Herbs are used throughout the world as an old form of health care. They have played a crucial role in the elaboration of modern medicine such as the conception of synthesized drugs. The medicinal plants have continued to maintain human health for many years [
In Morocco,
This work was undertaken to screen the phytochemicals of
Plant material was collected in December 2016 at 30 km East of Khouribga City, Morocco. The plant was authenticated by Dr. Mohammed Fanane (Department of Botany, Scientific Institute of Rabat, Morocco). Voucher specimens have been preserved in the herbarium of Scientific Institute of Rabat, Morocco, under # 101545. The rhizomes were washed with water and dried at room temperature in a shady and dry ventilated place.
Twenty five grams of dried rhizomes powder was boiled for 20 minutes at 100°C and cooled to room temperature. The solution was filtered and concentrated in a rotary vacuum evaporator to yield 5 g dry extract. The extracted material was suspended in distilled water.
Male adult Swiss mice with an average weight of 25 g were used for the current research. They were purchased from the animal colony of Pasteur Institute, Morocco. Mice were acclimatized in the animal holding with standard conditions; light/dark cycles (12/12 h), temperature (24±2°C) and air changes. Standard pellet diet was freely available to mice.
The mice were segregated into 4 groups of six mice, including a control group, and then deprived for 12 h of food. The RDA was administrated orally in three doses: 1, 2, and 4 g/kg following OECD, 2008, Guideline No. 425 [
The mice were segregated into 4 groups of six mice, three treated and one control group. In the treated groups, the RDA was administered repeatedly for 28 days in doses 0.5 g/kg/day, 1 g/kg/day and 1.5 g/kg/day following OECD, 1998, Guideline No. 407 [
On the head of the experiment period, the experimental animals were subjected to cerebral dislocation for blood collection using laboratory sample tubes. AST, ALT, LDH, urea, and creatinine were the biochemical parameters selected to be determined using an automated analyzer.
On the head of the experiment period, the animals were subjected to cerebral dislocation. Liver and kidney were collected for histopathological studies. The tissues were washed and fixed in 10% formaldehyde solution, dehydrated with alcohol and then enclosed in paraplast. Micrometer sections were conducted (5
Quantitative data were analyzed taking into account the mean ± standard error of the mean (SEM). The significance between means was assessed using one-way ANOVA. Tukey post hoc test was employed for multiple comparisons. Statistically, data showed to be significant when
The results of the phytochemical analysis of
Phytochemical analysis of
Polyphenol | Alkaloids | Flavonoids | Anthraquinone | Sterols and terpene | Saponins |
---|---|---|---|---|---|
++ | +++ | ++ | | | + |
+++: strong positive test; ++: positive test; +: low positive test;
In the first days of treatment, with a dose of 1 g/kg, a slight behavioral change was recorded in the treated mice, resulting in an accelerated running of 2 to 4 min compared to control group. Clinical symptoms such as diarrhea, lack of appetite, lethargy, salivation, the difficulty of locomotion, reduced activity, and convulsions were observed after animals dosing of doses of 2 and 4 g/kg of body weight. On the other hand, LD50 was determined at the highest dose administered of 4 g/kg (three over six mice were dead).
The clinical symptoms observed in treated animals were carefully registered during the whole period of feeding the RDA. With a dose of 0.5 g/kg, there were no visible toxic effects. We registered difficulty of locomotion, ataxia, restriction of food intake, and reduced activity in both groups given 1 and 1.5 g/kg
Throughout the whole period of dosing of RDA, there was no significant variation in the weight of fed animals with 0.5 g/kg/day (group 1) in comparison to control group (p>0.05). The fed animals with a dose of 1 g/kg/day (group 2), 1.5 g/kg/day (group 3) induced an important weight loss which began in the first week of dosing and attended to be very significant at the end of the treatment period (Figure
Effect of RDA at doses of 0.5 g/kg/day (group 1), 1 g/kg/day (group 2), and 1.5 g /kg/day (group 3) on the average weight of treated mice (results represent the means ± SEM).
