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Research Article | Open Access
Mun Fei Yam, Chung Pin Lim, Lee Fung Ang, Lip Yee Por, Siew Tung Wong, Mohd. Zaini Asmawi, Rusliza Basir, Mariam Ahmad, "Antioxidant and Toxicity Studies of 50% Methanolic Extract of Orthosiphon stamineus Benth", BioMed Research International, vol. 2013, Article ID 351602, 10 pages, 2013. https://doi.org/10.1155/2013/351602
Antioxidant and Toxicity Studies of 50% Methanolic Extract of Orthosiphon stamineus Benth
The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000 mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000 mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between both male and female treated rats in any doses tested. No abnormality of internal organs was observed between treatment and control groups. The oral lethal dose determined was more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000 mg/kg per day.
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 Orthosiphon stamineus Benth (Lamiaceae) or locally called Misai Kucing, has attracted much attention for research purposes. Orthosiphon stamineus is also found in other countries such as Thailand, Indonesia, and Europe. It is widely used for the treatment of many diseases, especially those affecting the urinary tract, diabetes mellitus, hypertension, rheumatism, tonsillitis, and menstrual disorders . The benefits of the traditional use of Orthosiphon stamineus have also been supported by the isolation and identification of several possible active chemical constituents from this plant, including flavonoids [2, 3], terpenoids [4, 5], saponins, hexoses, organic acids, caffeic acid derivatives, chromene, and myo-inositol [4–6]. Among the reported compounds, the most important components of Orthosiphon stamineus leaves are the polyphenols; polymethoxylated flavonoids such as sinensetin and eupatorine; caffeic acid derivatives, which include rosmarinic acid, cichoric acid, and caffeic acid .
Notwithstanding the widespread and long time usage of this plant, little toxicological information is available regarding the safety following chronic consumption of O. stamineus especially the bioactive 50% methanolic extract which has been reported to be effective in protection against alcohol-induced gastropathy, CCl4-induced liver damage, antipyretic, anti-inflammatory, and analgesic effects [7–10]. Currently, Malaysian authorities are paying more and more attention on the safety and potential toxicity of botanicals, including medicinal plants and edible materials. Therefore, the aims of the present study were to evaluate the antioxidant and provide scientific data on the safety of O. stamineus, focusing on the 14-day acute and 28-day subchronic toxicity of bioactive 50% methanolic extract of O. stamineus, using Sprague-Dawley (SD) rats, and hence, providing guidance for selecting a safe dose of O. stamineus in its use as a traditional medicine.
2. Materials and Methods
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).
2.2. Plant Materials and Extract
Orthosiphon stamineus was grown from cuttings using standard agronomic practices at Kepala Batas, Pulau Pinang, Malaysia. The leaves of the plant were collected after flowering. The leaves were identified by Mr. Adenan Jaafar, School of Biological Sciences, Universiti Sains Malaysia. A voucher specimen (number 10810) was deposited at the Herbarium of School of Biological Sciences, Universiti Sains Malaysia. The leaves were rinsed and dried in oven at 40°C for 2 days. The dried leaves were then ground by an electric grinder to a coarse powder and weighed. Subsequently, 300 g of powdered leaves of O. stamineus was extracted with 5 L methanol : water (50 : 50 v/v) at 60°C (for 8 hours) by maceration method. The resulting 50% methanolic extract of O. stamineus (MEOS) was concentrated using a Büchi-RE121 evaporator (Büchi Laboratorium-Technik, Switzerland) equipped with a Büchi-B169 vacuum system and then lyophilized in a Hetovac VR-1 (HETO Lab Equipment, Denmark) freeze dryer. The weight of the MEOS was recorded and the final plant-to-extract ratio was about 6%. The MEOS was then kept in desiccators in a refrigerator (0–4°C). The MEOS was freshly prepared daily by dissolving in distilled water.
2.3. HPLC Study
2.3.1. HPLC Instrumentation
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.
