Toxicity study of ethanolic extract of Acorus calamus rhizome

Abstract

Acorus calamus is widely used in traditional medicine in various ailments. However, there is no toxicological information available regarding its safety after exposure. The present study was designed to evaluate potential toxicity of an ethanolic extract of Acorus calamus Linn. rhizomes after acute and chronic administration in Wistar rats. In the acute toxicity study, female Wistar rats were treated with ethanolic extract by oral gavage at dose levels of 175, 550, 1750 and 5000 mg/kg body weight according to OECD 425. Animals were observed periodically during the first 24 h after administration of the extract, and daily thereafter for 14 days. In the chronic toxicity study, the ethanolic extract of Acorus calamus was administered orally at doses of 0, 200, 400 and 600 mg/kg body weight daily for 90 days in Wistar rats. The effects on clinical signs, body weight, food consumption, organ weight, haematology, clinical biochemistry, as well as histology, were studied. No mortality was observed, but clinical signs like abdominal breathing, piloerection and tremors were observed for 30 min in rats dosed with 1750 mg and 5000 mg/kg body weight of extract. No statistical significant data in body weight and feed consumption were observed. Haematological and biochemical analysis showed no marked differences in any of the parameters. Pathologically, neither gross abnormalities nor histopathological changes were observed. The ethanolic extract of A. calamus does not appear to have toxicity on acute and chronic administration in Wistar rats.

Full text

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International Journal of Green Pharmacy
| January-March 2012 | 38
Original article
Toxicity study of ethanolic extract of Acorus
calamus rhizome
Payal D. Shah, Mrunali Ghag1, Pradeep B. Deshmukh1, Yogesh Kulkarni2, Shrikant V. Joshi,
Bhavin A. Vyas, Dinesh R. Shah
Departments of Pharmacology, Maliba Pharmacy College, Tarsadi, Surat, 1Toxicology, Jai Research Foundation, Valvada, Vapi, Gujarat,
2Pharmacology, SPTM, SVKM’s NMIMS University, Mumbai, Maharashtra, India
Acorus calamus is widely used in traditional medicine in various ailments. However, there is no toxicological information available
regarding its safety after exposure. e present study was designed to evaluate potential toxicity of an ethanolic extract of Acorus
calamus Linn. rhizomes after acute and chronic administration in Wistar rats. In the acute toxicity study, female Wistar rats were
treated with ethanolic extract by oral gavage at dose levels of 175, 550, 1750 and 5000 mg/kg body weight according to OECD 425.
Animals were observed periodically during the first 24 h after administration of the extract, and daily thereafter for 14 days. In the
chronic toxicity study, the ethanolic extract of Acorus calamus was administered orally at doses of 0, 200, 400 and 600mg/kg body
weight daily for 90 days in Wistar rats. e effects on clinical signs, body weight, fo od consumption, organ weight, haematology, clinical
biochemistry, as well as histology, were studied. No mortality was observed, but clinical signs like abdominal breathing, piloerection
and tremors were observed for 30 min in rats dosed with 1750 mg and 5000 mg/kg body weight of extract. No statistical significant
data in body weight and feed consumption were observed. Haematological and biochemical analysis showed no marked differences
in any of the parameters. Pathologically, neither gross abnormalities nor histopathological changes were observed. e ethanolic
extract of A. calamus does not appear to have toxicity on acute and chronic administration in Wistar rats.
Key words: Acorus calamus, acute toxicity, chronic toxicity
Address for correspondence: Prof. Payal Shah, Maliba Pharmacy College, Gopal Vidyanagar, Bardoli-Mahuva Road, Tarsadi - 394 350,
Dist- Surat, Gujarat, India. E-mail: payaltina2003@yahoo.com
Received: 17-11-2011; Accepted: 27-01-2012
GP_205_11R5
EP corr done: (Thu 4/19/2012 3:23 PM) - 19 April, 2012
AP corr done: (GP_205_11R7) - 27 April, 2012
AP corr done: (Sat 4/28/2012 10:47 AM) - 30April, 2012
AP corr done: (Wed 5/2/2012 11:43 AM) - 02 May, 2012
Access this article online
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***
INTRODUCTION
Acorus calamus (L.) (Araceae) is a perennial, semiaquatic
and smelly plant found in the northern temperate and
subtropical regions of Asia, North America, and Europe.
