Acute and Chronic Toxicities of Pandanus Amaryllifolius Roxb . Water Extract from the Roots in Rats

Pandanus amaryllifolius Roxb. is one of the most valuable herbs that has been used as a medicine for diabetes mellitus. The study of the acute toxicity of the plant extract was conducted in male and female rats by a single oral administration of the test substance at a dose of 5 g/kg body weight. The toxic sign, general behaviors, mortality, and microanatomical characteristics of internal organs were examined at the end of experiment. The results showed no sign of differences as compared to the normal control rat. Thus a single oral administration of 5 g/kg body weight does of P. amaryllifolius extract significantly cause no toxicity. For the chronic toxicity test, after an oral feeding both male and female rats daily with the extract 1, 2, 4 and 8 g/kg body weight for 180 days, toxic signs, animal behavior and health monitoring were then investigated. There were no abnormalities in the test groups as compared to the controls. Furthermore, the rats of the test and control groups on day 180 and the satellite group on day 208 were analyzed by measuring their final body and organ weights, taking necropsy, and examining hematology, blood clinical chemistry, and microanatomy. Results did not show any differences except an increase in the body weight of the treated male rats when compared with the control groups. These results combined with the information of signs, behavior and health monitoring can lead to a conclusion that an oral administration of P. amaryllifolius extract at the doses of 1, 2, 4 and 8 g/kg body weight for 180 days does not produce chronic toxicity.


Introduction
Pandanus amaryllifolius Roxb.(Synonym P. odorus Ridl; Thai name: Toei-hom, family Pandanaceae) has been abundantly cultivated in Thailand.The leaves of P. amaryllifoliuscan are used as spice, food coloring and flavoring agents in culinary application.Root of P. amaryllifolius has long been reported as a remedy for diabetes mellitus.The ethanol extract from the leaf of P. amaryllifolius shows the antidiabetic effect on streptozotocininduced diabetic mice [1].The water extract of P. odorus root shows the hypoglycemic effect in diabetic rats [2][3].In an acute

Acute toxicity study
The acute oral toxicity was evaluated in rats as described by the Organization of Economic Co-operation and Development (OECD) Guideline for Testing of Chemicals [5] and World Health Organization (WHO) Guideline [6].The female rats were divided into two groups of ten animals (n = 5).The control group received a vehicle in a volume of 1 ml/kg body weight.The P. amaryllifolius extract was orally administered to rats using a single dose of 5 g/ kg body weight.The animals were monitored for appearance of signs of toxicity immediately after dosing and at 1, 2, 4, and 6 h after dosing.Observations were focused on changes in skin, fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems as well as somatomotor activity and behavioral pattern.The number of survivors were noted after 24 h and then maintained for a further 14 days with a once daily observation.The animals that died within this period were necropsies.Animals were weighed on day 0, and on days 7 th and 14 th .At the end of the experiment, all surviving animals were sacrificed with pentobarbital sodium.The vital organs including heart, lungs, livers, kidneys, spleen, adrenals, sex organs and brain were subjected to gross and histology examination.

Chronic toxicity study
The method was conducted according to the OECD Guideline for Testing of Chemicals [7] and WHO Guideline [6].Briefly, P. amaryllifolius extract at the doses of 1, 2, 4 and 8 g/kg body weight were administered to rats (ten males, ten females) once daily over 180 days, the control group received the vehicle.The satellite group (ten males, ten females) was given the extract at the dose of 8 g/kg body weight over 180 days and kept for other 28 days after the treatment in order to detect a delayed occurrence of toxic effect.All rats were observed for appearance of signs of toxicity or behavioral alterations during the experimental period.At the end of each experiment, the rats were fasted 12 hours, and then anesthetized with ether.Their blood was collected from a common carotid artery for hematological study, using Sysmex K-1000 Fully Automated Hematology Analyzer.The serum was separated from the blood and measured levels of glucose, blood urea nitrogen (BUN), creatinine, total protein, albumin, total bilirubin, direct bilirubin, alkaline phosphatase (ALP), serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamicpyruvic transaminase (SGPT), using the COBAS INTEGRA System.
After the blood collection, the animals were immediately sacrificed for tissue studies.The following tissues and organs were weighted, grossly examined, and then fixed in 10% buffered formaldehyde solution: heart, lungs, thymus, livers, kidneys, spleen, adrenals, small intestine, stomach and duodenum, muscle with sciatic nerve, thoracic spines, brain, eyes, sex organs, uterus and epididymis.The fixed organs from all animals were examined by histological method.

