Experimental and Toxicologic Pathology 63 (2011) 387–391

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Experimental and Toxicologic Pathology

journal homepage: www.elsevier.de/etp

Acute and subchronic oral toxicities of Calendula officinalis extract

in Wistar rats
Alicia Lagarto n, Viviana Bueno, Isbel Guerra, Odalys Valdés, Yamile Vega, Leonid Torres
Drug Research and Development Center, CIDEM, 17 No. 6208 e/ 62 y 64, Playa, Havana City, Cuba

a r t i c l e in fo abstract

Article history: We have studied the acute and subchronic oral toxicities of Calendula officinalis extract in male and
Received 13 February 2009 female Wistar rats. A single acute C. officinalis extract dose of 2000 mg/kg dissolved in distilled water
Accepted 28 February 2010 was administered by oral gavage for acute toxicity. Subchronic doses of 50, 250 and 1000 mg/kg/day
were administered in drinking water. The major toxicological endpoints examined included animal
Keywords: body weight, water and food intake, selected tissue weights, and histopathological examinations. In
Calendula officinalis addition, we examined blood elements: hematocrit, hemoglobin concentration, erythrocyte count, total
Acute and differential leukocyte count and blood clotting time and blood chemistry: glucose, total cholesterol,
Subchronic urea, total proteins, alkaline phosphatase, alanine aminotransferase (ALT) and aspartate aminotransfer-
Toxicity
ase (AST). In the acute study, there were no mortality and signs of toxicity. In the subchronic study,
Wistar rats
several of the blood elements were significantly affected in males and females after 90
days; hemoglobin, erythrocytes, leukocytes and blood clotting time. For blood chemistry parameters,
ALT, AST and alkaline phosphatase were affected. Histopathological examination of tissues showed
slight abnormalities in hepatic parenchyma that were consistent with biochemical variations observed.
These studies indicate that the acute and subchronic toxicities of C. officinalis extract are low.
& 2010 Elsevier GmbH. All rights reserved.

1. Introduction reduces the number of people experiencing severe dermatitis

(skin irritation, redness, pain) (Pommier et al., 2004).
Calendula officinalis (C. officinalis), also known as marigold, has C. officinalis is highly effective for the prevention of acute
been widely used on the skin to treat minor wounds, infections, dermatitis of grade 2 or higher and should be proposed for
burns, bee stings, sunburn, warts, and cancer. Most scientific patients undergoing postoperative irradiation for breast cancer
evidence regarding its effectiveness as a wound-healing agent is (Pommier et al., 2004). The methanolic extract and its 1-butanol-
based on animal and laboratory studies, while human research is soluble fraction from C. officinalis flowers were found to show a
virtually lacking. C. officinalis has many pharmacological proper- hypoglycemic effect, inhibitory activity of gastric emptying, and
ties. It is used for the treatment of skin disorders, pain and also as gastroprotective effect (Yoshikawa et al., 2001).
a bactericide, antiseptic and anti-inflammatory. Butanolic fraction Acute toxicity studies in rats and mice indicate that the extract
of C. officinalis possesses a significant free radical scavenging and is relatively nontoxic. Animal tests showed at most minimal skin
antioxidant activity (Cordova et al., 2002). irritation, and no sensitization or phototoxicity. Minimal ocular
C. officinalis flowers were believed to be useful in reducing irritation was seen with one formulation and no irritation with
inflammation, wound healing, and as an antiseptic. C. officinalis others. Six saponins isolated from C. officinalis flowers were not
was used to treat various skin diseases, ranging from skin mutagenic in an Ames test, and a tea derived from C. officinalis
ulcerations to eczema. Internally C. officinalis has been used for was not genotoxic in Drosophila melanogaster (Taylor and Francis
stomach ulcers and inflammation. The flavonoids, found in high Health Sciences, 2001).
amounts in C. officinalis, are responsible for its anti-inflammatory The use of C. officinalis by oral route in humans needs a safety
activity; triterpene saponins may also be important. C. officinalis evaluation for this route of administration. Available data are
also contains carotenoids. A study in women receiving radiation insufficient to support the safety of C. officinalis extract by oral
therapy to the breast for breast cancer reports that C. officinalis route. The overall objective of this investigation was to char-
ointment applied to the skin at least twice daily during treatment acterize the acute and subchronic oral toxicity of C. officinalis
extract by oral route in adult Wistar rats of both sexes. An
n
additional aim was to identify lowest-observed-adverse-effect
Corresponding author. Tel.: + 537 209 5451; fax: + 537 833 5556.
E-mail addresses: cidem@infomed.sld.cu,
levels (LOAELs) and no-observed-adverse-effect levels (NOAELs)
alicialp@infomed.sld.cu, cidem.cb@infomed.sld (A. Lagarto). for acute and subchronic C. officinalis extract exposure.

