Acute and chronic toxicities of Bacopa monnieri extract in Sprague-Dawley rats

Abstract

Background
Bacopa monnieri is a medicinal plant which has long been used in Ayurvedic medicines to augment brain function and to improve memory. The purpose of our study was to identify and evaluate possible toxic effects of B. monnieri extract in rats by assessing hematological, biochemical, and histopathological parameters.

Methods
Acute oral toxicity of Bacopa monnieri extract was studied in female rats by giving a single orally administered dose at a level of 5,000 mg/kg. The rats were monitored for toxic signs for 14 days. In the chronic toxicity test, groups of both female and male rats were given daily oral doses of B. monnieri extract at dose levels of either 30, 60, 300 or 1,500 mg/kg for 270 days. The behavior and health of the animals was then monitored. At the end of the observation period, the body and organ weights of the rats in each group were measured. Blood was collected and necropsy was performed to evaluate their hematology, blood clinical chemistry, and microanatomy.

Results
The acute toxicity test found no significant differences between the experimental and the control group rats. In the chronic toxicity test, animal behavior and health of the experimental groups were normal, just as in the control rats. All values of other parameters assessed remained within the normal range.

Conclusion
A single oral administration of B. monnieri extract at the dose of 5,000 mg/kg did not cause any serious undesirable effects. B. monnieri extract at doses of 30, 60, 300 and 1,500 mg/kg given for 270 days did not produce any toxicity in rats.

Full text

R E S E A R C H A R T I C L E Open Access
Acute and chronic toxicities of Bacopa
monnieri extract in Sprague-Dawley rats
Seewaboon Sireeratawong
1,2*
, Kanjana Jaijoy
3
, Parirat Khonsung
1
, Nirush Lertprasertsuk
4
and Kornkanok Ingkaninan
5
Abstract
Background: Bacopa monnieri is a medicinal plant which has long been used in Ayurvedic medicines to augment
brain function and to improve memory. The purpose of our study was to identify and evaluate possible toxic
effects of B. monnieri extract in rats by assessing hematological, biochemical, and histopathological parameters.
Methods: Acute oral toxicity of Bacopa monnieri extract was studied in female rats by giving a single orally
administered dose at a level of 5,000 mg/kg. The rats were monitored for toxic signs for 14 days. In the chronic
toxicity test, groups of both female and male rats were given daily oral doses of B. monnieri extract at dose levels of
either 30, 60, 300 or 1,500 mg/kg for 270 days. The behavior and health of the animals was then monitored. At the
end of the observation period, the body and organ weights of the rats in each group were measured. Blood was
collected and necropsy was performed to evaluate their hematology, blood clinical chemistry, and microanatomy.
Results: The acute toxicity test found no significant differences between the experimental and the control group rats.
In the chronic toxicity test, animal behavior and health of the experimental groups were normal, just as in the control
rats. All values of other parameters assessed remained within the normal range.
Conclusion: A single oral administration of B. monnieri extract at the dose of 5,000 mg/kg did not cause any serious
undesirable effects. B. monnieri extract at doses of 30, 60, 300 and 1,500 mg/kg given for 270 days did not produce
any toxicity in rats.
Keywords: Bacopa monnieri, Acute toxicity, Chronic toxicity
Background
Bacopa monnieri (L.) Wettst, known as Brahmi or water
hyssop, (Family Plantaginaceae), is a perennial creeper
found in marshy areas throughout Asia where it is used
in Ayurvedic medicine for enhancing memory and
improving brain function [1]. It has recently been re-
ported that B. monnieri extract has several pharmaco-
logical activities, e.g., a neuroprotective effect [2, 3],
ameliorating cognitive dysfunction [4–6], increasing
cerebral blood flow [7], enhancing the activity of anti-
oxidant enzymes and intracellular signaling pathways
[8], an antiparkinsonian agent [9], reducing blood pres-
sure [10], hepatoprotection [11], anti-fertility [12], anti-
addiction [13], antioxidant [14, 15], antidepressant [16],
anti-stress [17], anti-ulcer [18], anti-cancer [19], and
anti-inflammation [20].
Clinical studies have shown that B. monnieri reduces
the rate of memory loss of newly acquired information
[21], improves memory performance in older persons
[22], and enhances cognitive performance in humans
[23]. However, B. monnieri was also shown to cause side
effects in the gastrointestinal tract, i.e., nausea, increased
stool frequency and abdominal cramps [22]. Severe
liver toxicity has been reported in a woman after taking
several Ayurvedic herbs, including B. monnieri, for nine
months;however,aftershestoppedtakingtheherbs,
her liver function returned to normal [24]. Because of
the potential positive ethnobotanical and pharmaco-
logical applications of the plant, this study was designed
to investigate possible toxic effects of the B. monnieri
extract in rats.
* Correspondence: seewaboon@gmail.com
1
Department of Pharmacology, Faculty of Medicine, Chiang Mai University,
Chiang Mai 50200, Thailand
2
Division of Pharmacology, Department of Preclinical Science, Faculty of
Medicine, Thammasat University, Pathum Thani 12120, Thailand
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249
DOI 10.1186/s12906-016-1236-4
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Methods
Plant material and extract
The B. monnieri was collected in Phetchaburi Province,
Thailand, and was identified by Prof. Dr. Wongsatit
Chuakul. The voucher specimen (Phrompittayarat 001)
is maintained at the PBM Herbarium, Mahidol University,
Thailand. An aerial portion of B. monnieri was cut into
small pieces, dried (50 °C, 12 h.) and then pulverized. The
dried powder was percolated twice with 95 % ethanol at
the ratio of 1 g: 7 ml. The percolate was dried in a rotary
evaporator under reduced pressure. The yield was 10 % of
the fresh weight. The phytochemical study found the ex-
tract contained 6.25 % (w/w) of total saponins, a mixture
of 0.87 % of bacoside A
3
, 1.03 % of bacopaside I, 1.82 %
of bacopaside II, 0.8 % of bacopaside X, and 1.73 % of
bacopasaponin C. [25, 26]. The extract was kept in a
dark bottle in the refrigerator at 5 °C until used.
