A revival of interest in the use of phytomedicines has emerged worldwide for the management of a variety of diseases [
Gallic acid, quercetin, 2,2-diphenyl-2-picrylhydrazyl, dichloromethane, and polyvinylpyrrolidone (PVP) were purchased from Sigma Chemical Co, USA, and were used in
A rotatory evaporator (Buchi, B-480, UK) was used for the efficient removal of solvents from plant extracts. A microplate reader (SpectraMax Plus, USA) was used in the determination of the total antioxidant activity of the selected medicinal plant extracts. A UV-visible, double-beam spectrophotometer (UV-1800 Shimadzu, USA) was used in the spectrophotometric bioassays. Hematological parameters were analyzed using an automated hematology analyzer (Mindray BC 5150, China). Tissue processor (Shandon, UK), microtome (Thermo Fisher, Germany), and microscope (Olympus CX 21, Japan) were used in the preparation and evaluation of hematoxylin and eosin- (H and E-) stained tissue sections.
Leaves of
Leaves of
The extracted plant materials were dissolved in a relevant vehicle for the preparation of the human equivalent therapeutic dose in rats. The hexane and ethyl acetate extracts were dissolved in corn oil (vehicle 1), and the butanol extract was dissolved in 3% polyvinylpyrrolidone (vehicle 2). Accordingly, the equivalent human therapeutic doses of the selected extracts of
Healthy male and female rats of Wistar strain (150 ± 25 g, 10–12 weeks of age), purchased from the Medical Research Institute, Colombo, Sri Lanka, were used in the experiments. The animals were housed in standard environmental conditions at the animal house of the Faculty of Medicine, University of Ruhuna, Sri Lanka. They were maintained on a standard laboratory diet of pellets and water
Acute toxicity testing was performed for the selected plant extracts following the Organization for Economic Cooperation and Development (OECD) guideline 420, fixed-dose procedure [
Healthy Wistar rats were divided into four main groups, considering the average weight of animals. Each group consisted of ten animals including five male rats and five female rats. Group 1 rats, administered with equivalent volumes of distilled water once orally, served as the normal control. Group 2 and 3 rats were administered with the equivalent volumes of vehicle 1 (corn oil) and vehicle 2 (PVP), respectively. Animals of Group 4 were further divided into four subgroups (a–d;
Healthy Wistar rats were allotted to four groups, considering the average weight of animals. Each group consisted of ten animals including five male rats and five female rats. Group 1 rats, served as the untreated healthy control group, received distilled water daily. Group 2 and 3 were administered with the equivalent volumes of vehicle 1 (corn oil) and vehicle 2 (PVP), respectively. Group 4 consisted of four subgroups (a–d;
The body weight, consumption of food, and intake of water of each rat were assessed before the commencement of dosing, throughout the experimental period (28 days), and recorded at weekly intervals. The consumption of food and intake of water were calculated daily from the quantity of food, water supplied, and the amount remaining after 24 h.
The animals were sacrificed at the end of the study period of 28 days. Blood samples with a volume of 3.0 mL were collected by cardiac puncture for biochemical and hematological assessments. The heart, lung, small intestine, liver, spleen, and kidneys were excised for the assessment of the relative weight of organs and histopathological changes on H and E-stained sections.
Biochemical parameters including serum concentration of glucose [
Hematological analysis was performed using an automated hematology analyzer. Hemoglobin concentration, total red blood cell count, platelet count, red cell indices including packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, total white blood cell count, and white blood cell differential counts of the blood samples were measured.
The relative organ weight of heart, lung, small intestine, liver, spleen, and kidneys of each animal was calculated by dividing the weight of the organ by the body weight of the animal as follows:
The heart, small intestine, liver, spleen, and kidney tissues were fixed in 10% buffered formalin in labeled bottles. H and E-stained tissue sections of the experimental animals were prepared for the assessment of histopathology. The tissue sections were examined under a light microscope to detect pathological alterations in the vital organs upon the treatment with plant extracts. Features of cell injury, cellular vacuolization, pyknosis, hemorrhage, and the presence of inflammatory cell infiltrations were searched on H and E-stained sections. The observations were confirmed by a consultant histopathologist.
