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 ziziphus is a genus of about 40 species of spiny shrubs and small trees in the rhamnaceae family , distributed in the warm - temperate and subtropical regions throughout the world .
several pharmacological activities of plants in ziziphus genus have been shown in many scientific literatures , such as anti - ulcerogenic of z. lotus extract , antiproliferation of melanoma cells by z. jujube , and antiplasmodial and antimycobacterial activities of z. mauritiana .
z. attopensis is one of the plants in this genus and most commonly found in sparse forests , thickets , up to 1,500  m in laos and thailand . according to thai traditional folk medicine , the decoction of bark and
wood plant of z. attopensis has been commonly used as tonic , carminative , appetizer , and muscle analgesic .
the aim of the present study is therefore to evaluate the acute and chronic toxic effects of the water extract from the bark of z. attopensis in sd rats .
the plant material was cut into small pieces , dried in hot air oven at 50c for 6 hour , and ground into a fine powder .
a water decoction of herb is one of the effective methods used in ayurvedic and thai traditional medicine .
a decoction of z. attopensis dried bark was prepared as follows : one kilogram of bark powder of z. attopensis was suspended in fourteen liters of distilled water , brought to a strong boil , and then simmered at low heat for 3 - 4 hours .
male and female sprague - dawley ( sd ) rats , weighing 120140  g , were detained from the national laboratory animal center ( nlac ) , nakhon pathom .
they were housed under standard environmental conditions of temperature at  25  1c under a 12  h dark - light cycle and allowed free access to drinking water and standard pellet diet . all experimental protocols were approved by the animal ethics committee of the faculty of medicine , thammasat university ( no . 0001/2005 ) . according to the organization of economic co - operation and development ( oecd ) guideline for testing of chemicals   and the world health organization ( who ) guideline , acute oral toxicity was examined in male and female sd rats .
test substance at the dose of 5,000  mg / kg body weight was orally given once to one group of animals whereas the control group administered with vehicle .
body weight , body weight change , signs of toxicity , behaviors and mortality were observed for the initial 24th hour after administration and once daily for 14 days . at the end of the experiment period ,
all rats were sacrificed with overdose of pentobarbital sodium ( 100  mg / kg , intraperitoneal injection ) for necropsy examination .
the internal organs such as heart , lungs , livers , kidneys , spleen , adrenals , sex organs , and brain were performed on gross pathology and histopathological examination .
the animals were divided into six groups of twenty animals ( ten male and ten female ) .
test substance was administered orally at the doses of 1 , 2 , 4 , and 8  g / kg daily for 180 days , but the control group received an equal volume of vehicle . in order to access reversibility , the water extract was given to a group of rats ( satellite group ) at 8  g / kg once daily for 180 days and no treatment for the following 28 days . the toxic manifestations such as signs of toxicity , mortality , and the body weight changes were monitored daily [ 5 , 6 ] . at the end of each experiment ,
rats were anesthetized with pentobarbital sodium ( intraperitoneal injection , i.p . ) and blood collected with heparinized and nonheparinized tubes from common carotid artery for hematological and blood chemical studies , respectively .
after blood collection , the internal organs ( 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 ) were isolated and weighted to determine relative organs weights and observed for gross lesions .
the heparinized blood ( 2.5  ml ) was subjected to hematological tests using the sysmex k-1000 fully automated hematology analyzer for measuring white blood cell and differential leukocyte count , platelet , hematocrit , and hemoglobin contents .
the nonheparinized blood ( 5  ml ) was allowed to coagulate , then centrifuged , and the serum was separated .
serum was assayed for glucose , blood urea nitrogen ( bun ) , creatinine , total protein , albumin , total bilirubin , direct bilirubin , serum glutamic - oxaloacetic transaminase ( sgot ) , serum glutamic - pyruvic transaminase ( sgpt ) , and alkaline phosphatase ( alp ) .
samples of the internal organs were dehydrated by serial ethanol solution and enclosed with paraffin .
micrometer sections ( 5  m ) cut with microtome were stained with hematoxylin and eosin ( h&e ) and examined under a light microscope ; photomicrographs of the samples were recorded .