Animals
Male and female Sprague-Dawley rats, 4–5week-oldand
weighing 200–250 g, were purchased from the National
Laboratory Animal Center (NLAC), Nakorn Pathom,
Thailand. All rats were kept in an animal room at a con-
trolled temperature of 25 ± 1 °C and with a 12 h light-dark
cycle. A standard rat chow and water ad libitum were pro-
vided. The animals were acclimatized for one week before
the start of the experiment. The experimental protocols
were approved by the Animal Ethics Committee of Faculty
of Medicine, Thammasat University (AE011/2552).
Acute toxicity test
Following OECD Test Guideline 420, in the sighting
study female Sprague-Dawley rats received a starting
dose of 2,000 mg/kg of extract (Annex 2 - Flow chart of
the sighting study), but no toxicity was observed. A main
study starting dose of 5,000 mg/kg was the limit in the
acute toxicity study. The female rats were randomly
divided into control and treatment groups of five rats
each. Rats were fasted overnight prior to dosing on day 1.
Group 1, the control group, received distilled water
(1 mL/kg) and group 2, the test group, received B. mon-
nieri extract at a dose of 5,000 mg/kg in a constant vol-
umeof1mL/kgbodyweightbyoraladministration.
Observation of toxic signs was recorded systematically
for the first 30 min, then periodically during the first
24 h. At the end of the experiment the surviving rats
were kept for further 14 days to allow daily observation
of clinical signs of toxicity. The body weight of the animals
was measured prior to dosing and then weekly after that.
On day 15, the surviving animals were sacrificed using an
intraperitoneal overdose of pentobarbital sodium. Their
internal organs, including heart, lungs, liver, kidney,
spleen, adrenal glands, sex organs and brain, were excised,
and weighed and a gross pathological examination was
conducted. The organs were then fixed in a 10 % neutral
buffered formaldehyde solution for histopathological
examination [27, 28].
Chronic toxicity test
The chronic toxicity test was conducted in accordance
with WHO and OECD guidelines. The usual dose of B.
monnieri in food supplements for humans is 300 mg/day
or 5 mg/kg/day (the average body weight of a Thai adult
is about 60 kg). The dosage of the plant extract used in
rats, approximately 30 mg/kg/day, was calculated based
on body surface area [30]. Doses of 30, 60, 300 and
1,500 mg/kg/day of B. monnieri extract were used for
the chronic toxicity evaluation. The rats were divided by
sex into six groups, ten male and ten female each. All
the rats were orally administered either distilled water
(control group) or B. monnieri extract once daily for
270 days (experimental group). A satellite group received
adoseofB. monnieri extract of 1,500 mg/kg per day for
270 days after which the rats were then reared for an
additional 28 days with no B. monnieri extract to observe
their recovery and to identify any delayed effects of the
extract. Animals were weighed and observed daily for
toxicological signs, physiological and behavioral changes,
as well as mortality. The animals were anesthetized with
an intraperitoneal injection of pentobarbital sodium at the
end of the experiment. Blood samples were collected from
the common carotid artery with an EDTA tube for
hematological study and with a non-EDTA tube for bio-
chemical study. Hematological analysis using standard
techniques was performed for red blood cell count (RBC),
hemoglobin (HB), hematocrit (HCT), mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC),
platelet count (PLT), white blood cell count (WBC) as well
as neutrophil (PMN), lymphocyte (LYMP), monocyte
(MONO), eosinophil (EOS), and basophil (BASO) con-
centrations. Blood samples were biochemically analyzed
for levels of glucose (GLU), creatinine (CRE), blood urea
nitrogen (BUN), total protein (TP), albumin (ALB), total
bilirubin (T-BIL), direct bilirubin (D-BIL), alanine amino-
transferase (ALT), aspartate aminotransferase (AST), and
alkaline phosphatase (ALP). Finally, the rats were sacri-
ficed and their internal organs were excised, observed,
weighed and fixed in 10 % neutral buffered formaldehyde
solution for further pathological examination [28, 29].
Statistical analysis
One-way analysis of variance (ANOVA) and Dunnett’stest
were performed to determine statistical significance using
the SPSS program (version 22.0). In the acute toxicity
study, the data were analyzed using Student’st-test. Results
are expressed as mean ± standard error of mean (S.E.M.).
Pvalues less than 0.05 were considered significant.
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 2 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Results
A large single oral dose of B. monnieri extract (5,000
mg/kg) did not cause mortality in rats. Normal behavioral
patterns were observed and no changes of eyes, skin, fur,
mucous membranes, respiration, circulatory system, auto-
nomic or central nervous systems were found after that
dose. The body weight on day 14 of the extract-treated
rats had significantly increased compared to that of the
control group (Table 1). As shown in Table 2, the average
lung weight of treated rats was significantly greater than
in the control group, although physical appearance (color
and texture) was normal. No significant histopathological
change in the internal organs, including the liver and the
kidney, were observed (Fig. 1).
In the chronic toxicity study (Fig. 2) a small but sig-
nificant decrease in the body weight of female rats was
observed in the experimental group with some doses of
extract compared to the control group (distilled water)
measured on the same day. The organ weight of female
rats is presented in Table 3. Female B. monnieri extract-
treated rats showed the significant greater in the weight of
the brain (300 and 1,500 mg/kg), pancreas (1,500 mg/kg)
and ovary (60, 300 and 1,500 mg/kg), while a significant
smaller in liver weight (60 mg/kg) was observed. In male
B. monnieri extract-treated rats, significant greater in the
weight of the lungs (1,500 mg/kg), kidneys (30 mg/kg),
and epididymes (60 and 300 mg/kg) were observed. The
weight of the heart (satellite group) and liver (60 and
300 mg/kg) was significantly lower in male extract-treated
groups than in the control group (Table 4).
