Toxicity Assessment
Toxicity Assessment
Toxicity Assessment
Brazilian Journal of
Article
Pharmaceutical Sciences
http://dx.doi.org/10.1590/s2175-97902019000118224
Prabhat Upadhyay1, Rashmi Shukla2, Kavindra Nath Tiwari3, G P Dubey4, Sunil Kumar Mishra5*
Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 2Department of
1
Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 3Department of Botany,
MMV Banaras Hindu University Varanasi, India, 4Institute of Medical Sciences, Banaras Hindu University, Varanasi, India,
5
Department of Pharmaceutical, Engineering and Technology, Indian Institute of Technology, Banaras Hindu University,
Varanasi, India
The present study aimed to evaluate the safety of the alcoholic leaves extract of Reinwardtia indica
in Charles foster rats through an acute and sub-acute oral administration.For assessment of acute oral
toxicity test, ratswere orally treated with single dose of the alcoholic leaves extract of Reinwardtia indica
at the doses of 50, 250, 500, 1000 2000 and 5000 mg/kg. In sub-acute toxicity study, using the OECD
guidelines no. 407, the extract was administered at the doses of 50, 250, 500, 1000, 2000 mg/kg/day for
28 consecutive days and at the dose of 2000 mg/kg satellite group also used for 6 weeks.In acute toxicity
above mentioned doses neither showed mortality nor exterior signs of toxicity. In sub-acute, study no
significant changes found in haematological and biochemical level ofthe treated rat after 14 days and
28 days in comparison to control. The histopathology of rat brain, kidney, liver, and heart also showed
the no cellular changes after extract treated rat.The alcoholic leaves extract of Reinwardtia indica was
found non-toxic in single drug dose administration up to 5000 mg/kg (acute study) and in sub-acute
administration up to 2000 mg/kg.
management of lifestyle related disease or disorders like, of treatment, animals were weighed, marked and fasted
neurodegenerative, anti-depressant, cancer, anti-diabetic, overnight without suppression of water. Animals of the
metabolic disorders etc. (manuscript submitted). The aim control group received distilled water whereas the treated
of this study was to assess the acute and sub-acute toxicity groups received a single dose of 50, 250, 500, 1000, 2000
study in Charles foster rats at different dose level with and 5000 mg/kg body weight of the alcoholic extract
haematological and biochemical estimation from blood of Reinwardtia indica leaves (AERI) freshly prepared.
and histopathology of brain, kidney, heart & liver after After dosing, food was with held for a further 3–4 h while
14th & 28th days. animals were observed individually for clinical signs of
toxicity such as mortality, respiratory pattern, changes
MATERIAL AND METHODS in general behaviour, skin, eyes, fur and somatomotor
activity.
Plant material and preparation of extracts
Sub-acute toxicity study
R. indica was collected from Uttrakhand (2000- According to the OECD guidelines no. 407 with
2200 m altitude) the surroundings of the region of the slight modification, rats were divided into 6 groups of 6
Himalayan in the month of September to February. Plants animals and treated by gavage. Control group received
were flowering in winter (Paudel Chhetri et al., 2008; Bhat, distilled water while, the AERI treated groups received
Kumar, Bussmann, 2013). The specimen of plant part the extract once daily (10:00–1:00 pm) for 28 consecutive
leaves and stem were identified at the Herbarium (Voucher days at the doses of 50, 250, 500, 1000 and 2000 mg/kg.
specimen number Lina.2015/1) of the Department of Satellite group at the dose of 2000 mg/kg also used for
Botany, Institute of Science by Professor N. K. Dubey. 6weeks to observe the effect of extract in animals.
