a large number of the world 's populations , especially in developing countries , depend upon medicinal plants as an alternative and complimentary drugs therapy for various ailments . some of the most common practices involve the use of crude plant extracts , which may contain a broad diversity of molecules with often unknown biological effects . since the medicinal plants are being used indiscriminately without notifying to their possible unhealthy or toxic effects , the world health organization has recommended that traditional plants used for the treatment of diseases need further scientific investigation on their toxic side effects . plants produce bioactive compounds which act as defense mechanisms against any disease , and at the same time , may be toxic in nature . however , the general acceptability of herbal medicines has been limited by a lack of defined chemical characterization , dose regimen , and adequate toxicity data to evaluate their safety . therefore , it has become essential to assess the safety of plants used for medicinal purposes for possible toxicity . ocimum sanctum linn ( labiatae ) , known as holy basil , is a commonly used home remedy and has been advocated for various ailments like cold , fever , dysentery , hemorrhage and dyspepsia , glucoma , cataract , chronic conjunctivitis , and other painful eye diseases , as well as gastric and hepatic disorders in indigenous system of medicine . the plant is endowed with a variety of pharmacological properties including antistress , antifertility , immunoregulatory , hypoglycemic , antibacterial , antifungal , antiinflammatory , anti - carcinogenic , antioxidant , and cyclooxygenase inhibitory . os has been reported to show gastroduodenal ulcer protective , antisecretory , and gastric mucosal defense enhancing activities . the leaves of o. sanctum ( os ) contain a volatile oil composed of limonene , borneol , copaene , caryophyllene , and elemol , phenolic compounds ( rosmarinic acid , apigenin , cirsimaritin , and isothymusin ) , flavonoids ( orientin and vicenin ) , and aromatic compounds ( methyl chavicol and methyl eugenol ) . eugenol forms the major active constituent of os , even though other minor constituents like fixed oils and flavones have also been reported to have pharmacological activities . as to the best of our knowledge , there is no reference about the safe dosage of ocimum sanctum linn in traditional medicine so it was thought worthwhile to do the acute toxicity ( mortality and cns / ans toxicities ) in mice and subacute toxicity ( biochemical , hematological , and histopathological ) in rats . inbred charles - foster albino rats ( 160180  g ) and swiss albino mice ( 2025  g ) of either sex were obtained from the central animal house of the institute of medical sciences , banaras hindu university , varanasi . they were kept in the departmental animal house at 26  20c and relative humidity of 4456% , with light and dark cycles of 10 and 14  h , respectively , for one week before and during the experiments . animals were provided with standard rodent pellet diet ( pashu aahar vihar , ramnagar , varanasi ) and water ad libitum .  principles of laboratory animal care approval from the central animal ethical committee of the university was taken prior to the experimental work ( notification number - dean/2010 - 11/173 dated 23.07.2010 ) . the leaves of ocimum sanctum ( os ) ( ayurvedic gardens , banaras hindu university ) were collected during october - december and identified with the standard sample preserved in the department of dravyaguna , institute of medical sciences , banaras hindu university , varanasi . 50% ethanolic extract of os ( ose ) was prepared by adding 500  g of dried , crushed , and powdered leaves of os in 1000  ml of 50% ethanol in a round bottom flask and was kept at room temperature for 3 days in shade . the extract filtrate so obtained was pooled and evaporated on water bath till it dried . adult swiss albino mice of either sex , weighing between 20 and 25  g , fasted overnight and were used for acute toxicity study , as per the organization for economic co - operation and development ( oecd 423 ) guideline . four groups of mice ( n = 6 ) of both sexes was fasted overnight . the first control group mice received 0.5% carboxymethyl cellulose ( cmc ) suspension in distilled water while the other three groups received ose suspended in 0.5% cmc at doses of 200 , 600 , and 2000  mg / kg . the above doses were selected on the basis of our previous reported work on ose where 400  mg / kg was found to have good ulcer protective effects . animals were observed closely for first 4 hours , for any toxicity manifestation , like increased motor activity , salivation , convulsion , coma , and death . subsequently observations