2.3.2. Chromatographic Conditions
Chromatographic separations were achieved using an Agilent Eclipse Plus C18 (250 × 4.6 mm i.d.; 5 μm). A Zorbax guard fittings kit packed with replaceable Eclipse Plus C18 Guard column (12.5 × 4.6 mm i.d.; 5 μm) was used to protect the analytical column. A reverse-phase HPLC assay was carried out using an isocratic system with a flow rate of 1 mL/min, a column temperature of 25°C, and a mobile phase of acetonitrile : isopropyl alcohol : 20 mM phosphate buffer (NaH2PO4) (30 : 15 : 55 v/v), with pH adjusted to 3.5 using 85% phosphoric acid. The UV detection was set at 340 nm. The injection volume was 20 μL of solution. Total run time was less than 20 min for each injection. Data was acquired and processed with LC-Solution software. The peaks were detected at 340 nm and identified using reference standards of 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin. Sinensetin and eupatorin were selected as standards because these two compounds were found to be the bioactive ingredients in the plant [11–13].
2.4.1. Ferrous Ion Chelating Activity
Ferrous chelating activity was determined according to the method of Dinis et al. . MEOS was added to a solution of 0.05 mL ferrous chloride (FeCl2) (2 mM). The reaction was initiated by the addition of 0.2 mL ferrozine (5 mM) and the mixture was shaken vigorously and left standing at room temperature (24 ± 2°C) for 10 min. Absorbance of the solution was then measured spectrophotometrically (U-2000, Hitachi, Japan) at wavelength 562 nm. The percentage of inhibition of ferrozine-Fe2+ complex formation was calculated using the following formula: where was the absorbance of the control and was the absorbance in the presence of extracts. The experiment was run together with butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) as positive controls.
2.4.2. Hydroxyl Radical Scavenging Activity
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 . The reaction mixture consisted of 0.30 mL of 0.02 M sodium phosphate buffer (pH 7.0), 0.15 mL of 10 mM 2-deoxyribose, 0.15 mL of 10 mM FeSO4, 0.15 mL of 10 mM EDTA, 0.15 mL of 10 mM H2O2, 0.525 mL of H2O, and 0.075 mL of extract. The reaction mixture was incubated at 37°C for 2 h and the formed TBARS were measured. Seventy five milliliter of 2.8% trichloroacetic acid and 0.75 mL of 1.0% TBA in 50 mM NaOH were added to test tubes and boiled for 20 min. After cooling the mixture, absorbance was measured at 520 nm. The percentage of inhibition of inhibition rate of 2-deoxyribose oxidation by hydroxyl radical was given in the following formula: where was the absorbance of the control and was the absorbance in the presence of extracts.
2.4.3. Superoxide Anion Radical Scavenging Activity
Superoxide anion radical generated by the xanthine/xanthine oxidase system was determined spectrophotometrically by monitoring the product of nitroblue tetrazolium (NBT) . The reaction mixture consisted of 1.0 mL of 0.05 M phosphate buffer (pH 7.4), 0.04 mL of 3 mM xanthine, 0.04 mL of 3 mM EDTA, 0.04 mL of 0.15% bovine serum albumin, 0.04 mL of 15 mM NBT, and 0.04 mL of sample solution. After incubation at 25°C for 10 min, the reaction was initiated by adding 0.04 mL of 1.5 U/mL xanthine oxidase and carried out at 25°C for 20 min. After 20 min, the absorbance of the reaction mixture was measured at 560 nm. Decreased absorbance of the reaction mixture indicates increased superoxide anion radical scavenging activity.
2.4.4. Lipid Peroxidation Inhibition Study
Fresh livers were obtained from healthy male adult SD rats. They were then homogenized according to the method  for the preparation of 40% (w/v) liver homogenates in methanol. 80 μL of liver homogenate was mixed with 10 μL of Fenton’s reagent (containing 5 μL of 5 mM manganese chloride and 5 μL of 50 mM hydrogen peroxide) and 10 μL of various concentrations of MEOS (for control, the extract was replaced with distilled water). The mixture was incubated for 30 min at 37°C to produce lipid peroxidation. The mixture was then added with 900 mL FOX reagent (containing 49 mg ammonium ferrous sulfate in 50 mL of 250 mM H2SO4, 0.397 g of BHT, and 0.038 g of xylenol orange in 950 mL methanol). Absorbance of the mixture was read at 560 nm after 30 min of incubation at room temperature. The percentage of inhibition was calculated with the following equation: where was the absorbance in the presence of extracts and was the absorbance of the control.
