Comp Clin Pathol
DOI10.1007/s00580-012-1539-x
ORIGINAL ARTICLE
Acute and subacute toxicity study of andrographolide
bioactive in rodents: Evidence for the medicinal use
as an alternative medicine
Chellampillai Bothiraja & Atmaram P. Pawar & Vikas S.
Shende & Prajakta P. Joshi
Received: 4 April 2012 / Accepted: 12 June 2012
#Springer-Verlag London Limited 2012
Abstract Andrographolide, a diterpene bioactive of Androg-
raphis paniculata (Acanthaceae family), has long been used
in India and China as a complimentary medicine. Yet, there is
no detailed toxicological information to guarantee its safe use.
In the present study, acute and subacute toxicity of androgra-
pholide was assessed via oral route on rodents. In acute
toxicity tests, mice (n06/group/sex) received andrographolide
at oral doses of 1, 2, 3, 4, and 5 g/kg, whereas in subacute
acute tests Wistar rats (n06/group/sex) received 250 and
500 mg/kg for 21 consecutive days. Hematological, biochem-
ical, and histological analysis were performed in all animals.
No death or hazardous signs were observed from acute toxic-
ity study indicating that the LD50 of andrographolide by oral
treatment was greater than 5 g/kg body weight in both male
and female mice. No significant changes in body weight
gain, food intake, behavior, mortality, hematological, bio-
chemical, vital organ weight, and histopathology were ob-
served from subacute toxicity study. However, a significant
increase in white blood corpuscle (WBC) (714 %) and
lymphocyte (2124 %) counts coupled with a reduction in
C. Bothiraja (*) : P. P. Joshi
Department of Phytopharmaceutics,
Sharadchandra Pawar College of Pharmacy,
Otur,
Pune, 412409 Maharashtra, India
e-mail: pounbothi@yahoo.com
A. P. Pawar
Department of Pharmaceutics, Bharati Vidyapeeth University,
Poona College of Pharmacy,
Erandwane,
Pune, 411038 Maharashtra, India
V. S. Shende
Department of Pharmacology,
Sharadchandra Pawar College of Pharmacy,
Otur,
Pune, 412409 Maharashtra, India
urea (1724 %) suggested immune-stimulant and renal
protective activity of andrographolide. The data support
the safety of andrographolide which assure its medicinal
use as an alternative medicine.
Keywords Andrographolide . Acute toxicity . Subacute
toxicity . Rodents
Introduction
In recent years, there has been evidence for increasing popu-
larity of complementary and alternative medicine approaches
to health among the Americans. Despite this rapid growth,
there is limited evidence about the effectiveness and toxicity
of alternative medicine. Much more needs to be done to
develop the evidence base for herbals, botanicals, and dietary
supplements. Since ancient times, plants have been a source of
drugs, but scientific medicine tends to ignore the importance
of herbal medicine (Sofowora, 1982). The WHO suggested
that effective, locally available plants be used as substitutes for
synthetic drugs. Research work on medicinal plants has inten-
sified with exchange of information on these plants. Such
research will go a long way in the scientific exploration of
medicinal plants for the benefit of man and is likely to de-
crease the dependence on imported drugs (Amadou, 1998).
Andrographolide is a well-known diterpene lactone present
in Andrographis paniculata Nees (Acanthaceae family). Al-
though popular as king of bitters, it has several pharmacolog-
ical actions including analgesic, anti-pyretic, anti-inflammatory,
hepatoprotectant, anti-viral, anti-cancer, and hypoglycemic ac-
tivity (Bothiraja et al., 2009a, b; Shenv et al., 2002; Somenath
et al., 2007; Calabrese et al., 2000; Matsuda et al., 1994;
Zhang and Tan, 2000). Few toxicological studies have been
reported for andrographolide. When it was administered intra-
venously at a dose of 10 mg/kg in rabbits, it did not demonstrate

