The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of
Herbal medicines have received a great deal of attention as alternative medicines in recent years in Malaysia and are sold as dietary supplements. One of the Malaysian local herbs, scientifically known as
Notwithstanding the widespread and long time usage of this plant, little toxicological information is available regarding the safety following chronic consumption of
Ferrous chloride (FeCl2), hematoxylin, ferrozine, 2-deoxyribose, ethylenediaminetetraacetic acid (EDTA), ferrous sulfate (FeSO4), trichloroacetic acid (TCA), nitroblue tetrazolium (NBT), xanthine, xanthine oxidase sodium phosphate (dibasic), sodium phosphate (monobasic), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), manganese chloride, xylenol orange, manganese chloride, and sodium dihydrogen phosphate (NaH2PO4) were purchased from Sigma (St. Louis, MO, USA). Absolute alcohol and eosin were purchased from Riedel-de Haën (Seelze, Germany). Paraplast was purchased from Oxford Labware (St. Louis, MO, USA). Xylene was purchased from Fisher Scientific (Leics, UK). Disposable microtome blades 818 were purchased from LEICA (Germany). Thiobarbituric acid (TBA) was purchased from AppliChem (Darmstadt, Germany). 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin were obtained from Indofine Chemical Company (NJ, USA). HPLC-grade acetonitrile and isopropyl alcohol were purchased from Merck (Darmstadt, Germany).
HPLC analysis was performed using a Shimadzu-LC system (Shimadzu, Japan) equipped with a CBM-20A controller, LC-20AT pump, DGU-20A5 degasser, SIL-20A autosampler, SPD-20AV detector, and CTO-10ASvp column oven.
Chromatographic separations were achieved using an Agilent Eclipse Plus C18 (250 × 4.6 mm i.d.; 5
Ferrous chelating activity was determined according to the method of Dinis et al. [
Competition between deoxyribose and the extract against hydroxyl radical generated from the Fe3+/ascorbate/ethylenediaminetetraacetic acid (EDTA)/hydrogen peroxide (H2O2) system was measured to determine the hydroxyl radical scavenging activity of MEOS [
Superoxide anion radical generated by the xanthine/xanthine oxidase system was determined spectrophotometrically by monitoring the product of nitroblue tetrazolium (NBT) [
Fresh livers were obtained from healthy male adult SD rats. They were then homogenized according to the method [
Eight-week-old male and female Sprague-Dawley rats (160–190 g) were purchased from the Animal House, School of Pharmaceutical Sciences, Universiti Sains Malaysia. The rats were acclimatised to laboratory conditions for 7 days prior to performing the experiments. All rats were kept at 26 ± 3°C, with a light/dark cycle of 12 hours. The rats were housed in a single polycarbonate cage (3 rats per cage) with free access to food (normal laboratory chow, Gold Coin) and tap water
The experiment was performed according to the Organisation for Economic Cooperation and Development (OECD) revised up and down procedure for acute toxicity testing [
Experimental Sprague-Dawley rats of either sex were randomly assigned to 4 groups (
The excised organs were weighed individually. The relative organ weight index of each organ to its bodyweight was calculated as (weight of organ/bodyweight of rats on the day of sacrifice) × 100% [
Haematological and biochemical analyses were performed at the Pathology Laboratory, Lam Wah Ee Hospital, Penang. Complete blood cell counts were determined using a fully automated haematological analyser Abbott Cell-Dyn 3500 (Abbott Laboratories, IL, USA), while serum biochemistry tests were performed using a COBAS Integra 800 (Roche, Germany) [
Vital organs (kidney and liver) 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, USA) and cut into 5
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS). All the data are indicated as mean ± standard error of mean (SEM) and were analysed using one-way analysis of variance (ANOVA). Significant differences between the groups were determined using a Dunnett-comparison test with
The objective of HPLC analysis in this experiment was to standardize the MEOS using sinensetin, eupatorin, and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone. From the results, the percentages of sinensetin, eupatorin, and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone determined in MEOS were 1.12 ± 0.026%, 0.94 ± 0.025%, and 0.46 ± 0.016% (w/w), respectively.