Leaves were washed thoroughly under running tap water,
oven dried at 50–600C for two days finely ground using Body weight, food and water consumption
an electric blender and stored in plastic containers at room Body weights of the rat in all groups were recorded
temperature and in darkness until required for use. For the before administration of doses, further body weight
preparation of extract, the powdered sample of leaves (100 was taken weekly entire treatment and finally on the
g) were extracted by using a Soxhletapparatus with hot day of sacrifice. The amount of food and water intake
percolationmethodand absolute ethanol (250 ml) was used was recorded daily. The consumed amount of food and
as a solvent. The extract was then filtered and evaporated water were measured before provide to each group, their
to dryness at a 45oC with a rotary evaporator (Buchi R-210 remnants were calculated next day to get the differences,
Advanced, Switzerland) and named as AERI. which were recorded as daily food (gm./rat/day) and water
consumption (ml/rat/day).
Experimental design
Blood analysis
Animals
The protocol of this study was approved by Blood samples were collected from the retro-orbital
Institutional Animal Ethical Committee of Institute of region of the rats for measurement of haematological
Medical Sciences (BHU Varanasi, India). The inbreed (EDTA-coated tubes) and biochemical (dry tubes)
albino female rats of Charles foster strain (120-150 g) parameters after 14 days and 28 days.
was purchased from the Central Animal Facility of our
Institute and acclimatized in our laboratory conditions for Hematological analysis
7 days with free access to normal standard chow diet and
tap water. They were maintained under standard conditions The blood samples collected in heparinized tubes
of temperature (23±2 °C) and a relative humidity of 50%. were used for the hematological analyses. The following
parameters: red blood cell count (RBC), white blood
Acute toxicity cell count (WBC), neutrophils (NP), lymphocytes
The oral acute toxicity study was carried out (LC), monocytes (MC), eosinophils (EP), hemoglobin
according to the guideline Nr. 423 provided by the (Hb), platelets (PL) and packed cell volume (PCV) by
Organization of Economic Cooperation and Development automated analyzer (KX-21-Hematology-analyzer,
(OECD) for the acute toxicity class method (ATC) SysmexCorporation, USA).
procedure with slight modifications. Prior to administration
TABLE I - Behavioral responses and general appearance of rat treated with single dose of AERI in acute toxicity study
Observation Control group 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg 5000 mg/kg
Temperature Normal Normal Normal Normal Normal Normal Normal
Change in skin No effect No effect No effect No effect No effect No effect No effect
Eye color No effect No effect No effect No effect No effect No effect No effect
change
Food intake Normal Normal Normal Normal Normal Normal Normal
General Normal Normal Normal Normal Normal Normal Normal
physique
Diarrhea Not present Not present Not present Not present Not present Not present Not present
Coma Not present Not present Not present Not present Not present Not present Not present
Drowsiness Not present Not present Not present Not present Not present Not present Not present
Breathing not observed not observed not observed not observed not observed not observed not observed
difficulty
Sedation No effect No effect No effect No effect No effect Observed Observed
Tremor Not present Not present Not present Not present Not present Not present Not present
Death Alive Alive Alive Alive Alive Alive Alive
dose toxicity in rat (Koumba Madingou et al., 2016). But the essential medium for transport of nutrients and foreign
during dosing (28-day) and the recovery periods, there was bodies in the body. If extract shows the toxicity in the
no significant change in food and water intake i at different body, it directly affects its components such as red blood
dose treated groups as compared to their respective cells, white blood cells, platelets and haemoglobin. The
control (Table II). In satellite group, also rat treated with ranges of these components either decrease or increase
the highest dose (2000mg/kg b. wt.) did not show any significantly from normal.It indicated that the toxicity
significant change in food uptake and water intake. caused by the extract which can also affect the body
immune as well function of organs (Uddin et al., 2014).
Effect of AERI on haematological parameters Data of haematological analysis of 14th and 28th days were
given in Table III and Table IV. Results revealed that no
The study of haematological parameters indicates significant changes have been shown in the haematological
the toxic effects of the extract on the blood of rats due to analysis when compared with control. The same result also
changes in physiological or pathological level. Blood is found in the satellite treated group.
