Trends Research in Science and Technology (2010) 2 (1), 19-22 19

Safety Evaluation of Ethanol Extracts from Bosenbergia rotunda

(L.) Mansf. in Male Rats
Prakaimuk Saraithong, Supap Saenphet , Kanokporn Saenphet*
Department of Biology, Faculty of Science, Chiang Mai University,
Muang District, Chiang Mai 50200, Thailand

Abstract
This research was conducted to determine the toxicity of ethanol extracts from Bosenbergia rotunda on
male rats. Extracts at doses of 60, 120 and 240 mg/KgBW/day were orally administered to 27 male rats for 60
days, while a control group received distilled water. The body weight, hematological and blood biochemical
values as well as histopathologic architecture of the kidney and liver were examined. The results showed that
the extracts had no effect on the body weight, regardless of the dosage used. All hematological parameters,
including the total white blood cell count, the differential white blood cell count, packed red cell volume (PCV)
and hemoglobin were the same as those of the control. The kidney and liver functions, including blood urea
nitrogen (BUN), creatinine (Crea), aspatate aminotransferase (AST) and alanine aminotransferase (ALT), were
within the normal range. No histopathologic changes of liver or kidney were observed. It can be concluded that
ethanol extracts of B. rotunda at all doses used in the present study have no toxic effect on male rats.

Key words: Boesenbergia rotunda, Safety, Test, Rats

Introduction
Boesenbergia rotunda, so called “krachai” in Thai,
is a deciduous plant belonging to the Family
Zingiberaceae. It is known in every Thai kitchen as
its rhizome is commonly used as an ingredient in many
dishes. In addition to improving the flavor of food, B.
rotunda offers many important health benefits, for
instance, relief of stomachache, anti-flatulence, the
eradication of ringworm and the promotion of
appetite [1]. Besides being used as an indispensable
spice and herbal medicine, Thai people also
extensively use “krachai” as an aphrodisiac agent. It
has been mentioned in a Thai medicinal textbook that
B. rotunda has vigor and sexual enhancing properties
[2]. Although B. rotunda has long been consumed, its
safety has not been established in scientific studies.
Due to the growing demand for herbal products with
the potential to improve human health, enhance vigor,
as well as promote sexual efficacy, this present study
was designed to evaluate the safety of B. rotunda
through the assessment of sub-chronic toxicity. The
evaluated parameters included toxicology signs, body
* E-mail: k_saephet@yahoo.com
weight, hematological tests and blood biochemical
assays.
Materials and Methods
Extraction of B. rotunda
B. rotunda was collected from Chiang Mai province,
Thailand, identified and deposited in the Herbarium
of the Department of Biology, Chiang Mai University.
The rhizomes were sliced and dried at 60° C and then
ground to a fine powder. Ethanol extract was
prepared by soxhlet extraction using 50% ethanol and
then evaporated by rotary evaporation. The crude
extract was kept in a dry place and used after prepa-
ration at the required doses in distilled water.
Animals
Male Wistar rats (Rattus norvegicus), approxi-
mately 6 weeks of age with weights between 240-250
g. were used in the present investigation. The animals
were purchased from the National Laboratory Animal
Center, Thailand. They were allowed to acclimatize
in the departmental animal facility for one week prior

