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International Journal of Universal Pharmacy and Bio Sciences 4(1): January-February 2015

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093***

ICV 5.13*** Pharmaceutical Sciences RESEARCH ARTICLE……!!!

ACUTE AND SUB-CHRONIC ORAL TOXICITY OF STANDARDIZED

WATER EXTRACT OF MATRICARIA CHAMOMILLA L. IN MOROCCO

Ghizlane Hajjaj1*

, Amina Bounihi1, Mouna Tajani

2, Layachi Chebraoui

3, Mounia Bouabdellah

3,

Nadia Cherradi4, Rouas Lamiaa

4, Yahia Cherrah

1, and Amina Zellou

1

1*Laboratory of Pharmacology and Toxicology, Department of Drugs Sciences, Faculty of

Medicine and Pharmacy, Mohammed V Souissi University, Rabat, Morocco. 2Department of Biology, Faculty of Sciences, Ibn Tofail University, Kenitra, Morocco.

3 Central Laboratory of Biochemistry, Ibn Sina Hospital Rabat, Morocco

4 MD, Department of Pathology, Children's Hospital, Faculty of Medicine and Pharmacy of the

Mohammed V Souissi University, Rabat, Morocco.

KEYWORDS:

Acute toxicity,

Matricaria chamomilla

L., Biochemical

parameters, water extract,

Hematological parameter.

For Correspondence:

Ghizlane Hajjaj*

Address: Laboratory of

Pharmacology and

Toxicology, Department

of Drugs Sciences,

Faculty of Medicine

and Pharmacy,

Mohammed V Souissi

University, Rabat,

Morocco.

Email:

[email protected]

om

ABSTRACT

The objective of this study was to evaluate the acute and Sub-chronic

toxicity (90 days) of Matricaria chamomilla L. water extract

(MCWE) via oral route in male Wistar rats. For the acute toxicity

study, MCWE administrated to mice at doses ranging from 300 to

2000 mg/kg. General behavior, adverse effects, mortality were

recorded for up to 14 days post treatment for this study, the median

lethal dose (LD50) of MCWE was found to be higher than 2g/kg.

In the Sub-chronic toxicity study, MCWE was administrated orally

to rats at doses of 300 and 600 mg/kg for 90 days. Animal body

weight was recorded weekly. MCWE at different doses used, did not

induce any statistically significant changes in body weight gain or

organs weight compared to control group. It is also showed no

significant alteration in hematological parameters in treatment

groups. However, biochemical parameters showed a significant

(p<0.05) decrease in blood sugar and rise in serum urea suggesting

degenerative changes in the kidney. Histopathological analyses,

revealed adverse changes in the architecture of kidney, lung and

spleen for the treated groups. In this study we can then conclude that,

no-observed adverse effect level (NOAEL) for repeated-doses

administration of MCWE was considered to be under 3OO mg/kg

body weight however MCWE may have long term toxic effects on

the kidney, lung and spleen. Further studies in non-rodents must be

performed to prove its safety.

mailto:[email protected]

mailto:[email protected]



INTRODUCTION:

More than 35,000 species of plants are used worldwide mainly in the food industry, herbal

medicine, cosmetics, perfumery, pharmaceutical, hygiene, with a turnover of approximately 19

billion dollars [1]. Morocco is known for its rich vegetation and plant biodiversity [2] due to its

geographical and climatic conditions. It is one of the Mediterranean countries with a long

tradition in the field of phytotherapy [3, 4].

The use of medicinal plants and preparations derived from them as dietary supplements,

nutraceuticals, functional foods and herbal medicinal products has become more widely accepted

in developing countries. Therefore, it is important to evaluate the adverse effects of these plants

and their preparations.

In Morocco it has been estimated that approximately 7000 plant species and sub-species grow

wild, 950 of which are endemic [5,6]. Among these, many species are aromatic or medicinal

plants and are used locally in Morocco’s rich and widely used traditional medicine system. It has

been estimated that about 231 local plant species present phytotherapeutic properties used by the

local population to treat a variety of diseases [7, 8].

Matricaria chamomilla L. (MC) commonly known as Babonj in Morocco belongs to the

Asteraceae family it has been extensively used in traditional medicine for hay fever,

inflammation, muscle spasms, menstrual disorders, ulcers, wounds, gastrointestinal disorders,

rheumatic pain, and hemorrhoids. In the form of an aqueous extract Matricaria chamomilla L.

has been frequently used as a mild sedative to calm nerves and reduce anxiety, to treat hysteria,

nightmares, insomnia and other sleep problems [9].