0940-2993/$ - see front matter & 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.etp.2010.02.015
388 A. Lagarto et al. / Experimental and Toxicologic Pathology 63 (2011) 387–391

2. Materials and methods 2000 mg/kg body weight C. officinalis powder. All rats were
monitored continuously for 24 h after dosing for signs of toxicosis.
2.1. Test substances For the remainder of the 14-day study period, animals were
monitored daily for any additional behavioral or clinical signs of
C. officinalis extract toxicity and weekly for changes in body weight. Rats were
Batch: 05001 euthanized on day 14 by lethal dose of sodium pentobarbital, and
Coming to UCTB Tecnologı́as Básicas, CIDEM organs and tissues were examined macroscopically for toxicant-
The plant materials were collected in winter (January and induced changes (OECD, 2001).
February) at the EEPM ’’Dr. Juan Tomás Roig’’. Voucher specimens
were kept in Herbal ’’Dr. Juan Tomás Roig’’ of EEPM (No. 4625).
They were dried in a solar dryer and were chopped in a windmill
with mesh size of the particles not greater than 5 mm. Aqueous 2.4. Subchronic toxicity test
extract of flowers of C. officinalis was obtained by decoction of
plant material for 45 min and the ratio of solvent volume to the Animals were exposed to C. officinalis extract in drinking water for
weight of the plant material was 3:1. The extract was dried by a 90-day period as described by Mosberg and Hayes (1989)
atomization used a spray dry equipment. considering the body weight and water consumption of two previous
Qualitative flavonoids identification was performed by thin weeks. The concentrations were calculated in mg/500 ml so that
layer chromatography. One gram of test material was extracted dosages delivered were therefore 50, 250 and 1000 mg/kg/day.
with 10% sulfuric acid in methanol. The filtrated was extracted Concentrations of C. officinalis extract in drinking water were adjusted
with ethyl acetate, organic fraction was concentrated to dryness every 7 days to achieve the targeted dose level. Animals were
and residual was dissolved in 1 ml of ethanol. Reference maintained on an ad libitum diet and tap water throughout the test,
substance was dissolved in 1% ethanol. A mixture of ethyl acetate: with body weight, food and water consumption monitored weekly,
formic acid: acetic acid: water (10:1:1:2) was used as mobile and behavioral and clinical signs daily.
phase. Application volume was 5–10 ml and a mixture of 10% Blood samples were collected from abdominal aorta under
oxalic acid: 10% boric acid in absolute ethanol (9:1) at 105 oC was anesthesia of sodium pentobarbital 40 mg/kg on day 28 of dosing
%
used as developer. As a result two green phosphorescent stains to 2 treated groups (5 males and 5 females) administered with
were observed at 365 nm with the same Rf value as that of the high dose and 2 control groups for hematology and chemical
reference. biochemistry. At the completion of the subchronic study, blood
Quercetin content was determined by high-performance liquid samples were collected from all surviving animals as described
chromatography (HPLC). HPLC was performed on Lichrocart RP- above and serum obtained for hematological and biochemical
18 column (250  4 mm2, 5 mm) with a 5% formic acid as mobile analyses.
phase A and methanol as mobile phase B. The chromatography Hematology included determination of hematocrit by micro-
conditions were: mobile phase flow 1 ml/min, mobile phase A 75% hematocrit capillaries, hemoglobin concentration by diagnostic
for 5 min, from 75% to 45% at 20 min, from 45% to 75% at 24 min, kit produced by Biologic Products Inc. Havana, Cuba, erythrocyte
keeping at 75% 2 min; UV detector of l ¼370 nm and injection and leukocyte counts in Neubauer chamber, differential leukocyte
volume 20 ml. Quercetin content in dry powder was count by extension in microscopy slides and blood clotting time
0.024 70.0008% w/w. by addition of calcium chloride to citrated blood. Clinical
biochemistry included glucose, total cholesterol, urea, alanine
2.2. Animals aminotransferase (ALT), aspartate aminotransferase (AST), total
proteins and alkaline phosphatase were measured using diag-
Animal care was in conformity with Canadian Council for nostic kit produced by Biologic Products Inc. Havana, Cuba. The
Animal Care guidelines. All animals used in this study were absorbance values were determined in a spectrophotometer
outbred Wistar rats, purchased from Biologic Control Laboratory Spectronic Genesys 2.
CIDEM (Havana, Cuba). Female rats weighing 150–200 g were After collecting blood samples, rats were euthanized by
used in an acute toxicity assay. Male and female rats used in a exsanguinations. Selected organs for weight were liver, kidneys,
subchronic toxicity assay were 6 weeks old at the onset of dosing. adrenals, spleen, brain, heart, ovaries and testes. Selected organs
Rats were housed by sex in polycarbonate cages with ad libitum (heart, kidneys, liver, spleen, brain, lungs, stomach, intestines,
access to standard diet EMO 1001 (Centro Nacional para la thymus, adrenals, thyroid, parathyroid, trachea, nasal structures,
Producción de Animales de Laboratorio, Havana, Cuba) and tap pancreas, salivary glands, cervical ganglio, gonads, prostate, and
water. Animals were maintained under a light:dark schedule of seminal vesicle) were removed, fixed, sectioned, and stained for
12 h:12 h at a temperature of 20 1C. In the acute study, 3 male and histopathological examination.
3 female rats were randomly assigned to 2 groups treated with A satellite group of 10 animals per sex was used in the top dose
2000 mg/kg of C. officinalis extract. In the subchronic study, 50 group for observation, after the treatment period, of reversibility
male and 50 female rats were randomly assigned to 5 groups of 10 or persistence of any toxic effects. In these groups blood sample
animals each (control, low, medium, high dose and satellite and organs were taken 28 days after the end of the 90-day
group). Animals were allowed a 2-week acclimatization period treatment period (OECD, 1998).
prior to toxicity testing during which body weight and food and
water consumption were monitored.

