Ayurvedic and traditional medical practitioners of Sri Lanka use the decoction of the immature inflorescence of
The coconut palm,
Preliminary phytochemical screening of the IC revealed that it contains a high amount of proanthocyanidins. The extraction, purification, and characterization of ethyl acetate soluble proanthocyanidins (EASPA) of the IC have previously been reported [
Medicinal plants are used for the prevention and treatment of various diseases worldwide. They are a rich source of diverse bioactive compounds that provide unlimited opportunities for the discovery of novel drugs and herbal remedies [
All chemicals and solvents used for the study were of AR grade and purchased from Sigma-Aldrich, Germany. Commercially available test kits (ALP, ALT, glucose, cholesterol, and urea) purchased from Sigma-Aldrich, France were used to analyze biochemical parameters.
Konelab 20XT auto analyzer analyzed biochemical parameters. Blood samples in the tubes were centrifuged by Harmonic series centrifuge. Light microscope (OLYMPUS CH-2) with ×4 and ×10 objective lens was used to examine the histopathological slides. Haematological parameters were analyzed using BCC 3000B, DIRUI three part Haematological analyzer. 13C NMR spectrum was obtained in deuterated methanol for purified EASPA with a Bruker Avance AV- 500 Nuclear Magnetic Resonance Spectrometer at 600 MHz.
Fresh immature inflorescences of the healthy adult
Extraction and purification of EASPA of the IC was carried out according to previously published methods [
Crude EASPA (1.00 g) was purified by column chromatography using Sephadex LH-20. The nonproanthocyanidin phenolics were eluted first with ethanol. Proanthocyanidin phenolics were then eluted with 70% aqueous acetone. Prussian blue and acid catalyzed cleavage tests were used to examine the collected fractions. Collected fractions, which gave positive results for both tests were combined. The acetone was removed
Female Wistar albino rats (Species—
Acute oral toxicity study of EASPA was performed using female Wistar rats according to the Organization for Economic Co-operation and Development (OECD) guideline 423 [
The percentage of body weight change was calculated according to the following equation [
On the 14th day, all the rats were kept fasting overnight. On the 15th day, they were weighed and sacrificed by overdose inhalation of the anesthetic ether. Blood samples were collected by cardiac puncture for haematological and biochemical analyses. This was followed by histopathological studies.
Each blood sample (obtained under Section 2.6.) was divided into two separate tubes, with and without the anticoagulant, ethylenediamine–tetraacetate (EDTA). The blood samples in the tubes without the anticoagulant were kept for 2 hours until complete clotting was observed. Afterwards, tubes were centrifuged at 4000 rpm at 4°C for 10 minutes. Serum was then separated and subjected to analysis of biochemical parameters, alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea, glucose, and cholesterol. Biochemical parameters were analyzed by standard methods using test kits with a Konelab auto analyzer. The blood samples in the tubes with the anticoagulant (EDTA) were immediately analyzed for haematological parameters (red blood count (RBC), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, white blood count (WBC), lymphocyte, monocyte, basophil, and neutrophil) using a haematological analyzer [
At the end of the acute toxicity study (on the 15th day), all the rats in control and test groups were sacrificed by overdose inhalation of the anesthetic ether. Necropsy and gross examination on internal organs (liver, heart, kidney, uterus, and spleen) were carried out. The organs were observed macroscopically for any abnormalities and presence of lesions. Afterwards, the internal organs were dissected, cleaned of any fat and weighed to obtain the absolute weight of the organs. Relative organ weight (ROW) of different organs was calculated using the equation given below according to previously published methods [
Afterwards, organs were fixed in 10% buffered formalin solution and preserved for subsequent histopathological examinations. The preserved organs were taken out from the 10% buffered formalin solution and processed routinely and embedded in paraffin wax. Histology sections at a thickness of 4
Subacute toxicity study of EASPA was carried out using female Wistar rats according to the OECD guideline 407 [
At the end of the subacute toxicity study (on the 29th day), rats in all groups who were kept fasting overnight, were weighed and sacrificed by an overdose inhalation of the anesthetic ether. Blood samples of rats in all groups were collected by cardiac puncture for haematological [red blood count (RBC), haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, white blood count (WBC), lymphocyte, monocyte, basophil, and neutrophil) and biochemical (alkaline phosphatase (ALP), alanine aminotransferase (ALT), serum urea, serum glucose, and cholesterol] analyses, similar to the procedure described under Section 2.6.1.
