Safety assessment of standardised methanol extract of Cinnamomum burmannii.
ARTICLE INFOKeywords:
Cinnamomum burmannii
Acute toxicity
Sub-chronic toxicity
ABSTRACT
The present study aims to evaluate the safety of methanol extract of Cinnamomum burmannii (MECB) by acute 14-day (single dose) and sub-chronic 28-day (repeated doses) oral administration to Sprague-Dawley rats. Our results showed that no toxicity was found in either acute or sub-chronic toxicity studies. MECB (containing 0.07% and 0.20% (w/w) of coumarin and trans-cinnamaldehyde, respectively), which was given orally at doses of 500, 1000 and 2000 mg/kg caused neither visible signs of toxicity nor mortality. No significant differences were observed in general condition, growth, organ weight, hematological parameters, biochemical values, or the gross and microscopic appearance of the organs from the treatment groups as compared to the control group. In conclusion, MECB did not cause any mortality nor did it cause any abnormalities in the necropsy and histopathology findings of treated rats. The [LD.sub.50] for the MECB was found to be more than 2000 mg/kg. No adverse effects were observed in the treated rats at all the doses tested. The no-observed-adverse-effect level (NOAEL) for the 28-day study was determined to be 2000 mg/kg body weight/day.
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Introduction
A number of medicinal plants have a long history of traditional use, and they have played many important roles for humans since the dawn of civilisation (Pankaj et al. 2009). These medicinal plants have been proven by pharmacological research to serve as potential sources of bioactive compounds used to treat many illnesses and diseases (Pankaj et al. 2009). These bioactive compounds are actually combinations of secondary products present in the plant. They have been used as food preservatives, pharmaceuticals, alternative medicines and natural therapies (Bishnu et al. 2009) for centuries. These compounds are mostly alkaloids, steroids, tannins, phenolic compounds, flavonoids, resins and fatty acids (Bishnu et al. 2009). These compounds are odorous, complex, volatile compounds produced by special cells or groups of cells and concentrated in one particular region of plant such as the leaves, bark and stems (Mounia et al. 2006).
Cinnamon is one medicinal plant that is presently receiving much attention but has yet to be fully investigated. It is a tree-like shrub classified under Lauraceae family (Abdul 2009). The genus Cinnamomum, which comprises approximately 250 species, is distributed mostly in Asia and Australia. Cinnamon leaves and bark are normally used as spices and in the production of essential oils. They provide a variety of oils with different aromatic characteristics and compositions to the flavour industry (Jayaprakasha et al. 2007). According to literature, several species of Cinnamomum are of medical importance and exhibit various pharmacological and biological effects (Pankaj et al. 2009; Mounia et al. 2006; Pawar and Thaker 2006).
Cinnamomum burmannii Blume, which is also known as Indonesian cassia, and its major components that have been isolated, (E)-cinnamaldehyde and proanthocyanidins, have been reported to possess significant in vitro antibacterial properties against five common food-borne pathogenic or faecal indicator bacteria (Shan et al. 2007). Moreover, the ethanol extract of Cinnamomum burmannii has been shown to control food-borne pathogens and inhibit lipid oxidation (Shan et al. 2009). Cao et al. (2007) showed that compounds present in Cinnamomum extract may be used in the treatment of diabetes by increasing the amount of anti-inflammatory protein tristetraprolin, insulin receptor (3 and glucose transporter 4 in a 3T3-L1 adipocyte model. Despite its many medical uses, little toxicological information is available regarding its safety following repeated consumption. Currently, the safety and potential toxicity of the botanicals including medicinal plants and edible materials are of concern to Malaysian authorities. Therefore, the present study was undertaken to determine the possible harmful effects of methanol extract of Cinnamomum burmannii (MECB) after acute 14-day and sub-chronic 28-day oral administration to Sprague-Dawley rats.
Materials and methods
Experimental animals
Eight-week-old male and female Sprague-Dawley rats (180-200g) were used for the acute 14-day and sub-chronic 28-day toxicological studies. The rats were purchased from the Animal House, School of Pharmaceutical Sciences. Universiti Sains, Malaysia. The animals were acclimatised to laboratory conditions for 7 clays prior to performing the experiments. Three rats were housed in a single polycarbonate cage with free access to food (normal laboratory chow, Gold Coin) and tap water ad libitum. Food and water were added each day and changed every 2-3 days. All rats were kept at 26 [+ or -] 3[degrees]C, with a light/dark cycle of 12 h and humidity around 30%. All the procedures in this study were performed according to the Animal Ethics Guidelines of Universiti Sains. Malaysia.
Plant materials and extract
The bark of Cinnamomum burmannii was collected from the Agroforestry Garden (Parak) at Sungai Dareh village, Pulau Pun-jung Sub-District, Dharmasraya District, West Sumatra Province, Indonesia, and was authenticated by Mr. Onrizal S. Hut, Forestry Department. Agricultural Faculty, Universitas Sumatera Utara. The specimen was collected and compared to the existing voucher specimens at the Forestry Department. Agricultural Faculty, Universitas Sumatera Utara, and examined macroscopically and microscopically. The bark was dried, ground into powder and subsequently extracted with methanol using Soxhlet's apparatus. The resulting extract was concentrated using a rotary-evaporator and then freeze dried. Lastly, the weight of the extract was taken and recorded. The final plant-to-extract ratio was about 25.2%. (252g of powdered MECB was obtained from the extraction of 1 kg of powdered bark). The extract used in this experiment was prepared daily by suspending it in 1% fresh carboxymethylcellulose (CMC).