2.3. Acute toxicity test 2.5. Statistics

Animals were fasted for 16 h prior to dosing. Female and male Data were analyzed using the one-way analysis of variance
were dosed by oral gavage, using a stainless steel feeding needle, (ANOVA) and Duncan test for body weight, food consumption,
with 20 ml/kg body weight of aqueous solution containing 10% blood data and relative organ weight data. Probability values of
(w/v) of C. officinalis powder. The dose delivered was therefore po0.05 were considered statistically significant.
 A. Lagarto et al. / Experimental and Toxicologic Pathology 63 (2011) 387–391 389

3. Results neutrophil counts were significantly increased and lymphocytes

counts were significantly decreased in males and a slight increase in
3.1. Acute toxicity test monocyte counts in females was observed in the high dose group.

We did not observe any signs of toxicity during the acute 3.6. Hematology and biochemistry
investigation. Normal tendency in body weight was observed and
none of the tissues examined showed any significant changes. All blood parameters tested appeared to be affected by
subchronic dosing with C. officinalis (Table 2). Hemoglobin
3.2. Subchronic toxicity test concentration was significantly decreased in females at dosages
of 250 and 1000 mg/kg/day. Hematocrit level was significantly
For brevity, results are restricted to those parameters for depressed in the male high dose group. Erythrocyte and leukocyte
which significant differences existed between groups. levels, in both males and females, were significantly increased in a
dose-dependent manner and blood clotting time was significantly
3.3. Behavioral observations prolonged in males at 250 and 1000 mg/kg/day. Table 3 shows
mean corpuscular hemoglobin concentration (MCHC) was
Two animals were sacrificed of the low and medium dose significantly decreased in females at dosages of 250 and
groups (one animal each) due to unhealthy state of the animal. 1000 mg/kg/day. The increase in erythrocyte and decrease in
In gross necropsy were observed labyrinthitis. Sings of toxicity hemoglobin concentration were not reversible. Hemoglobin
were not observed during the 90-day experimental period. concentration in males decreased 28 days after the treatment
period and hematocrit increased in females (Table 4).
3.4. Consumption and body weight trends
Table 3
MCHC values in female rats following 90 days of exposure to C. officinalis extract in
Mean water and food consumption and body weight trends of the subchronic study and those in the satellite group after 28 days of recovery.
all groups were similar.
Group Mean 7SD (n¼ 9–10 animals)
3.5. Results at 28 day of dosing
Control 43.7 7 3.9
50 mg/kg/day 39.4 7 3.9
There was no treatment-related changes in biochemistry results. 250 mg/kg/day 34.07 3.5nn
Total leukocyte count was significantly increased in females and 1000 mg/kg/day 35.4 7 4.3nn
Satellite 33.8 7 5.8nn
differential leukocyte count was significantly fluctuated in both
sexes by dosing with C. officinalis extract. Table 1 shows that nn
p o 0.01, significantly different from control.

Table 1
Total and differential leukocyte counts at 28 days of treatment with C. officinalis extract in the subchronic study.