The results of biochemical parameters were examined, including ALT, AST, urea, creatinine, and LDH, represented in Figure
Effect of RDA on AST, ALT, LDH, urea, and creatinine after 28 days of treatment (group 1: 0.5 g/kg/day; group 2: 1g/kg/day; group 3: 1.5 g/kg/day) (results represent the means ± SEM).
The findings of biochemical parameters showed a significant augmentation of AST measured in treated group 2 (1 g/kg/day) and 3 (1.5 g/kg/day) in comparison to control group (p<0.05). There was no significant change remarked in group 1 fed with the lowest dose (0.5 g/kg/day) (p>0.05) (Figure
Results of Tukey test for comparisons between groups regarding biochemical parameters variation (Subacute Toxicity).
Chemical parameters | control group vs. | control group vs. group 2 | control group vs. group 3 | group 1 vs. | group 1 vs. | group 2 vs. |
---|---|---|---|---|---|---|
AST | 0.99 | 0.001 | 0.001 | 0.001 | 0.001 | 0.99 |
ALT | 1 | 0.98 | 0.02 | 0.004 | 0.003 | 0.005 |
Urea | 0.99 | 1 | 0.00 | 0.99 | 0.00 | 0.00 |
Creatinine | 0.98 | 0.96 | 0.03 | 0.99 | 0.05 | 0.06 |
LDH | 0,85 | 0.01 | 0.53 | 0.01 | 0.83 | 0.81 |
Control: normal diet; group 1: normal diet + 0.5 g/kg/day extract; group 2: normal diet + 1 g/kg/day extract; group 3: normal diet + 1.5 g/kg/day extract.
The histopathological injuries detected in kidney tissues of treated mice, such as tubular necrosis, hyaline necrosis, inflammatory cell infiltration, and cell congestion, were the main tissue damage which became very detectable in the kidney of the mice fed with increasing doses of RDA as shown in Table
Lesion scores of the kidney for all groups analyzed using ANOVA test (subacute toxicity).
Histopathology of kidney | Histopathology scores of observation | ANOVA test for global comparison of organ lesions among groups | |||
---|---|---|---|---|---|
(mean) per each group | |||||
control group | group 1 | group 2 | group 3 | ||
Tubular necrosis | 0 | 0 | 0.11±0.01 | 0.25±0.05 | 0.006 |
Hyaline necrosis | 0 | 0 | 0.05±0.05 | 0.19±0.04 | 0.04 |
Coagulation necrosis | 0 | 0 | 0 | 0.22±0.17 | 0.31 |
Inflammatory infiltrate | 0 | 0 | 0.35±0.15 | 0.60±0.10 | 0.02 |
Extended coagulation | 0 | 0 | 0 | 0 | 1 |
Altered tissue architecture | 0 | 0 | 0 | 0 | 1 |
Cell vacuolization | 0 | 0 | 0 | 0.25±0.25 | 0.47 |
Tubular degeneration | 0 | 0 | 0.20±0.20 | 0.31±0.19 | 0.28 |
Cell congestion | 0 | 0 | 0.10±0.03 | 0.17±0.02 | 0.04 |
Tubular atrophy | 0 | 0 | 0 | 0 | 1 |
Interstitial fibrosis | 0 | 0 | 0 | 0 | 1 |
Vascular dilation | 0 | 0 | 0 | 0.16±0.03 | 0.006 |
Control: normal diet; group 1: normal diet + 0.5 g/kg/day extract; group 2: normal diet + 1 g/kg/day extract; group 3: normal diet + 1.5 g/kg/day extract.
Considering the major histopathological alterations detected in the kidney of treated mice, such as tubular necrosis, hyaline necrosis, inflammatory cell infiltration, and vascular dilatation, no significant difference was shown between control and fed mice with 0.5 g/kg/day (group 1), nor 1 g/kg/day (group 2). For comparisons between treated groups in terms of announced histopathological injuries, no significant difference was remarked between group 1 (0.5 g/kg/day) and group 2 (1 g/kg/day) (p>0.05). Considering groups 2 and 3, a significant difference for vascular dilatation injury was observed (Tables
Results of Tukey test for comparisons between groups regarding lesions in the kidney (subacute toxicity).