The presence of bioactive phytoconstituents including phenolic compounds, tannins, flavonoids, steroid glycosides, alkaloids, terpenoids, coumarins, and saponins was tested in the selected extracts of
Thin-layer chromatography (TLC) profiles were developed for the selected extracts considering the polarity of solvents. The solvent systems which showed fine separations with a maximum number of components were selected for the development of TLC fingerprint.
Total polyphenol content in the selected plant extracts was estimated using the Folin–Ciocalteu method with some modifications [
Quantification was done with respect to the standard curve of gallic acid in a range of 0–1 mg/mL (
The total flavonoid content of plant extracts was determined by the method of Siddhuraju and Becker [
The flavonoid content was calculated using a standard of quercetin solution in the range of 0–125
The assay was performed according to the method of Blois [
Percentage DPPH radical scavenging activity was calculated as follows:
Data were statistically analyzed using SPSS software 22.0. Quantitative data were expressed as mean ± SEM. One-way ANOVA followed by Dunnet’s post hoc test was used for multiple comparisons, and the values of
The potential toxic effects of several Sri Lankan medicinal plants detailed in traditional pharmacopoeias have been reported using
Laboratory animals are widely used for the scientific evaluation of the safety of novel therapeutics prior to clinical trials [
The administration of a single oral dose of plant extracts at the therapeutic dose induced neither mortality nor treatment-related signs of toxicity in experimental animals throughout the study period of 14 days. Further, body weight, consumption of food, and intake of water were not affected by the administration of plant extracts. In addition, the selected extracts of
However, a 14-day behavioral toxicity study is inadequate to confirm the complete toxicity profile of a therapeutic agent [
Change in body weight of Wistar rats administered with the hexane, ethyl acetate, butanol, and aqueous extracts of
Biochemical parameters in serum after treatment with selected plant extracts of
Normal control | Vehicle corn oil | Vehicle PVP | Hexane extract | Ethyl acetate extract | Butanol extract | Aqueous extract | |
---|---|---|---|---|---|---|---|
BUN (mmol/L) | 8.12 ± 0.50 | 6.89 ± 0.89 | 7.25 ± 0.49 | 7.84 ± 1.07 | 7.22 ± 0.49 | 8.34 ± 0.61 | 8.37 ± 0.27 |
Creatinine ( | 66.39 ± 2.23 | 55.25 ± 3.51 | 56.75 ± 2.46 | 58.87 ± 1.71 | 54.10 ± 2.79 | 56.75 ± 2.43 | 55.47 ± 3.86 |
Fasting plasma glucose (mmol/L) | 5.48 ± 0.14 | 4.50 ± 0.23 | 5.16 ± 0.44 | 4.85 ± 0.43 | 4.07 ± 0.22a | 4.82 ± 0.41 | 4.89 ± 0.40 |
Total cholesterol (mmol/L) | 1.73 ± 0.08 | 1.47 ± 0.16 | 1.56 ± 0.10 | 1.96 ± 0.08 | 1.51 ± 0.16 | 1.72 ± 0.10 | 1.92 ± 0.11 |
Triglycerides (mmol/L) | 1.25 ± 0.09 | 0.86 ± 0.04b | 0.88 ± 0.09a | 1.00 ± 0.05 | 1.12 ± 0.11 | 0.95 ± 0.12 | 0.96 ± 0.08 |
Total protein (g/L) | 76.11 ± 2.48 | 75.64 ± 3.61 | 80.00 ± 3.77 | 70.08 ± 2.22 | 70.56 ± 2.87 | 68.26 ± 1.24 | 68.65 ± 1.62 |
AST (U/L) | 109.07 ± 12.02 | 76.7 ± 9.41 | 76.48 ± 7.11 | 101.28 ± 14.70 | 81.94 ± 3.93 | 66.00 ± 17.52a | 85.56 ± 3.28 |
