herbal plants have been used as medicines for thousands of years , and ; some specific plants have been used for particular ailments .
medicinal plants are sources of raw materials for both traditional systems of medicine and modern medicine
. these medicines are also in great demand in the developed world for primary health care because of their efficacy , safety and fewer lesser side effects .
traditionally , herbs and herbal products have been considered to be nontoxic and have been used by the general public and traditional medicinal doctors worldwide to treat a range of ailments .
however , the fact that something is natural does not necessarily make it safe or effective .
the active ingredients of plant extracts are chemicals that are similar to those in purified medications , and they have the same potential to cause serious adverse effects . whilst the literature documents severe toxicity resulting from the use of herbs , on many occasions the potential toxicities of herbs and herbal products have not been recognized .
pistacia integerrima j. l. stewart ex brandis ( pi ) is a species of pistachio tree , commonly called zebrawood , native to asia .
it is one of the important plants of indian traditional medicine and belongs to the family anacardiaceae .
the phytochemical constituents of pi have been reported to be tannins , essential oils and resins .
the oil contains alpha - pinene ( 25% ) , camphene ( 27% ) , di - limonene ( 4%  5% ) , 1:8-cineol ( 10% ) , caprylic acid ( 15% ) , alpha - terpineol ( 20% ) and aromadendrene ( 4%  5% ) .
the two triterpenic acids are ketocarboxylic and appear to be identical to the alpha and the beta acids [ 3 , 4 ] .
pi is used for a variety of purposes in india , including timber , dye , and fodder ; it is also used as a herbal remedy for many ailments including coughing , asthma , fever , vomiting , diarrhea , loss of appetite , nose bleeds , snakebites , and gastrointestinal and liver disorders [ 5 - 8 ] .
pi is reported to be useful as an anti- inflammatory , and an anti - diabetic agent and as a blood purifier [ 9 - 11 ] .
galls excrescences are formed by insects on the leaves , petioles and branches of plants .
these leaf galls are harvested and used to make kakad shringi ; a herbal medicine used to treat diarrhea in northern india .
pi leaf galls were collected in the month of july from the nadaun block of the hamirpur district , himachal pradesh .
ravindra pandey , department of natural products research , columbia institute of pharmacy , raipur , and a voucher specimen , i.e. , leaf galls , with voucher number 0342 , was deposited in the herbarium of the institute .
the powdered material was soaked in methanol , distilled water , chloroform , ethyl acetate and n - hexane for one week and subjected to extraction until exhaustion of the plant material .
male wistar rats with weights in the range of 160  200 g were used for the experiments , which were carried out according to a protocol approved by the institutional animal ethics committee , columbia institute of pharmacy , raipur , chhattisgarh , india ( 1321/ac/10/cpcsea , dated- 01/28/2010 ) .
the animals were housed in polycarbonate cages in a room with a 12-hour day - night cycle , a temperature of 22  2c , and a humidity of 45%  64% . during the entire experimental period , animals were fed with a balanced commercial pellet diet ( ashirwad industries , mohali , india ) and were allowed ad libitum access to water and normal saline .
the acute toxicity studies were conducted as per guideline 425 of the organization of economic and cooperation development ( oecd ) , where a dose limit of 2,000 mg / kg of body weight was used .
animals were observed individually after dosing at least once during the first 30 minutes , periodically during the first 24 hours , with special attention given during the first 4 hours and daily thereafter , for a total of 14 days .
individual body weights were recorded once a week as well as on the day the experiment ended .
changes in skin , fur , eyes , mucous membranes secretions , excretions , and autonomic activity ( e.g. lacrimation , piloerection , pupil size , unusual respiratory pattern ) were recorded , as were changes in gait , posture , and response to handling , as well as the presence of clonic or tonic movements .
the sub - acute toxicity studies were conducted as per the guideline 407 of the oecd ; this provides information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time .
four groups of animals were selected , group 1 being the normal control and groups 2 , 3 and 4 being treated daily with pi at doses of 250 , 500 , and 1,000 mg / kg of body weight , respectively , for 28 days .
all animals were monitored for toxic manifestations such as change in body weight and , mortality .
after 28 days all surviving animals were fasted overnight , after which blood samples were collected for hematological and biochemical analyses .
animals were sacrificed after blood collection , and internal organ were removed and preserved in 10% formalin for histological examination .