Thin layer chromatography (TLC) fingerprint analyses
Coumarin and trans-cinnamaldehye (Sigma-Aldrich, St. Louis, MO, USA) were selected as marker compounds and the following samples were prepared for TLC analyses.
Sample A: 1 g powdered dry cinnamon bark was shaken with 10 ml dichloromethane for 15 min. The extract was filtered and evaporated to dryness. The residue was then dissolved in 1 ml toluene. Sample B: 1 g MECB was shaken with 10 ml dichloromethane for 15 min. The extract was filtered and evaporated to dryness. The residue was then dissolved in 1 ml toluene. Sample C: 20 mg of MECB dissolved in 1 ml methanol. Sample D: 1 mg/ml of coumarin in methanol. Sample E: 1 mg/ml of trans-cinnamaldehyde in methanol.
Chromatography was performed on silica gel 60F254 TLC plates (10cm x 10cm; 0.25 mm layer thickness; Merck). Samples (20 [micro]l) were applied on the TLC plates. A mixture of toluene: ethyl acetate (93:7) was used as mobile phase. TLC plate development was performed by using a glass tank, which had been pre-saturated with mobile phase for 2 h. The solvent was allowed to run up the plate to a height of 8 cm. TLC analyses were made under room temperature. After development, the TLC plates were dried and the components were visualised by UV light at 254 nm, 366 nm and under visible.
High-performance liquid chromatography (HPLC) analysis of coumarin and trans-cinnamaldehye
The separation of coumarin and trans-cinnamaldehye from MECB was performed by using a Shimadzu-LC system (Shimadzu Corporation, Kyoto, Japan) equipped with a CBM-20A controller, LC-20AT pump, DGU-20A5 degasser, SIL-20A auto-sampler, SPD-20AV detector, and CTO-10Asvp column oven at 30 [degrees]C. Agilent Eel ipse Plus C18 column (250mm x 4.6mm i.d.; 5 [micro]m) and Zorbax guard fittings kit packed with replaceable Eclipse Plus C18 Guard column (12.5 mm x 4.6 mm i.d.; 5 [micro]m) were used in the analysis. The separations were done by using isocratic mobile phase consisted of 0.04% (v/v) glacial acetic acid in water:acetonitrile (40:60). The flow rate was 1 ml/min. Injection volume was 20 [micro]I and the UV detector was set at wavelength of 280 nm. The mobile phase was filtered under vacuum through 0.45 [micro]m membrane filter (Merck, Darmstadt, Germany) prior to HPLC analysis. All chromatographic operations were carried out at ambient temperature.
Sample solution (100 [micro]g/ml) was prepared by dissolving the dried extract in HPLC grade methanol. Standard coumarin and trans-cinnamaldehye solutions in the range of 0.78-100 [micro]g/ml were prepared and injected into HPLC system to establish a calibration curve. Peak height versus marker concentration was subjected to least square linear regression analysis and the slope, intercept and correlation coefficient for the calibration curve were determined. The concentration of coumarin and trans-cinnamaldehye from the MECB was expressed in percentage per mg dry weight of extract (Celeghini et al. 2001). All measurements were performed in triplicates.
Acute toxicity study
This study was performed according to the Organisation for Economic Cooperation and Development (OECD) revised up and down procedure for acute toxicity testing (OECD Guideline 425, 2001). In this study, a limit dose of 2000 mg/kg of MECB was administered orally by gavage to five healthy female adult Sprague-Dawley rats. The rats were fasted overnight prior to dosing and weighed before the extract was administered. The administration volume was adjusted between 1-2 ml for each and every rat. A single oral dose of 2000 mg/kg of the extract was given to the rat on the first day of the experiment, and the rat was observed for mortality and clinical signs of toxicity for the first hour, then hourly for 3 h and then periodically throughout 48 h. Other rats were dosed sequentially at 48-h intervals if the first rat survived after 48 h of treatment. All of the experimental animals were maintained under close observation for 14 days. The number of mortality within the study period was recorded. The [LD.sub.50] was predicted to be above 2000 mg/kg if three or more rats survived (Mohamed et al. 2011).
