3.80∗
Urea (mmol/L)	5.30 ± 0.54	4.47 ± 0.69∗	4.97 ± 1.15∗	4.32 ± 0.61∗
Uric acid (mmol/L)	45.50 ± 4.80	38.25 ± 10.40∗	35.50 ± 9.00∗	33.25 ± 6.20∗
Na+ (mmol/L)	141.00 ± 2.58	142.50 ± 4.43	142.50 ± 1.20	142.50 ± 6.40
K+ (mmol/L)	4.12 ± 0.15	4.20 ± 0.08	4.32 ± 0.09	4.05 ± 0.25
Ca2+ (mmol/L)	2.12 ± 0.22	2.22 ± 0.28	2.30 ± 0.81	2.27 ± 0.09
Mg2+(mmol/L)	0.82 ± 0.12	0.82 ± 0.12	0.87 ± 0.09	0.87 ± 0.05
Cl− (mmol/L)	101.50 ± 2.60	102.25 ± 4.34	101.00 ± 1.80	102.25 ± 2.98
PO4- (mmol/L)	2.30 ± 0.29	2.40 ± 0.29	2.47 ± 0.29	2.42 ± 0.09

Results are expressed as mean ± SD (n=8).

∗denotes level of significant difference when compared to the control group as determined using a one-way ANOVA followed by Dunnett's multiple comparison tests.

Lipid profiles (TC, TG, HDL-C, and LDL-C) (Figure 3) and atherogenic indices (CRR, AC, AIP, and CRI-2) (Table 7) are reliable markers for cardiovascular diseases. Methanolic extract of pomelo significantly reduced the serum TG levels and all atherogenic indices at the three doses. Rats administered with 1000 mg/kg dose had significantly lower TC and LDL-C levels and significantly higher HDL-C level.

Figure 3

The effects of different concentrations of pomelo methanolic extracts on lipid profiles. Results are expressed as mean ± SD (n=8). ∗ denotes level of significant difference compared to control group (p< 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests.

Table 7

The effects of different concentrations of pomelo methanolic extracts on atherogenic indices.

Biochemical parameter	Control	250 mg/kg	500 mg/kg	1000 mg/kg
CRR	1.81 ± 0.05	1.51 ± 0.05∗	1.49 ± 0.05∗	1.37 ± 0.08∗
AC	0.81 ± 0.04	0.50 ± 0.05∗	0.49 ± 0.05∗	0.47 ± 0.07∗
AIP	0.36 ± 0.01	0.29 ± 0.08∗	0.28 ± 0.05∗	0.27 ± 0.05∗
CRI-2	0.35 ± 0.03	0.31 ± 0.05∗	0.31 ± 0.05∗	0.19 ± 0.02∗

Results are expressed as mean ± SD (n=8). ∗ denotes level of significant difference when compared to control group (p < 0.05) and it was determined using one-way ANOVA followed by Dunnett's multiple comparison tests.

Pomelo extract had a positive effect on serum iron profile. Pomelo extract at 1000 mg/kg dose significantly increased Fe level. However, although the other two lower doses (250 and 500 mg/kg) increased Fe level as well, it did not reach statistical significance. This was similarly seen for TIBC (Figure 4).

Figure 4

The effects of pomelo methanolic extract on serum iron profile. Results are expressed as mean ± SD (n=8). ∗ denotes level of significant difference when compared to control group (p < 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests.

Pomelo extract did not significantly affect pancreatic function. Moreover, the extract showed no significant changes on serum amylase and lipase levels indicating that it is not toxic to the liver (Table 8). In addition, there was a positive effect of the extract on glycemic status where rats administered with 1000 mg/kg dose had significantly reduced blood glucose levels (Figure 5) although this effect was not seen for the extract administered in the lower doses.

Table 8

The effects of different concentrations of pomelo methanolic extracts on pancreatic function.

Biochemical parameter	Control	250 mg/kg	500 mg/kg	1000 mg/kg
Amylase (U/L)	730.5 ± 30.96	749.5 ± 51.09	740.0 ± 46.56	725.0 ± 73.78
Lipase (U/L)	32.25 ± 3.40	30.75 ± 2.36	29.75 ± 2.63	31.25 ± 2.06

Results are expressed as mean ± SD (n=8).

No significant changes were found when all treatment groups compared against control group (p < 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests.