Statistical analysis
Results were expressed as mean ± standard error of mean (S.E.M.).Statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc least-significant difference (LSD) test.The data obtained from acute toxicity studies were analyzed using Student's t-test.P values less than 0.05 were considered significant.

Results and Discussions
The acute toxicity study aims to investigate animal responses after a single dose administration of P. amaryllifolius extract.The results of this study provided early information for safety evaluation and then appropriate dosage for further chronic toxicity assessment.Signs and symptoms of indicated organ systems, body weight, organ weight and histological data were collected and analyzed to investigate the acute toxicity of this extract.
The result of our study found that all rats did not exhibit signs of toxicity or mortality after a single oral administration of P. amaryllifolius extract at the dose of 5 g/kg.The body weight gain and internal organ weights were observed since a decrease in both parameters would indicate the presence of toxicity.The body weight on the 7 th and 14 th day of female rats received the extract did not different from that of the control group Table 1.At the end of this study, the average weight of the internal organs and condition of the color and the texture were normal and comparable to the control group Table 2.In addition, the histological examination of the lung, heart, liver, spleen, adrenal grand, kidney, thymus, stomach and duodenum, small intestinal, ovary, uterus, muscle and nerve, thoracic spine, eyes and brain were normal in both the control and the treated groups.Furthermore, the histological examinations in both control and treated groups revealed normal results of these following organs: the lung, heart, liver, spleen, adrenal grand, kidney, thymus, stomach and duodenum, small intestinal, ovary, uterus, muscle and nerve, thoracic spine, eyes and brain.As shown in Fig. 1, as compared to the control, no abnormality was found in vital organs, liver and lung of rats after receiving the extract.According to the OECD guideline for testing of chemicals (2001) [5], the results of acute toxicity suggested that P. amaryllifolius extract is reasonably nontoxic.
Because signs, symptoms, organ weight and body weight become a preliminary evidence for evaluation of sample's acute toxicity, the initial assessment of chronic toxicity was similarly undertaken.After administration of P.amaryllifolius, we observed the signs and symptoms of indicated organ systems, and weighted the body and organs before examining the histological outcomes.Results of the chronic toxicity showed that administration of the extract at doses of 1, 2, 4 and 8 g/kg daily over 180 days does not cause mortality and behavioral changes.In female treated rats, the body weight and internal organ weight were not different from those of the control group Table 3-4.The body weight of male rats was found to be statistically different from that of the control group Table 5.However, the weights of some internal organs of male rats were found to be statistically different from those of the control group Table 6.On the other hands, the gloss examinations did not show any abnormality of animal tissues in all groups and in both sexes.Thus, these results may be due to variation of the size and/or weight of animals' organs [8].
Taken the above results together, it could be concluded that P. amaryllifolius extract had no chronic toxicity on animals.
To confirm these findings, the histology examination of all animal organs was determined whether or not the toxicity of P. amaryllifolius was present.A satellite group was given the P. amaryllifolius extracting at 8 g/kg daily over 180 days followed by no treatment for 28 days.A satellite group was given the P. amaryllifolius extracting at 8 g/kg daily over 180 days followed by no treatment for 28 days.* Significantly different from control, p<0.05.and risks of any disease.Hematological parameters provide vital information regarding the status of bone marrow activity and intravascular effects such as anemia or hemolysis.The lower level of WBC is likely caused by some immune diseases, viral infections or hematologic diseases such as a plastic anemia or bone marrow suppression, which resulting in the decrease of RBC, platelets and WBC.In addition, leukemia could be one of the factors leading to normal or reduced WBC known as a leukemic leukemia.However, the bacterial infection usually results in WBC to increase dramatically which requires a differential count of WBC to establish diagnosis.Moreover, high numbers of WBC could represent leukemia, but white blood cells were needed to be considered thoroughly.Some of the hematological values of female and male rats were slightly different from those of the control group Table 7-8 whereas the differential white blood cells count values showed no difference between the control and the treated groups Table 9-10.However, the slight change in all of the parameters is not much significant because variations of the animals must be taken into account [9][10][11].Therefore, the results indicated that P. amaryllifolius did not cause chronic toxicity on either blood cells or the hematopoietic production that causes abnormalities on animals.