ALT (U/L) | 50.17 ± 2.22 | 39.46 ± 2.77 | 38.99 ± 2.40 | 46.56 ± 6.11 | 41.61 ± 2.95 | 38.53 ± 6.66 | 43.18 ± 2.29 |
ALP (U/L) | 187.74 ± 16.80 | 195.76 ± 16.10 | 183.40 ± 18.27 | 224.26 ± 17.20 | 189.68 ± 18.44 | 218.36 ± 17.44 | 214.12 ± 37.01 |
6.76 ± 0.63 | 4.86 ± 1.25 | 4.75 ± 1.16 | 3.62 ± 0.43 | 5.21 ± 1.62 | 4.17 ± 0.56 | 4.75 ± 0.34 |
Values are expressed as mean ± SEM of ten animals in each group. Results are significant compared to the untreated control group at a
The kidney and the liver are the vital organs to show toxic effects upon exposure to potential toxic substances as these organs are primarily involved in the detoxification process [
An increased serum concentration of liver enzymes such as ALT and AST is a reliable indicator of liver toxicity. These changes occur in the blood due to changes in hepatic cellular permeability or necrosis and cellular injury [
The fasting blood glucose values and lipid profile parameters were estimated in experimental rats for the assessment of metabolic states in relation to carbohydrate and lipid metabolism, respectively. Repeated oral administration of the plant extracts resulted in a decrease in the level of glucose in plant-treated rats with a significant reduction in the ethyl acetate extract-treated group (
Hematological parameters in whole blood after treatment with selected plant extracts of
Normal control | Vehicle corn oil | Vehicle PVP | Hexane extract | Ethyl acetate extract | Butanol extract | Aqueous extract | |
---|---|---|---|---|---|---|---|
RBC (×106/ | 7.52 ± 0.14 | 7.97 ± 0.19 | 8.20 ± 0.25 | 8.26 ± 0.22 | 8.46 ± 0.11a | 8.48 ± 0.17a | 8.19 ± 0.18 |
Hb (g/dL) | 13.81 ± 0.25 | 14.04 ± 0.10 | 13.86 ± 0.14 | 14.88 ± 0.47 | 15.06 ± 0.29a | 14.38 ± 0.29 | 14.44 ± 0.23 |
PCV (%) | 50.70 ± 1.03 | 53.74 ± 0.98 | 53.86 ± 1.56 | 53.10 ± 1.88 | 55.48 ± 0.84 | 56.16 ± 1.01a | 53.84 ± 0.93 |
PLT (×103/ | 258.30 ± 17.83 | 240.00 ± 12.19 | 243.00 ± 18.23 | 337.00 ± 23.30a | 188.40 ± 5.49a | 275.20 ± 21.36 | 282.60 ± 12.57 |
WBC (per mm3) | 2.40 ± 0.48 | 2.24 ± 0.48 | 2.72 ± 0.57 | 4.46 ± 0.70 | 4.00 ± 0.20 | 3.64 ± 0.38 | 4.58 ± 0.77 |
Neutrophils (%) | 18.33 ± 1.32 | 18.20 ± 2.48 | 16.80 ± 2.44 | 18.60 ± 0.68 | 20.00 ± 3.78 | 21.00 ± 4.16 | 18.40 ± 2.42 |
Lymphocytes (%) | 73.70 ± 2.34 | 79.00 ± 1.79 | 73.20 ± 2.22 | 73.80 ± 2.22 | 74.80 ± 3.17 | 71.00 ± 3.83 | 78.40 ± 2.50 |
Eosinophils (%) | 1.80 ± 0.59 | 1.20 ± 0.20 | 1.20 ± 0.20 | 1.60 ± 0.68 | 0.80 ± 0.37 | 0.80 ± 0.20 | 1.20 ± 0.37 |
Basophils (%) | 1.70 ± 0.26 | 3.00 ± 0.00 | 2.40 ± 0.40 | 1.80 ± 0.58 | 2.40 ± 0.75 | 2.20 ± 0.20 | 1.60 ± 0.51 |
Monocytes (%) | 1.86 ± 0.34 | 1.20 ± 0.20 | 1.00 ± 0.00 | 0.60 ± 0.24 | 1.20 ± 0.37 | 1.40 ± 0.24 | 0.40 ± 0.40a |
MCV (fL) | 66.83 ± 0.27 | 66.88 ± 0.31 | 65.72 ± 0.17 | 65.76 ± 0.26 | 67.26 ± 0.26 | 66.60 ± 0.10 | 65.82 ± 0.41 |
MCH (pg) | 18.13 ± 0.13 | 17.66 ± 0.22 | 17.56 ± 0.14 | 17.80 ± 0.10 | 18.22 ± 0.08 | 17.76 ± 0.08 | 17.82 ± 0.09 |
MCHC (g/dL) | 27.23 ± 0.20 | 26.42 ± 0.23a | 26.64 ± 0.12 | 27.04 ± 0.15 | 27.22 ± 0.11 | 26.78 ± 0.06 | 27.10 ± 0.14 |
Values are expressed as mean ± SEM of ten animals in each group. Results are significant compared to the untreated control group at a