Several studies on pharmacological activities of MC indicated that it has several therapeutic

activities in both in vitro and in vivo tests, for example, antioxidant, analgesic, antiviral, anti-

inflammatory, antidiabetic , antiproliferative , antibacterial , Sedative and hepatoprotective

activities [10,11,12]. However, only limited toxicity data of this plant has been reported.

The data of acute and sub-chronic toxicity on MC should be obtained in order to increase the

confidence in its safety to human, particularly for the development of pharmaceutical products.

All new pharmaceutical drugs are tested for their safety prior to their use in human volunteers

and patients. A key stage in ensuring the safety of drugs is to conduct toxicity study in

appropriate animal models.

http://www.hindawi.com/journals/aps/2012/270824/



In the present study, we investigated for the first time acute and sub-chronic toxicity of the

standardized water extract of Matricaria chamomilla L. leaves and stems in Morocco.

MATERIALS AND METHODS:

Plant Preparation:

Fresh plants of Matricaria chamomilla L. were purchased at a farmer’s market in Hay Nahda-

Rabat on spring season between March and June in 2013. The plants were identified and

authenticated at the Department of Plant Biology, Ibn Tofail University, Morocco. A voucher

specimen (N° Rab78995) was deposited in the Herbarium of Botany Department of Scientific

Institute of Rabat.

Then, samples of MC were well cleaned and washed with water and then dried in the shade and

at room temperature. After this period, leaves and stems of the plant have been grinded and

transformed to powder. The powder was preserved in clean plastic containers, kept away from

light, heat and moisture.

Plant Extraction:

A 200 g of powdered leaves and stems of MC were blended with 1 litre of distilled water for a

period of 24 hrs with agitation at room temperature.

After, the extracts were taken and filtered by using whatman filter paper. The extracts were

concentrated using a rotary evaporator at 40°C under reduced pressure. The filtrate was

evaporated to dryness using a rotary evaporator. The extract was then stored below ambient

temperature. This yielded a brown residue of 16.76 % W/W extract with reference to dry starting

material which was stored in an air tight bottle and kept in a refrigerator at 4 °C.

Animal procedures:

Adult female Swiss mice weighing between (20-30 g) were used to calculate LD50 and female

and male wistar rats with an average weight of 180- 220 g were used in sub-chronic

toxicity study.

The rodents were obtained from the animal centre of Mohammed V-Souissi, Medicine and

Pharmacy Faculty Rabat, Morocco. They were housed in clean polypropylene cages and

maintained under standard conditions of light (12 hours with alternative day/night cycles),

relative humidity (60-70%) and temperature (26 ± 1 °C). The animals were fed daily with rodent

pellet diet and tap water ad-libitum under strict hygienic conditions. The animals were treated

according to directives of the Official Journal of the European Community about the care and of

the use of the animals of laboratory [13, 14].



Acute Toxicity Study:

Acute oral toxicity study of the Matricaria chamomilla L. water extract was performed

according to OECD 425 guidelines [15].

Four groups of Adult female Swiss mice (n = 3) were kept fasting for 3- 4 hr and provided only

water. One control group treated with the vehicle (distilled water), three groups treated with

MCWE at 300 and 2000 mg/kg body weight by gastric intubations. The Rodents were observed

for behavioral and neurological symptoms continuously for the first 4 h after dosing. The number

of survivors was noted after 24 h and these animals were then maintained for further 14 days

with observations made daily.

If mortality was observed in two out of three mice, then the dose administered was assigned as

toxic dose. If mortality was observed in one mouse, the same dose was repeated again to confirm

the toxic dose.

No mortality or morbidity was observed in MCWE treated rodents up to 2000 mg/kg. Hence 300

mg/kg and 600 mg/kg doses were taken for efficacy studies.

Evaluation of chronic toxicity:

According to the WHO guideline and the OECD 452 [16, 17], 36 Wistar rats were randomly

assigned into three groups (n=12), six females and six males in each group. The extract was

administered orally at concentrations of 300 and 600 mg/kg to the two subsequent treatment

groups for consecutive 90 days by gavage daily, while the control group received distilled water.

Animals were fasted 3h prior to dosing to facilitate administration of the complete dose. The

choice of doses used was based on LD50 values.

All rats were weighed weekly and monitored daily for any behavioural changes, respiratory

pattern, cardiovascular signs, motor activities, reflexes, change in skin and fur, mortality.

Blood samples for hematological and blood chemical analyses were realized at D0 and 1, 2 and 3

months.

All rats were later sacrificed at the 12th week, after the last dose for internal macroscopic, organ

weights, necropsy, and histopathological parameters.