The effect of B. monnieri extract on hematological
parameters is depicted in Tables 5 and 6. In the female
satellite group, the values of RBC, HB, HCT, and MCV
were significantly increased. However, MCH, MCHC
and PLT levels were significantly lower than those of the
control group. Moreover, the amounts of WBC and
LYMP were significantly increased in female rats treated
with B. monnieri extract at doses of 1,500 and 300 mg/kg,
respectively. In the male extract-treated groups, the levels
of RBC (30 mg/kg) and MCHC (satellite group) were
significantly decreased, whereas the level of HCT was
significantly increased in the satellite group. The amount
of WBC, NEU and LYMP was significantly increased in
male rats treated with B. monnieri extract at doses of
300 and 1,500 mg/kg.
Clinical blood chemistry parameters were used to clar-
ify the effect of B. monnieri extract on the liver (SGPT,
SGOT, TP, ALB, ALP, T-BIL and D-BIL), kidney (BUN
and CRE), and pancreatic (GLU) function. As shown in
Table 7, the level of GLU was significantly decreased in
female rats treated with B. monnieri extract at doses of
60, 300 and 1,500 mg/kg. A small but significant
increase of CRE levels and a decrease of TP levels were
observed in the female satellite group. In male rats, the
level of AST was significantly decreased in groups
treated with B. monnieri extract at doses of 60, 300 and
1,500 mg/kg. In the male satellite group, a small but
significant increase of BUN, ALT and ALP levels was
found when compared to those of the control group.
No significant histopathological change in the internal
organs was found in either the control or the extract-
treated rats (Fig. 3).
Discussion
Toxicity testing is intended to provide information on
the safety of an herbal product before further evaluation
of its benefits in clinical trials. The appropriate period of
administration of the test product to animals is deter-
mined by the anticipated period of clinical use in
humans [28]. The acute oral toxicity test is used for
evaluating any adverse effects appearing within a short
time after a single large oral dose of the test substance
or after multiple doses given within 24 h. The B. mon-
nieri extract at a dose of 5,000 mg/kg was selected for
acute toxicity testing as an initial dose of 2,000 mg/kg
did not cause any observable signs or symptoms of tox-
icity. The results showed that B. monnieri did not cause
death or result in any other signs of toxicity. There was
a slight difference in body weight between the control
and the treated groups at the beginning of experiment,
but it was not statistically significant. However, a small
Table 1 Body weight of female rats in acute toxicity test
Body weight (g)
Day 0 Day 7 Day 14
Control 218.00 ± 3.74 215.00 ± 2.24 233.00 ± 2.00
B. monnieri extract
5,000 mg/kg
234.00 ± 4.30 255.00 ± 6.12 260.00 ± 7.58*
Values are expressed as mean ± S.E.M., n=10
*Significantly different from control, p< 0.05
Table 2 Organ weight (g) of female rats in acute toxicity test
Organ weight (g)
Control B. monnieri extract 5,000 mg/kg
Brain 1.79 ± 0.08 1.73 ± 0.03
Lung 1.27 ± 0.02 1.44 ± 0.06*
Heart 0.84 ± 0.05 0.92 ± 0.03
Liver 6.74 ± 0.29 9.10 ± 0.61
Pancreas 0.62 ± 0.04 0.86 ± 0.08
Spleen 0.65 ± 0.04 0.82 ± 0.04
Adrenal 0.04 ± 0.00 0.04 ± 0.00
kidney 0.83 ± 0.02 0.86 ± 0.02
Ovary 0.07 ± 0.00 0.07 ± 0.00
Uterus 0.49 ± 0.02 0.47 ± 0.04
Values are expressed as mean ± S.E.M., n=10
*Significantly different from control, p< 0.05
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 3 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

yendiKreviL
Control
B. monnieri
5,000 mg/kg
Fig. 1 Histopathology of the liver and kidney of rats in acute toxicity test at 40x magnification (haematoxylin-eosin stain)
Fig. 2 Body weight of rats treated with B. monnieri in chronic toxicity. aFemale. bMale. *Significantly different from control, p< 0.05
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 4 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

but significant difference in body weight was observed
on day 14. The small increase in body weight of the
treated animals can still be accepted as normal, as it can
occur due to differences in food consumption.
Neither gross nor histopathological abnormalities were
observed in any of the internal organs, including the
liver and kidney of B. monnieri-treated rats. This sug-
gests that B. monnieri extract is nontoxic at a dose of
5,000 mg/kg. The dose of B. monnieri extract used in
this study is approximately 1,000 times higher than that
generally used in humans (5 mg/kg/day). This indicates
that B. monnieri is quite safe for human use.
A chronic toxicity study normally takes 9 to 12 months
as some substances may not display toxicity immediately,
but it may appear after repeated exposure. According
to WHO guidelines, the period of testing administra-
tion in animals should be based on the expected period
of clinical use of that substance in humans. The
repeated oral administration of the test substance for
9-12 months in animals has been suggested to be com-
parable to administration for more than 6 months in
humans [28].
The objective of chronic toxicity studies is to characterize
the profile of the impact of prolonged and repeated
exposure to a substance in a mammalian species over a
significant portion of the average life span of that spe-
cies. The study evaluates the impact on target organs
including possible accumulation of the substance. The
results obtained can then be used as supporting data
for designing further clinical trials. The dosage of plant
extract for chronic toxicity testing in rats in this study
was calculated according to Reagan-Shaw et al. [30].
The B. monnieri extract at doses of 30, 60, 300 and
1,500 mg/kg used in this study are equivalent to 6, 12,
60, and 300 times of the usual B. monnieri dose in
humans, respectively.