It is six feet tall, aromatic herb with creeping rhizomes.
The leaves are long, slender, sword-shaped and simple,
arising alternately from the horizontal rhizomes. These
are longitudinally fissured with nodes, somewhat
vertically compressed and spongy internally. Flowers
are small and fragrant with pale green spadix; fruits
are three-celled eshy capsule.[1-7]
All parts of the plant contain volatile oil having
terpenoids, calamine, calamenol, calamenone, eugenol,
camphene, pinene and asaronaldehyde. Acorafuran
is a sesquiterpenoid found in calamus oil.[8-11] The
rhizomes are utilized extensively by the Chinese,
Indians and American Indians as well as by other
cultures.[9] Its roots and rhizomes are used in treatment
of various ailments including mental disorders,
such as hysteria, insanity, insomnia, melancholia,
neurasthenia, epilepsy, diarrhoea and asthma.[12,13] The
leaves extract of Acorus calamus were studied for anti-
inammatory activity on keratinocyte HaCaT cells.[14]
The roots and rhizomes extracts of Acorus calamus
have been reported with various pharmacological
activities such as analgesic,[12] cardiovascular,[15,16]
anticonvulsant,[17] hypolipidemic,[18] antispasmodic,[19]
anti-inammatory,[20] antibacterial,[21] antiulcer and
cytoprotective activity.[12] In most of studies the roots
and rhizomes extracts of A. calamus reported for its
CNS activities.[12,13,22-24]
In an earlier report, it has been shown that
ethanolic extract of rhizome of this plant possesses
immunosuppressive,[25] sedative, analgesic, moderately
hypotensive and respiratory depressant properties.
[15]
Acorus calamus extract is also used in traditional
Chinese prescription and its beneficial effects on
memory disorder, learning performance,[25] lipid
peroxide[26] content and its senescence eect have been
reported.
Despite the wide use of A. calamus extract in traditional
medicine, no study has been reported in the scientic
literature about its toxicity. Hence, the present study

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Shah, et al.: Toxicity of Acorus calamus
International Journal of Green Pharmacy | January-March 2012 |
39
was carried out to determine acute and chronic toxicity of
the ethanolic extract of rhizomes of A. calamus (EAC) in
Wistar rats.
MATERIALS AND METHODS
Plant Material
A. calamus rhizomes were obtained from the local market
of Surat, Gujarat, India. The samples were identied
and authenticated by Dr. Minoo Parabia (Professor of
Botany) Veer Narmad South Gujarat University, Surat,
Gujarat, India. The voucher specimen (R001) is kept in
the Pharmacology Department at SPTM, SVKM’s NMIMS
University, Mumbai. The plant material was shade-
dried, milled to powdered form and stored in airtight
containers.
Preparation of EAC
Rhizome powder of A. calamus was extracted with 95%
ethanol (Qualigens ne chemicals, Mumbai) for 12 hours
in Soxhlet extractor. The obtained extract was concentrated
using rotary vacuum evaporator (BUCHI, Japan) at 40-
60°C. The concentrated semisolid extract was stored in
refrigerator at 2-8°C till further use. The yield (w/w) of the
ethanolic extract was found to be 27%.
Phytochemical Analysis
Preliminary phytochemical investigations of EAC for the
presence of active phytoconstituents such as carbohydrates,
alkaloids, proteins, volatile oils, triterpenes, avonoids,
saponins, phenols, resins and tannins were carried out using
the methods previously described.[27]
Preparation of Dosing Solution
EAC (200,400,600 mg) was dissolved in 10 ml of 0.5% carboxy
methyl cellulose (CMC) each to obtained concentration of
20, 40, 60 mg/ml, respectively, and administered orally at
the dose volume of 10 ml/kg body weight to achieve the
dose level of 10 mg/kg body weight.