Sub-chronic toxicity study
Sprague-Dawley rats of either sex were divided randomly into four groups (n = 12; six males and six females per group, a total of 48 rats), and their weights were recorded. MECB (prepared in 1% CMC) was administered daily by oral gavage for 28 clays in single doses of 500 mg/kg (group I), 1000 mg/kg (group II) and 2000 mg/kg (group III) while the control rats (Group IV) received only the vehicle (1% CMC in distilled water). Toxic manifestations and mortality were monitored daily for 28 clays. The bodyweights of all the rats were measured and recorded at the end of every week. After 28 days of treatment, all rats were anaesthetised using C[O.sub.2]. Blood samples were collected via cardiac puncture and transferred into non-heparinised and EDTA-containing tubes for both biochemical and hematological analyses, respectively (Rosidah et al. 2009). Thereafter, the rats were sacrificed by cervical dislocation. Brain, heart, lungs, thymus, liver, kidneys, adrenal glands, sex organs (ovaries and uterus for female rats; testes for male rats), spleen, stomach, and gut (begin from small intestine until the end of large intestine) of all experimental rats were excised, weighed, and examined macroscopically. The vital organs (lungs, kidneys, liver, stomach and brain) were then preserved in 10% formalin for histopathological study (Mohamed et al. 2011).
Relative organ weight
The excised organs were weighed individually. The index of each organ to its bodyweight ratio (relative organ weight) was calculated as (weight of organ/bodyweight of rats on the day of sacrifice) x 100% (Rosidah et al. 2009). The balance employed was purchased from Mettler-Toledo Group, Model Dragon 204 (Ohous Corporation, Navigator N2B110, Switzerland, d = 0.1 g).
Blood analyses
Hematological and biochemical analyses were performed at the Pathology Laboratory, Lam Wah Ee Hospital, Penang. Complete blood cell counts were determined using a fully automated hematological analyser Abbott Cell-Dyn 3500 (Abbott Laboratories. IL, USA) while serum biochemistry tests were performed using a COBAS Integra 800 (Roche, Germany) (Rosidah et al. 2009).
Histopathological study
All tissues were processed using a Citadel 1000 histokinette (Shandon Scientific Ltd., Cheshire, UK). These tissues were embedded in paraffin using a Histo-Center II-N (Barnstead/Thermolyne Corp., Dubuque, IA) and cut into 5-[micro]m-thick sections with a Reichert-Jung Histocut 820 II (Cambridge Instrument GmbH, Nussloch, Germany). The sections were later stained with hematoxylin and eosin and observed microscopically (Mohamed et al. 2011).
Statistical analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS). All the data are indicated as mean [+ or -] standard error of mean (S.E.M.) and were analysed statistically using one-way analysis of variance (ANOVA). Significant differences between the control and experimental groups were determined using a Dunnett-comparison test with p<0.05 taken as significant.
Results
TLC fingerprint analyses
Fig. 1 shows the TLC fingerprint of the markers and cinnamon samples. Mobile phase of toluene: ethyl acetate (93:7) gave a good resolution of the coumarin and trans-cinnamaldehye at Rf of 0:43 and 0.55, respectively. Coumarin was observed as a dark spot at 254 nm without any spray reagent (Fig. 1a) and at 366 nm when the plate was sprayed with 5% potassium hydroxyl solution (Fig. 1c). Trans-cinnamaldehye was observed as a brown spot under visible light when the plate was sprayed with anisiadehyde-sulphuric acid reagent (Fig. 1b) and dinitrophenylhydrazine reagent (Fig. 1d), respectively.
[FIGURE 1 OMITTED]
HPLC analyses
Fig. 2 shows the HPLC chromatogram of the standard markers and the extract. Coumarin and trans-cinnamaldehye were identified by comparing the retention time of standards with that of the extract. The retention time ([t.sub.r]) determined for coumarin and trans-cinnamaldehyde standards were 6.924 min and 10.923 min, respectively, while that of the MECB were 6.925 min and 10.925 min, respectively. Quantification was performed on the basis of linear calibration plots of the UV absorption peak height at 280 nm against concentration. Linear correlation between the peak height and concentrations gave correlation coefficient of 0.9999 which indicates a good linearity. The coumarin and trans-cinnamaldehyde levels in the extract were found to be 0.07% and 0.20% (w/w) of dry extract, respectively.
[FIGURE 2 OMITTED]
Acute toxicity study
In the acute 14-day toxicity study, the MECB, given at a close of 2000 mg/kg did not cause any visible signs of toxicity or mortality to the rats. All five female SD rats treated orally with the extract at the same close survived until the end of the experiment. No death was recorded.
Sub-chronic toxicity study
In sub-chronic 28-day toxicity study, no observable changes occurred in the general behaviours of the rats treated with different doses (500, 1000 and 2000 mg/kg) of MECB compared to the control group. No significant changes were detected in either the bodyweights (Fig. 3) or relative organ weights (data not shown) of all the rats. No death was recorded in either sex of both the control and treatment groups at any of the doses tested, during or after the course of the experiment. The controls and treated rats appeared uniformly healthy at the end of the experiment and throughout the 28 clays treatment period.
[FIGURE 3 OMITTED]
Blood analyses
No significant differences were observed in any of the hematological parameters tested (red blood cell count (RBC), haemoglobin concentration (Hgb), haematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total white blood cell count (WBC), or white blood cell differential count) in all of the treated rats compared to control rats (Table 1). Meanwhile, analysis of biochemical parameters (alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), creatinine, bilirubin, urea, sodium, potassium and chlorine) showed no significant difference in any of the parameters tested between the control and the treated groups in either sex of the rats (Table 2).