Parameter Male Female

Control 1000 mg/kg/day Control 1000 mg/kg/day

Total leukocyte count (cell/mm3) 2330 7 869 3617 7813 2717 7 539 6570 7876nnn
Differential count of leukocytes (%)
Neutrophils 12.8 7 3.1 19.5 73.9n 14.67 3.4 15.2 73.8
Lymphocytes 85.2 7 3.3 75.8 73.6nn 84.47 3.4 82.6 73.8
Monocytes 1.27 0.8 1.8 70.8 0.87 0.4 1.6 70.5n

Note: values represent the mean 7 standard deviation (n¼ 5).

n
po 0.05, significantly different from controls.
nn
po 0.01, significantly different from controls.
nnn
po 0.001, significantly different from controls.

Table 2
Selected hematological parameters following 90 days of exposure to C. officinalis extract in the subchronic study.

Parameter Control 50 mg/kg/day 250 mg/kg/day 1000 mg/kg/day Control range

Male
Hemoglobin (mmol/l) 13.07 1.4 13.2 7 0.8 13.17 1.2 12.8 70.5 12.4–13.8
Hematocrit (%) 59.6 7 3.4 59.7 7 1.8 55.87 2.3 47.6 74.8nn 52–58
Erythrocyte count (cellx103/mm3) 2128 7 64 2903 7 51nn 3994 7 81nn 4167 770nn 2585–5214
Leukocyte count (cell/mm3) 9494 7 400 10,321 7 359nn 15,112 7 459nn 19,500 7280nn 5465–8288
Blood clotting time (s) 105.3 7 16.0 147.9 7 23.9nn 194.3 7 24.1nn 284.3 720.6nn 94–109

Female
Hemoglobin (mmol/l) 12.4 7 0.4 12.1 7 0.7 9.97 1.1nn 9.9 71.0nn 9.7–11.8
Hematocrit (%) 46.2 7 3.4 47.2 7 4.3 46.87 2.0 45.3 71.9 45–47
Erythrocyte count (cell  103/mm3) 2128 7 44 2879 7 57nn 40427 48nn 4216 794nn 2149–3027
Leukocyte count (cell/mm3) 9367 7 328 10,9007 245nn 15,535 7 351nn 19,485 7354nn 6918–9136
Blood clotting time (s) 121.6 7 27.7 143.6 7 28.4 100.97 13.7 112.2 713.9 102–144

Note: values represent the mean 7 standard deviation (n¼ 10). Control ranges represent historical control data.
nn
po 0.01, Significantly different from controls.
390 A. Lagarto et al. / Experimental and Toxicologic Pathology 63 (2011) 387–391

Table 4
Selected hematological and biochemical parameters in the satellite group after 28 days of recovery following 90 days of exposure to C. officinalis in the subchronic study.

Parameter Male Female

Control 1000 mg/kg/day Control 1000 mg/kg/day

Hematocrit (%) 59.6 7 3.4 54.7 7 5.4 46.2 7 3.4 52.1 7 3.9nn
Hemoglobin (mmol/l) 12.1 7 0.5 10.5 7 0.8nn 12.4 7 0.4 10.5 7 0.2nnn
Erythrocyte count (cell  103/mm3) 5366 7 187 8021 7 446nnn 5146 7 256 7860 7 534nnn
Leukocyte count (cell/mm3) 7610 7 1361 6050 7 1784 6788 7 1644 4758 7 1465
Blood clotting time (s) 105.3 7 16.0 98.6 7 26.2 139.1 7 39.5 103.2 7 61.9
ALT (U/l) 98.6 7 56.1 40.7 7 11.3nn 15.6 7 4.1 5.8 7 4.1nnn
AST (U/l) 159.6 7 18.9 139.3 7 25.8 108.7 7 18.7 124.6 7 30.6

Note: values represent the mean 7 standard deviation (n¼10).

nn
po 0.01, Significantly different from controls.
nnn
po 0.001, Significantly different from controls.

Table 5
Selected biochemical parameters following 90 days of exposure to C. officinalis extract in the subchronic study.

Parameter Control 50 mg/kg/day 250 mg/kg/day 1000 mg/kg/day Control range

Male
ALT (U/l) 62.5 7 11.0 42.3 7 13.2 33.5 7 8.3 3.1 7 1.4nnn 39–65
AST (U/l) 164.6 7 22.5 163.6 7 18.5 136.4 7 34.9 137.8 7 27.9 154–194

Female
ALT (U/l) 18.2 7 2.6 31.9 7 5.9nn 30.7 7 6.0nn 15.8 7 4.3 13–22
AST (U/l) 108.7 7 18.7 148.9 7 23.1n 146.7 7 22.8n 124.9 7 30.6 91–126

Note: values represent the mean 7 standard deviation (n¼10). Control ranges represent historical control data.
n
p o 0.05, significantly different from controls.
nn
po 0.01, significantly different from controls.
nnn
po 0.001, significantly different from controls.