Acute Toxicity Study
Female Wistar rats, 8-10 weeks old (150-200 g) were
obtained from Breeding Facility of Jai research foundation,
Vapi, India. Animals were kept under controlled
environmental conditions (22±0.5°C, relative humidity
65-67%, 6 am to 6 pm alternate light–dark cycles, food
and water ad libitum) in polypropylene cages covered
with stainless steel grid and an autoclaved clean rice husk
breeding. The animals were allowed to acclimatize for
seven days prior to the commencement of experiment.
The animal protocol was approved by the Institutional
Animal Ethical Commiee (IAEC) protocol number R-290
as per provisions of Commiee for the Purpose of Control
and Supervision of Experimental Animals (CPCSEA),
New Delhi, India.
Rats had free access to drinking water ltered through
an Aquaguard water lter system tap water and nutrilab
rodent feed, except overnight fasting before the treatment
with a single dose of the EAC. EAC was suspended in
0.5% CMC on the day of the experiment. Dose progression
slope 2 of OECD 425[28,29] was followed throughout the
study with the starting dose of 175 mg/kg body weight
to 5000 mg/kg body weight. The rats were observed for
signs of toxicity and mortality at 0.5, 1, 2, 3, 4 and 6 hours
post dosing. Subsequently, the rats were observed twice
a day for morbidity and mortality for a period of 14 days
following oral dosing. The clinical signs were recorded
once a day. Individual animal body weight was recorded
prior to dosing on day 0, 7 and 14. At the end of the 14 days
observation period, all the treated rats were euthanised
by carbondioxide asphyxiation. They were subjected to
gross pathological examination, consisting of external
examination and opening of the abdominal and thoracic
cavities. Abnormalities, if any were recorded. etal50 Journal
of Applied ToxicologyThe LD50 values were determined
according to the method of Dixon’s maximum likelihood
method using soware (AOT 425 StatPgm).
Chronic Toxicity Study
Wistar albino rats of either sex, 5-6 weeks old (100-190 g),
were housed in polypropylene cages covered with stainless
steel grid top (one in a cage) under the standard conditions.
The animals were divided into four groups (I, II, III and
IV) of ten rats each (ve females and ve males). EAC was
suspended in 0.5% CMC on the day of the experiment,
and was given daily in morning by oral gavage for 60 days
at doses of 200 mg/kg (Group II), 400 mg/kg (Group III),
and 600 mg/kg (Group IV), while the control rats (Group
I) received only the vehicle. Toxic manifestations and
mortality were monitored for 60 days. Clinical signs were
recorded immediately aer dosing, if any. Body weight,
feed consumption were measured weekly. At the end of
study, blood samples were obtained by the retro-orbital
puncture under light diethyl ether anaesthesia, with or
without anticoagulant (ethylenediamine tetraacetate).
Blood with the anticoagulant was used immediately for
the determination of haematological parameters, while
blood without the anticoagulant was centrifuged at 1789 × g
for 10 min at 4°C, and the serum obtained was stored at
−20°C until analyzed for biochemical parameters. Aer
blood collection, rats were sacriced by carbon dioxide
asphyxiation. All organs were examined grossly, weighed
and collected for histopathological examination.[30]
Haematology
The haematological analysis was performed using
automatic haematological analyzer (Sysmex, Japan). Total
red blood cells (RBC), leukocyte (WBC), haematocrit,
haemoglobin, platelet count, cloing time, erythrocyte

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Shah, et al.: Toxicity of Acorus calamus
International Journal of Green Pharmacy
| January-March 2012 | 40
indices: -mean corpuscular haemoglobin (MCH), mean
corpuscular volume (MCV), mean corpuscular haemoglobin
concentration (MCHC) and dierential leucocyte count,
reticulocyte count (Leishman’s stain) of blood sample were
recorded.
Clinical biochemistry
Glucose, total cholesterol, triglyceride, alanine
aminotransferase (ALT), aspartate aminotransferase (AST),
alkaline phosphatase, gamma glutamyltranspeptidase
(GGT), total protein, total bilirubin, albumin, globulin,
albumin:globulin ratio (A/G ratio), total creatinine, blood
urea nitrogen (BUN), urea, calcium and phosphorus were
determined using auto analyzer (BT-2000, Japan).