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 20 Saraithong, P. et al.
to the day of the experiment. They had access to water
and a standard diet (C.P. 082). The study room was
maintained at approximately 25 ± 2 °C in a 12 hour
light/dark cycle. The Animal Use and Care Regula-
tions of the Department of Biology, Faculty of
Sciences, Chiang Mai University, were followed (Re
004/08).
Sub-chronic toxicity test
The rats were randomized into 4 groups with 9
rats in each group. They were then treated orally for
60 days with B. rotunda extract at doses of 60, 120
and 240 mg/kg respectively. Controls were treated with
distilled water. The body weights of the rats were
recorded weekly. At the end of the treatment period,
blood samples were collected for examination of the
following blood chemistry profiles:
- Blood chemistry : aspatate aminotransferase
(AST), alanineaminotrans-ferase (ALT), blood urea
nitrogen (BUN) and creatinine (Crea). (These chem-
istries were measured by automate (Synchron C5X,
Beckman) with the cooperation of the Clinical
Chemistry Department, Faculty of Associated
Medical Science, Chiang Mai University.)
- Hematological test: hematocrit (PVC), total
white blood cell count and differential cell count.
(These counts were detected by a routine complete
blood cell count method [7].
The animals were then sacrificed and examined
for changes in internal organs. The liver and kidney
were removed and fixed in Bouin’s solution for histo-
Table 1. Differential white blood cell counts of male rats received 60, 120 and 240 mg/kg of
B. rotunda extract for 60 days as compared to control (Mean ± SD).
Concentration of Differential white blood cell counts (cells/ml)
B.Rotunda extract
Neutrophil Eosinophil
0 mg/kg (Control) 230,985 ± 105,217 12,530 ± 8,397
60 mg/kg 229,734 ± 59,633 ns 8,759 ± 5,022 ns
120 mg/kg 228,011 ± 71,379 ns 12,146 ± 6,604 ns
240 mg/kg 199,935 ± 101,719 ns 10,938 ± 6,257 ns
ns = not significantly different between groups
À È“1-92 20
pathological examination using a routine histology
technique with Hematoxyline and Eosin staining.
Statistical Analysis
Data were expressed as mean ± standard devia-
tion. The significance of difference between the means
was determined by One-way Analysis of Variance
(ANOVA). The observed significance was then
confirmed using the Least Significant Difference
(LSD) test.
Results and Discussion
All rats treated with all doses of B. rotunda
extract survived through the experiment. No signs of
sub-chronic toxicity were observed during the period
of 60 days.
The results of hematological values are presented
in Table 1. and Table 2. The values of hematocrit,
hemoglobin, total white blood cell count and differ-
ential white blood cell count were similar in all groups
and they were in normal ranges for Wistar rats [3].
These results indicate normal function of the hemato-
logical system.
There were no differences in any of the blood
biochemical values between the extract-treated groups
and the control group. Levels of AST, ALT, BUN and
Crea did not alter [Table 3.] and they remained at
normal levels [3]. These results indicate that the liver
and kidney functions were not affected.
Basophil Lymphocyt Monocyte
501 ± 745 126,336 ± 78575 4,546 ± 3,210
886 ± 1,690 ns 09,534 ± 68068 ns 4,026 ± 3,347 ns
1,046 ± 972 ns 112,895 ± 56677 ns 3,401 ± 2,173 ns
1,836 ± 1,535 ns 112,300 ± 91,119 ns 3,291 ± 3,161 ns
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 Safety Evaluation of Ethanol Extracts from Bosenbergia rotunda (L.) Mansf. in Male Rats 21

Table 2. Hematocrit, hemoglobin and total white blood cells of male rats received 60, 120
and 240 mg/kg of B. rotunda extract for 60 days as compared to control (Mean ± SD).

Concentration of Total White Blood Cells

B.Rotunda extract Hematocrit (%) Hemoglobin (g%)
(cells/ml)

0 mg/kg
(Control) 41.49 ± 2.94 14.74 ± 1.11 3800 ± 1486
ns ns
60 mg/kg 42.84 ± 2.54 15.23 ± 0.67 3675 ± 1100 ns
ns ns
120 mg/kg 41.86 ± 1296 15.05 ± 0.90 3575 ± 1296 ns
240 mg/kg 40.58 ± 3.27 ns 14.89 ± 0.87 ns 3288 ± 1947 ns

ns = not significantly different between groups

Table 3. Blood biochemistry values of male rats received 60, 120 and 240 mg/kg of B. rotunda
extract for 60 days as compared to control (Mean ± SD).