Table 3 Organ weight (g) of female rats in chronic toxicity test
Control B. monnieri extract
30 mg/kg 60 mg/kg 300 mg/kg 1,500 mg/kg Satellite group
Brain 1.97 ± 0.02 1.94 ± 0.03 1.94 ± 0.03 2.07 ± 0.04* 2.08 ± 0.02* 2.01 ± 0.03
Lung 2.16 ± 0.26 2.16 ± 0.26 2.00 ± 0.14 2.14 ± 0.13 2.32 ± 0.19 2.00 ± 0.13
Heart 1.27 ± 0.05 1.22 ± 0.03 1.24 ± 0.04 1.38 ± 0.09 1.36 ± 0.06 1.31 ± 0.05
Liver 9.90 ± 0.32 9.55 ± 0.28 8.71 ± 0.30* 9.72 ± 0.23 9.33 ± 0.29 9.54 ± 0.27
Spleen 0.83 ± 0.03 0.81 ± 0.03 0.74 ± 0.03 0.83 ± 0.03 0.84 ± 0.04 0.79 ± 0.04
Pancreas 1.03 ± 0.09 1.09 ± 0.08 1.21 ± 0.14 1.34 ± 0.13 1.42 ± 0.14* 0.98 ± 0.12
Adrenal 0.04 ± 0.00 0.04 ± 0.00 0.05 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00
kidney 1.27 ± 0.02 1.28 ± 0.02 1.20 ± 0.03 1.22 ± 0.02 1.24 ± 0.04 1.25 ± 0.03
Ovary 0.07 ± 0.00 0.08 ± 0.00 0.09 ± 0.01* 0.09 ± 0.01* 0.09 ± 0.01* 0.07 ± 0.00
Uterus 1.18 ± 0.14 1.36 ± 0.33 1.01 ± 0.07 0.96 ± 0.09 0.86 ± 0.05 1.13 ± 0.10
A satellite group was given the B. monnieri extract at 1,500 mg/kg daily over 270 days followed by no treatment for 28 days
Values are expressed as mean ± S.E.M., n= 10. *Significantly different from contro l, p< 0.05
Table 4 Organ weight (g) of male rats in chronic toxicity test
Control B. monnieri extract
30 mg/kg 60 mg/kg 300 mg/kg 1,500 mg/kg Satellite group
Brain 2.11 ± 0.03 2.16 ± 0.03 2.16 ± 0.04 2.14 ± 0.04 2.17 ± 0.04 2.11 ± 0.03
Lung 2.34 ± 0.10 2.60 ± 0.13 2.58 ± 0.14 2.58 ± 0.20 3.59 ± 0.21* 2.36 ± 0.13
Heart 1.84 ± 0.07 1.89 ± 0.05 1.76 ± 0.03 1.79 ± 0.06 1.97 ± 0.07 1.67 ± 0.06*
Liver 15.80 ± 0.44 16.80 ± 0.34 14.22 ± 0.23* 14.09 ± 0.56* 14.61 ± 0.61 14.75 ± 0.37
Pancreas 0.97 ± 0.06 1.05 ± 0.04 0.99 ± 0.04 1.05 ± 0.04 0.98 ± 0.04 0.96 ± 0.03
Spleen 1.27 ± 0.04 1.21 ± 0.12 1.41 ± 0.14 1.08 ± 0.14 1.54 ± 0.13 0.94 ± 0.09
Adrenal 0.04 ± 0.00 0.04 ± 0.00 0.05 ± 0.00 0.04 ± 0.00 0.04 ± 0.00 0.04 ± 0.00
Kidney 1.86 ± 0.04 2.03 ± 0.06* 1.82 ± 0.04 1.76 ± 0.04 1.76 ± 0.06 1.74 ± 0.03
Testis 2.13 ± 0.03 2.11 ± 0.09 2.11 ± 0.03 2.13 ± 0.03 2.05 ± 0.07 2.00 ± 0.04
Epididymis 0.86 ± 0.02 0.93 ± 0.03 0.99 ± 0.03* 1.04 ± 0.06* 0.93 ± 0.03 0.87 ± 0.02
A satellite group was given the B. monnieri extract at 1,500 mg/kg daily over 270 days followed by no treatment for 28 days
Values are expressed as mean ± S.E.M., n= 10. *Significantly different from contro l, p< 0.05
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 5 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

In this study, no changes in animal behavior or any
toxic signs were observed in the B. monnieri-treated rats.
Body weight and internal organ weight were the main
parameters used in the assessment of toxicity. Reduction
of those two parameters can be used as a sensitivity
index of toxicity [31–33]. In this study, the animals were
weighed daily throughout the experimental period.
Although the weight of some groups of rats decreased
or increased slightly, the differences were very small
and remained within normal limits. Monitoring the
health of the animals during the entire 270 day period
found no signs of morbidity or diseases. Rats of both
sexes appeared healthy as evidenced by the normal ap-
pearance of their general behavior, respiratory patterns,
cardiovascular signs, motor activities, reflexes and lack
of abnormal changes of skin or fur.
Table 5 Hematological parameters of female rats in chronic toxicity test
Hematological
parameter
Control B. monnieri extract
30 mg/kg 60 mg/kg 300 mg/kg 1,500 mg/kg Satellite group
RBC (x10
6
/μl) 8.96 ± 0.27 9.03 ± 0.37 8.80 ± 0.13 8.56 ± 0.20 8.98 ± 0.18 9.92 ± 0.20*
HB (g/dl) 16.36 ± 0.37 16.22 ± 0.30 16.24 ± 0.22 15.84 ± 0.29 16.41 ± 0.27 17.50 ± 0.26*
HCT (%) 50.00 ± 1.60 50.80 ± 2.12 48.80 ± 0.73 48.60 ± 1.05 50.20 ± 0.89 56.60 ± 0.98*
MCV (fl) 55.70 ± 0.30 56.10 ± 0.43 55.40 ± 0.27 56.50 ± 0.31 55.80 ± 0.59 57.00 ± 0.30*
MCH (pg) 18.30 ± 0.22 18.09 ± 0.44 18.41 ± 0.25 18.56 ± 0.27 18.30 ± 0.31 17.61 ± 0.13
MCHC (g/dl) 32.83 ± 0.45 32.18 ± 0.79 33.13 ± 0.38 32.82 ± 0.37 32.75 ± 0.38 30.90 ± 0.17*
PLT (x10
5
/μl) 0.83 ± 0.05 0.85 ± 0.05 0.77 ± 0.02 0.73 ± 0.02 0.72 ± 0.09 0.68 ± 0.04*
WBC (x10
3
/μl) 5.18 ± 0.34 4.96 ± 0.38 5.87 ± 0.36 6.32 ± 0.61 7.03 ± 0.63* 6.27 ± 0.67
NEU (x10
3
/μl) 1.37 ± 0.16 1.28 ± 0.15 1.30 ± 0.14 1.53 ± 0.42 1.41 ± 0.19 2.03 ± 0.26