were made at regular intervals for 24  h. the animals were under further investigation up to a period of 14 days and the number of mice that died within the study period was noted . the repeated doses ( 28 days ) for oral toxicity studies were carried out in rats according to the oecd test guideline 407 . rats were divided randomly into 4 groups of 6 animals each ( 3 males and 3 females ) . after an overnight fast , animals in group i received 0.5% cmc suspension ( control group ) , whereas rats in groups ii , iii , and iv ( test groups ) received ose suspended in 0.5% cmc at the doses of 200 , 400 , and 800  mg / kg body weight , respectively . doses of ose and cmc were administered daily by oral gavage in the volume of 10  ml / kg body weight , once daily for 28 consecutive days . the animals were observed daily for any abnormal clinical signs and death during the study period . body weights were measured and recorded at the beginning and then after every week of the experiment . at the end of the study , all animals fasted overnight ( water ad libitum ) and , on 29th day , the animals were weighed . blood was collected from retroorbital technique with or without edta for haematological and biochemical analysis , respectively . the animals were sacrificed with an overdose of ether and other body organs were taken out for detailed weight and histopathological changes . haemoglobin , total leukocyte count , and differential leukocyte count ( polymorph , lymphocyte , and eosinophil )   were determined in control and ose - treated groups . the serum was carefully aspirated with a pasteur pipette into sample bottles for the various biochemical assays . assay kits ( span diagnostic reagent kit and precichem diagnostic kit ) were employed for aspartate transaminase ( ast ) , alanine transaminase ( alt ) , alkaline phosphate ( alp ) , creatinine , blood glucose , total protein , and total cholesterol and bilirubin analysis was determined in the serum following the procedure described in the kits . the rats were quickly dissected and the liver , kidneys , stomach , spleen , lung , heart , and testis or ovary were excised and weighed . the specimens for histopathology were fixed in 10% neutral , buffered formalin for 18  h at 4c . 3 - 4  m in thickness of each specimen of liver , kidney , heart , spleen , and testis / ovary was cut and stained with hematoxylin and eosin stain following the standard laboratory procedures . the stained sections were examined under microscope for any cellular damage or change in morphology of that particular tissue . statistical comparison was performed using one way analysis of variance ( anova ) followed by dunnett 's test for multiple comparisons . all statistical analysis was performed using spss statistical version 16.0 software package ( spss inc . the limit dose of 2000  mg / kg did not cause death or any toxic signs in treated male and female mice . all six mice were normal throughout the study and survived until the end of the 14-day experiment period . oral administration of ose at doses of 200 , 400 , or 800  mg / kg body weight daily for 28 day , did not produce any mortality . little or no change was observed in body weight , food consumption , and water intake in ose ( 200 , 400 , and 800  mg / kg)-treated groups compared with control group after 28 days of study period in rats ( tables 1 and 2 ) . hematological parameters like mean haemoglobin content , wbc , rbc , and differential cell counts were not significantly different with ose - treated rats from control group ( table 3 ) . biochemical parameters for liver and kidney function test like aspartate transaminase ( ast ) , alanine transaminase ( alt ) , alkaline phosphatase ( alp ) , creatinine , albumin , blood glucose , total protein , total cholesterol , and bilirubin did not show any difference with the above doses of ose compared to control group ( table 3 ) . the organs like liver , kidney , heart , spleen , and testis or ovary isolated in various groups did not reveal any abnormalities in their gross examinations and difference in their mean weights both in treated and control groups ( table 4 ) . the histological studies with liver , spleen , kidney , heart , and testis / ovary did not reveal any pathological changes after treatment even with higher dose of 800  mg dose of ose when administered for 28 days ( figures 1 , 2 , 3 , 4 , 5 , and 6 ) . the administration of herbal preparations without any standard dosage , coupled with a scarcity of adequate scientific studies on their safety , has raised concerns regarding their toxicity . recently we reported teratological effects of asparagus racemosus in rats which have been advocated in indigenous system of medicine during pregnancy and lactation , which thus indicated