abnormal cardiovascular responses (Guo et al., 1988), and oral
administration of 1 g/kg in rabbits for 7 days did not affect body
weight, blood counts, and liver or kidney function (Akbarsha et
al. 1990). An oral dose of 50 mg/kg per day to rats for seven to
30 consecutive days caused a significant induction of the liver
microsomal drug-metabolizing enzymes aniline hydroxylase,
N-demethylase, and O-demethylase. However, a single dose
only caused inhibition of aniline hydroxylase activity
(Choudhury and Poddar, 1984). Andrographolide (200, 600,
and 2,000 mg/kg) did not induce progesterone-mediated ter-
mination of pregnancy in rats (Panossian et al., 1999). A study
pointed, andrographolide produces toxic effect to a male
reproductive system and confirmed possible prospective use
of andrographolide as a male contraceptive (Akbarsha and
Murugaian, 2000).
Although medicinal use of andrographolide is wide-
spread, the literature lacks detailed hematological and bio-
chemical toxicological information. Realizing the need to
evaluate the potential toxicity of andrographolide, the pres-
ent study aimed at investigating the acute and subacute
toxicity of andrographolide on rodents. The ensuing results
shall bring forth the judicious use of the valuable phytome-
dicine, andrographolide, as an alternative medicine.
Materials and methods
Materials
The fresh leaf material of A. paniculata was obtained from
Shri Shail medicinal farm, Nagpur, Maharashtra, India in the
months of JulyAugust 2011. The plant was identified by
Dr. Patil N., Department of Botany, Pune University, Pune,
India. The voucher specimen (DG-0012) documenting its
collection is on deposit at the Department of Botany, Pune
University, Pune, India. Andrographolide (95.9 %) was
isolated from powdered leaves according to our previous
reported method (Bothiraja et al., 2009a, 2011).
Animals
Healthy male and female mice (2025 g) and Wistar rats
(200220 g) were supplied by Laxshmi Biofarms (Alephata,
Pune, India). They were breed in the animal facility and
housed in a temperature- (1822 C; 12 h light/dark cycle)
and humidity- (4555 %) controlled environment with free
access to water and food. The studies were performed accord-
ing to Organization for Economic Cooperation and Develop-
ment (OECD 2001) guidelines. The study protocols were
approved by the Institutional Animal Ethics Committee of
Sharadchandra Pawar College of Pharmacy, Pune, India (pro-
tocol number: 1197/2011/34). The animals were fasted but
provided free access to water overnight before the study.
Comp Clin Pathol
Acute toxicity study in mice
The mice were randomly divided into six groups (n06/sex).
The first group (control group) received distilled water
orally. Groups 26 were orally treated with andrographolide
(suspended in 0.5%w/v carboxymethylcellulose) with doses
1, 2, 3, 4, and 5 g/kg of body weight, respectively. The
animals were continuously observed for general behavioral
changes, sign of toxicity, and mortality for 1 h after treat-
ment and then intermittently for 4 h and thereafter over a
period of 24 h. Mice were further observed for up to 14 days
for behavioral change and sign of toxicity.
Subacute toxicity study in rats
Experimental
The Wistar rats were randomly assigned to three groups by
sex (n06). The first group received distilled water orally
(control group). Groups 2 and 3 were orally treated with
andrographolide (suspended in 0.5%w/v carboxymethylcel-
lulose) with doses of 250 mg/kg and 500 mg/kg, respective-
ly, for 21 consecutive days. All animals were supplied with
feed and water ad libitum during the testing periods. The
clinical sign was observed daily for physiological and be-
havioral changes. Toxic manifestations such as toxicity and
mortality were observed for signs of abnormalities. Body
weight changes were recorded weekly, and food consump-
tion and water intake were monitored daily. At the end of
treatment, the animals were fasted overnight, but allowed
access to water ad libitum. They were then anaesthetized by
ether inhalation and blood samples were collected by retro-
orbital puncture (Waynforth, 1980) using capillary tubes
with or without the anticoagulant.
Hematological analysis
The blood samples collected with the anticoagulant were
used immediately for the determination of hematological
parameters such as red blood corpuscles (RBC) count, white
blood corpuscles (WBC) count, hemoglobin (Hb), hemato-
crit (HCT), mean corpuscular volume (MCV), mean corpus-
cular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), and platelet count were performed
using a veterinary blood cell counter (ERMA Inc., PCE 210
Vet). The differential leukocyte count was performed with
an optical microscopy after staining.
Biochemical analysis
The blood samples collected without the anticoagulant was
centrifuged at 10,000 rpm for 10 min at 4 C to separate the
serum. The serum was analyzed for biochemical parameters
Comp Clin Pathol
such as glucose, urea, aspartate aminotransferase (AST),
alanine aminotransferase (ALT), cholesterol, alkaline phos-
phatase (ALP), total protein, and albumin employing stan-
dard diagnostic kits (Aspen Laboratory, Delhi, India). The
difference between total protein and albumin values were
taken as a globulin value.
Necropsy
At the end of the protocol, the animals were anaesthetized by
ether inhalation and necropsy was performed on randomly
selected two animals of each group per sex to analyze the
macroscopic external features of vital organs such as liver,
kidney, and stomach. These organs were carefully removed
and weighed. Organ weights were expressed in relative terms
(gram per 100 g of body weight). The collected organs were
fixed in 10 % buffered formalin and embedded in paraffin.
Histology sections (5 m thick) were stained with hematoxy-
lin and examined under a light microscope (Olympus CH02).
Statistical analysis
Results are presented as mean standard error mean.
Results were analyzed by one-way ANOVA and Dunnetts
post hoc test. Only the values with P<0.05 were considered
as significant.
Results
Acute toxicity in mice
No death or hazardous sign on tested animals were recorded
during first 24 h as well as 14 days of observation after oral
treatment of andrographolide at the doses of 1, 2, 3, 4, and
5 g/kg, respectively.
Fig. 1 Body weight gain
curves of male (a) and female
(b) rats treated orally with the
andrographolide by oral route
for 21consecutive days. All
values are expressed as mean
SD, n06
Subacute toxicity in rats
General signs and body weight
The daily oral treatment with andrographolide at dose of
250 mg/kg and 500 mg/kg for 21 days did not produce any
death or hazardous sign such as piloerection, alteration in
the locomotor activity, or other physiological activities as
compared to the control animals. Likewise, no significant
changes were recorded in body weight gain of control and
treated rats (Fig. 1). Other parameters such as feed and water
intake did not show any significant differences in either the
control or the treated group (data not presented).
Hematological and biochemical parameters
The effects of andrographolide on the hematological and
biochemical parameters are given in Tables 1 and 2. No
significant differences were observed for all the parameters
in treated groups compared with that of the control group.
However, there was significant (P<0.05) induction in WBC
(714 %) and lymphocytes (2124 %) counts in the treated
animals compared to that of the control group. Also, there was
a significant reduction (P<0.05) in the mean urea (1724 %)
levels in treated animals as compared to control animals.
Necropsy
There were no significant differences observed between
the control and treated groups with respect to the rela-
tive organ weights of the male and female rats (Table 3).
The macroscopic examination of the organs did not
show significant changes in color and texture when
compared with the control group. Microscopic analysis
of the histological sections of the vital organ (Fig. 2)
did not show evidence of any histological abnormalities
 Comp Clin Pathol