An elevation in total white blood cell counts was noticed in all groups of experimental rats treated with the plant extracts compared to the untreated control group, even though the changes were not significant (
Relative weight of organs (g/g BW) after treatment with selected plant extracts of
Normal control | Vehicle corn oil | Vehicle PVP | Hexane extract | Ethyl acetate extract | Butanol extract | Aqueous extract | |
---|---|---|---|---|---|---|---|
Heart | 0.33 ± 0.01 | 0.33 ± 0.01 | 0.35 ± 0.01 | 0.32 ± 0.01 | 0.32 ± 0.00 | 0.32 ± 0.01 | 0.34 ± 0.01 |
Liver | 2.68 ± 0.03 | 2.60 ± 0.04 | 2.61 ± 0.04 | 2.62 ± 0.05 | 2.57 ± 0.05 | 2.74 ± 0.04 | 2.71 ± 0.07 |
Small intestine | 1.78 ± 0.09 | 1.43 ± 0.12 | 1.73 ± 0.12 | 1.51 ± 0.07 | 1.52 ± 0.15 | 1.48 ± 0.09 | 1.48 ± 0.09 |
Lungs | 0.54 ± 0.01 | 0.47 ± 0.02a | 0.50 ± 0.02 | 0.51 ± 0.02 | 0.49 ± 0.00 | 0.49 ± 0.02 | 0.52 ± 0.01 |
Spleen | 0.25 ± 0.00 | 0.24 ± 0.01 | 0.25 ± 0.01 | 0.24 ± 0.01 | 0.26 ± 0.01 | 0.24 ± 0.01 | 0.24 ± 0.01 |
Kidneys | 0.59 ± 0.01 | 0.56 ± 0.01 | 0.55 ± 0.01 | 0.55 ± 0.01 | 0.52 ± 0.01a | 0.54 ± 0.01 | 0.57 ± 0.01 |
Values are expressed as mean ± SEM of 10 animals in each group. Results are significant compared to the untreated control group at a
The histological findings of the present study corroborated the results of biochemical parameters. Compared to the untreated control group, light microscopic appearance of H and E-stained sections of the heart, liver, small intestine, spleen, and kidney of experimental rats showed normal structure following repeated administration of the plant extracts. Figures
Photomicrographs of H and E-stained cardiac tissues of the different groups of experimental rats (×400). Healthy control group (a), vehicle 1: corn oil (b), vehicle 2: PVP (c), groups of animals treated with the hexane (d), ethyl acetate (e), butanol (f), and aqueous (g) extracts of
Photomicrographs of H and E-stained liver tissues of the different groups of experimental rats (×400). Healthy control group (a), vehicle 1: corn oil (b), vehicle 2: PVP (c), groups of animals treated with the hexane (d), ethyl acetate (e), butanol (f), and aqueous (g) extracts of
Photomicrographs of H and E-stained sections of the small intestine of the different groups of experimental rats (×400). Healthy control group (a), vehicle 1: corn oil (b), vehicle 2: PVP (c), groups of animals treated with the hexane (d), ethyl acetate (e), butanol (f), and aqueous (g) extracts of
Photomicrographs of H and E-stained sections of spleen of the different groups of experimental rats (×400). Healthy control group (a), vehicle 1: corn oil (b), vehicle 2: PVP (c), groups of animals treated with the hexane (d), ethyl acetate (e), butanol (f), and aqueous (g) extracts of
Photomicrographs of H and E-stained sections of kidney tissues of the different groups of experimental rats (×400). Healthy control group (a), vehicle 1: corn oil (b), vehicle 2: PVP (c), groups of animals treated with the hexane (d), ethyl acetate (e), butanol (f), and aqueous (g) extracts of
The findings of the preliminary phytochemical screening of the selected leaf extracts of
Phytoconstituents present in the selected extracts of
Hexane extract | Ethyl acetate extract | Butanol extract | Aqueous extract | |
---|---|---|---|---|