Figure 5

The effects of different concentrations of pomelo methanolic extracts on blood glucose levels. Results are expressed as mean ± SD (n=8). ∗ denotes level of significant difference when compared to control group (p < 0.05) as determined by using a one-way ANOVA followed by Dunnett's multiple comparison tests.

3.5. Histopathological Examination

Histopathological examination of the liver, kidney, lung, intestine, brain, stomach, pancreas, and heart did not show any morphological and pathological changes following administration of methanolic extract of pomelo in all three doses (250, 500, and 1000 mg/kg) for 21 days when compared with the control (Figure 6) [only the figure for the highest dose (1000 mg/kg) was shown]. This fact again confirmed its safety on these organs.

Figure 6

Histopathological photomicrographs of control and treatment group (1000 mg/kg) (6100 X magnification, scale bar: 20 μm). No morphological and pathological changes were observed between the control and pomelo extract administered groups, where C=control. Figure 6 is reproduced from Sudip Paul et al. (2017) [under theCreative Commons Attribution License/public domain].

4. Discussion

To our knowledge, our study is the first to investigate on subacute toxicity of pomelo. We found that not only pomelo extract does not confer hepato- and renal toxicities, but it also ameliorates serum liver biomarkers (ALT, ALP, LDH, and GGT), serum creatinine, urea, uric acid, TG, glucose and atherogenic indices including CRR, CRI-2, AC, and AIP as compared to control. In addition, the highest dose (1000 mg/kg) confers a significant increase in iron, TIBC, TC, LDL-C, and HDL-C levels. The chemical constituents in the fruit or fruit extract (also known as secondary metabolites) have been reported to be biological active ingredients responsible for different activities, such as antioxidant, antimicrobial, antifungal, anti-inflammatory, and anticancer effects in many previous studies [35-37].

Polyphenols are the vital phytochemicals to prevent body from oxidative damage [38]. The findings of this study indicate that pomelo is a rich natural source of polyphenols and flavonoids as well as ascorbic acid. Additionally, the antioxidant activities of methanolic extract of pomelo were evaluated by FRAP and DPPH assays. FRAP assays primarily measure the abilities of antioxidants to reduce ferric tripyridyltriazine (Fe3+) into a ferrous form (Fe2+), whereas DPPH scavenging assay measures percentages of radical scavenging activity[39]. The DPPH radical scavenging activity and high FRAP value of pomelo extract confirmed the high antioxidant potential of pomelo.

Body weight of animals, relative organ weight profile, and percent water content are important parameters for toxicological study. This is due to the fact that toxic agents or substances are responsible for abnormal metabolic reactions in the body that may alter those parameters. Body weight is closely correlated with food consumption. In the presence of toxicity, the normal diets of animals tend to be affected causing a reduction in the body weight. Based on the toxicity study conducted, no significant difference was observed in body weight gain among the control and test groups during the experimental period indicating that the extract is safe at the investigated doses.

Another parameter, the relative organ weights are widely accepted in the assessment of test study-associated toxicities [40] because it can reduce the variability of data and increase the chances of detecting treatment effects. In fact, the liver, kidney, heart, and brain are among the most important organs in animals. Abnormality in liver weight may suggest treatment related changes, including hepatocellular hypertrophy [41] while alterations in heart weight may be related to myocardial hypertrophy [42]. Changes in kidney weight may cause renal toxicity or chronic nephropathy and changes in brain weight is associated with neurotoxicity [43, 44]. In this study, there was no significant change in relative organ weight of all organs for all treatment groups when compared with control group, indicating that the methanolic pomelo extract did not confer any toxic effects on the major organs.

The percentage of water content is another important factor in assessing the toxic effect of drug in animals. Adult human body consists of approximately 55% to 75% of water [45]. Dehydration accounts for the loss of water in the body, while edema results from deposition of excess water in body tissues [46]. After feeding the rats with pomelo extract for 21 days, there was no significant alteration in percent water content in the different organs of rats again, indicating its safety.

Assessment of hematological parameters is a good index of physiological and pathological status in humans as well as other animals. Hematological parameters provide important information about abnormalities in body metabolic processes and the response of the body to injures or lesions, deprivation and stress [47]. Additionally, the extent of toxic effect of toxic materials can be determined early by the assessment of hematological parameters [48]. The common hematological parameters measured are WBCs, neutrophils, lymphocytes, monocytes, eosinophils, RBCs, HGB, HCT, MCV, MCH, MCHC, PLT, RDW-SD, RDW-CV, PDW, MPV, P-LCR, and PCT [49]. Again, our study indicates that administration of pomelo extract caused no abnormal changes in any of the investigated hematological parameters.