Table 1 Effect of andrographolide on hematological parameters of Wistar rats

Sex AG dose Hb RBC WBC HCT MCV MCH MCHC Platelets N L M

(mg/kg) (g/dL) (106/mm3) (103/mm3) (%) (m3) (pg) (%) (105/mm3) (%) (%) (%)

Control 12.50.2 6.50.3 7.60.2 32.30.7 49.41.3 19.20.5 39.60.4 6.740.24 21.80.8 57.32.8 1.00.2
Male 250 11.80.4 6.40.2 7.90.4* 31.50.4 49.21.7 18.10.3 38.60.2 6.420.22 23.51.2 67.51.3* 3.00.3
500 11.60.2 6.20.4 8.20.3* 33.40.6 51.51.2 20.40.4 39.20.3 6.260.41 25.31.6 69.4.7* 3.50.1
Control 12.60.3 6.40.2 7.40.3 31.70.3 48.71.4 19.30.2 39.40.5 6.520.35 21.20.3 55.22.2 2.40.2
Female 250 12.10.2 6.30.3 8.10.5* 31.30.5 51.41.6 19.10.6 39.60.4 6.130.23 25.51.3 66.7.3* 4.70.6
500 12.10.5 6.20.3 8.50.2* 31.50.4 47.51.4 18.70.5 39.30.2 6.150.23 27.61.7 69.52.1* 4.30.1