Pathological examination
Gross necropsy
All animals in the study were subjected to detailed gross
necropsy, which includes careful examination of the external
surface of the body, all orices and the cranial, thoracic
and abdominal cavities and their contents. The adrenals,
testes, urinary bladder, thyroid and parathyroid, kidneys,
brain, heart, spleen, liver, lungs and thymus of all animals
were trimmed of any adherent tissue, as appropriate, and
their wet weight taken as soon as possible aer dissection
to avoid drying.
Histopathology
Histopathological examination of control and high dose
group was performed for all gross lesions and for brain,
spinal cord, stomach, small and large intestines, liver,
kidneys, adrenals, spleen, heart, thymus, thyroid, trachea
and lungs, gonads, accessory sex organs (e.g., uterus,
prostate), urinary bladder, lymph nodes, peripheral
nerve (sciatic or tibial) preferably in close proximity
to the muscle, and a section of bone marrow by xing
immediately in 10% formalin for routine histopathological
examination. Tissues were embedded in paran wax;
sections of tissues were cut at 3-5 m thickness with
microtome and stained with haematoxylin and eosin for
microscopical examination.
Statistical Analysis
Data were expressed as mean±S.E.M and assessed
statistically by appropriate statistical methods (Bartlet’s,
ANOVA, Dunnett‘s ‘t’ test and Student‘t’ test) were
employed to assess the signicance among dierent groups
with signicance level of P<0.05.
RESULTS
Phytochemical Analysis
Presence of carbohydrates, alkaloids, proteins, volatile oils,
triterpenes, avonoids, saponins, resins and tannins were
indicated by phytochemical investigations of EAC.
Acute Toxicity Study
The results of the acute toxicity study of EAC are presented
in Table 1. No mortality or change in body weight was
observed in rats al dose level of the A. calamus extract
550 mg/kg, and 750 mg/kg body weight. Some clinical signs
such as tremors, pilo erection and abdominal breathing were
observed immediately aer the oral dosing of 1,750 and
5,000 mg/kg body weight of EAC but no mortality or change
in body weight were observed. No signicant changes were
observed in gross necropsy on day 14.
The acute toxicity data indicated that the calculated LD50
value (Dixons likelihood method) for the oral doses of the
EAC was found to be more than 5,000 mg/kg body weight.
Chronic Toxicity Study
Clinical signs
No clinical signs like tremors, convulsions, piloerection,
aggression, lethargy, abdominal breathing, gait and
licking were observed throughout the study period of
90 days.
Body weight and feed consumption
Changes in body weight and feed consumption of control
and extract treated rats are presented in Figures 1 and 2
and Figures 3 and 4, respectively. Rats in control group
gained weight with time (as expected), with no signicant
dierence in feed consumption and weight gain at the end
of 90 day. Moreover, no mortality was recorded up to the
dose of 600 mg/kg, during the 90-day treatment.
Haematological parameters
The values for the haematological parameters in treated
and control rats are shown in Table 2. Level of total RBC,
leukocyte (WBC), hematocrit, and hemoglobin, platelet
count, cloing time, Erythrocyte indices: -mean corpuscular
haemoglobin (MCH), mean corpuscular volume (MCV),
mean corpuscular haemoglobin concentration (MCHC)
and Differential Leucocyte Count, Reticulocyte count
(Leishman’s stain) on chronic oral administration of EAC
(daily for 90 days) did not exhibit any signicant changes
in any of hematological parameters. All values remained
Table 1: Clinical signs of toxicity observed during
acute oral toxicity study of ethanolic extract of Acorus
calamus in Wistar rats
Dose (mg/kg
body weight)
Latency Symptoms
17 5 - None
550 - None
1750 - Piloerection, abdominal breathing
5000 - Tremor, Piloerection, abdominal breathing
5000 - Tremor, Piloerection, abdominal breathing
5000 - Tremor, Piloerection, abdominal breathing
No symptoms observed during the observation period; latency – Time of death after
the dose

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Shah, et al.: Toxicity of Acorus calamus
International Journal of Green Pharmacy | January-March 2012 |
41
Figure 1: Effect of ethanolic extract of Acorus calamus (EAC) on body weight in
male rat in chronic toxicity study. Each point represents Mean±S.E.M.