On the 29th day of the subacute toxicity study, all the rats in control and test groups were sacrificed using an overdose inhalation of the anesthetic ether and blood samples were collected by cardiac puncture method. Necropsy and gross examination of internal organs (liver, heart, kidney, uterus, and spleen) were carried out. Then, internal organs (liver, heart, kidney, uterus, and spleen) were dissected, cleaned of any fat, weighed, and observed macroscopically. Relative organ weight was also calculated. Finally, they were fixed in 10% buffered formalin solution for histopathological examination similar to the procedure described under Section 2.6.2.
All the qualitative data were expressed as mean ± Standard Error of Mean (SEM). Every statistical analysis was performed with one-way analysis of variance (ANOVA) followed by the
Extraction and purification of EASPA of the immature IC were carried out with minor modifications according to previously published methods. Crude proanthocyanidins extracted to aqueous acetone (70%) containing 0.1% ascorbic acid was partitioned into ethyl acetate to yield ethyl acetate soluble proanthocyanidins (EASPA) which after solvent removal yielded crude EASPA as a light brown powder in 0.20% by weight to the fresh inflorescence. In the purification of EASPA by Sephadex LH-20 column chromatography, nonproanthocyanidin phenolics were first eluted with ethanol and subsequently, EASPA that was adsorbed to Sephadex LH-20 was eluted with 70% aqueous acetone. All the fractions eluting from the column were analyzed for the presence of phenolics by the Prussian blue test and proanthocyanidins by the acid catalyzed cleavage test. Acetone fractions, which gave positive results for both the tests were combined and acetone was removed under reduced pressure. The resulting aqueous fraction upon freeze-drying yielded the purified EASPA as an off white powder in 0.063% by weight to the fresh inflorescence. The purified EASPA was characterized by 13C NMR spectroscopy. The 13C NMR spectrum was similar to what is reported previously and showed signals characteristic for epicatechin units indicating that EASPA is composed mainly of epicatechin units [
In the acute toxicity study, single oral administration of EASPA at the dose level of 2000 mg/kg body weight to rats did not result in any mortality. Any of the treated rats did not exhibit any visible signs of toxicity or behavioral changes and were found to be normal throughout the 14-day study period similar to rats of the control group. Mean body weight of test and control group rats are given in Figure
Effect of EASPA on mean body weight in acute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
Effect of EASPA on percentage body weight gain in acute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
Effect of EASPA on serum haematological parameters in acute toxicity study.
Parameters | Unit | Control | 2000 mg/kg |
---|---|---|---|
WBC | 109/L | 6.10 ± 1.06 | 6.15 ± 0.53 |
RBC | 1012/L | 7.48 ± 0.42 | 7.08 ± 0.08 |
Haemoglobin | g/dL | 15.35 ± 0.83 | 14.45 ± 0.07 |
Haematocrit | % | 43.2 ± 2.78 | 41.7 ± 0.42 |
MCV | fL | 117.55 ± 0.72 | 117.9 ± 0.69 |
MCH | pg | 20.0 ± 0.07 | 20.45 ± 0.32 |
MCHC | g/dL | 34.1 ± 0.48 | 34.62 ± 0.62 |
PLT | 109/L | 912.25 ± 67.43 | 908.5 ± 48.73 |
Lymphocytes | 109/L | 5.65 ± 0.98 | 5.65 ± 0.50 |
Monocytes | 109/L | 0.3 ± 0.06 | 0.3 ± 0.03 |
Granules | 109/L | 0.15 ± 0.03 | 0.2 ± 0.00 |
RDW-CV | % | 12.27 ± 0.23 | 12.4 ± 0.06 |
MPV | fL | 12.0 ± 0.28 | 11.8 ± 0.33 |
PCT | % | 0.57 ± 0.04 | 0.54 ±0.04 |
Values are expressed as a mean ± SEM (
WBC: Total white blood cell count; RBC: total red blood cell count; PLT: platelet count; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; RDW-CV: red blood cell distribution width; MPV: mean platelet volume; PCT: procalcitonin.