TABLE II - Effect of AERI on food intake and water consumption Effect of AERI on biochemical parameters
by rat during 28 days treatment and recovery period (satellite
group)
The body has two essential and vital organs for
proper function are liver and kidney. The function of liver
Average food Average water and kidney are different one is used for metabolism of
Treatment intake intake
intake and other one is used for excretion of waste product.
(g/da/rat) (mL/day/rat
To assess the toxicity of any new compound it is necessary
Control 4.29 ± 1.90 4.81 ± 1.20 to know the status of these two vital organs, which can be
50 mg/kg 4.52 ± 1.61 3.81 ± 1.94 checked through biochemical estimation without sacrifice
250 mg/kg 4.89 ± 1.31 4.26 ± 1.92 the rat. For liver function assessment mainly used the
500 mg/kg 3.28 ± 1.02 4.26 ± 1.68 SGOT, SGPT and ALP however for kidney function
1000 mg/kg 4.24 ± 1.08 4.65 ± 1.32 assessments mainly used urea and creatinine. After intake
2000 mg/kg 4.39 ± 1.94 4.83 ± 1.53 of any compound or molecules if any changes have been
seen in above mentioned biomarkers from normal range,
Satellite control 4.23 ± 1.56 4.12 ± 1.81
showed the toxicity of the compounds or molecules
Satellite (2000 mg/kg) 4.45 ± 1.71 4.91 ± 1.01 (Kuatsienu, Ansah, Adinortey, 2017).
TABLE III - Hematological parameters of rat treated with the different dose level of AERI in sub-acute toxicity after 14th day
Parameter Normal range Control 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg Satellite
Hemoglobin 10.2-46.6 15.73±1.48 14.11±1.08 14.41±1.56 13.65±0.39 14.65±1.84 13.59±1.21 15.15±1.08
(%)
Toat RBC (106/ 5-10 9.14±0.29 9.80±0.65 9.40±0.14 9.83±0.27 9.93±0.47 9.02±0.07 9.90±0.77
µL)
WBC (103/µL) 6-15 11.41±2.42 9.08±2.96 9.05±0.16 10.68±1.32 8.17±1.28 9.29±1.50 13.56±1.08
Platelets 782-985 909.30±22.04 862.06±51.55 895.17±80.75 908.50±60.85 920.77±44.17 931.18±23.42 981.16±70.16
(103/L)
PVC (%) 39-49 48.05±1.23 45.16±3.01 44.58±2.05 41.90±2.04 42.13±2.02 44.32±1.05 48.89±1.14
LC (%) 55-95 71.09±1.52 74.38±4.36 72.24±1.06 72.91±2.06 77.78±1.98 84.39±2.13 93.03±1.23
NP (%) 10-40 13.23±1.64 14.48±3.60 14.04±3.16 60.41±1.14 16.15±2.06 23.58±2.16 36.90±5.20
MV (%) 1-4 1.05±0.01 1.28±0.25 1.18±1.30 1.38±0.28 2.23±0.06 1.82±0.18 2.95±0.88
EP (%) 0-4 1.86±0.84 1.06±0.28 1.40±0.30 1.12±0.90 2.09±0.08 2.02±0.19 2.90±0.84
TABLE IV - Hematological parameters of rat treated with different dose level of AERI in sub-acute toxicity after 28th day
Normal
Parameter Control 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg Satellite
range
Hemoglobin (%) 10.2-46.6 16.02±1.91 14.64±1.62 15.60±0.79 14.83±1.24 15.84±1.72 14.07±1.96 15.15±1.08
Toat RBC 5-10 10.90±0.07 9.88±0.45 9.84±0.25 9.93±0.05 9.98±0.82 9.54±0.65 9.90±0.77
(106/µL)
WBC (103/µL) 6-15 12.94±1.74 9.85±0.69 10.02±0.49 1384±1.73 11.63±0.49 10.04±1.18 13.56±1.08