All values are expressed as mean SD, n06

AG andrographolide, Hb hemoglobin, RBC red blood corpuscles, WBC white blood corpuscles, HCT hematocrit, MCV mean corpuscle volume,
MCH mean corpuscle hemoglobin, MCHC mean corpuscle hemoglobin concentration, N neutrophils, L lymphocytes, M monocytes
*
P<0.05 (statistically significant compared with control)

such as inflammation, necrosis, degeneration, cellular
infiltration, vascular changes, and hemorrhage or cellu-
lar abnormalities. Hepatocytes in liver, glomeruli, and
distal and proximal tubules in kidney and epithelium in
stomach appeared normal in andrographolide-treated
animals.
Discussion
Toxicology tests in animals and humans are usually carried
out on substances thought to have therapeutic potentials.
This is more so necessary in ensuring the quality of herbal
medicines, since not all of them are as safe as they are
frequently claimed (Capasso et al., 2000). It can be harmful
to take herbal medicines without being aware of their po-
tential adverse effects. Although the public and some health
care professionals believe that herbal medicines are relative-
ly safe because they are natural, there is remarkably little
data to support this assumption (Rodriguez-Fragoso et al.,
2008). In addition, there are reports on the toxicities of some
medicinal plants/phytoconstituents.
Phytoconstituents have been used as alternatives to allo-
pathic medicines in many countries. Despite widespread
use, few scientific studies have been undertaken to ascertain
safety and efficacy of phytoconstituents (Grac et al., 2007).
Both oral acute and subacute toxicity evaluations are neces-
sary for a complete toxicity study. The use of andrographo-
lide in this study was prompted as the dosage form of the
phytomedicine or traditional Chinese medicines (Bothiraja
and Pawar 2011a, b, Bothiraja et al. 2012).
The present study showed that oral administration of
andrographolide in acute dose up to 5 g/kg did not produce
any sign of toxicity or death in rats, suggesting a lethal dose
50 % (LD50) above 5 g/kg. According to Kennedy et al.
(1986), a chemical substance with a LD50 within the range
of 515 g is considered practically nontoxic. In fact, the
LD50 value of andrographolide could not be detected from
the test dose via oral route. However, the maximum tolerat-
ed dose (5 g/kg) was estimated and suggested andrographo-
lide could be regarded as nontoxic from acute ingestion.
An acute toxic study provides a guideline for selecting
doses for subacute toxic study (1/10th and 1/20th of maxi-
mum dose in mice) which may be more clinically relevant

Table 2 Effect of andrographo-

lide on serum enzymes and bio- Parameters Control AG 250 mg/kg AG 500 mg/kg
chemical levels of Wistar rats
Male Female Male Female Male Female

ALT (IU/L) 36.63.3 28.53.1 36.82.7 27.33.5 35.62.9 29.52.4

AST (IU/L) 45.32.5 30.41.6 47.53.2 32.53.4 46.22.6 31.52.8
ALP (IU/L) 101.44.2 110.83.2 103.52.1 109.42.8 100.53.1 111.64.3
All values are expressed as
Cholesterol (mg/dL) 91.12.6 84.33.9 89.63.5 87.83.2 92.82.7 88.32.8
mean SD, n06
Plasma glucose (mg/dL) 98.43.3 89.62.7 97.83.2 91.42.7 99.73.6 88.92.6
AG andrographolide, ALT
alanine aminotransferase, AST Total protein (gm/dL) 6.90.3 7.40.2 6.40.7 6.70.4 6.80.6 7.60.3
aspartate aminotransferase, Albumin (gm/dL) 3.80.1 3.80.7 3.20.4 3.40.1 3.50.4 3.90.1
ALP alkaline phosphatase Globulin (gm/dL) 3.10.6 3.60.3 3.10.7 3.30.4 3.30.8 3.70.3
*
P<0.05 (statistically significant Urea (mg/dL) 46.94.2 41.73.6 38.42.4* 35.73.2* 36.52.6* 34.53.6*
compared with control)
Comp Clin Pathol