Figure 2: Effect of ethanolic extract of Acorus calamus (EAC) on body weight in
female rats in chronic toxicity study. Each point represents Mean±S.E.M
Table 2: Effect of ethanolic extract of Acorus calamus on haematological parameters in chronic toxicity study for
90 days
Parameter Male Female
Control EAC (mg/kg body weight) Control EAC (mg/kg body weight)
200 400 600 200 400 600
RBC count (106/µl) 7.48±0.23 7.68±0.17 7.59±0.31 7.29±0.26 6.16±0.13 6.11±0.08 6.23±0.19 6.19±0.26
Reticulocyte (%) 1.5±0.06 1.44±0.05 1.44±0.06 1.45±0.05 1.71±0.03 1.69±0.04 1.7±0.04 1.69±0.05
Hb (g/dl) 14.6±0.17 14.62±0.14 14.58±0.47 14.6±0.38 13.18±0.36 13.14±0.08 13.24±0.24 13.16±0.18
HCT (%) 40.78±0.82 40.60±0.48 38.68±1.32 40.8±1.23 37.28±1.07 37.32±0.19 37.22±0.93 37.28±1.23
MCV (fl) 54.64±1.26 55.72±0.80 57.08±1.18 56.08±0.76 60.46±0.45 57.82±0.86 56.64±1.19 56.08±0.76
MCH (pg) 19.56±0.52 18.02±0.25 19.44±0.51 20.06±0.25 21.38±0.17 20.48±0.21 20.6±0.57 20.06±0.25
MCHC (g/dl) 35.82±0.31 36.28±0.25 35.79±0.59 35.82±0.30 29.16±6.44 35.44±0.30 36.34±0.52 35.82±0.30
WBC count (103/µl) 4.92±0.51 5.28±0.22 5.38±0.45 4.44±0.51 2.94±0.34 3.04±0.18 4.26±0.66 3.66±0.62
Lymphocyte (%) 71.6±1.69 75.6±0.75 73.4±1.29 72.4±1.03 76.6±2.11 72.8±1.39 72.4±2.73 70.4±1.69
Neutrophil (%) 26.6±1.03 23.2±0.86 25.6±1.44 25.8±0.58 22.4±2.50 25.2±1.16 27±2.49 28±1.87
Monocyte (%) 1.6±0.75 0.6±0.40 0.8±0.49 1±0.77 0.4±0.40 1.8±0.66 0.6±0.25 1.2±0.58
Basophil (%) 0±0.00 0±0.00 0±0.00 0±0.00 0±0.00 0±0.00 0±0.00 0±0.00
Eosinophil (%) 0.2±0.20 0.6±0.00 0.2±0.00 0.8±0.00 0.6±0.40 0±0.00 0.2±0.00 0.4±0.00
Platelet (103/µl) 707±122.18 699±62.69 685.6±93.02 702±52.79 631.4±54.25 690.2±42.43 662±64.65 702±52.79
Clotting time (seconds) 120±13.42 120±13.42 108±7.35 120±0.00 114±11.23 144±11.23 120±9.49 108±7.35
Values are expressed as Mean±S.E.M (n=5). *P<0.05; EAC – Acorus calamus; RBC – Red blood corpuscles; Hb – Haemoglobin; HCT – Haematocrit; MCV – Mean corpuscular
volume; MCH – Mean corpuscular haemoglobin; MCHC – Mean corpuscular haemoglobin concentration; WBC – White blood cells
within physiological range throughout the treatment
period.
Biochemical parameters
The values for biochemical parameters in treated and control
rats are presented in Table 3. Chronic oral administration
of EAC (daily for 90 days) did not show any signicant
changes in any of the biochemical parameters. All values
remained within physiological range throughout the
treatment period.
Pathological examination
There was no signicant dierence between the control
and treated groups in the organ weights of male and
female rats [Table 4]. No alternations were detected
in pathological examination of the tissues during the
microscopic examination of the internal organs. No
histopathological ndings could be aributed to EAC and
no dierences found in the organ morphology across the
treated groups.