Table 1 Hematological values of rats treated with methanol extract
of Cinnamomum burmannii for 28 days. Data are expressed as mean
[+ or -] S.E.M.
Unit Treatment for 28
days
Control Methanol
extract of
Cinnamomum
burmannii
500 mg/kg
Male
White blood [10.sup.9]/l 16.63 [+ or -] 16.82 [+ or
cell count 1.04 -] 1.06
Neutrophils [10.sup.9]/l 4.93 [+ or 4.90 [+ or
-] 0.15 -] 0.13
Lymphocytes [10.sup.9]/l 8.08 [+ or -] 8.43 [+ or
0.29 -] 0.21
Monocytes [10.sup.9]/l 0.78 [+ or -] 0.81 [+ or
0.02 -] 0.02
Eosinophils [10.sup.9]/l 0.07 [+ or -] 0.07 [+ or
0.00 -] 0.00
Basophils [10.sup.9]/l 0.50 [+ or -] 0.53 [+ or
0.02 -] 0.02
Red blood cell [10.sup.12]/l 8.13 [+ or -] 8.08 [+ or
count 0.15 -] 0.13
Haemoglobin g/L 14.58 [+ or -] 14.52 [+ or
0.30 -] 0.23
Haematocrit [10.sup.9]/l 0.67 [+ or -] 0.66 [+ or
0.02 -] 0.01
Mean red blood fl 82.62 [+ or -] 82.18 [+ or
cell volume 0.95 -] 1.56
Mean Pg 17.97 [+ or -] 18.07 [+ or
corpuscular Hb 0.18 -] 0.18
Mean g/l 21.77 [+ or -] 21.90 [+ or
corpuscular Hb 0.20 -] 0.12
concentration
Platelets [10.sup.9]/l 1047.67 [+ or 1037.00 [+
-] 21.38 or -] 21.25
Mean platelet fl 8.19 [+ or -] 8.22 [+ or
cell volume 0.22 -] 0.20
Female
White blood [10.sup.9]/l 8.66 [+ or -] 6.73 [+ or
cell count 1.52 -] 0.93
Neutrophils [10.sup.9]/l 2.14 [+ or -] 1.51 [+ or
0.53 -] 0.28
Lymphocytes [10.sup.9]/l 5.83 [+ or -] 4.81 [+ or
0.89 -] 0.74
Monocytes [10.sup.9]/l 0.39 [+ or 0.33 [+ or
-] 0.10 -] 0.07
Eosinophils [10.sup.9]/1 0.03 [+ or -] 0.02 [+ or
0.01 -] 0.01
Basophils [10.sup.9]/1 0.27 [+ or -] 0.17 [+ or
0.07 -] 0.06
Red blood cell [10.sup.12]/l 7.61 [+ or -] 7.45 [+ or
count 0.14 -] 0.16
Haemoglobin g/L 14.01 [+ or -] 14.20 [+ or
0.12 -] 0.35
Haematocrit [10.sup.9]/l 0.69 [+ or -] 0.69 [+ or
0.01 -] 0.02
Mean red blood fl 90.90 [+ or -] 92.92 [+ or
cell volume 1.19 -] 1.06
Mean Pg 18.44 [+ or -] 19.02 [+ or
corpuscular Hb 0.24 -] 0.32
Mean g/l 20.29 [+ or -] 20.48 [+ or
corpuscular Hb 0.05 -] 0.12
concentration
Platelets [10.sup.9]/l 1099.80 1115.00 [+
[+ or -] 23.62 or -] 16.36
Mean platelet fl 8.28 [+ or -] 8.20 [+ or
cell volume 0.15 -] 0.30
1000 mg/kg 2000
mg/kg
Male
White blood 16.92 [+ or -] 16.72 [+
cell count 1.08 or -]
1.02
Neutrophils 5.10 [+ or -] 5.39 [+
0.11 or
-] 0.11
Lymphocytes 8.84 [+ or -] 8.61 [+
0.17 or -]
0.25
Monocytes 0.85 [+ or -] 0.88 [+
0.03 or -]
0.02
Eosinophils 0.07 [+ or -] 0.07 [+
0.00 or -]
0.00
Basophils 0.53 [+ or -] 0.53 [+
0.01 or -]
0.02
Red blood cell 8.18 [+ or -] 8.22 [+
count 0.18 or -]
0.08
Haemoglobin 14.85 [+ or -] 14.82 [+
0.20 or -]
0.13
Haematocrit 0.68 [+ or -] 0.67 [+
0.01 or -]
0.01
Mean red blood 83.85 [+ or -] 81.50 [+
cell volume 1.43 or -]
1.03
Mean 18.07 [+ or -] 18.00 [+
corpuscular Hb 0.24 or
-] 0.19
Mean 21.92 [+ or -] 21.85 [+
corpuscular Hb 0.10 or
concentration -] 0.11
Platelets 1062.33 [+ or -] 1057.50
22.42 [+ or -]
13.59
Mean platelet 8.34 [+ or -] 8.27 [+
cell volume 0.24 or -]
0.21
Female
White blood 10.14 [+ or -] 6.99 [+
cell count 2.05 or -]
1.94
Neutrophils 2.39 [+ or -] 1.44 [+
0.63 or -]
0.28
Lymphocytes 5.16 [+ or -] 5.03 [+
1.62 or -]
1.72
Monocytes 0.38 [+ or -] 0.30 [+
0.07 or -]
0.07
Eosinophils 0.03 [+ or -] 0.01 [+
0.01 or -]
0.00
Basophils 0.22 [+ or -] 0.21 [+
0.04 or -]
0.05
Red blood cell 7.81 [+ or 7.89 [+
count -] 0.13 or -]
0.20
Haemoglobin 14.13 [+ or -] 13.43 [+
0.39 or -]
1.08
Haematocrit 0.70 [+ or -] 0.71 [+
0.01 or -]
0.01
Mean red blood 89.48 [+ or -] 90.32 [+
cell volume 1.59 or -]
1.62
Mean 18.05 [+ or -] 17.16 [+
corpuscular Hb 0.35 or -]
1.55
Mean 20.23 [+ or -] 19.02 [+
corpuscular Hb 0.48 or -]
concentration 1.68
Platelets 1093.75 1105.05
[+ or -] 16.16 [+ or -]
28.57
Mean platelet 8.14 [+ or -] 7.53 [+
cell volume 0.22 or
-] 0.15
Table 2 Biochemical values of rats treated with methanol extract of
Cinnamomum burmannii for 28 days. Data are expressed as mean
[+ or -] S.E.M.