the hematology parameters measured for the sub - acute toxicity study of pi were white blood cell count ( wbc ) , red blood cell count ( rbc ) , hemoglobin ( hb ) , hematocrit ( hct ) , mean corpuscular volume ( mcv ) , mean corpuscular hemoglobin ( mch ) , mean corpuscular hemoglobin concentration ( mchc ) , platelets ( plt ) and lymphocyte ( lymp ) . clinical biochemistry analysed to investigate the major toxic effects on tissues and
specifically , the effects on the kidneys and the liver were performed on blood samples obtained from of all animals .
biochemical parameters measured for the sub - acute toxicity study of pi were glucose , albumin , urea , total protein ( tp ) , serum glutamate oxaloacetate transaminase ( sgot ) and serum glutamate pyruvate transaminase ( sgpt ) .
histological examinations were performed on the tissues , which had been preserved in 10% formalin solution . in this study , all data are expressed as means  standard errors of the mean ( sems ) .
the results were analyzed statistically through graph pad prism 6.0 by using the twoway analysis of variance ( anova ) , followed by the bonferroni post - test to calculate the level of significance .
the number of animals was six ( n = 6 ) ; statistically significant difference in the outcomes , when compared to the control group , were indicated as follows : p < 0.05 , p < 0.01 , p < 0.001 .
an investigation of acute toxicity is an initial step in the characterization of the biological effect of any substance and is necessary for conducting any biological experiment .
no deaths or hazardous signs were recorded in the rats during the 14 days of observation after acute treatment by an oral route with pi at doses of 50 , 300 , and 2,000 mg / kg of body weight . in sub - acute toxicity investigation , no toxicity and no deaths were recorded during the 28 days of treatment by an oral route with pi in doses of 250 , 500 , and 1,000 mg / kg of body weight .
no significant changes were observed in the body weights of the both control and the experimental groups of animals ( figs .
the hematological profiles of the animals revealed no significant change in any of the clinical parameters ( tables 1,2 ) .
the mchcs for the experimental animals of both sexes were higher than those for the control animals , but the values were within the normal range , demonstrating the methanolic extract of pi exhibited no toxicity . during the biochemical analyses ,
all the parameters were in the normal range , and no significant changes were noted when the values of the parameters for the different dose groups were compared with the values for the control group .
slight variations were observed in parameters such as glucose ( glu ) , urea , sgpt , sgot in the 1,000 mg / kg of body weight dose group , but the values were in the normal range ( tables 3,4 ) .
the organs were found to be devoid of any sign of toxicity , and no significant changes in any of the organ weights were observed ( tables 5,6 ) .
the histopathological examination of the kidney , lungs and liver were normal in both the control and the treated groups .
the research protocol was envisaged and designed to evaluate the toxicity profile of methanolic extract of pi with a main focus on observing the normal physiological conditions of experimental animals after treatment the with extract .
thus , because of the advancement of research in this area , a wide range of parameters and experimental targets were studied to assess the overall profile of the pi extract to determine if any toxicity was present .
the various hematological and biochemical parameters evaluated for the rats showed no abnormalities , and the values were within normal physiological limits and were comparable to the values in the vehicle - treated ( control ) animals .
the pi extract is seen to have had no , toxic effect on any of the observed blood parameters , wbc , rbc , hb , hct , mch , mchc , plt and lymp , for the different dose levels .
regardless of the doses of the pi extract the blood parameters were within the normal range . in ( fig .
2 ) , small differences in the levels of the biochemical parameters sgpt and sgot can be seen .
higher doses of the pi extract , i.e. 500 and 1,000 mg / kg of body weight , both caused increase in the sgpt and the sgot levels , but these increases did not take the levels outside the normal range .
histopathological evaluation showed no adverse effects of pi extract on the dissected organs ; swelling , atrophy , and hypertrophy were not observed .
the results of the urine analysis showed no signs of toxicity in the urine ; the parameters being within the normal range ; creatinine , ph , color , quantity of urine . however , pus cells , sperms , bacteria , and ketone bodies were found , but rarely .
no statistically significant differences were found in any of the parameters between the control and the treated groups with respect to various vital organs , and no visceral abnormalities were seen in any of the groups .
all animals survived until the scheduled euthanasia and no significant gross pathological alterations were found in the internal organs .