LYMP (x10
3
/μl) 3.18 ± 0.39 3.40 ± 0.24 4.27 ± 0.28 9.59 ± 5.25* 5.31 ± 0.50 3.63 ± 0.58
MONO (x10
3
/μl) 0.15 ± 0.03 0.23 ± 0.06 0.21 ± 0.06 0.27 ± 0.07 0.19 ± 0.03 0.23 ± 0.04
EOS (x10
3
/μl) 0.04 ± 0.02 0.04 ± 0.03 0.09 ± 0.03 0.04 ± 0.02 0.06 ± 0.02 0.03 ± 0.02
BASO (x10
3
/μl) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
A satellite group was given the B. monnieri extract at 1,500 mg/kg daily over 270 days followed by no treatment for 28 days
RBC red blood cell count, HB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular
hemoglobin concentration, PLT platelet count, WBC white blood cell count, NEU neutrophil, LYMP lymphocyte, MONO monocyte, EOS eosinophil, BASO basophil
Values are expressed as mean ± S.E.M., n= 10. *Significantly different from contro l, p< 0.05
Table 6 Hematological parameters of male rats in chronic toxicity test
Hematological
parameter
Control B. monnieri extract
30 mg/kg 60 mg/kg 300 mg/kg 1,500 mg/kg Satellite group
RBC (x10
6
/μl) 9.76 ± 0.18 8.53 ± 0.89* 10.03 ± 0.30 9.53 ± 0.30 9.60 ± 0.21 10.73 ± 0.20
HB (g/dl) 16.99 ± 0.23 16.73 ± 0.50 16.92 ± 0.43 16.73 ± 0.51 16.77 ± 0.41 17.82 ± 0.28
HCT (%) 52.00 ± 0.91 51.00 ± 1.86 52.90 ± 1.55 50.60 ± 1.58 50.50 ± 1.30 57.50 ± 0.98*
MCV (fl) 53.00 ± 0.21 53.40 ± 0.31 52.60 ± 0.27 53.00 ± 0.30 52.50 ± 0.27 53.50 ± 0.31
MCH (pg) 17.38 ± 0.17 17.54 ± 0.09 16.85 ± 0.15 17.54 ± 0.11 16.41 ± 1.03 16.56 ± 0.12
MCHC (g/dl) 32.80 ± 0.28 32.91 ± 0.26 32.07 ± 0.25 33.16 ± 0.19 33.18 ± 0.22 30.92 ± 0.12*
PLT (x10
5
/μl) 0.95 ± 0.03 0.86 ± 0.03 0.97 ± 0.03 0.93 ± 0.04 1.85 ± 0.89 0.89 ± 0.04
WBC (x10
3
/μl) 7.70 ± 0.42 7.23 ± 0.69 9.52 ± 0.40 11.62 ± 1.44* 10.01 ± 0.72* 7.79 ± 0.60
NEU (x10
3
/μl) 1.37 ± 0.16 1.80 ± 0.23 2.12 ± 0.22 2.89 ± 1.19* 2.18 ± 0.26 2.10 ± 0.26
LYMP (x10
3
/μl) 5.94 ± 0.28 5.14 ± 0.52 6.90 ± 0.29 8.08 ± 0.50* 7.31 ± 0.50* 5.31 ± 0.55
MONO (x10
3
/μl) 0.27 ± 0.06 0.21 ± 0.07 0.30 ± 0.07 0.49 ± 0.17 0.45 ± 0.12 0.35 ± 0.06
EOS (x10
3
/μl) 0.11 ± 0.05 0.08 ± 0.03 0.20 ± 0.08 0.06 ± 0.05 0.07 ± 0.03 0.02 ± 0.02
BASO (x10
3
/μl) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
A satellite group was given the B. monnieri extract at 1,500 mg/kg daily over 270 days followed by no treatment for 28 days
RBC red blood cell count, HB hemoglobin, HCT hematocrit, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular
hemoglobin concentration, PLT platelet count, WBC white blood cell count, NEU neutrophil, LYMP lymphocyte, MONO monocyte, EOS eosinophil, BASO basophil
Values are expressed as mean ± S.E.M., n= 10. *Significantly different from contro l, p< 0.05
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 6 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

The internal organs were weighed and examined for
gross pathology. Some test substances may harm tissues
at the cellular level but not show any observable abnor-
malities. For this reason histopathological examination
should be carried out to identify any cellular damage of
the internal organs or tissues. In this study, internal
organs including the brain, lungs, heart, liver, kidney,
pancreas, spleen, stomach, duodenum, small intestine,
and sex organs were examined. The examination found
no significant histopathological changes in these organs
or tissues. Although some statistically significant but
minor differences in the body weight and the weight of
some of the internal organs were observed, the values of
those parameters were in normal range.
Hematological examination is one of the methods used
to evaluate aspects of health status that may not be
visible during physical examination [34]. This is important
becausethehematopoieticsystemisverysensitiveto
toxicsubstances.Itcanbeaffectedbytheingestionof
some toxic plants [35]. In particular, hematological
parameters can provide important information about
bone marrow activity as well as about intravascular ef-
fects such as hemolysis and anemia [36]. Morphological
examination of RBC and WBC can identify their pro-
ductionordestruction.BASA,EOS,LYMP,MONO,
and PMN are also used to assess the effect of test sub-
stances on immune systems [36–38]. Our results found
significant differences in some hematological parame-
ters, but values remained within normal limits [39].