Unit Treatment
for 28
days
Control Methanol
extract of
Cinnamomum
burmannii
500 mg/kg 1000 2000
mg/kg mg/kg
Male
AST(U/L) u/i 126.00 [+ 128.00 [+ 127.00 132.33
or -] or -] 4.55 [+ or [+ or
4.97 -] -] 2.06
3.67
ALT(U/L) u/i 74.50 [+ 74.67 [+ or 74.83 73.83 [+
or -] -] 2.59 [+ or or -]
3.80 -] 2.63
3.17
Urea (mmol/l) u/i 7.40 [+ or 7.25 [+ or 7.52 [+ 7.08 [+
-] 0.31 -] 0.22 or -] or -]
0.36 0.31
Creatinine [mu]mol/1 34.83 [+ 36.17 [+ or 35.17 36.83 [+
([mu]mol/l) or -] -] 1.01 [+ or or -]
1.30 -] 0.70
0.70
ALP (U/L) [mu]mol/l 290.50 [+ 300.00 [+ 300.50 310.83
or or -] 11.48 [+ or [+ or
-] 13.16 -] -] 11.25
12.98
Bilirubin mmol/l 1.80 [+ or 1.80 [+ or 1.80 [+ 1.80 [+
([mu]mol/l) -] 0.00 -] 0.00 or -] or -]
0.00 0.00
Sodium g/l 143.17 [+ 141.83 [+ 141.67 142.17
(mmol/l) or -] or -] 0.60 [+ or [+ or
0.40 -] -] 0.48
0.95
Potassium g/l 4.72 [+ or 4.75 [+ or 4.88 [+ 4.78 [+
(mmol/l) -] 0.22 -] 0.14 or or -]
-] 0.14 0.08
Chlorine g/l 100.83 [+ 101.67 [+ 103.83 103.83
(mmol/l) or -] or -] 0.67 [+ or [+ or
0.98 -] -] 1.08
0.83
Female
AST(U/L) U/I 135.25 [+ 127.00 [+ 133.33 129.50
or -] or -] 4.42 [+ or [+ or
2.97 -] -] 5.66
4.78
ALT (U/L) U/I 54.00 [+ 56.80 [+ or 56.33 56.00 [+
or -] -] 1.34 [+ or or -]
2.46 -] 2.60
2.87
Urea (mmol/l) U/I 8.53 [+ or 7.04 [+ or 6.50 [+ 5.48 [+
-] 0.11 -] 0.07 or -] or -]
0.23 0.40
Creatinine [mu]mol/1 49.00 [+ 46.80 [+ or 46.00 42.50 [+
([mu]mol/l) or -] -] 1.10 [+ or or -]
1.76 -] 1.96
1.00
ALP (U/L) [mu]mol/l 233.33 [+ 236.00 [+ 236.00 229.50
or -] or -] 6.04 [+ or [+ or
2.05 -] -] 4.66
6.91
Bilirubin mmol/l 1.80 [+ or 1.80 [+ or 1.80 [+ 1.80 [+
([mu]mol/l) -] 0.00 -] 0.00 or -] or -]
0.00 0.00
Sodium g/l 144.00 [+ 144.00 [+ 143.20 140.75
(mmol/l) or -] or -] 0.29 [+ or [+ or
0.29 -] 0.18 -] 0.61
Potassium g/l 4.54 [+ or 4.16 [+ or 3.95 [+ 4.65 [+
(mmol/l) -] 0.15 -] 0.05 or -] or -]
0.02 0.41
Chlorine g/l 102.66 [+ 105.20 [+ 105.25 104.00
(mmol/l) or -] or -] 0.67 [+ or [+ or
0.22 -] -] 0.58
0.20
Histopathological study
No lesions or pathological changes attributable to treatment with MECB were observed in the organs of either sex of the treated rats (data not shown), compared to their respective normal groups.