Platelets (103/L) 782-985 922.70±34.02 959.17±63.22 900.15±84.89 885.11±49.56 881.92±46.11 977.56±70.56 981.16±70.16
PVC (%) 39-49 49.9±2.16 47.15±1.22 46.50±2.14 44.64±3.18 45.07±1.09 46.89±2.41 48.89±1.41
LC (%) 55-95 76.10±3.68 78.43±3.58 73.59±2.15 77.54±2.98 89.98±2.17 91.03±3.23 93.03±1.23
NP (%) 10-40 25.32±1.88 23.12±1.80 28.50±1.10 33.66±1.12 13.34±2.89 32.89±1.28 369.90±5.20
MV (%) 1-4 1.50±0.15 1.53±0.16 1.48±0.12 2.05±0.15 2.36±0.12 2.19±0.54 2.95±0.88
EP (%) 0-4 1.93±0.11 1.90±0.35 1.94±0.38 1.97±0.73 2.80±0.61 2.69±0.24 2.90±0.84
The results suggested from our study that the levels tested doses in comparison to control. The satellite group
or activities of biochemical parameters in animals after 14th after 6 weeks also showed the no significant changes in
& 28th days and found no significant variations occurred the above mentioned biochemical with compare to control
in SGOT, SGPT, ALP, Urea and creatinine levels at all (Table V and Table VI)
TABLE V - Biochemical estimation from blood serum of rats after 14th day’s treatment at the different dose level in the subacute
toxicity study
Parameter Normal range Control 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg Satellite
SGOT (U/L) 54-298 153.6±16.70 155.33±20.49 163±33.34 162.5±35.21 163.33±33.59 215.5±33.59 99.02±3.09
SGPT (U/L) 17-77 43.5±7.77 43±9.9 38.16±4.68 49.16±6.06 42±8.72 51.16±7.72 49.57±0.19
ALP (U/L) 64-128 87.83±10 97.66±7.56 94.16±9.21 97.83±8.68 90.16±5.98 93±7.93 103.97±3.18
Creatinine 0.2-0.9 0.34±0.035 0.36±0.04 0.34±0.04 0.35±0.03 0.35±0.02 0.36±0.02 0.28±0.026
(mg/dL)
Urea (U/L) 35-96 52.5±5.07 56.5±6.9 59.5±6.4 76±5.76 52±4.18 57.5±6.31 43±5.41
BUN (mg/dL) 8-33 24.51±2.36 26.38±3.23 27.78±2.98 32.06±4.02 24.28±1.95 26.85±2.94 20.08±2.47
TABLE VI - Biochemical estimation from blood serum of rats after 28th day’s treatment at different dose level in sub- acute toxicity
study
Parameter Normal range Control 50 mg/kg 250 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg Satellite
SGOT (U/L) 54-298 163.5±32.5 167.16±32.74 183.33±24.8 178.66±19.2 192.5±35.47 208.66±15.99 99.02±3.09
SGPT (U/L) 17-77 45.5±8.65 46±7.94 39.83±6.19 59.33±6.15 46.66±7.27 53.5±7.18 49.57±0.19
ALP (U/L) 64-128 95.33±6.71 101±7.5 97.66±9.13 106.16±5.59 99.5±3.39 111.16±9.3 103.97±3.18
Creatinine 0.2-0.9 0.39±0.05 0.38±0.023 0.47±0.05 0.8±0.04 0.44±0.05 0.45±0.02 0.28±0.026
(mg/dL)
Urea (U/L) 35-96 65.16±5.66 68.66±8.61 66.83±6.71 81±6.22 61.66±6.09 65.66±8.85 43±5.41
BUN (mg/dL) 8-33 30.43±2.64 37.82±2.9 32.21±3.13 35.49±2.69 28.79±2.84 30.66±4.13 20.08±2.47
Histopathology
Estimated dose
FIGURE 1 - Body weight assessment of treated rats in sub-acute
toxicity study. The 1/10th and 1/20th of maximum dose from
FIGURE 2 - Histopathology (H&E stain10x) of rat tissues of treated groups rats after 14th days.
FIGURE 3 - Histopathology (H&E stain10x) of rat tissues of treated groups rats after 28th days.
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