Effect of extract on average body weight of rats:

The animals were weighed individually at the beginning of the study and at weekly intervals till

the end of the study. Heart, lung, liver and kidney and spleen weights of all rodents were

measured immediately after post treatment sacrifice.



Blood Sample collection:

Rodents were kept fasted after a 16-18 hours overnight afterwards, blood samples were collected

from each animal from the retro-orbital sinus under diethyl ether anesthesia at D0, months 1, 2

and 3.

The blood samples were put in EDTA tubes and used for determining the biochemical

parameters and into heparinised tubes for hematological parameters.

Determination of hematological parameters:

The blood samples were analyzed for red blood cell (RBC) count, hemoglobin (Hb) levels,

hematocrit (HCT), platelets (PLT), White blood cell (WBC) count, differential WBC count

[Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E)], mean corpuscular volume

(MCV), mean corpuscular hemoglobin (MH) and mean corpuscular hemoglobin concentration

(MCHC).

Determination of serum biochemical parameters:

The blood samples were centrifuged at 4000 rpm for 10 minutes to obtain serum for the

following investigations:

Alanine amino transferase (ALT), Aspartate amino transferase (AST), Urea, Creatinine, Glucose.

Histopathology:

All rats were sacrificed at the end of the study period and subjected to detailed gross necropsy.

Vital organs such as heart, kidneys, liver, lung and spleen were isolated from all rats, weighed

and examined for any large lesions. All tissues were preserved in 10% buffered formaldehyde

solution for histopathological examination. The tissues were embedded in paraffin, and then

sectioned, stained with haematoxylin and eosin and were examined microscopically.

Statistical analysis:

Statistical evaluation was performed using Student’s test followed by one-way ANOVA analysis

of variance. All results are presented as Mean ±S.E.M. Results were considered significant at p <

0.05.

RESULTS:

Acute toxicity:

Oral administration of the MCWE up to dose 2000 mg/kg in mice did not cause any mortality or

any toxicity during the experimentation period However, mice after receiving MCWE exhibited

a slight reduction in the locomotors activity.



The MC at 2000mg/kg body weight orally is safe for consumption and for medicinal uses in

according with the OECD guidelines No.423.

Sub-Chronic Toxicity :

Effect of the oral MC water extract on the general behaviour of the rats:

Daily oral administration of MCWE for 90 days at doses 300 and 600 mg/kg body weight did not

show any physiological activities or other general behavior, no obvious symptoms of toxicity or

mortality during the entire period of the experiment. Both the control and treated rats appeared

consistently healthy throughout the 90 day period of study.

Body weight and weight of organs

The body weights of control and MC water extract treated rats are presented in Table 3.

In both sexes of rats no significant difference body weight gain was noted between the control

and any of the treated groups at any time period.

The sub-chronic oral ingestion of MCWE over 90 days caused no significant changes in the

weights of the organs (heart, spleen, lungs, kidneys and liver) in the treated as compared to the

control rats Table 4.

Hematological parameters:

The effect of sub-chronic administration of MCWE on Hematological parameters is presented in

Table 1.

There were no significant changes in hematological parameters (RBC, Hb, HCT, PLT, WBC,

Neutrophils (N), Lymphocytes (L), Monocytes (M), Eosinophils (E), MCV, MCH and MCHC)

in any of the tested groups as compared to control group in given dose and duration of the study.

Biochemical parameters:

Sub-Chronic oral administration of MCWE (300 and 600 mg/kg) did not cause significant

changes in the levels of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and

Creatinine as shown in Table 2.

However, blood glucose level was significantly decreased in treated animals (P < 0.05) as

compared to the control group up to 90 days of treatment On the other hand, there was a

significant rise in urea in the treated groups as compared to the control group.

Histopathological changes:

Rats treated sub-chronically with 300 or 600 mg/kg of MCWE did not show any adverse effect

on the histoarchitecture of the heart and the liver.



However, after 90 days of treatment with the MCWE, spleen Red pulp congestion, and

pulmonary vascular congestion as well as kidney glomerular ischemia and tubular necrosis were

observed starting from 300 mg/Kg and 600 mg/Kg, as illustrated in Figure 1.

DISCUSSION:

Toxicity tests are essentially performed on either mice or rats because of their availability, low

cost and the wealth of toxicology data in the literature already available for these species [16].

For the acute oral toxicity study in mice, general behavior adverse effects and mortality were

determined for up to 14 days. There was no mortality and clinical signs of toxicity observed at

the doses of 300 and 2000 mg/kg of MCWE. The MCWE with an LD50 > 2000 mg/kg is

considered to be non-toxic through acute exposure in mice.