DISCUSSION
Phytotherapeutic products are, many times, mistakenly
regarded as safe because they are “natural”. Nevertheless,
those products contain bioactive principles with potential
to cause adverse effects. A Word Health Organization
(WHO) survey indicated that about 70-80% of the world’s
populations rely on non-conventional medicine, mainly
of herbal source, in their primary healthcare. In addition,
100
120
140
160
180
200
220
240
260
280
300
0714 19 21 28 35 42 49 56 63 70 77 84 90
Body weight (gm)
Days
Vehical control
200 mg/kg
400 mg/kg
600 mg/kg
100
150
200
250
300
350
400
450
0714 19 21 28 35 42 49 56 63 70 77 84 90
Body weight (gm)
Days
Vehical control
200 mg/kg
400 mg/kg
600 mg/kg

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Shah, et al.: Toxicity of Acorus calamus
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| January-March 2012 | 42
Table 3: Effect of ethanolic extract of Acorus calamus on biochemical parameters in chronic toxicity study for 90 days
Parameter Male Female
Control EAC (mg/kg body weight) Control EAC (mg/kg body weight)
200 400 600 200 400 600
GLU (mg/dL) 65±10.08 63.44±5.59 67.34±7.60 68.12±4.81 72.2±8.03 71.72±3.63 71.28±11.48 71.42±8.09
CHO (mg/dL) 79.04±4.98 83.94±3.11 83.9±2.97 83.7±3.60 85.44±9.10 89.58±11.29 87.66±11.59 90.18±2.50
TRIG (mg/dL) 109.5±16.26 103.08±13.24 109.46±11.96 105.44±17.68 83.46±5.20 83.22±8.93 89.3±24.45 75.2±6.40
ALT (IU/L) 57.44±12.41 56.1±5.04 54.42±2.61 56.74±3.60 38.96±0.72 36.16±3.90 42.22±4.06 61.94±2.67
AST (IU/L) 35.8±7.66 34.28±5.45 35.22±4.72 35.22±5.24 34.1±8.46 35.28±3.87 35.84±1.56 34±4.96
ALP (IU/L) 98.48±5.31 118. 0 8 ±11.11 114.7±5.89 111. 2 8 ±19.10 88.16±5.78 85.2±13.01 89.44±5.88 89.84±6.77
GGT (IU/L) 2.4±0.75 2.1±0.10 2.22±0.14 1.92±0.33 0.48±0.24 1.66±1.00 2.22±1.28 0±0.00
T.PRO (g/dL) 7.06±0.30 7.36±0.12 8.84±0.33 8.08±4.73 9.04±0.14 8.24±0.30 8±0.53 8.28±0.22
T.BIL (mg/dL) 0.06±0.04 0.04±0.00 0.06±0.00 0.08±0.00 0.28±0.07 0.26±0.04 0.4±0.07 0.24±0.05
ALB (g/dL) 5.22±0.12 5.24±0.07 5.14±0.16 5.24±0.04 6.56±0.10 6.18±0.17 5.72±0.31 6.38±0.14
GLB (g/dL) 1.84±0.19 2.12±0.07 1.82±0.18 7.84±4.74 2.5±0.13 2.32±0.16 2.28±0.26 2.48±0.12
ALB:GLB 2.92±0.26 2.59±0.07 2.89±0.11 2.66±0.38 2.66±0.16 2.45±0.17 2.56±0.21 2.36±0.10
CRE (mg/dL) 0.74±0.04 0.68±0.04 0.8±0.06 0.94±0.05 1.08±0.07 1.06±0.03 1.12±0.07 0.88±0.06
BUN (mg/dL) 22.2±0.97 22.22±2.01 22.9±2.42 22.38±1.14 28.26±2.80 30.12±1.86 30.18±1.80 28.26±0.91
UREA (mg/dL) 47.52±2.05 47.56±4.32 47±4.16 47.58±2.57 60.46±5.98 64.48±3.99 64.58±3.81 60.5±1.95
CAL (mg/dL) 9.58±0.27 9.84±0.34 9.58±0.32 9.62±0.31 11.66±0.29 10.74±0.29 10.54±0.20 10.78±0.28
PHO (mg/dL) 7.06±0.60 8.12±0.46 8.08±0.18 8.14±0.18 5.4±0.47 5.66±0.54 5.68±1.14 5.54±0.05
Values are expressed as Mean±S.E.M (n=5). *P<0.05. EAC – Acorus calamus; GLU – Glucose; CHO – Total cholesterol; TRIG – Triglyceride; ALT – Alanine aminotransferase;