Ferrozine can quantitatively form the complexes with Fe2+. Chelating activity of the MEOS with ferrous ion was 1.6% at 0.125 mg/mL. In addition, there was an increase of chelating activity to 30% at 2 mg/mL which was slightly lower than positive control (BHA and BHT) (Figure
Ferrous ion chelating activity of MEOS (
The hydroxyl radical scavenging activity was investigated by using the Fenton reaction. As shown in Figure
Hydroxyl radical scavenging activity of MEOS (
Superoxide anion radical scavenging activity of MEOS was determined by the xanthine oxidase system. Figure
Superoxide radical scavenging activity of MEOS (
Antilipid peroxidation activity of MEOS was determined by the FOX method. IC50 of liver homogenate lipid peroxidation of MEOS was 0.34 ± 0.024 mg/mL.
In this experiment, oral administration of 5000 mg/kg of MEOS did not cause any visible signs of toxicity to the rats. No changes were observed in the behavior and mortality of the animals over the 14 days. All five female SD rats survived until the end of the experiment with no mortality were recorded. The LD50 determined was greater than 5000 mg/kg.
In subchronic toxicity study, there were no observable changes in the general behaviour of all the treated rats (groups 1, 2, and 3) as compared to the control group. No significant changes were detected in either the bodyweights (Figure
(a) Relative organs weight of male rats orally treated daily with 50% methanolic extract of
% organ weight/bodyweight | Treatment for 28 days | |||
---|---|---|---|---|
|
50% methanolic extract of |
|||
1250 mg/kg | 2500 mg/kg | 5000 mg/kg | ||
Male | ||||
Brain |
|
|
|
|
Heart |
|
|
|
|
Liver |
|
|
|
|
Thymus |
|
|
|
|
Spleen |
|
|
|
|
Kidney (right) |
|
|
|
|
Kidney (left) |
|
|
|
|
Adrenal gland |
|
|
|
|
Lungs |
|
|
|
|
Testis (right) |
|
|
|
|
Testis (left) |
|
|
|
|
Stomach |
|
|
|
|
Stomach (empty) |
|
|
|
|
Gut |
|
|
|
|
Gut (empty) |
|
|
|
|
Data are expressed as mean ± SEM.
% organ weight/bodyweight | Treatment for 28 days | |||
---|---|---|---|---|
|
50% methanolic extract of |
|||
1250 mg/kg | 2500 mg/kg | 5000 mg/kg | ||
Female | ||||
Brain |
|
|
|
|
Heart |
|
|
|
|
Liver |
|
|
|
|
Thymus |
|
|
|
|
Spleen |
|
|
|
|
Kidney (right) |
|
|
|
|
Kidney (left) |
|
|
|
|
Adrenal gland |
|
|
|
|
Lungs |
|
|
|
|
Ovaries |
|
|
|
|
Uterus |
|
|
|
|
Stomach |
|
|
|
|
Stomach (empty) |
|
|
|
|
Gut |
|
|
|
|
Gut (empty) |
|
|
|
|
Data are expressed as mean ± SEM.
The effect of daily oral administration of MEOS on the bodyweight of (a) male and (b) female rats (
There were no significant differences observed in any of the haematological 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 any of the treated rats as compared to control rats (Table
(a) Haematological values of male rats treated with 50% methanolic extract of
Unit | Treatment for 28 days | ||||
---|---|---|---|---|---|
Control | 50% methanolic extract of |
||||
1250 mg/kg | 2500 mg/kg | 5000 mg/kg | |||
Male | |||||
White blood cell count | 109/L |
|
|
|
|
Neutrophils | 109/L |
|
|
|
|
Lymphocytes | 109/L |
|
|
|
|
Monocytes | 109/L |
|
|
|
|
Eosinophils | 109/L |
|
|
|
|
Basophils | 109/L |
|
|
|
|
Red blood cell count | 1012/L |
|
|
|
|
Hemoglobin | g/L |
|
|
|
|
Hematocrit | % |
|
|
|
|
Mean red blood cell volume | fL |
|
|
|
|
Mean corpuscular Hb | pg |
|
|
|
|
Mean corpuscular Hb concentration | g/L |
|
|
|
|
Platelets | 109/L |
|
|
|
|
Mean platelet cell volume | fL |
|
|
|
|
Data are expressed as mean ± SEM.