Discussion
Cinnamon is commonly used in the spice and flavour industries (Jayaprakasha et al. 2007). Often, the powder of Cinnamon bark is used in the preparation of many types of desserts, spicy candies and liqueurs, as well as in tea (Khalid et al. 2007). Despite its widespread uses, the potential toxicity of this plant must be investigated thoroughly to provide information on the safety use of the plant. Therefore, the present study was conducted to determine the possible harmful effects of Cinnamomum burmannii to experimental animals at the specific doses chosen. The toxicity of MECB in rats was evaluated by both acute 14-day and sub-chronic 28-day toxicological studies.
There are various literature reports on the beneficial effects of trans-cinnamaldehyde (Zhang et al. 2010) and coumarin. Coumarin was selected as one of the markers since it has been reported as one of the toxic constituents of extracts derived from the stem bark of Cinnamomum species (Woehrlin et al. 2010). Comprehensive report on coumarin metabolism, toxicity and carcinogenicity and its relevance for human risk assessment has been described (Lake 1999). However, literature reports by Woehrlin et al. (2010) showed that there is a wide variation of coumarin levels in the genus Cinnamomum even within a single tree. Therefore, the objectives of the chromatographic analyses were to determine the levels of coumarin and trans-cinnamaldehyde in the MECB before administration to the rats and to ensure the safe utilisation of Cinnamomum burmannii stem barks as spices and favouring agent. The levels of coumarin (0.07%, w/w) and trans-cinnamaldehyde (0.20%, w/w) determined were consistent with the report by Abraham et al. (2010) which showed that coumarin levels in Cinnamomum burmannii (cassia sticks) analysed by the German control authorities was below 1000 mg/kg. Tolerable daily intake (TDI) of 0.1 mg of coumarin per kg body weight was derived by European Food Safety Authority (EFSA 2004).
According to Mohamed et al. (2011), in an acute 14-day toxicity study, a specific range of doses was selected and performed on experimental animals. The purpose was to determine the proper dose(s) for the subsequent sub-chronic 28-day toxicological study. In this experiment, MECB, at a single close of 2000 mg/kg, was administered orally to five female Sprague-Dawley rats. The rats were observed daily for any signs of toxicity and disturbance of general behaviours during the entire period of the experiment. Based on observations, the extract did not cause any signs of toxicity in the treated rats. The general behaviours of the treated rats were no different from the controls. Thus, this study indicated that the MECB produced little or no short-term effects on the treated rats. No significant mortality or alteration on the behaviour patterns of the treated rats were observed compared to their respective controls. This suggested that the MECB did not induce acute toxicity at the dose tested. The [LD.sub.50] of the extract is found to be more than 2000 mg/kg.
In sub-chronic 28-day toxicity study, the rats were treated orally by gavage with the MECB at different doses (500, 1000 and 2000 mg/kg) daily for 28 days. According to Teo et al. (2002), slight changes in bodyweight gain and internal organ weight will be detected if the rats were exposed to potential toxic substances. From the results, however, the bodyweight of treated and control rats was similar (Fig. 3) with no significant difference detected. The present study showed that the bodyweight of all treated rats increases gradually throughout the experimental period, and the increase during the 28-day toxicity study was not significantly different compared to the control group (p>0.05). In addition, no significant differences were observed in gross anatomy, weight, size or colour by microscopic examination of the internal organs between the treated and control groups (data not shown). The results obtained were comparable to those in the acute toxicity. Both control and treated rats of both sexes appeared generally healthy during and throughout the experimental period. No mortality was recorded and no toxicity signs were detected in any of the treated rats.
According to Olson et al. (2000), hematological, gastrointestinal and cardiovascular adverse effects in animals have the highest overall concordance of toxicity with humans. Adeneye et al. (2006) also claimed that the haematopoietic system in animals can be served as an important index in the physiological and pathological status for both animals and humans. This is because the haematopoietic system is very sensitive to toxic compounds. Toxic materials will tend to affect and cause a certain degree of damage to the haematopoietic system and cause changes to the hematological parameters. Therefore, several important hematological parameters were selected and included in this study to evaluate the toxicity of MECB. From the results obtained and analysed statistically, between the rats fed with MECB and the control group (Table 1) in either sex, no significant differences were observed in any of the selected parameters. Therefore, this indicated that the MECB did not affect the haematopoiesis in rats and did not interfere or cause any damage to the circulating red blood cells, white blood cells or platelets.