Effect of EASPA on serum biochemical parameters in acute toxicity study.
Group | Unit | Control | 2000 mg/kg |
---|---|---|---|
ALP | U/L | 229.25 ± 19.78 | 245.67 ± 25.94 |
ALT | U/L | 45.125 ± 0.63 | 93.93 ± 34.57 |
Glucose | mg/dL | 91.46 ± 12.11 | 56.42 ± 7.26 |
Cholesterol | mg/dL | 50.82 ± 1.27 | 48.96 ± 1.34 |
Urea | mmol/L | 3.55 ± 0.16 | 3.23 ± 0.20 |
Values are expressed as a mean ± SEM (
Effect of EASPA on relative organ weight in acute toxicity study.
Organ | Control | 2000 mg/kg |
---|---|---|
Liver | 4.44 ± 0.03 | 4.39 ± 0.03 |
Kidneys | 0.40 ± 0.02 | 0.42 ± 0.00 |
Heart | 0.34 ± 0.01 | 0.35 ± 00 |
Spleen | 0.29 ± 0.00 | 0.30 ± 0.00 |
Uterus | 0.65 ± 0.02 | 0.71 ± 0.04 |
Values are expressed as a mean ± SEM (
In the subacute toxicity study, oral administration of EASPA daily for 28 days at doses of 1.75 mg/kg, 3.5 mg/kg, 7 mg/kg, and 14 mg/kg body weight to rats did not result in any mortality. Rats of any of the test groups did not show any visible signs of toxicity such as changes in skin, eyes, fur, and mucous membranes. Further, these rats did not show any behavioral changes including salivation, sleep, coma, lethargy, and diarrhea and were found to be normal throughout the study period compared to the control group. The effect of EASPA on the mean body weight of rats in all test groups is given in Figure
Effect of EASPA on mean body weight in the subacute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
Effect of EASPA on percentage body weight gain in the subacute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
Effect of EASPA on average food intake in the subacute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
Effect of EASPA on average daily water intake (mL/rat/week) in the subacute toxicity study. Values are expressed as a mean. The error bars represent the standard error of the mean (
The effect of oral administration of EASPA daily for 28 days on haematological and biochemical parameters which were determined at the end of the study period in treated and control group rats are summarized in Tables
Effect of EASPA on serum haematological parameters in the subacute toxicity study.