RBCs and its indices (MCV, MCH, and MCHC) are among the most useful indicators in the early diagnosis of anemia [50]. RBC, hematocrit counts, and hemoglobin concentration are essential to assess the effect of chemical substances on hematopoietic system [51]. In case of hypoxia, dehydration, renal cell carcinoma, pulmonary fibrosis and/or polycythemia vera hemoglobin, and hematocrit level tend to increase in the presence of toxic compound but in the presence of decrease with bleeding, hemolysis, leukemia, anemia, bone marrow failure, malnutrition, and overhydration [52, 53].

In toxicity studies, excess level of WBC indicates the impact of chemicals in inducing the immune response of treated animals. Thus, WBCs and its differential count including neutrophils, lymphocytes, eosinophils, monocytes, and basophils play important role in the immune system to combat infections where the overproduction of leukocytes can be an important biomarker for inflammation and stress-related disorders [54]. On the other hand, significant decrease in WBCs count indicates lower production of leukocytes (leucopenia) which indicates the body being less able to fight against infections [55]. In fact, platelet count and its indices (MPV and PDW) are the key biomarkers for early diagnosis of atherosclerosis, thromboembolic, and ischemic heart disease [56-58]. Thrombocytopenia is a condition of abnormally low number of platelets while thrombocytosis is a condition of abnormally high number of platelets in the circulatory system [55, 59], both of which can be stimulated by toxic compounds. Nevertheless, our hematological findings demonstrated that administration of pomelo extract causes no toxic effect on the animals again indicating that the extract is safe.

Elevated serum activities of enzymes produced by the liver, kidney, heart, and pancreas indicate their leakage into the blood stream as a result of necrosis of the tissues [60]. The liver is the major organ of animal body that performs approximately 500 vital metabolic functions including transforming and cleaning of foreign substances [61]. The levels of serum ALT, AST, ALP, GGT, TB, TP, ALB, GLB, and albumin-globulin (A/G) ratio are among the most sensitive biomarkers of liver damage or toxicity [62, 63]. Likewise, ALT and AST are the major intracellular enzymes of the liver. Elevation of their levels in the blood indicates hepatocyte injuries [64] while the levels of ALP and GGT are increased in serum in the presence of hepatobiliary damage [65]. Although the increased activity of AST and LDH in serum also indicates myocardial damage, hepatocellular injuries may also occur since both enzymes are found in heart and liver [63]. Moreover, serum elevation of both ALT and AST occurs only after the permanent damage of hepatocellular membrane [66]. Lower concentrations of serum TP and albumin levels also tend to provide information on the severity of liver necrosis [67]. In fact, elevated level of TB is an important biochemical marker to the severity of necrosis as it is a measure of binding, conjugation, and excretory capacity of the hepatocytes [68].

In our study, the fruit extract exerted no adverse effect on all parameters for liver function markers. Furthermore, their levels were significantly decreased at the highest dose of pomelo extract (1000 mg/kg), which may be contributed by the high presence of phenolic compounds [69] that may confer some protective effects. Moreover, in this study, the levels of TP, ALB, GLB, and A/G remained unchanged at the three dose levels suggesting that pomelo extract is not toxic on normal hepatic excretory and synthetic functions.

The kidneys play several vital roles in maintaining good health by filtering waste materials from the blood and expelling the products from the body as urine. Besides, the kidneys also control fluid balance and minerals levels and maintain some hormonal activity [70]. Kidney function is normally assessed by measuring serum creatinine, urea, and uric acid levels [71] where abnormal changes tend to occur when there is marked damage to functional nephrons [72]. In this study, all three doses of pomelo extracts significantly ameliorated renal function biomarkers when compared to normal controls. Hence it is hypothesized that pomelo extract may be beneficial for individuals with kidney disease.

Electrolytes such as Na+, K+, Cl−, Mg2+, PO4+, and Ca2+ are chemicals found in the blood and other bodily fluids which are essential for the maintenance of bodily physiological functions, including regulation of pH balance, fluid balance, blood pressure, nerve transmission, muscle contraction, and energy production [73]. If the levels of these electrolytes become significantly low, muscle spasms, weakness, convulsions, changes in blood pressure, and nervous system disorders may occur [74, 75]. Kidneys, being the excretory organ, maintain the electrolyte balance. All three doses of the extract did not alter body electrolytes balance significantly and therefore it is plausible that pomelo extract do not affect body's homeostatic function.