Clinical blood chemistry examination was performed
to quantify any toxic effects on pancreas function
(GLU), kidney function (BUN, CRE) and liver function
(SGOT, SGPT, ALP, TP, ALB, T-BIL and D-BIL). Plasma
GLU is monitored to demonstrate the effect of a test
substance on glucose metabolism. In terms of kidney
function, the most common cause of acute or chronic
renal failure is elevated BUN and increased CRE. Liver
function is measured using a blood test which evaluates
various functions of the liver, e.g., metabolism, detoxifi-
cation, storage, and production of several vital protein
components of blood plasma. Conditions commonly
associated with testing for abnormalities of liver function
Table 7 Biochemical parameters of female and male rats in chronic toxicity test
Biochemical
parameter
Control B. monnieri extract
30 mg/kg 60 mg/kg 300 mg/kg 1,500 mg/kg Satellite group
Female rats
GLU (mg/dl) 177.30 ± 5.66 159.90 ± 7.50 147.50 ± 6.55* 134.10 ± 8.50* 132.20 ± 7.96* 170.3 ± 8.85
BUN (mg/dl) 16.12 ± 1.99 16.54 ± 0.41 15.76 ± 1.23 16.47 ± 0.44 27.50 ± 11.31 21.32 ± 0.53
CRE (mg/dl) 0.56 ± 0.02 0.57 ± 0.01 0.55 ± 0.01 0.58 ± 0.01 0.60 ± 0.02 0.61 ± 0.02*
TP (g/dl) 6.29 ± 0.08 6.20 ± 0.08 6.15 ± 0.09 6.22 ± 0.12 6.16 ± 0.10 5.86 ± 0.03*
ALB (g/dl) 3.20 ± 0.03 3.27 ± 0.03 3.27 ± 0.03 3.20 ± 0.05 3.12 ± 0.04 3.18 ± 0.05
T-BIL (mg/dl) 0.10 ± 0.01 0.10 ± 0.01 0.11 ± 0.01 81.18 ± 81.09 0.10 ± 0.00 0.25 ± 0.02
D-BIL (mg/dl) 0.18 ± 0.02 0.17 ± 0.02 0.23 ± 0.03 0.28 ± 0.08 0.22 ± 0.03 0.11 ± 0.01
AST (U/L) 195.60 ± 26.12 220.20 ± 22.37 162.70 ± 9.09 213.40 ± 21.86 178.20 ± 21.36 204.80 ± 21.98
ALT (U/L) 57.60 ± 9.00 77.70 ± 10.96 49.70 ± 5.35 73.40 ± 9.83 61.10 ± 9.88 63.20 ± 7.79
ALP (U/L) 43.20 ± 2.92 40.60 ± 3.03 41.90 ± 3.22 48.00 ± 7.90 48.10 ± 6.03 43.10 ± 3.94
Male rats
GLU (mg/dl) 209.10 ± 8.67 209.40 ± 15.59 181.40 ± 18.86 219.60 ± 14.18 196.40 ± 9.49 174.20 ± 8.07
BUN (mg/dl) 17.52 ± 0.46 18.25 ± 0.45 17.29 ± 0.63 16.59 ± 0.32 16.94 ± 0.50 19.62 ± 0.31*
CRE (mg/dl) 0.57 ± 0.03 0.56 ± 0.02 0.54 ± 0.02 6.09 ± 5.55 0.53 ± 0.01 0.61 ± 0.02
TP (g/dl) 6.11 ± 0.12 5.85 ± 0.15 6.19 ± 0.09 6.11 ± 0.10 6.37 ± 0.13 6.25 ± 0.18
ALB (g/dl) 3.07 ± 0.05 3.03 ± 0.06 3.06 ± 0.03 3.07 ± 0.05 3.10 ± 0.04 3.21 ± 0.06
T-BIL (mg/dl) 0.20 ± 0.03 0.18 ± 0.02 0.18 ± 0.02 0.14 ± 0.02 0.16 ± 0.02 0.18 ± 0.02
D-BIL (mg/dl) 0.09 ± 0.01 0.09 ± 0.00 0.10 ± 0.01 0.09 ± 0.00 0.08 ± 0.01 0.18 ± 0.07
AST (U/L) 193.00 ± 13.81 158.60 ± 9.13 152.30 ± 13.61* 142.60 ± 8.27* 146.80 ± 10.34* 201.80 ± 22.31
ALT (U/L) 63.20 ± 6.35 61.90 ± 5.82 54.60 ± 5.27 51.30 ± 2.86 54.40 ± 3.99 83.40 ± 9.42*
ALP (U/L) 55.80 ± 2.51 53.20 ± 2.28 52.90 ± 1.59 55.10 ± 2.61 61.10 ± 2.22 64.90 ± 3.40*
A satellite group was given the B. monnieri extract at 1,500 mg/kg daily over 270 days followed by no treatment for 28 days
GLU glucose, BUN blood urea nitrogen, CRE creatinine, TP total protein, ALB albumin, T-BIL total bilirubin, D-BIL direct bilirubin, AST aspartate aminotransferase,
ALT alanine aminotransferase, ALP alkaline phosphatase
Values are expressed as mean ± S.E.M., n= 10. *Significantly different from contro l, p< 0.05
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

yendiKreviL
Control
B. monnieri
30 mg/kg
B. monnieri
60 mg/kg
B. monnieri
300 mg/kg
B. monnieri
Fig. 3 Histopathology of the liver and kidney of rats in chronic toxicity test at 40x magnification (haematoxylin-eosin stain)
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

include gallbladder disease, hepatitis, fatty liver, cirrhosis,
infectious mononucleosis and alcoholism [38, 40]. In this
study, significant differences in some clinical blood
chemistry values were observed in both sexes between
the experimental and the control groups. However, the
alteration of these parameters was minor and remained
within the normal range [41]. The data suggest that
B. monnieri extract does not alter the function of the
pancreas, liver or kidney.
The satellite-treated group was assessed for reversibi-
lity of the toxic effects of the test substance and for the
occurrence of delayed toxic effects. In this study, the
satellite-treated group received B. monnieri extract at a
dose of 1,500 mg/kg/d for 270 days. The rats were then
kept for an additional 28 days post treatment. No toxic
signs or symptoms or any abnormalities were detected.
Taken together, all of our results from both the acute
and the chronic toxicity tests indicate that the B. monnieri
extract is fairly non toxic for chronic use.
Conclusion
B. monnieri extract did not cause any signs of toxicity
or any other symptoms in the acute and chronic oral
toxicity tests of both female and male rats. That indicates
that B. monnieri extract is relatively non-toxic. Further
study regarding the toxicology of this extract should be
conducted in non-rodent species or in humans.