Table 6
Heart weights (% total body weight) of male rats following 90 days of exposure to
C. officinalis extract in the subchronic study.

Group Mean7SD (n¼ 9–10 animals)

Control 0.2694 70.0232

50 mg/kg/day 0.3226 70.0279n
250 mg/kg/day 0.3065 70.0559
1000 mg/kg/day 0.2776 70.0272
n
p o 0.05, significantly different from control.

Of the biochemical parameters tested, ALT and AST appeared

Fig. 1. Photomicrograph showing liver from representative animal. (A) Control
to be affected by subchronic dosing with C. officinalis (Table 5).
liver with normal histology. (B) Mononuclear cells increase in portal triad with
ALT levels were significantly decreased in males in a dose- lymphocytes prevalence observed in high dose groups. (C) Lymphocyte aggrega-
dependent manner and significantly increased in females at tion close to centrilobular vein observed in one animal of female high dose group
dosages of 50 and 250 mg/kg/day. AST levels were significantly (hematoxylin and eosin stain).
increased in females at doses of 50 and 250 mg/kg/day. The
decrease in ALT levels in male animals was not reversible and the increased mononuclear cells in portal triads with lymphocytes
increased ALT level in female animals was not found 28 days after prevalence. Activated Kupffer cells, inflammatory cells and focal
treatment (Table 4). necrosis and lymphocytes aggregation were observed in only 1 of
10 high-dose females (Fig. 1). Focal hepatocellular atrophy was
3.7. Organs weight observed in 8 of 10 low-dose females, and 1 of 10 satellite
females. Perivascular aggregation of lympho-reticular cells in
portal triad was observed in 2 of 10 satellite males. There were no
Minor but statistically significant increases were observed in
treatment-related lesions in other tissues.
the relative heart weight (as % of total body weight) of low-dose
male rats compared with control group, but not in the mid or high
dose group (Table 6).
4. Discussion
3.8. Histopathology
Exposure of Wistar rats to single po dose of C. officinalis extract
Slight histological changes were observed in the liver, with 2 of did not produce any treatment-related effect. The effects of
10 high-dose both sexes, and 3 of 10 satellite females exhibiting C. officinalis extract exposure in subchronic regimen varied by sex
 A. Lagarto et al. / Experimental and Toxicologic Pathology 63 (2011) 387–391 391

but generally affect hematology and biochemical parameters, and alterations that were observed in the satellite group. Histological
induced slight abnormalities in liver tissues. changes were agreed with biochemical variations observed that
Decreases in hemoglobin concentration in female medium and were of low degree and reversible. In cell cultures, C.officinalis
high dose groups were within normal ranges for this species. extracts stimulate cell proliferation and cellular metabolism by
Iron deficiency is reported to cause decreased MCHC values the increase in mitochondrial dehydrogenase activity without
(Suber, 1989) and could be the cause for decreased hemoglobin cells toxicity (Matysik et al., 2005). This effect could be
concentration. Erythrocyte count variations were not reversible. responsible for cell proliferation observed in the liver. The
The increase of erythrocyte count could be due to excessive increase in Kupffer cell activity could be an answer to some toxic
diuresis caused by the administration of the extract. We had metabolite, but this alteration was observed in only one animal of
obtained a marked diuretic effect of C. officinalis extract at a dose female high dose group. Histopathological abnormalities were not
of 50–450 mg/kg in rats (data not published). In that case observed in the heart. Increase in relative heart weight in the low-
hemoglobin and hematocrit should be increased too and these dose group could be due to experimental variation.
effects were not observed. On the contrary, hemoglobin level Thus the NOAEL for acute gavage dosing to adult Wistar rats of
decreases in females probably by iron deficiency and this effect both sexes was 2000 mg/kg due to absence of toxic effect in the
was observed 28 days after the exposure period even in males. acute toxicity test. The LOEL for histopathological changes in the
The decrease observed in hematocrit level in male high dose liver, AST and ALT activities and hematological variations was
group could be due to experimental variations and was not 50 mg/kg/day for subchronic exposure in both sexes of Wistar
considered to be treatment related. rats. These effects were relatively mild and would not be expected
Leukocytes counts were affected in both sexes, which might be to have a significant impact on the health of the animal.
related to histopathological alterations observed in the liver.
Blood clotting time was significantly prolonged in male medium
and high dose groups. The effect observed was shortterm.
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