Histopathology of kidney | control group vs. group 1 | control group vs. group 2 | control group vs. group 3 | group 1 vs. group 2 | group 1 vs. group 3 | group 2 vs. group 3 |
---|---|---|---|---|---|---|
Tubular necrosis | 1 | 0.19 | 0.008 | 0.11 | 0.008 | 0.58 |
Hyaline necrosis | 1 | 0.73 | 0.049 | 0.73 | 0.049 | 0.19 |
Coagulation necrosis | 1 | 1 | 0.38 | 1 | 0.38 | 0.38 |
Inflammatory infiltrate | 1 | 0.15 | 0.03 | 0.15 | 0.03 | 0.33 |
Extended coagulation | 1 | 1 | 1 | 1 | 1 | 1 |
Altered tissue architecture | 1 | 1 | 1 | 1 | 1 | 1 |
Cell vacuolization | 1 | 1 | 0.35 | 1 | 0.35 | 0.53 |
Tubular degeneration | 1 | 0.74 | 0.47 | 0.74 | 0.47 | 0.93 |
Cell congestion | 1 | 0.22 | 0.06 | 0.22 | 0.06 | 0.52 |
Tubular atrophy | 1 | 1 | 1 | 1 | 1 | 1 |
Interstitial fibrosis | 1 | 1 | 1 | 1 | 1 | 1 |
Vascular dilation | 1 | 1 | 0.009 | 1 | 0.009 | 0.009 |
Control: normal diet; group 1: normal diet + 0.5 g/kg/day extract; group 2: normal diet + 1 g/kg/day extract; group 3: normal diet + 1.5 g/kg/day extract.
No histopathological injuries were detected in the kidney of fed animals with 0.5 g/kg in comparison to the control group (Figure
Sections of kidney parenchyma of control mice (H&E x 40). (a), (b), and (c) are kidney sections of normal tissues of three mice selected randomly from the control group.
Sections of kidney parenchyma of mice fed with 1 g/kg/day (H&E, x 40). (a) Tubular necrosis; (b) inflammatory infiltrate; (c) tubular degeneration.
Sections of kidney parenchyma of mice fed with 1.5 g/kg/day (H&E, x40). (a) Inflammatory infiltrate (circle); (b) renal necrosis; (c) inflammatory infiltrate (circle) and tubular necrosis (arrow); (c) tubular degeneration (arrow) and inflammatory infiltrate (circle); (e) inflammatory infiltrate (circle) and tubular necrosis (arrow); (f) cell congestion.
The histopathological changes recorded in liver tissue of treated animals, such as hepatocellular necrosis, lobular necrosis, cell vacuolization, and inflammatory infiltrate, were the mean tissue damage, which became very remarkable in the liver of fed mice with increasing doses of RDA as reported in Table
Lesion scores of the liver for all groups analyzed using ANOVA test (subacute toxicity).
Histopathology of liver | Histopathology scores of observation (mean) per each group | ANOVA test for global comparison of organ lesions among groups | |||
---|---|---|---|---|---|
control group | group 1 | group 2 | group 3 | ||
Hepatocellular necrosis | 0 | 0.001±0.0007 | 0.45±0.05 | 0.35±0.05 | 0.002 |
Lobular necrosis | 0 | 0 | 0.13±0.01 | 0 | 0.00 |
Altered tissue architecture | 0 | 0 | 0 | 0 | 1 |
Cell vacuolization | 0 | 0 | 0.02±0.02 | 0.25±0.05 | 0.008 |
Cell congestion | 0 | 0 | 0 | 0 | 1 |
Inflammatory infiltrate | 0 | 0.01±0.01 | 0.17±0.12 | 0.5±0.10 | 0.03 |
Dilatation of sinusoids | 0 | 0 | 0 | 0 | 1 |
Kupffer cell hypertrophy | 0 | 0 | 0 | 0 | 1 |
Hepatic steatosis | 0 | 0 | 0 | 0 | 1 |
Cholestasis | 0 | 0 | 0.17±0.17 | 0.35±0.05 | 0.13 |
Haemorrhagic foci | 0 | 0 | 0 | 0 | 1 |
Disruption of blood vessel | 0 | 0 | 0 | 0 | 1 |
Control: normal diet; group 1: normal diet + 0.5 g/kg/day extract; group 2: normal diet + 1 g/kg/day extract; group 3: normal diet + 1.5 g/kg/day extract.