Abbreviations
ALB, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST,
aspartate aminotransferase; BASO, basophil; BUN, blood urea nitrogen; CRE,
creatinine; D-BIL, direct bilirubin; EOS, eosinophil; GLU, glucose; HB, hemoglobin;
HCT, hematocrit; LYMP, lymphocyte; MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular
volume; MONO, monocyte; PLT, platelet; PMN, neutrophil; RBC, Red blood cell
count; T-BIL, total bilirubin; TP, total protein; WBC, white blood cell count
Acknowledgements
This study was supported by the National Research Council of Thailand (NRCT).
Funding
This study was granted from the National Research Council of Thailand (NRCT).
Availability of data and materials
Data are all contained within the paper.
Authors’contributions
All authors designed and coordinated all laboratory experiments. All authors
conducted, analyzed, interpreted the results. All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
All experimental protocols on animals in this study were complied with
the standards for the care and use of experimental animals and were
approved by the Animal Ethics Committee of Faculty of Medicine,
Thammasat University, Thailand (AE011/2552).
Author details
1
Department of Pharmacology, Faculty of Medicine, Chiang Mai University,
Chiang Mai 50200, Thailand.
2
Division of Pharmacology, Department of
Preclinical Science, Faculty of Medicine, Thammasat University, Pathum Thani
12120, Thailand.
3
McCormick Faculty of Nursing, Payap University, Chiang
Mai 50000, Thailand.
4
Department of Pathology, Faculty of Medicine, Chiang
Mai University, Chiang Mai 50200, Thailand.
5
Bioscreening Unit, Department
of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical
Sciences and Centre of Excellence for Innovation in Chemistry, Naresuan
University, Phitsanulok 65000, Thailand.
Received: 25 October 2015 Accepted: 21 July 2016
References
1. Sivarajan VV. Balachandran I. Ayurvedic dr ugs and their plant sources.
New Delhi: Oxford and IBH Publishing Co; 1994. p. 97.
2. Thomas RB, Joy S, Ajayan MS, Paulose CS. Neuroprotective Potential of
Bacopa monnieri and Bacoside A against dopamine receptor dysfunction
in the cerebral cortex of neonatal hypoglycaemic rats. Cell Mol Neurobiol.
2013;33(8):1065–74.
3. Saini N, Singh D, Sandhir R. Neuroprotective effects of Bacopa monnieri in
experimental model of dementia. Neurochem Res. 2012;37(9):1928–37.
4. Le XT, Pham HT, Do PT, Fujiwara H, Tanaka K, Li F, Van Nguyen T, Nguyen
KM, Matsumoto K. Bacopa monnieri ameliorates memory deficits in olfactory
bulbectomized mice: possible involvement of glutamatergic and cholinergic
systems. Neurochem Res. 2013;38(10):2201–15.
5. Piyabhan P, Wetchateng T. Cognitive enhancement effects of Bacopa
monnieri (Brahmi) on novel object recognition and VGLUT1 density in the
prefrontal cortex, striatum, and hippocampus of sub-chronic phencyclidine
rat model of schizophrenia. J Med Assoc Thai. 2013;96(5):625–32.
6. Piyabhan P, Wetchateng T, Sireeratawong S. Cognitive enhancement effects
of Bacopa monnieri Brahmi) on novel object recognition and NMDA receptor
immunodensity in the prefrontal cortex and hippocampus of sub-chronic
phencyclidine rat model of schizophrenia. J Med Assoc Thai. 2013;96(2):231–8.
7. Kamkaew N, Scholfield CN, Ingkaninan K, Taepavarapruk N, Chootip K.
Bacopa monnieri increases cerebral blood flo w in rat independent of
blood pressure. Phytother Res. 2013;27(1):135–8.
8. Priyanka HP, Bala P, Ankisettipalle S, Thyaga Rajan S. Bacopa monnieri and
L-deprenyl differentially enhance the activities of antioxidant enzymes and
the expression of tyrosine hydroxylase and nerve growth factor via ERK 1/2
and NF-kB pathways in the spleen of female Wistar rats. Neurochem Res.
2012;38(1):141–52.
9. Jadiya P, Khan A, Sammi SR, Kaur S, Mir SS, Nazir A. Anti-parkinsonian
effects of Bacopa monnieri: insights from transgenic and pharmacological
Caenorhabditis elegans models of Parkinson’s disease. Biochem Biophys
Res Commun. 2011;413(4):605–10.
10. Kamkaew N, Scholfield CN, Ingkaninan K, Maneesai P, Parkington HC, Tare
M, Chootip K. Bacopa monnieri and its constituents is hypotensive in
anaesthetized rats and vasodilator in various artery types. J Ethnopharmacol.
2011;137(1):790–5.
11. Menon BR, Rathi MA, Thirumoorthi L, Gopalakrishnan VK. Potential effect of
Bacopa monnieri on nitrobenzene induced liver damage in rats. Indian J
Clin Biochem. 2010;25(4):401–4.
12. Singh A, Singh SK. Evaluation of antifertility potential of Brahmi in male
mouse. Contraception. 2009;79(1):71–9.
13. Sumathi T, Balakrishna K, Veluchamy G, Niranjali Devaraj S. Inhibitory effect
of Bacopa monniera on morphine induced pharmacological effects in mice.
Nat Prod Sci. 2007;13(1):46–53.
14. Russo A, Borrelli F, Campisi A, Acquaviva R, Raciti G, Vanella A. Nitric oxide-
related toxicity in cultured astrocytes: Effect of Bacopa monniera. Life Sci.
2003;73:1517–26.
15. Pawar R, Gopalakrishnan C, Bhutani KK. Dammarane triterpene saponin from
Bacopa monniera as the superoxide Inhibitor in polymorphonuclear cells.
Planta Med. 2001;67:752–4.
16. Sairam K, Dorababu M, Goel RK, Bhattacharya SK. Antidepressant activity
of standardized extract of Bacopa monniera in experimental models of
depression in rats. Phytomedicine. 2002;9:207–11.