Analyses of several biochemical parameters were also included in this toxicity study. According to Kaneko et al. (1997), when the cells were exposed to toxic substances, these toxic substances will cause a certain degree of damage to the cells and causing cellular injury. Hence, the cellular permeability of the injured cells is altered and releases the biochemical substances into the blood stream. These biochemical substances such as the transaminase enzymes (ALT. AST and ALP) are mostly found in the cytoplasm and mitochondria of many cells (chiefly in the heart muscle, liver and skeletal muscles) but less in the kidney, pancreas and erythrocytes (Costa et al. 2008). Therefore, these biochemical parameters were selected in such a way that an increase in the transaminase enzymes and total bilirubin levels in blood samples directly indicates hepatic toxicity (Costa et al. 2008), while the changes in the levels of creatinine, urea, sodium, potassium and chloride indicate kidney toxicity (Marcelo et al. 2002). We focused on these selected parameters because among all the organs, kidneys and liver are the first organs to show toxicity when they are exposed to potential toxic substances. However, based on the statistical analyses of these biochemical parameters, no significant difference was observed in any of the parameters tested for either the treated or normal rats. These data suggested that the MECB did not induce or cause any damage to the liver and kidneys of the rats and can be considered non-toxic to these animals.
Organ-to-bodyweight ratio (relative organ weight) is another indicator used to evaluate toxicity of toxic substances in rats (Rosidah et al. 2009; Teo et al. 2002). The weight of the damaged organ(s) will either increase (swell) or decrease and ultimately alter their organ-to-bodyweight ratio, when compared to their respective controls. The data obtained showed that no significant difference exists in the organ-to-bodyweight ratios between all the treatment groups and their respective control groups, in either male or female rats.
The toxicology profile of MECB was further studied by histopathological analysis and microscopic examination on those isolated organs from both the treated and control rats. Results showed that there were no significant changes or damages observed in the morphology of all the isolated vital organs, either from the rats ingested with MECB or normal rats. The isolated organs showed normal architecture (data not presented). This again suggested that daily oral administration of the MECB for 28 days did not cause any detrimental changes or morphological disturbances to the rats or to their vital organs.
In general, MECB did not induce either acute 14-day or sub-chronic 28-day toxicity in rats at the doses employed. The NOAEL of MECB determined was 2000 mg/kg bodyweight/day (Copplestone 1988).
Conclusion
Oral administration of MECB (containing 0.07% and 0.20% (w/w) of coumarin and trans-cinnamaldehyde, respectively) at the closes of 500, 1000 and 2000 mg/kg bodyweight/day to both male and female Sprague-Dawley rats did not cause any mortality. The extract did not induce either acute 14-day or sub-chronic 28-day toxicity in rats. In addition, all hematological and biochemical parameters of the treated groups (both sexes) were no different compared to those in the control groups. Furthermore, no significant differences were observed in the body or organ weights, nor did the MECB cause abnormalities in necropsy and histopathological findings. In this preclinical study, the MECB did not cause toxicity for 28 days and no adverse effects were observed in any of the treated rats at all the doses tested. Therefore, the NOAEL for the sub-chronic 28-day study was determined to be 2000 mg/kg bodyweight/day, the highest close tested.
Conflicts of interest statement
I declare that no competing interests existed for the authors or the institute before, during and after preparing and submitting this paper for review.
Acknowledgement
This study was supported by USM-Short Term Research Grant Scheme (304/PFARMAS1/6311040).
References
Abdul, R.J., 2009. Effects of cinnamon on blood glucose and lipids levels in diabetic patients (Type 1). African Journal of Biochemistry Research 3 (5), 181-184.
Abraham, K., Woehrlin, F., Lindtner, O., Heinemeyer, G., Lampen, A., 2010. Toxicology and risk assessment of coumarin: focus on human data. Molecular Nutrition and Food Research 54, 228-239.
Adeneye, A.A., Ajagbonna, O.P., Adeleke, T.I., Bello, S.O., 2006. Preliminary oral toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats. Journal of Ethnopharmacology 105, 374-379.
Bishnu, J., Sunil, L., Anuja, S., 2009. Antibacterial property of different medicinal plants: Ocimum sanctum, Cinnamomum zeylanicum, Xanthoxylum armatum and Origanum majorana. Kathmandu University Journal of Science. Engineering and Technology 5 (1), 143-150.
Cao, H.P., Polansky, M.M., Anderson, R.A., 2007. Cinnamon extract and polyphenols aVect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3-L1 adipocytes. Archives of Biochemistry and Biophysics 459, 214-222.
Celeghini, R.M.S., Vilegas, J.H.Y., Lancas, F.M., 2001. Extraction and quantitative HPLC analysis of coumarin in hydroalcoholic extracts of Mikania glomerata Spreng. ("guaco") leaves. Journal of the Brazilian Chemical Society 12 (6), 706-709.
Copplestone, J.F., 1988. The development of the WHO recommended classification of pesticides by hazard. Bulletin of the World Health Organization 66, 545-551.
Costa, S.J.H., Lima, C.R., Silva, E.J.R., Araiujo, A.V., Fraga, M.C.C.A., Ribeiro, E., Ribeiro, A., Arrucla, A.C., Lafayette, S.S.L., Wanderley, A.G., 2008. Acute and subacute toxicity of the Carapa guianensis Aublet (Meliaceae) seed oil. Journal of Ethnopharmacology 116, 495-500.
EFSA, 2004. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the commission related to coumarin. EFSA Journal 104, 1-36.