Hematologic data were useful for diagnosis and investigation of initial abnormality to demonstrate healthy status of animals
The liver, kidney and pancreas are major vital organs.The liver plays a role in synthesis and delivery of various compounds, especially glucose, protein and lipid to be utilized in the body.While essential vitamins and minerals are accumulated in the liver, hepatotoxic chemicals or metals are kept before being excreted out of the body.One of the kidneys' main functions is to produce and control hormone synthesis, in particular hormones related to calcium and erythrocyte production.These hormones are able to collect nourishing substances which are being reabsorbed by renal tubules.Additionally, apart from excretion of wasted products, the kidney can control the balance of fluids and electrolytes.
The clinical blood chemistry values were used to analyze kidney function (BUN and creatinine) and liver function (glucose, total protein, albumin, total and direct bilirubin, SGOT, SGPT and ALP).In both female and male satellite groups, some clinical blood chemistry values such as creatinine, BUN, SGOT and ALP were statistically different from those of the control group.In general, if the clinical blood chemistry values differ more or less than one fold from the normal values, abnormality of kidney, liver and pancreas's function should be noted [11][12][13][14][15].However, in our study, the observed difference was less than one fold Table 11-12, suggesting normal function of the organs.The results can be concluded that P. amaryllifolius extract had no chronic toxicity on liver and kidney.
The histological examination of cells and tissues is an important procedure in chronic toxicity evaluation.Although there were no significant changes in the following parameters: symptoms, body weight, organ weight, gloss examination, hematology and blood chemistry profiles; the histological examination pointed out that cells and tissues of the animals significant altered from baselines after administration of P. amaryllifolius.In this study, the histological features of the lung, heart, spleen, adrenal grand, thymus, stomach and duodenum, small intestinal, ovary, uterus, A satellite group was given the P. amaryllifolius extract at 8 g/kg daily over 180 days followed by no treatment for 28 days.* Significantly different from control, p<0.05.

Acute and Chronic Toxicities of Pandanus Amaryllifolius Roxb. Water Extract from the Roots in Rats
Copyright: © 2016 Sireeratawong et al.

Figure 1 :
Figure 1: Histology of female kidney and liver in control and P. amaryllifoliustreated groups in acute toxicity.Section was stained with haematoxylin and eosin.No significant damage was detected in any treatment group.

Figure 2 :Figure 3 :
Figure 2: Histology of male kidney and liver in control and P. amaryllifolius treated groups in chronic toxicity.Section was stained with haematoxylin and eosin.No significant damage was detected in any treatment group.

Table 1 :
Body weights of female rats in acute toxicity of the water extract from the root of P.

Table 2 :
Organ weights of female rats in acute toxicity of the water extract from the root of P. amaryllifolius.

Table 3 :
Body weights of female rats in chronic toxicity of the water extract from the root of P. amaryllifolius.Values are expressed as mean ± S.E.M., n = 10.A satellite group was given the P. amaryllifolius extracting at 8 g/kg daily over 180 days followed by no treatment for 28 days.

Table 4 :
Organ weights of female rats in chronic toxicity of the water extract from the root of P. amaryllifolius.

Table 5 :
Body weights of male rats in chronic toxicity of the water extract from the root of P. amaryllifolius.

Table 6 :
Organ weights of male rats in chronic toxicity of the water extract from the root of P. amaryllifolius.Values are expressed as mean ± S.E.M., n = 10.A satellite group was given the P. amaryllifolius extracting at 8 g/kg daily over 180 days followed by no treatment for 28 days.* Significantly different from control, p<0.05.

Table 7 :
Hematological values of female rats in chronic toxicity of the water extract from the root of P. amaryllifolius.

Table 8 :
Hematological values of male rats in chronic toxicity of the water extract from the root of P. amaryllifolius

Table 9 :
White blood cell count values of female rats in chronic toxicity of the water extract from the root of P. amaryllifolius

Table 10 :
White blood cell count values of male rats in chronic toxicity of the water extract from the root of P. amaryllifolius.

Table 11 :
Clinical blood chemistry values of female rats in chronic toxicity of the water extract from the root of P. amaryllifolius.Values are expressed as mean ± S.E.M., n = 10.A satellite group was given the P. amaryllifolius extract at 8 g/kg daily over 180 days followed by no treatment for 28 days.

Table 12 :
Clinical blood chemistry values of male rats in chronic toxicity of the water extract from the root of P. amaryllifolius.Values are expressed as mean ± S.E.M., n = 10.A satellite group was given the P. amaryllifolius extracting at 8 g/kg daily over 180 days followed by no treatment for 28 days.* Significantly different from control, p<0.05.