17. Chowdhuri DK, Parmar D, Kakkar P, Shukla R, Seth PK, Srimal RC. Anti-stress
effects of bacosides of Bacopa monnieri: modulation of HSP to expression,
Sol and cytochrome P450 activity in rat brain. Phytother Res. 2002;16:639–45.
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 9 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

18. Sairam K, Rao VC, Babu DM, Goel KR. Prophylactic and curative effects of
Bacopa monniera in gastric ulcer models. Phytomedicine. 2001;8:423–30.
19. Elangovan V, Govindasamy S, Ramamoorthy N, Balasu-bramanian K. In vitro
studies on the anticancer activity of Bacopa monniera. Fitoterapia. 1995;66:211–5.
20. Jain P, Khanna NK, Trehan T, Pendse VK, Godhwani JL. Anti-inflammatory
effects of an Ayurvedic preparation, Brahmi Rasayan,inrodents.IndianJ
Exp Biol. 1994;32:633–6.
21. RoodenrysS,BoothD,BulzomiS,PhippsA,MicallefC,SmokerJ.
Chronic Effects of Brahmi (Bacopa monnieri)onHumanMemory.
Neuropsychopharmacology. 2002;27:279–81.
22. Morgan A, Stevens J. Does Bacopa monnieri improve memory performance
in older persons? Results of a randomized, placebo-controlled, double-blind
trial. J Altern Complement Med. 2010;16(7):753–9.
23. Pase MP, Kean J, Sarris J, Neale C, Scholey AB, Stough C. The cognitive-
enhancing effects of Bacopa monnieri: a systematic review of randomized,
controlled human clinical trials. J Altern Complement Med. 2011;18(7):647–52.
24. Teschke R, Bahre R. Severe hepatotoxicity by Indian Ayurvedic herbal
products: a structured causality assessment. Ann Hepatol. 2009;8(3):258–66.
25. Phrompittayarat W, Putalun W, Tanaka H, Wittaya-areekul S, Jetiyanon K,
Ingkaninan K. An enzyme-linked immunosorbant assay using poly clonal
antibodies against bacopaside I. Anal Chim Acta. 2007;584:1–6.
26. Phrompittayarat W, Putalun W, Tanaka H, Wittaya-areekul S, Jetiyanon K,
Ingkaninan K. Determination of pseudojujubogenin glycosides from Brahmi
based on immunoassay using a monoclonal antibody against bacopaside I.
Phytochem Anal. 2007;18:411–8.
27. Organization of Economic Co-operation and Development. The OECD
guideline for testin g of chemic al: 420 Acute Oral Toxicity–Fixed Dose
Method. Paris France; 2001a. Available at http://www.oecd-ilibrary.org/
environment/test-no-420-acute-oral-toxicity-fixed-dose-procedure_
9789264070943-en. Accessed 25 Nov 2015.
28. World Health Organization (WHO). General guidelines for methodologies
on research and evaluation of traditional medicine. EDM/TRM/2000.1.
Switzerland; 2000. Available at http://apps.who.int/medicinedocs/en/d/
Jwhozip42e/. Accessed 25 Nov 2015.
29. Organization of Economic Co-operation and Development. The OECD
guideline for testing of chemical: 452 Chronic Toxicity Studies. France; 1981.
Available at http://www.oecd-ilibrary.org/environment/test-no-452-chronic-
toxicity-studies_9789264071209-en. Accessed 25 Nov 2015.
30. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human
studies revisited. FASEB. 2008;22:659–61.
31. Tofovic SP, Jackson EK. Effects of long-term caffeine consumption on
renal function in spontaneously hypertensive heart failure prone rats.
J Cardiovascular Pharmacol. 1999;33:360–6.
32. Raza M, Al-Shabanah OA, El-Hadiyah TM, Al-Majed AA. Effect of prolonged
vigabatrin treatment on hematological and biochemical parameters in
plasma, liver and kidney of Swiss albino mice. Sci Pharm. 2002;70:135–45.
33. Teo S, Stirling D, Thomas S, Hoberman A, Kiorpes A, Khetani V. A 90-day oral
gavage toxicity study of D-methylphenidate and D. L-methylphenidate in
Sprague-Dawley rats. Toxicology. 2002;179:183–96.
34. Kronfeld DS, Medway W. Blood chemistry. In: Medway W, Prier JE, Wilkinson JS
(12th eds.): Textbook of Veterinary Clinical Pathology. Baltimore: Williams &
Wilkins Co.; 1969.
35. Adeneye AA, Ajagbonna OP, Adeleke TI, Bello SO. Preliminary toxicity and
phytochemical studies of the stem bark aqueous extract of Musanga
cecropioides in rats. J Ethnopharmacol. 2006;105(3):374–9.
36. Gregg LV. Anemias and polychythenias. In: Hematology techniques and concepts
for veterinary technicians. U.S.A: Iowa State University Press; 2000. p. 95–101.
37. Williams JW, N elson DA, Morris MW. Examination of the blood. In: William JW,
editor. Hematology international. 4th ed. New York: Mc Graw-Hell; 1990. p. 9–24.
38. Levine BS. Animal clinical pathology. In: Derelanko MJ, Hollinger MA, editors.
CRC Handbook of Toxicology. 2nd ed. U.S.A: CRC Press; 2002. p. 742–68.
39. National laboratory animal center. Hematological data of NLAC-MU
laboratory animals. Mahidol University. Thailand. http://www.nlac.mahidol.ac.
th/acth/index.php/quality-control/health-report/27-quality-control#Anchor3.
Accessed 23 July 2016.
40. Young LY, Holland EG. The clinical use of drugs: Interpretation of clinical
laboratory tests. 6th ed. Vancouver: Lippincott Williams & Wilkins; 1995. p. 4–20.
41. National laboratory animal center. Clinical chemistry of NLAC-MU laboratory
animals. Mahidol University. Thailand. http://www.nlac.mahidol.ac.th/acth/
index.php/quality-control/health-report/27-quality-control#Anchor3.
Accessed 23 July 2016.
• We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
www.biomedcentral.com/submit
Submit your next manuscript to BioMed Central
and we will help you at every step:
Sireeratawong et al. BMC Complementary and Alternative Medicine (2016) 16:249 Page 10 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com