Concentration of
BUN (mg/dl) Crea (mg/dl) AST(IU/l) ALT(IU/L)
B.Rotunda extract

0 mg/kg
(Control) 27.60 ± 6.19 0.66 ± 0.05 124 ± 22 42.6 ± 13
ns ns ns ns
60 mg/kg 26.80 ± 4.21 0.64 ± 0.05 130 ± 11 40.5 ± 4.4
120 mg/kg 29.60 ± 1.82 ns 0.66 ± 0.09 ns 129 ± 21 ns 44.0 ± 12.7 ns
240 mg/kg 27.40 ± 4.98 ns 0.68 ± 0.11 ns 129 ± 15 ns 39.0 ± 2.45 ns

ns = not significantly different between groups, BUN = Blood Urea Nitrogen , Crea = Creatinine,
AST= Aspatate Aminotransferase , ALT= Alanineaminotransferase

Upon gross examination of the internal organs, no
abnormal signs were observed. Histopathological
examinations were performed on the liver and
kidney. No remarkable histopathological findings were
observed in these two organs.
There were no differences in the body weights
between the extract-treated groups and the control
group after 60 days of treatment [Figure 1.]. During
the experiment, the rats in each group did not show
abnormal signs of appearance, behavior or diet.

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 22
500
450
Body weights (g)
400
350
300
250
200
1 2 3 4
Figure 1. The body weights mean of male rats received 60, 120 and 240 mg/kg of B. rotunda
extract for 60 days as compared to control (0 mg/kg).
The toxicities of some aphrodisiac plants such as
gallic acid, Butea superba, and Kaempferia parviflora,
have been reported previously [4-6]. Those toxicities
included the alteration of leucopoiesis, micronucleus
induction, vacuolization in hepatocytes as well as the
deviation of blood biochemical profiles from a nor-
mal range. The normal values of data obtained from
our study scientifically supports the concept that the
use of B. rotunda in daily meals and in the form of
herbal beverages at the doses investigated in this ex-
periment is safe for humans. Since consumption of
health products based directly on natural materials
has shown a rapid sales growth in Thailand recently,
continued extensive research on such materials is
needed. The development of non-harmful plants as
new natural products with particular medicinal im-
portance will be a valuable asset in medical treat-
ments.
Acknowledgements
Thanks to the Faculty of Science, Chiang Mai
University for their financial support.
À È“1-92 22
Saraithong, P. et al.
control
60mg/kg
120mg/kg
240mg/kg
5 6 7 8 9 10
Weeks
References
1. Chomchalow, N., Bansiddhi, J. & MacBaine, C.
(2003) Amazing Thai Spices. Bangkok,
Horticultural Research Institute, Department
of Agriculture and Horticultural Science
Society.
2. Wutythamawech, W. (2000) Encyclopedia of Thai
herbs I. Bangkok: Phet 69 Printing. (in Thai).
3. Sharp, P.E. & La Regina, M.C. (1998)
The laboratory rats. Boca Raton,CRC. Press.
4. Niho, N. (2001) Subchronic toxicity study of gallic
acid by oral administration in F344 rats. Food
Chem Toxicol 39, 1063-1070.
5. Pongpanparadon, A., Aritajat, S. & Saenphet, K.
(2002) The toxicity of Butea superba Roxb.
SEAJ. Trop. Med. Publ. Health (suppl) l ,
171-173.
6. Sudwan, P., Saenphet, K., Saenphet, S. &
Suwansirikul, S. (2006) Effect of Kaempferia
parviflora Wall.Ex.Baker on sexual activity
of male rats and its toxicity. SEAJ. Trop.
Med. Publ. Health 37 (suppl) 3, 210-215.
7. Dacie, J.V. & Lewis, S.M. (1984) Practical
haematology. 6th ed. London, Churchill
livingstone.
1/10/10, 5:51 PM