AST – Aspartate aminotransferase; ALP – Alkaline phosphatase; GGT – Gamma glutamyltranspeptidase; T.PRO – Total protein; T.BIL – Total bilirubin; ALB – Albumin;
GLB – Globulin; CRE – Creatinine; BUN – Blood urea nitrogen; CAL – Calcium; PHO – Phosphorous
Figure 3: Effect of ethanolic extract of Acorus calamus (EAC) on feed consumption
in male rats in chronic toxicity study. Data are expressed as Mean±S.E.M
Figure 4: Effect of ethanolic extract of Acorus calamus (EAC) on feed consumption
in female rats in chronic toxicity study. Data are expressed as Mean±S.E.M
Table 4: Effect of ethanolic extract of Acorus calamus on organ weight (g) in chronic toxicity study for 90 days
Parameter Male Female
Control EAC (mg/kg body weight) Control EAC (mg/kg body weight)
200 400 600 200 400 600
Adrenals 0.092±0.01 0.091±0.01 0.09±0.01 0.085±0.01 0.108±0.01 0.105±0.01 0.119±0.01 0.11±0.01
Testes 3.446±0.05 3.339±0.11 3.125±0.12 3.054±0.14 - - - -
Urinary bladder 0.142±0.01 0.219±0.06 0.129±0.01 0.135±0.02 0.093±0.01 0.1±0.01 0.089±0.00 0.125±0.02
Thyroid and parathyroid 0.027±0.00 0.025±0.00 0.033±0.00 0.036±0.00 0.032±0.00 0.026±0.00 0.022±0.00 0.025±0.01
Kidneys 2.41±0.07 2.296±0.09 2.364±0.15 2.304±0.06 1.471±0.05 1.681±0.07 1.516±0.06 1.527±0.06
Brain 2.132±0.05 2.052±0.06 2.048±0.03 2.095±0.04 1.921±0.05 1.933±0.02 1.89±0.03 1.97±0.08
Heart 1.192±0.09 1.008±0.03 1.102±0.04 1.13±0.03 0.779±0.01 0.746±0.03 0.713±0.03 0.738±0.02
Spleen 0.646±0.04 0.572±0.02 0.507±0.03 0.633±0.05 0.427±0.01 0.533±0.06 0.408±0.02 0.424±0.02
Liver 10.854±0.31 10.555±0.29 10.124±0.14 10.408±0.51 6.713±0.46 7.331±0.11 7.312±0.47 6.704±0.14
Lungs 1.773±0.09 1.885±0.15 3.088±0.38 3.308±0.18 1.48±0.13 1.444±0.06 1.347±0.08 1.45±0.13
Thymus 0.506±0.04 0.423±0.02 0.511±0.07 0.457±0.06 0.395±0.02 0.432±0.03 0.394±0.03 0.422±0.06
Values are expressed as Mean±S.E.M (n=5). *P<0.05; EAC – Acorus calamus
0
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7142128354249566370778490
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Vehical control
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600 mg/kg
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7142128354249566370778490
Feed consumption (gm)
Days
Vehical control
200 mg/kg
400 mg/kg
600 mg/kg

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International Journal of Green Pharmacy | January-March 2012 |
43
the poor pharmacovigilance services in this area make
it dicult to determine the frequency of adverse eects
caused by the use of phytotherapeutic products. Thus,
all the “natural” products used in therapeutics must be
submied to ecacy and safety tests by the same methods
used for new synthetic drugs.[31]
Although medicinal plants may produce several biological
activities in humans, but very lile is known about their
toxicity and the same applies for Acorus calamus.