in summary , our acute and sub - acute toxicity studies of wistar rats revealed no toxicological symptoms or mortality at any of the dose levels of methanolic extract of pi used in this research .
daily treatments with pi at intervals of 24 hours in doses of 250 , 500 , and 1,000 mg / kg of body weigh p.o . had no adverse effect on body weight .
the various hematological and biochemical parameters evaluated in the rats showed no abnormalities , and the values were within the normal physiological limits and were comparable to the values for the vehicle - treated ( control ) animals .
the methanolic extract of pi was found to be non - toxic based on the results of the oral acute and sub - acute toxicity tests performed on rats in this research . however , an evaluation of chronic toxicity is needed to determine the long - term safety of the extract .
values are expressed as means  sems ( number of animals , n = 6 ) ; and were significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with the control group . sems , standard errors of the mean ; wbc , white blood cell ; rbc , red blood cell , hb , haemoglobin ; hct , hematocrit ; mcv , mean corpuscular volume ; mch , mean corpuscular haemoglobin ; mchc , mean corpuscular hemoglobin concentration ; plt , platelets ; lymp , lymphocyte .
values are expressed as means  sems ( number of animals , n = 6 ) ; and were significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with the control group .
sems , standard errors of the mean ; wbc , white blood cell ; rbc , red blood cell , hb , haemoglobin ; hct , hematocrit ; mcv , mean corpuscular volume ; mch , mean corpuscular haemoglobin ; mchc , mean corpuscular hemoglobin concentration ; plt , platelets ; lymp , lymphocyte .
values were expressed as means  sems ( n = 6 ) and were significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with control group .
sems , standard errors of the mean ; glu , glucose ; tp , total protein ; alb , albumin ; sgpt , serum glutamate oxaloacetate transaminase ; sgot , serum glutamate pyruvate transaminase .
values were expressed as means  sems ( n = 6 ) and were significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with control group .
sems , standard errors of the mean ; glu , glucose ; tp , total protein ; alb , albumin ; sgpt , serum glutamate oxaloacetate transaminase ; sgot , serum glutamate pyruvate transaminase .
values were expressed as means  sems ( n = 6 ) and were significantly different at p < 0.05 ,
values were expressed as means  sems ( n = 6 ) and were significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with control group .
n = 6 ) and were ; significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with the control group .
values are expressed as means  sems ( number of animals , n = 6 ) and were ; significantly different at p < 0.05 , p < 0.01 , and p < 0.001 , when compared with the control group .
 objectives : the goals of this research were to evaluate acute ( single - dose ) and sub - acute ( repeated - dose ) toxicity profiles of methanolic extract of pistacia integerrima j. l. stewart ex brandis ( pi ) for wistar rats and to assess the safety profile of pi by observing physiological changes , mortality , changes in body weight , the histopathology of body organs , the hematology and the biochemistry of the animals.methods:the toxicity profile of pi was evaluated using wistar rats of both sexes . 
 animals were divided into four groups : group 1 ; control group ( normal saline ) , group 2 ; pi-1 ( 250 mg / kg ) , group 3 ; pi-2 ( 500 mg / kg ) , group 4 ; pl-3 ( 1,000 mg / kg ) . 
 an acute - toxicity study in which animals received a single dose of pi extract ( 2,000 mg/ kg ) and were then observed for 14 days for changes in skin , fur , eye color , mucous membrane secretions and excretions , gait , posture , and tonic or clonic movements was performed according to guideline 425 of the organization of economic and corporation development ( oecd ) . in the repeated - dose toxicity study ( oecd  407 ) animals received a daily dose of pi extract for 28 days ( 4 weeks ) . the parameters observed in this study include body weight , hematology and biochemistry of the animals.results:in the acute toxicity study , no mortalities or changes in behavior were noted in the animals . 
 the repeated - dose toxicity study was also devoid of any toxicity in the animals during the 28 days of testing with pi extract . 
 the extract did not alter- the body weight , hematology or biochemistry of the animals . 
 the methanolic extract of pi was to be found safe to the no - observed - adverse - effect - level ( noael ) for the single- dose and repeated - dose toxicity tests in rats.conclusion:the methanolic extract of pi was devoid of toxicity ; hence , it can be used for various ayurvedic preparations and treatments of diseases . 