For the lowest dose tested (0.5 g/kg/day), no histopathological changes were detected in the liver compared to the control group (Figure
Sections of liver parenchyma of control mice (H&E, x40). (a), (b), and (c) are liver sections of normal tissues of three mice selected randomly from the group of control mice.
Sections of liver parenchyma of fed mice with 1 g/kg/day (H&E, x40). (a) Inflammatory infiltrates; (b) hepatic necrosis; (c) hepatic necrosis (arrow) and inflammatory infiltrates (circle).
Histologic section of liver tissue of treated mice with 1.5 g/kg/day (H&E, x40). (a) Hepatic cholestasis; (b) inflammatory infiltrate (green circle) and hepatic cholestasis (blue circle); (c) hepatic necrosis; (d) inflammatory infiltrate; (e) hepatic necrosis (arrow) and inflammatory infiltrate (circle); (f) hepatic cholestasis (blue arrow) and hepatic necrosis (green arrow).
Since ancient time, people believe that the use of herbal medicines is safe because of their natural origin. In Morocco, there are no regulatory requirements for the majority of medicinal plants sold by herbalist, including adherence to information in pharmacopeias [
Studying the acute toxicity of RDA was useful to assess its toxicity at single doses. As reported in the literature, there are very little reported cases of acute human or animal poisoning by
The clinical symptoms occurred in mice under subacute toxicity conditions like convulsions, the difficulty of locomotion, weight loss, and hypoactivity are probably related to toxic properties of RDA [
Urea is the final nitrogenous excretion derived from protein amino acid catabolism and it is produced in the liver. In kidney the urea is filtered from serum, it is the common biochemical indice measured for evaluating kidney function on the basis of urea concentration in serum, and it is largely used for the diagnosis of acute kidney failure [
Transaminases (ALT, AST) are enzymes found inside living cells, especially in the liver and muscles which intervene in a multitude of biological reactions [
Lactate dehydrogenase (LDH) is a biomarker of tissue damage because this enzyme is normally found in most tissues of the organism and only in small amounts in the blood circulatory. When the tissues are damaged, the cells release the LDH causing an increase in its concentration in the blood circulatory. The dosage of LDH is generally required in the diagnosis of tissue lesions [
The histopathological injuries reported in the kidney of treated animals in this current work such as nephropathy, necrosis, tubule-interstitial fibrosis, inflammatory infiltrate, altered tissue architecture, tubular degeneration, tubular necrosis, and cell congestion are in confirmation with the biochemical alterations of urea and creatinine, and even the histologic changes observed in liver tissue, such as necrosis, lymphocytic infiltrate, hepatic cholestasis, tubular necrosis, foci of hemorrhage, cell congestion, and altered tissue architecture, are in accordance with serum parameters variation registered in this research work (AST, ALT, LDH).
The histopathological and biochemical findings reported in the present study were in confirmation with kidney failure occurring in Belgium patients adopting weight loss regimes including species of
The obtained results in the present work were comparable to those reported in the literature [
Aristolochic acids contained from genus
Biologically, aristolochic acids open experimental pathways for conceptualizing the mechanism by which the renal fibrogenesis is involved in human pathology. The identification of complications related to treatment with medicinal plants puts several questions in terms of health safety [
This study showed that the rhizomes decoction of
All data are available in the following labs: Laboratory of Chemistry Biochemistry, Nutrition, and Environment, Faculty of Medicine and Pharmacy, University Hassan II, Casablanca, Morocco, Laboratory of Biochemistry, Environment, and Agri-food, Faculty of Science and Technology University Hassan II, Mohammedia, Morocco, Environment and Health Team, Department of Biology, Multidisciplinary Faculty of Safi, Cadi Ayyad University, Safi, Morocco, Laboratory of Botany, Faculty of Sciences, University of Abdelmalek Essaadi Tetouan, Morocco, and Life Science Division, National Centre for Energy, Sciences, and Nuclear Techniques, Rabat, Morocco.
We declare that there are no conflicts of interest.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RG-1440-100.