Usually acute (single dose) toxicity study is carried out
on laboratory animals by using high dose (sucient to
produce death or morbidity) of the substance in question
and/or based on previous report on its toxicity or toxicity
of structurally related compounds. There was no previous
report on toxicity of EAC. Therefore, the doses starting at
175, 550, 1,750 and limit dose 5,000 mg/kg were selected for
acute toxicity study, as per OECD 425. No mortality was
observed in animal at all selected dose levels.[29]
Some signs of toxicity were observed on administration of
doses 1,750 and 5,000 mg/kg body weight Table 1, but they
were all reversible in a maximum period of 24 h aer the
administration of the extract. The LD50 value was found to
be more than 5,000 mg/kg body weight for EAC.
There is no real correlation between acute dose LD50 and
prediction of adverse eects of chronic daily dosing. In
addition, the LD50 in animals does not predict the human
lethal dose of a drug or the symptomatology of acute
poisoning aer overdose. Nevertheless, the acute dose
study provides a guidance for selecting doses for the
chronic low-dose study, which may be more clinically
relevant.[32]
Therefore, on basis of clinical observations of acute
toxicity study of ethonolic extract of Acorus calamus, doses
200 mg/ kg, 400 mg/kg and 600 mg/kg were selected for
chronic toxicity study.
No mortality or clinical signs were observed with the above
mentioned dose levels. However, it is very important as
it indicates the capability of reaction of the organism that
received the drug.
No statistical significant reduction in body weight
Figures 1 and 2 and feed consumption Figures 3 and 4,
respectively, were observed. Body weight changes if any
are an indicator of adverse side eects, as the animals
that survive cannot lose more than 10% of the initial body
weight. The determination of such parameters is important
in the study of safety of a product with therapeutic purpose,
as proper intake of nutrients and water are essential
to the physiological status of the animals and to the
accomplishment of the proper response to the drug tested
instead of a “false” response due to improper nutritional
conditions.[33]
Aer 90 days of treatment, there were no signicant changes
in the hematological parameters between control and
treated groups [Table 2].
The results indicate that the Acorus calamus extract was
neither toxic to the circulating red cells, white blood
cells and platelets nor interfered with their production.
Haematopoiesis and leucopoiesis were also not aected
even though the haematopoietic system is one of the most
sensitive targets for toxic compounds and an important
index of physiological and pathological status in man and
animals.[34,35] Therefore, it plausible to assume that the extract
is not haematotoxic.
All biochemical parameters were in range for 90 days
indicating no sever liver or kidney damage [Table 3].
Signicant changes in enzymes like ALP, AST and ALT
represent liver impairment, since these are important
indices of liver toxicity.[29] Serum cholesterol and proteins
are mainly regulated via synthesis in the liver and increase
or decrease in serum concentrations of constituents suggests
liver toxicity.
Kidney toxicity has also been reported after use of
phytotherapeutic products what makes essential its
evaluation. In that case, creatinine and urea determinations are
critical as these substances are markers of kidney function.[32]
This was also confirmed through histopathological
examination of the kidney.
The organ weight examinations of important organs like
adrenals, testes, urinary bladder, pancrease, thyroid and
parathyroid, kidneys, brain, heart, spleen, liver, lungs and
thymus indicated no signicant dierence [Table 4].
Further, the safety of EAC in rats was supported by
histopathological examinations, suggesting that there was
no dierence or sever damage in all important organs.
The studies carried out suggest that in 600 mg/kg dose,
the extract seems to be safe. Thus, considering that Acorus
calamus extract to be used for undetermined time, further
studies in non-rodents must be performed to prove its safety.
CONCLUSION
The present investigation demonstrates that the traditional
medicine, the ethanolic extract of Acorus calamus lack
potential toxicity, as it neither cause any lethality nor

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Shah, et al.: Toxicity of Acorus calamus
International Journal of Green Pharmacy
| January-March 2012 | 44
changes the general behaviour in both the acute and chronic
toxicity studies.
ACKNOWLEDGMENT
Authors are thankful to Jai Research Foundation, Vapi, Gujarat,
India for all support and help for research work.
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How to cite this article: ***
Source of Support: Jai Research Foundation, Conict of Interest: None
declared.
???
Dear sir,
What should we consider to maintain uniformity throught the article:
RBC – Red blood corpuscles or RBC – Red blood cells
WBC – White blood corpuscles or WBC – White blood cells