Table 3 Effect of andrographolide on relative organ weight of Wistar group after 21-day period of daily treatment points out that
rats
andrographolide has no effect on health status, growth, or
Sex AG dose (mg/kg) Relative organ weight development of animals.
(g/100 g body weight) The hematopoeitic system, being one of the most sensitive
targets of toxic chemicals, is an important index of physiolog-
Liver Kidney Stomach
ical and pathological status of human and animals. In this
Control 14.971.32 1.610.07 1.100.06 study, the test group of animals did not show any significant
Male 250 14.690.72 1.520.13 1.040.13 variation in the hematological parameters except WBC and
500 14.511.12 1.540.14 1.030.08 lymphocyte count. The prolonged dosing of rats with androg-
Control 14.841.01 1.410.09 1.000.03 rapholide appeared to have beneficial effects on their hemato-
Female 250 15.201.32 1.590.32 1.140.14 poietic system. A significant and dose-dependent increase in
500 15.360.81 1.630.05 1.150.12 the WBC and lymphocyte count was noted in both sexes of
treated animals. This supported immunostimulant activity of
All values are expressed as mean SD, n06 andrographolide as was observed by Basak et al. (1999).
AG andrographolide There were no observed differences in the liver function-
ing of the treated and untreated control animals. On admin-
(Mehta et al., 2009). Thus, derived doses of 250 and istration of andrographolide, the serum levels of marker
500 mg/kg failed to produce toxicity in male and female enzyme such as ALT, AST, and ALP remain unchanged,
rats. The high dose (500 mg/kg/day) in the subacute toxicity indicating no severe tissue damage. The elevated levels of
test was used according to the subacute guidelines (OECD, these enzymes are clinically measurable indications of po-
2001; Bautista et al., 2004). A lower dose of 250 mg/kg/day tential risks to normal liver function (Hilaly et al., 2004).
was also used to determined dose-related toxic effects. The other parameters associated with liver function, albu-
Changes in body weight have been used as an indicator min, globulin, and total protein, were normal compared to
of adverse effects of drugs and chemicals (Hilaly et al., control group. Even blood glucose and cholesterol lev-
2004). There were no observed changes in feed and water els were within their normal ranges. However, there was
consumption. This implies that dosing with andrographolide a significant decrease in urea levels as compared to
may not have affected their appetite and feed conversion control animals supporting renal protective activity of
rates adversely. There was no digestive distribution like andrographolide (Rao, 2006).
diarrhoea or vomiting when served equally. Absence of Further, there were no findings of pathological nature in the
significant changes in the general behavior and body weight histological profiles of liver, kidney, and stomach of treated
gain of rats in the treated groups as compared to the control groups. Moreover, theses organs were not subjected to major

Fig. 2 Histology of the liver,

kidney, and stomach of the
control (a) and those exposed to
250 mg/kg (b) and 500 mg/kg
(c) of andrographolide for
21 days on Wistar male rats. In
liver: hepatocyte (black arrow),
hepatic cords (yellow arrow),
vascular (red arrow); in kidney:
tubules (black arrow),
glomeruli (yellow arrow),
vascular (red arrow); in
stomach: epithelium (black
arrow)

injury as there was no significant alteration in liver-to-body
weight ratio, kidney-to-body weight ratio, and stomach-to-
body weight ratio indicating safety of andrographolide.
Conclusion
The study concluded that oral administration of androgra-
pholide did not develop adverse effects in rodents. This study
envisages the safety of andrographolide and is a testimony
for its medicinal use as a complementary and alternative
medicine. Chronic toxicity, mutagenicity, and carcinogenic-
ity studies are necessary to further support the safe use of
andrographolide.
Acknowledgments The authors are thankful to All India Council for
Technical Education (AICTE), New Delhi, India for providing financial
support in the form of a Junior Research Fellowship (JRF) to Miss.
Prajakta Joshi. The authors are thankful to the Principal, Sharadchandra
Pawar College of Pharmacy, Otur, Pune, India for infrastructural facilities.
Conflict of interest The author(s) declare(s) that they have no con-
flicts of interest to disclose.
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