Parameters | Unit | Control | 1.75 mg/kg | 3.5 mg/kg | 7 mg/kg | 14 mg/kg |
---|---|---|---|---|---|---|
WBC | 109/L | 6.20 ± 1.61 | 5.60 ± 0.56 | 6.40 ± 0.53 | 7.20 ± 0.35 | 5.50 ± 0.04 |
RBC | 1012/L | 6.12 ± 0.32 | 6.89 ± 0.08 | 6.24 ± 0.21 | 6.66 ± 0.10 | 6.20 ± 0.13 |
Haemoglobin | g/dL | 11.70 ± 0.37 | 12.80 ± 0.68 | 12.90 ± 0.49 | 13.40 ± 0.17 | 13.00 ± 0.08 |
Haematocrit | % | 33.10 ± 1.46 | 37.00 ± 0.47 | 34.90 ± 1.47 | 35.90 ± 0.40 | 34.60 ± 0.71 |
MCV | fL | 109.43 ± 0.78 | 107.45 ± 0.49 | 111.70 ± 0.93 | 107.80 ± 0.44 | 111.60 ± 0.17 |
MCH | pg | 19.55 ± 0.86 | 20.10 ± 0.14 | 20.30 ± 0.28 | 20.20 ± 0.08 | 21.00 ± 0.62∗ |
MCHC | g/dL | 35.8 ± 1.25 | 37.42 ± 0.53 | 36.95 ± 0.31 | 37.3 ± 0.17 | 37.65 ± 1.02∗ |
PLT | 109/L | 558.07 ± 109.03 | 495.33 ±74.24 | 522.00 ± 10.7 | 431.00 ± 69.3 | 535.00 ± 17.4 |
Lymphocytes | 109/L | 5.5 ± 0.20 | 5.87 ± 0.21 | 5.90 ± 0.49 | 5.70 ± 0.45 | 5.1.00 ± 0.04 |
Monocytes | 109/L | 0.37 ± 0.06 | 0.40 ± 0.05 | 0.30 ± 0.04 | 0.30 ± 0.04 | 0.20 ± 0.00 |
Granules | 109/L | 0.20 ± 0.00 | 0.26 ± 0.02 | 0.20 ± 0.00 | 0.20 ± 0.00 | 0.20 ± 0.00 |
RDW-CV | % | 23.90 ± 0.54 | 23.75 ± 0.19 | 23.40 ± 0.26 | 23.70 ± 0.31 | 23.30 ± 0.13 |
MPV | fL | 10.60 ± 0.12 | 10.40 ± 0.20 | 10.00 ± 0.26 | 10.00 ± 0.08 | 10.10 ± 0.13 |
PCT | % | 0.30 ± 0.05 | 0.25 ± 0.03 | 0.26 ± 0.01 | 0.21 ± 0.03 | 0.26 ± 0.00 |
Values are expressed as a mean ± SEM (
RBC: Total red blood cell count; WBC: total white blood cell count; PLT: platelet count; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; RDW-CV: red blood cell distribution width; MPV: mean platelet volume; PCT: procalcitonin.
Effect of EASPA on serum biochemical parameters in the subacute toxicity study.
Biochemical parameters | Unit | Control | 1.75 mg/kg | 3.5 mg/kg | 7 mg/kg | 14 mg/kg |
---|---|---|---|---|---|---|
ALP | U/L | 226.83 ±15.14 | 246.50 ± 9.75 | 245.17 ± 3.00 | 244.00 ± 3.96 | 260.83 ± 5.59 |
ALT | U/L | 72.31 ± 3.26 | 68.35 ± 4.05 | 70.08 ± 10.00 | 74.90 ± 12.23 | 64.55 ± 8.07 |
Glucose | mg/dL | 75.24 ± 10.03 | 80.9 ± 10.28 | 83.12 ± 22.28 | 74.38 ± 7.48 | 82.06 ± 5.05 |
Cholesterol | mg/dL | 62.95 ± 3.56 | 63.51 ± 1.78 | 64.16 ± 2.13 | 55.03 ± 0.99 | 60.08 ± 0.77 |
Urea | mmol/L | 7.03 ± 0.48 | 5.91 ± 0.16 | 6.03 ± 0.14 | 6.32 ± 0.23 | 5.95 ± 0.23 |
Values are expressed as a mean ± SEM (
Vital internal organs such as liver, kidney, spleen, heart, and uterus in rats of all test groups did not show any abnormality in color and texture compared to those of control group rats in gross examinations during necropsy at the end of the study period. The ROW of dissected organs (liver, kidney, spleen, heart, and uterus) in treated and control group rats which was recorded during necropsy is shown in Table
Effect of EASPA on relative organ weights in the subacute toxicity study.