In the Sub-chronic toxicity study, the MCWE was administered to rats orally at doses of 300 and

600 mg/kg daily for 12 weeks. Both female and male rats administered with the MCWE (300

and 600 mg/kg) did not show any toxicity during the experimentation period.

Moreover, no lethality was recorded for any dose up to the maximum of 600 mg/kg (NOAEL)

during the 90 days of treatment.

Generally, the reductions in body weight gain and internal organ weights are simple and

sensitive indices of toxicity after exposure to toxic substances [17]. In both sexes of rats, body

and organ weights of treatment rats did not produce any statistically significant difference

relative to that of control. However, slight changes were found in the weights of organs that may

due to the random variation in size of organs in each animal [18].

Hematological studies easily reveal anomalies in body metabolic processes, and the blood profile

usually furnishes vital information on the response of the body to injury, deprivation and/or

stress. The hematological parameters showed no significant changes in the treated rats compared

to the control rats, respectively indicating that MCWE had no toxicological effects on the

circulating blood cells on their production. The serum biochemical parameters were studied to

evaluate the possible alterations in hepatic and renal functions influenced by MCWE.

It is well known that almost all drugs, chemicals and xenobiotics are eliminated through renal

excretion hence it was found necessary to estimate the effects of MCWE on kidney functions.

Daily oral administration of MCWE for up to 90 days did not result a significant changes in the

biochemical parameters except for a significant decrease in plasma glucose levels and rise in

urea. However we observed a significant increase in serum Urea levels at different doses (300



and 600mg/kg) especially at the second month of treatment when compared to control group.

This may be associated with kidney dysfunction most likely by renal filtration mechanism and

probably indicates that chronic exposure of MCWE at different doses for up to 90 days may

interfere with the capacity of the kidney to excrete this metabolite as suggested by Bailey C. J.

in the Potential new treatments for Type 2 diabetes [19].

MCWE caused a reduction in plasma glucose demonstrating that the plant has hypoglycemic

activity, this confirming a previous report of hypoglycemic activity of Matricaria resulting from

inhibition of the enzyme glycogen phosphorylase [20].

In the last experiment, necropsy and histopathological examinations were performed to further

confirm whether or not the organs or tissues had been damaged. The results showed macroscopic

or microscopic changes in these internal organs or tissues (lungs, spleen and kidney) in treated

rats with MCWE. The lesions include: glomerular ischemic lesion with sickle cell anemia and

minimal focal tubular necrosis when compared to control rat kidney and

pulmonary vascular congestion when compared to control rat lungs and spleen Red pulp

congestion when compared to rat spleen.

These changes will interfere with ability of the kidney to carry out its normal excretory roles.

This may have contributed to the high levels of urea seen in the blood of rats treated with

MCWE at the end of this study. Also the kidney is highly susceptible to toxicants because a high

volume of blood flows through it and it filters large amounts of toxins which can concentrate in

the kidney tubules [21].

In conclusion, at the doses consumed empirically in traditional Moroccan medicine, Matricaria

chamomilla L. appears to be relatively nontoxic in short term therapies. However, the changes

must be taken into consideration it can cause at long term lungs and kidney toxicity;

Furthermore, determination of food intake and water consumption is important in the study of

safety of a product with therapeutic purpose.

Competing interests

The authors declare that they have no competing interests.

ACKNOWLEDGMENTS

The authors wish to thank all the individuals and institutions who made this survey possible.



Table 1- Hematological values of rats in the chronic toxicity study of Aqueous Extract of

Matricaria chamomilla L.

Aqueous Extract of Matricaria chamomilla L.