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 study were analyzed using student 's t - test .
single oral administration of za extract at a dose of 5  g / kg did not show visible signs of toxicity , abnormal behaviors , and mortality .
neither body weight nor internal organ weight of treated rats was significantly changed relative to that of the control group ( data not shown ) . furthermore , gross and pathological examinations of the internal organs revealed no pathological abnormality . according to oecd guideline   and kennedy et al .
, substances that explain the median lethal dose ( ld50 ) higher than 5  g / kg body weight by oral route can be considered practically nontoxic .
thus , it can be concluded that za extract is absent of the acute oral toxicity .
the rats in chronic toxicity study daily received the za extract at the doses of 1 , 2 , 4 , and 8  g / kg for 180 days .
no clinical toxicity signs and mortality were observed in the treated group compared to the control group .  
figure 1 shows the effects of the za extract on body weight and body weight gain of female and male rats .
the body weight gain of the male rats treated with za extract at 1 , 2 , and 4  g / kg was significantly decreased as compared with that of the control group
. however , food intake of all groups was not significantly altered , and there was no sign of morbidity and diseases after monitoring animal health in the entire period of 180 days .
although the weights of some internal organs of male rats ( data not shown ) were found to be statistically different from the control groups , they did not exhibit any gross morphological changes .
the hematopoietic system is very sensitive to toxic compounds and can be altered by the ingestion of some toxic plants .
the changes in the hematological system have a higher predictive value for human toxicity , when the data are translated from animal studies .
the status of bone marrow activity and intravascular effects were monitored by hematological examination . moreover ,
the morphological examination of red blood cells and white blood cells indicates their production or destruction .
white blood cell counts ( basophil , eosinophil , lymphocyte , monocyte , and neutrophil ) are also performed in order to evaluate immune system [ 911 ] . in the present study ,
some of the hematological values of female and male treated rats were slightly different from those of the control group ( tables 1 and 2 ) ; nonetheless , the differential white blood cell count values showed no difference between the control and the treated groups ( data not shown ) . because all values lay within the normal limits [ 12 , 13 ] .
thus , the changes in all of the criteria do not suggest that za extract produces chronic toxicity .
clinical blood chemistry examination was further performed in order to intensely evaluate any toxic effects on the pancreas function ( glucose ) , kidney function ( bun , creatinine ) , and liver function ( sgot , sgpt , alp , total protein , albumin , and total and direct bilirubin ) [ 11 , 14 ] . in both female and male groups , some clinical blood chemistry values such as total protein , albumin , bilirubin , sgot , and alp were statistically different from that of the control group ( tables 3 and 4 ) . nevertheless , these values are within the normal range [ 1518 ] .
bilirubin levels increase in anemia , that is , a result of a hemolytic process .
the increased levels of sgot and sgpt in the blood are associated with the damage of hepatic cells . moreover ,
the increase of serum protein level is an indication of the tissue injury and reflection of hepatic toxicity .
this observation of a major increase in the levels of liver enzymes may indicate the hepatotoxicity of za .
the histopathological analysis of the liver showed no alteration in the liver structure of za - treated rats ( figures 2 and 3 ) . in conclusion , the present study demonstrated that the water extract from the bark of z. attopensis did not produce any toxic signs and symptoms of acute and chronic oral toxicity tests .
moreover , it did not cause any lethality nor produced any remarkable hematological , and blood chemical adverse effects both in chronic toxicity studies in sd rats .
we studied an acute and chronic oral toxicity of the extract from ziziphus attopensis ( za ) in male and female sd rats according to the oecd guidelines . after a single oral administration of za 5  g / kg body weight 
 , measurement of the body and organs , necropsy , and health monitoring were performed . 
 the body and organ weights and behavior were not changed relative to the control rats indicating that za does not produce acute toxicity . 
 the chronic toxicity was determined by oral feeding both male and female rats daily with za at the doses of 1 , 2 , 4 , and 8  g / kg body weight for 180 days . 
 body weight changes , hematological and biochemical parameters , organ weights , gross finding , and histopathology examination were monitored during the experimental period . 
 the results did not show any differences from the control groups . 
 analyses of these results with the information of signs , behavior , and health monitoring can lead to a conclusion that the long - term oral administration of za for 180 days does not cause chronic toxicity .