Group | Liver | Kidneys | Heart | Spleen | Uterus |
---|---|---|---|---|---|
Control | 4.63 ± 0.13 | 0.79 ± 0.00 | 0.37 ± 0.01 | 0.25 ± 0.00 | 0.91 ± 0.03 |
1.75 mg/kg | 4.49 ± 0.15 | 0.79 ± 0.02 | 0.34 ± 0.01 | 0.24 ± 0.00 | 0.83 ± 0.05 |
3.5 mg/kg | 4.53 ± 0.15 | 0.8 ± 0.00 | 0.35 ± 0.01 | 0.23 ± 0.00 | 0.92 ± 0.00 |
7 mg/kg | 4.40 ± 0.28 | 0.82 ± 0.02 | 0.34 ± 0.01 | 0.25 ± 0.01 | 0.98 ± 0.00 |
14 mg/kg | 3.42 ± 0.29 | 0.73 ± 0.01 | 0.35 ± 0.00 | 0.23 ± 0.00 | 0.82 ± 0.02 |
Values are expressed as a mean ± SEM (
Light microscopic examinations of histopathological sections of vital organs (liver, kidney, heart, spleen, and uterus) in test and control group rats are shown in Figure
Effect of EASPA on histomorphologies of vital organs in test group rats in subacute toxicity study (H and E Stain, 100x). [(a), (b), (c), (d), (e): liver; (f), (g), (h), (i), (j): kidney; (k), (l), (m), (n), (o): spleen; (p), (q), (r), (s), (t): heart; (u), (v), (w), (x), (y): uterus].
Practitioners in Ayurvedic and traditional medicine use the IC for the treatment of menorrhagia in Sri Lanka. According to preliminary phytochemical investigation, the IC contains a high amount of proanthocyanidins. The extraction, purification, and characterization of EASPA of the IC have previously been reported [
Generally, a higher dose of the test material is selected for the acute toxicity study. In this study, limit dose, 2000 mg/kg body weight was selected as described in the OECD guideline 423 [
In acute toxicity study, rats treated at the dose level of 2000 mg/kg body weight did not reveal any mortality, visible signs of toxicity or behavioral changes similar to that of control group rats during the 14-day study period. Similar observations were made for rats of all four treated groups in the subacute toxicity study during the 28-day study period. Mortality and clinical signs of toxicity are not only important observations in toxicity studies but also indicators of toxicity effects induced by the test material [
Body weight change is a sensitive indication of the general health status of animals [
In toxicity studies, serum biochemistry analyses play a major role in evaluating the possible toxic effects induced by the oral treatment of the test material [
In toxicity studies, haematology analyses also play a major role in evaluating the possible toxic effects induced by the oral treatment of the test material [
The liver, kidney, spleen, heart, and uterus are primary organs, which are affected by metabolic reactions caused by toxic compounds [
Considering the results of the acute toxicity study, it is possible to suggest that a single oral administration of EASPA to rats was well tolerated up to the dose level of 2000 mg/kg body weight. Therefore, it is possible to suggest that the LD50 of EASPA is above 2000 mg/kg body weight via oral route. According to the Globally Harmonized System of Classification and Labeling of Chemicals under OECD guideline, 423, EASPA can be classified into the category 5 (LD50 > 2000 mg/kg), which was the lowest toxicity class in the classification. According to results of the subacute toxicity study, the oral administration of EASPA to rats daily for 28 days at 1.75, 3.5, 7, and 14 mg/kg body weight dose levels is safe.
This study provides valuable data on acute and subacute toxicity studies of EASPA of the immature inflorescence of
The data used to support the findings of this study are included within the article. The 13C NMR spectrum of EASPA is available on request.
The authors declare that they have no conflicts of interest.
This work was financially supported by the University of Sri Jayewardenepura, Sri Lanka (Research Grant ASP/01/RE/SCI/25). Spectroscopic facilities provided by the Natural Products Center, University of Arizona, USA is greatly appreciated.