Lipid profiles are biochemical tests routinely performed to assess the atherogenic status of cardiovascular diseases (CVD) and coronary artery diseases (CAD). It includes serum TG, TC, and its subfractions HDL-C and LDL-C. Alterations in lipid metabolism, including hypertriglyceridemia, have been associated with CVD and CAD [76] where high plasma TG, TC, LDL-C, and low HDL-C levels contribute to atherosclerosis, hypertension, obesity, and diabetes mellitus [77-79]. In fact, atherogenic indices (CRR, ACC, RIP, and CRI-2) derived from lipid profiles are directly correlated with the risk of cardiovascular diseases. The higher the atherogenic indices are, the higher the risk of cardiac-related disease is [77, 78]. In our study, methanolic extract of pomelo significantly reduced serum TG levels and all atherogenic indices when administered in three different doses. Rats administered with 1000 mg/kg dose had significantly decreased TC and LDL-C level and increased HDL-C level indicating the protective effect of pomelo fruit against cardiovascular disease. Additionally, high plasma HDL-C level exert a protective effect by reversing cholesterol transport because it scavenges excess cholesterol from the peripheral tissues of the body [80].

Serum Fe and TIBC are important constituents of iron profile. In fact, iron deficiency is the most common micronutrient deficiency worldwide that leads to iron deficiency anemia. Low levels of Fe and increased levels of TIBC suggest that total iron body stores are low which are signs of iron deficiency anemia and polycythemia vera [81]. In addition, increased Fe and decreased TIBC levels may indicate anemia of chronic disease such as pernicious anemia, hemolytic anemia, sickle cell anemia, hemochromatosis, and chronic liver disease [82]. Our findings suggest that pomelo extract significantly increased iron levels and decreased TIBC when administered at the highest dose (1000 mg/kg) which further supports the potential application of pomelo for preventive effects against iron deficiency-related disorders.

Serum amylase and lipase activities are two major indicators of acute pancreatitis since these enzymes are synthesized and secreted into the intestinal lumen and are released into the circulation in catalytically active form [83]. Serum lipase activity is a more reliable diagnostic marker for acute pancreatitis because its lipolytic activity potentially can be assayed rapidly [84, 85]. In our study, pomelo extract did not affect pancreatic function. Moreover, the extract significantly reduced lipase activity.

Estimation of blood glucose level is the first step in diagnosing diabetes mellitus, as it is a reliable marker that increases in diabetes patient after meals are taken [86]. Medicinal plants and their products have been considered as an excellent source of alternative medicine by virtue of their active phytochemical constituents. These constituents can improve diabetes by increasing insulin secretion, inhibiting intestinal glucose absorption, reducing oxidative stress, and facilitating metabolites in insulin dependent processes [87]. Overall, methanolic extract of pomelo showed positive effects on the glycemic status where rats administered with the highest dose had significantly reduced blood glucose levels.

Histopathological examination of tissue can help in understanding the relationship between the concentrations of toxins in the body and their effect on the target organs. Not only that, histopathology can be used to identify toxic mechanism and predict safe concentrations of substances. Tissue necrosis mainly occurs in liver, kidney, heart, pancreas, stomach, spleen, lung, and brain which are the common investigated organs. In our study, there were no remarkable morphological and pathological changes in the major organs following administration of pomelo extract at the three doses for 21 days indicating that the extract is safe. We have also analysed the male and female sexes separately and in all of the parameters, there were no significant changes in the findings indicating that the effect is not gender-specific or is influenced by hormones. However, further studies both in vitro and in vivo are warranted to classify the individual active ingredients by using different solvents and elucidate the exact molecular mechanism behind its beneficial actions on liver function, renal function, lipid profile, and cardiac and pancreatic functions.

5. Conclusion

Pomelo is a rich source of natural antioxidant as indicated by the high content of polyphenols, flavonoids, ascorbic acid, DPPH free radical scavenging activities, and FRAP values. The in vivo study indicates that the fruit is nontoxic, safe, and beneficial to be consumed at the investigated doses. Oral administration of pomelo extracts up to 1000 mg/kg causes no physiological and biochemical abnormalities over three-week treatment period where there were no histopathological changes in major organs suggesting its safety. Biochemical studies indicate that pomelo extract can enhance liver, cardiac, and pancreatic functions while supplying a good source of iron.