Jayaprakasha, G.K., Negi, P.S., Jena, B.S., Jagan, M.R.L., 2007. Antioxidant and antimu-tagenic activities of Cinnamomum zeylanicum fruit extracts. Journal of Food Composition and Analysis 20, 330-336.
Kaneko, J.J., Harvey, J.W., Bruss, M.L., 1997. Clinical Biochemistry of Domestic Animals. Academic Press, San Diego. USA. pp. 932.
Khalid, S.A., Dilshad, A., Saifeldein, B.A., Abdullah, H.A., 2007. Nutritive value, levels of polyphenols and anti-nutritional factors in Sri Lankan cinnamon (Cinnamomum zeyalnicum) and Chinese cinnamon (Cinnamomum cassia). Food Science and Agricultural Research Center, King Saud Universiti, 5-21.
Lake, B.C., 1999. Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment. Food and Chemical Toxicology 37, 423-453.
Marcelo, A.R., Emy, L.I., Renata, G., Roberto, K.N.C., Silvana, M.C., Joao, C.P.M., Ciomar, A.B., 2002. Toxicological studies on Stryphnodendron adstringens. Journal of Ethnopharmacology 83, 101 -104.
Mohamed, E.A.H., Lim, C.P., Ebrika, O.S., Asmawi, M.Z., Sadikun, A., Yam, M.F., 2011. Toxicity evaluation of a standardised 50% ethanol extract of Orthosiphon Stamineus. Journal of Ethnopharmacology 133, 358-363.
Mounia, O., Stephane, C., Linda, S., Monique, L., 2006. Antimicrobial effects of selected plant essential oils on the growth of a Pseudomonas putida strain isolated from meat. Meat Science 73, 236-244.
OECD., 2001. OECD guideline 425: acute oral toxicity--up-and-down procedure. OECD Guidelines for the Testing of Chemicals, vol. 2. Organization for Economic Cooperation and Development, Paris, France.
Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G., Bracken, W., Dorato, M., Deun, K.V., Smith, P., Berger, B., Heller, A., 2000. Concordance of toxicity of pharmaceuticals in humans and in animals. Regulatory Toxicology and Pharmacology 32, 56-67.
Pankaj, G., Abhishek, C., Kaushik, P., 2009. Laboratory evaluation of crude extracts of Cinnamomum tamala for potential antibacterial activity. Electronic Journal of Biology 5 (4), 75-79.
Pawar, V.C., Thaker, V.S., 2006. In vitro efficacy of 75 essential oils against Aspergillus niger. Mycoses 49 (4), 316-323.
Rosidah, Yam, M.F., Amirin, S., Mariam, A., Gabriel, A.A., Zaini Mohd., A., 2009. Toxicology evaluation of standardized methanol extract of Gynura procumbens. Journal of Ethnopharmacology 123, 244-249.
Shan, B., Cai, Y.Z., Brooks, J.D., Corke, H., 2009. Antibacterial and antioxidant effects of five spice and herb extracts as natural preservatives of raw pork. Journal of the Science of Food and Agriculture 89, 1879-1885.
Shan, B., Cai, Y.Z., Brooks, J.D., Corke, H., 2007. Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): activity against foodborne pathogenic bacteria. Journal of Agricultural and Food Chemistry 55, 5484-5490.
Teo, S., Strlig, D., Thomas, S., Hoberman, A., Kiorpes, A., Khetani, V., 2002. A 90-days oral gavage toxicity study of D-methylphenidate and D.L-methylphemdate in Sprague-Dawley rats. Toxicology 79, 183-196.
Woehrlin, F., Fry, H., Abraham, K., Preiss-Weigert, 2010. Quantification of flavoring constituents in cinnamon: high variation of coumarin in Cassia Bark from the German retail market and in authentic samples from Indonesia. Journal of Agricultural and Food Chemistry 58, 10568-10575.
Zhang, J., Liu, L., He, Y., Kong, W., Huang, S., 2010. Cytotoxic effect of trans-cinnamaldhyde on human leukemia K 562 cells. Acta Pharmacologica Sinica 31, 861-866.
Mariam Ahmad (a), *, Chung Pin Lim (a), *, Gabriel Akyirem Akowuah (b), Nur Najihah Ismail (a), Mohd. Akmal Hashim (a), Sook Yee Hor (a), Lee Fung Ang (a), Mun Fei Yam (a)
(a) School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia
(b) University College Sedaya International, 56000 Kuala Lumpur. Malaysia
* Corresponding authors at: School of Pharmaceutical Sciences. Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Tel.: +60 4 6533888x4962; fax: +60 4 6570017.
E-mail addresses: mariam@usm.my (M. Ahmad). Colin_lim85@yahoo.com (C.P. Lim).
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| Author: | Ahmad, Mariam; Lim, Chung Pin; Akowuah, Gabriel Akyirem; Ismail, Nur Najihah; Hashim, Mohd. Akmal; H |
|---|---|
| Publication: | Phytomedicine: International Journal of Phytotherapy & Phytopharmacology |
| Article Type: | Report |
| Geographic Code: | 9MALA |
| Date: | Sep 15, 2013 |
| Words: | 6345 |
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