Treatment time

1 month 2 months 3 months

parameters Control 300mg/kg 600mg/kg 300mg/kg 600mg/kg 300mg/kg 600mg/kg

RBC 7.89 ± 0.73 7.7 ± 0.60 7.69 ± 0.71 7.66 ± 0.35 8.14 ± 0.64 8.08 ± 0.6 8.38 ± 0.86

Haemoglobin 14.58 ±

0.73

14.55 ±

0.26

14.26 ±

0.31

14.07 ±

0.74

14.25 ±

0.54

14.8 ± 0.64 14.71 ± 0.64

Haematocrit 39.75 ±

0.79

40.08 ±

0.48

40.93 ±

0.65

40.75 ±

0.77

41.65 ±

0.82

42.65 ±

0.93

42.26 ± 0.97

Platelets 623 ± 63 594 ± 71 585 ± 87 631 ± 63 597 ± 93 599 ± 81 597 ± 91

WBC 10.54 ± 2.1 10.62 ±

0.45

11.13 ±

0.88

11.06 ± 0.8 11.75 ±

0.70

10.83 ±

0.97

11.85 ± 0.45

Neutrophils 20.75 ±

1.89

20.8 ± 0.32 20.62 ±

0.88

21.12 ± 0.2 20.28 ±

0.96

21.17 ±

0.29

20.34 ± 1.08

Lymphocytes 70.51 ±

2.66

70.98 ±

2.06

70.56 ±

2.01

70.81 ±

2.77

69.03 ±

3.07

68.1 ± 3.61 67.91 ± 3.93

Monocytes 2.03 ± 0.17 2.01 ± 0.08 1.98 ± 0.09 2.05 ± 0.18 2.00 ± 0.18 2.00 ± 0.3 2.03 ± 0.33

Eosinophils 1.7 ± 0.43 1.7 ± 0.37 1.7 ± 0.36 1.73 ± 0.34 1.73 ± 0.35 1.75 ± 0.32 1.75 ± 0.34

MCV 51.47 ±

1.25

51.8 ± 0.79 51.7 ± 0.84 51.02 ±

1.65

51.37 ±

0.86

51.92 ±

1.98

51.5 ± 0.84

MCH 18.34 ±

0.69

17.75 ±

0.63

18.15 ±

0.65

17.87 ±

0.66

17.97 ±

0.82

18.46 ±

0.88

18.33 ± 1.82

MCHC 34.17 ±

0.47

34.2 ± 0.36 34.3 ± 0.36 34.03 ±

0.77

34.46 ±

0.88

34.2 ± 0.49 34.01 ± 0.87



Table 2- Biochemical values of rats in the chronic toxicity study of Aqueous Extract of


Aqueous Extract of Matricaria chamomilla L.

Treatment time

1 month 2 months 3 months

parameters Control 300mg/kg 600mg/kg 300mg/kg 600mg/kg 300mg/kg 600mg/kg

ASAT (U/I) 84 ± 2.1 85 ± 1.9 83 ± 1.7 83 ± 2.0 85 ± 2.1 84 ± 1.9 85± 2.3

ALAT (U/I) 65.83 ± 4.3 61.33 ± 4.6 65.66 ±

3.65

64.55 ±

4.15

65.16 ±

4.18

66.55 ±

4.11

64.83 ±

3.22

Urea (mg/l) 0.26 ± 0.05 0.26 ± 0.06 0.28 ± 0.03 0.25 ± 0.09 0.26 ±

0.07*

0.34 ± 0.04 0.35 ±

0.06*

Creatinine (mg/l) 5 ± 0.63 4.73 ± 0.42 4.96 ± 0.27 5.03 ± 0.28 5.11 ± 0.20 4.88 ± 0.49 4.92 ± 0.17

Glucose (g/l) 1.52 ± 0.12 1.02 ± 0.08* 1.15 ±

0.11*

1.08 ± 0.4* 1.11 ±

0.39*

1.07 ±

0.45*

1.12 ±

0.35*



Table 3- Body Weights of rats in the chronic toxicity study of Aqueous Extract of Matricaria

chamomilla L.

Table 4- Organ Weights of rats in the chronic toxicity study of Aqueous Extract of


organs control Aqueous Extract of Matricaria

chamomilla L.

300mg/kg 600mg/kg

Lungs 1.81 ± 0.05 1.80 ± 0.10 1.80± 0.5

Heart 0.88 ± 0.09 0.87 ± 0.05 0.88 ± 0.09

Liver 10.43 ± 0.24 10.42 ± 0.08 10.10 ± 0.14

Kidney 0.85 ± 0.03 0.85 ± 0.06 0.86± 0.04

Spleen 0.72 ± 0.02 0.71 ± 0.04 0.74 ± 0.06

Group

control AEMC 300mg/kg .p.o. AEMC 600mg/kg .p.o.

0 191.86 ± 19.74 192.25 ± 15.16 191.20 ± 11.80

1 225.97 ± 18.91 217.71 ± 13.14 221.71 ± 15.97

2 255.93 ± 24.29 252.51 ± 20.11 249.59 ± 20.21

3 282.62 ± 30.83 274.92 ± 17.53 271.29 ± 31.55



(T) (MC1) (MC2)

(A)

(B)

(C)

(D)

(E)

Figure 1- Photomicrographs of the sections of the heart (A), liver (B), spleen (C), lungs (D),

kidney (E) of control (T) and AEMC administered at the dose 300mg/kg (MC1) and the dose

600mg/kg (MC2). Sections were stained with hematoxylin and eosin.



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