Unit | Treatment for 28 days | ||||
---|---|---|---|---|---|
Control | 50% methanolic extract of |
||||
1250 mg/kg | 2500 mg/kg | 5000 mg/kg | |||
Female | |||||
White blood cell count | 109/L |
|
|
|
|
Neutrophils | 109/L |
|
|
|
|
Lymphocytes | 109/L |
|
|
|
|
Monocytes | 109/L |
|
|
|
|
Eosinophils | 109/L |
|
|
|
|
Basophils | 109/L |
|
|
|
|
Red blood cell count | 1012/L |
|
|
|
|
Hemoglobin | g/L |
|
|
|
|
Hematocrit | % |
|
|
|
|
Mean red blood cell volume | fL |
|
|
|
|
Mean corpuscular Hb | pg |
|
|
|
|
Mean corpuscular Hb concentration | g/L |
|
|
|
|
Platelets | 109/L |
|
|
|
|
Mean platelet cell volume | fL |
|
|
|
|
Data are expressed as mean ± SEM.
Biochemical values of male and female rats treated with 50% methanolic extract of
Unit | Treatment for 28 days | ||||
---|---|---|---|---|---|
Control | 50% methanolic extract of |
||||
1250 mg/kg | 2500 mg/kg | 5000 mg/kg | |||
Male | |||||
Aspartate transaminase (AST) | U/L |
|
|
|
|
Alanine aminotransferase (ALT) | U/L |
|
|
|
|
Urea | U/L |
|
|
|
|
Creatinine |
|
|
|
|
|
Alkaline phosphatase (ALP) |
|
|
|
|
|
Bilirubin | mmol/L |
|
|
|
|
Sodium | g/L |
|
|
|
|
Potassium | g/L |
|
|
|
|
Chlorine | g/L |
|
|
|
|
Female | |||||
Aspartate transaminase (AST) | U/L |
|
|
|
|
Alanine aminotransferase (ALT) | U/L |
|
|
|
|
Urea | U/L |
|
|
|
|
Creatinine |
|
|
|
|
|
Alkaline phosphatase (ALP) |
|
|
|
|
|
Bilirubin | mmol/L |
|
|
|
|
Sodium | g/L |
|
|
|
|
Potassium | g/L |
|
|
|
|
Chlorine | g/L |
|
|
|
|
Data are expressed as mean ± SEM.
No lesions or pathological changes were observed in the organs of either sex of the MEOS treated rats as compared to their respective control groups.
Antioxidant capacity is one of the commonly used parameters to determine the bioactive components which exhibited pharmacological activities.
In acute 14-day toxicity study, a limited dose was selected and performed on the experimental rats. Its purpose was to determine a proper range of doses to be used in the subsequent subchronic 28-day toxicological study [
During the subchronic 28-day toxicity study, the rats were treated orally with different doses of MEOS (1250, 2500, and 5000 mg/kg/day) for 28 days. The results obtained were comparable to those in the acute toxicity study. 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. Both evaluation of the acute and subchronic toxicity of MEOS had indicated that a single oral administration of MEOS up to 5000 mg/kg dose caused neither visible signs of toxicity nor mortality to experimental rats. Hence, the LD50 of MEOS determined is more than 5000 mg/kg.
Generally, an increase or decrease in bodyweight of an animal has been used as an indicator of adverse effect of drugs and chemicals [
In addition to bodyweight, relative organ weight has also been used as another basic indicator to determine whether the rats have been exposed to harmful agents [
Analysis of blood parameters in animal studies could be very useful when evaluating the risk of human toxicity as the changes in the haematological system provide a predictive value for toxicity [
Several important biochemical parameters were also included in this toxicity study. These biological parameters are indicators for organ toxicity. For example, kidney functions were evaluated by means of serum urea and creatinine. Increase of blood creatinine has been shown to be a good indicator of negative impact in kidney functions [
Histopathological studies were conducted on vital organs of all the rats. According to Wang et al., impaired organs often have abnormal atrophy [
This finding is comparable with Mohamed et al.’s [
Mariam Ahmad declares that no competing interests existed for the authors or the institute before, during, and after preparing and summiting this paper for review.
This study was funded by Fundamental Research Grant Scheme (FRGS) 203/PFARMASI/6711306 and FRGS/FP31-2012A, UMRG Program (Sub-Program 3/RP003C-13ICT), and High Impact Research Grant (UM.C/625/1/HIR/MOHE/FCSIT/15) from Universiti Sains Malaysia, University of Malaya, and Ministry Education Malaysia.