Phenolic compounds | + | ++ | +++ | ++++ |
Tannins | − | − | + | + |
Flavonoids | − | − | ++ | + |
Steroid glycosides | − | − | − | + |
Coumarins | − | − | − | − |
Terpenoids | − | − | + | + |
Saponins | − | − | − | + |
Alkaloids | − | − | − | − |
The presence of phytoconstituents is expressed as + whereas the absence of phytoconstituents is expressed as −. The degree of presence is expressed as follows: +: present and in mild level; ++: present and in moderate level; +++: present and in higher level; ++++: present and in abundant level.
TLC profiles were developed for the selected extracts of
TLC profiles for hexane, ethyl acetate, butanol, and aqueous extracts of
Table
Total polyphenol content, total flavonoid content, and total antioxidant potential of the leaf extracts of
Plant extract | Total polyphenol content (mg gallic acid/g of extract) | Total flavonoid content (mg quercetin/g of extract) | Total antioxidant capacity by DPPH assay; IC50 ( |
---|---|---|---|
Hexane | 0.17 ± 0.07 | 56.82 ± 3.57 | 451.49 ± 17.95 |
Ethyl acetate | 23.09 ± 0.09 | 54.81 ± 1.45 | 235.57 ± 4.71 |
Butanol | 3.56 ± 0.19 | 61.52 ± 0.24 | 124.48 ± 1.15 |
Aqueous | 0.89 ± 0.02 | 4.62 ± 0.02 | 267.70 ± 4.88 |
Antioxidants in medicinal plants play a major role in the defense against oxidative damage caused by reactive oxygen species [
In DPPH radical scavenging assay, low IC50 values represent a high level of antioxidant activity and
Values are expressed as mean ± SD of three measurements in each group. Data are expressed as milligrams of gallic acid equivalents per gram of extract and milligrams of quercetin equivalents per gram of extract for total polyphenol content and total flavonoid contents, respectively. The total antioxidant capacity is expressed in terms of IC50 (
Findings of the present study revealed neither mortality nor behavioral changes in the acute and 28-day repeated dose oral toxicity assessment of hexane (55 mg/kg), ethyl acetate (75 mg/kg), butanol (60 mg/kg), and aqueous (140 mg/kg) plant extracts of
Analysis of variance
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Blood urea nitrogen
2,2-Diphenyl-2-picrylhydrazyl
Gamma-glutamyl transferase
Hematoxylin and eosin
Hemoglobin concentration
High-density cholesterol
Concentration of the extract/reference compound at 50% inhibition
Low-density lipoprotein cholesterol
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Mean corpuscular volume
Organization for Economic Cooperation and Development
Packed cell volume
Platelet count
Polyvinylpyrrolidone
Red blood cell count
Standard deviation
Standard error of the mean
Statistical Package for Social Sciences
Thin-layer chromatography
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
White blood cell count.
The data used to support the findings of this study are included within the article.
The authors declare no conflicts of interest.
The authors are thankful to Mr. O. M. P. Chaminda, Faculty of Medicine, University of Ruhuna, for the technical assistance provided. This work was financially supported by the National Science Foundation research grant (RG/2016/HS-03) and University Grants Commissions special allocation for strengthening research grant, University of Ruhuna, Sri Lanka (RU/PG-R/16/14).