that herbal drugs are not safe as thought otherwise . to the best of our knowledge , there are no published studies on ocimum sanctum toxicological profile following subacute exposure . in oral acute toxicity study , as high dose of ose at 2000  mg / kg did not show any observable toxic effects in the mice in terms of any deaths or abnormal symptoms which points to its being nontoxic and safe in mice . subacute toxicity study in rats with 200 , 400 , and 800  mg / kg of ose when administered for 28 days did not produce any mortality , change in food and water intake , body and organ weights , or histopathological changes in the organs like liver , kidney , spleen , heart , testis or ovary further strengthen the safety profile of ose . subacute studies in rats did not show any change in hematological , liver functions and spleen with 800  mg / kg ose when administered for 28 days . the hematopoietic system / bone marrow is one of the most sensitive targets for toxic compounds and an important index of physiological and pathological status in man and animal . analysis of blood parameters is relevant to risk evaluation as the changes in the hematological system have a higher predictive value for human toxicity , when the data are translated from animal studies . subacute exposure of rat to the lower doses of the ose produced small and transient changes in some biochemical and hematological parameters . liver is a site of cholesterol disposal or degradation and its major site of synthesis . in the same perspective since no significant changes were observed in glucose and cholesterol levels this study , it suggests that ose had no effect on the lipid and carbohydrate metabolism in rats . further , drugs showing any toxicity in liver affect the transaminases , ( aspartate aminotransferase ) ast , and ( alanine amino transferase ) alt which are well - known enzymes used as good indicators of liver function   and biomarkers predicting possible toxicity . generally , any damage to the parenchymal liver cells results in elevations of both transaminases in the blood . in our study , both ast and alt did not show any treatment related increase even at the 800  mg / kg dose compared to the control group . in addition , ast found in the serum is of both mitochondrial and cytoplasmic origin and any rise can be taken as a first sign of cell damage that leads to the outflow of the enzymes into the serum . thus , no significant increases observed in alt and ast activities strongly suggest that the subacute administration of ose did not alter the hepatocytes and consequently the metabolism in the rats . further , ose did not show any histological changes in spleen and liver indicating no effect on reticuloendothelial system . ose neither showed any significant difference in the weight of the organs or color of organs nor affected the histopathological changes in organs like heart and testis / ovary indicating least cumulative toxic effects on reproductive and cardiac tissues . further , no change was also observed in kidney function as evidenced by little or no change in serum creatinine level as well as histological changes in treated kidneys . this study provides valuable data on the acute and subacute oral toxicities profile of ocimum sanctum linn leaves ( a common household remedy for number of ailments ) that could be very useful in its future clinical study . the 50% ethanol extract of ocimum sanctum leaves seemed to be nontoxic as was seen after its acute and subacute oral administrations . further , teratogenic , mutagenic and carcinogenic studies with this plant are needed to complete the safety profile of this plant .
the present study was aimed to study the acute and subacute toxicity studies with orally administered 50% ethanolic leaves extract of ocimum sanctum linn ( ose ) . in acute toxicity tests , four groups of mice ( n = 6/group / sex ) 
 were orally treated with doses of 200 , 600 , and 2000  mg / kg , and general behavior , adverse effects , and mortality were recorded for up to 14 days . in subacute toxicity study , rats received ose by gavage at the doses of 200 , 400 , and 800  mg / kg / day ( n = 6/group / sex ) for 28 days , and biochemical , hematological , and histopathological changes in tissues ( liver , kidney , spleen , heart , and testis / ovary ) were determined . 
 ose did not produce any hazardous symptoms or death and cns and ans toxicities in the acute toxicity test . 
 subacute treatment with ose did not show any change in body weight , food and water consumption , and hematological and biochemical profiles . 
 in addition , no change was observed both in macroscopic and microscopic aspects of vital organs in rats . 
 our result showed that ocimum sanctum extract could be safe for human use .