2.5. Toxicology Studies
2.5.1. Experimental Animals
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 ad libitum. All the procedures were performed according to the Animal Ethics Guidelines of Universiti Sains Malaysia.
2.5.2. Acute Toxicity Study
The experiment was performed according to the Organisation for Economic Cooperation and Development (OECD) revised up and down procedure for acute toxicity testing . All the rats were fasted overnight and weighed before the extract was administered. A maximum dose of 5000 mg/kg of MEOS was administered by oral gavage to 5 healthy female adult Sprague-Dawley rats. The administration volume was adjusted between 1 and 2 mL for each and every rat. After administration of MEOS to the first rat, the rat was observed for clinical signs of toxicity for the first hour, then hourly for three hours, and then periodically throughout 48 hours. Other rats were administrated sequentially at 48-hour intervals if the first rat survived after 48 hours of treatment. All the experimental rats were maintained under close observation throughout the 14 days and the number of mortality was recorded. The LD50 was predicted to be above 5000 mg/kg if three or more rats survived after the experimental period .
2.5.3. Subchronic Toxicity Study
Experimental Sprague-Dawley rats of either sex were randomly assigned to 4 groups (; six males and six females per group), and their weights were recorded. Different doses of MEOS (1250, 2500, or 5000 mg/kg) were prepared in distilled water and administered daily as single doses to different groups of rats: group 1 (5000 mg/kg), group 2 (2500 mg/kg), and group 3 (1250 mg/kg), while group 4 (control) received only distilled water. Toxic manifestations and mortality were monitored daily for 28 days. The bodyweights of all the rats were measured and recorded at the end of every week and the treated rats were anesthetised using CO2 after 28 days of treatment. Blood samples were collected via cardiac puncture and transferred into nonheparinised and EDTA-containing tubes for both biochemical and haematological analyses, respectively . Thereafter, the rats were killed by cervical dislocation. All organs, brain, heart, lungs, thymus, liver, kidneys, adrenal glands, sex organs (ovaries and uterus for female rats; testes for male rats), spleen, stomach, and gut (begining from small intestine until the end of large intestine), of all experimental rats were excised, weighed, and examined macroscopically. Vital organs such as lungs, kidneys, livers, and stomach were then preserved in 10% formalin for histopathological study .
2.5.4. Relative Organ Weight
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% .
2.5.5. Blood Analyses
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) .
2.5.6. Histopathological Study
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 μm thick sections with a Reichert-Jung Histocut 820 II (Cambridge Instrument GmbH, Nussloch, Germany). All the sections were stained with haematoxylin and eosin and examined microscopically .
2.6. Statistical Analysis
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 taken as significant.
3.1. HPLC Analysis
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.
3.2. Antioxidant Activities
3.2.1. Ferrous Ion Chelating Activity
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 1).
3.2.2. Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activity was investigated by using the Fenton reaction. As shown in Figure 2, addition of less than 2 mg/mL of MEOS effectively inhibited the formation of hydroxyl radicals linearly up to 72%.
3.2.3. Superoxide Anion Radical Scavenging Activity
Superoxide anion radical scavenging activity of MEOS was determined by the xanthine oxidase system. Figure 3 showed the percentage inhibition of superoxide radical generated by BHT, BHA, and MEOS. The inhibition activity of MEOS reached 63% when 2 mg/mL of extract was used.
3.2.4. Lipid Peroxidation Inhibition Study
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.
3.3. Toxicology Studies
3.3.1. Acute Toxicity Study
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.
3.3.2. Subchronic Toxicity Study
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 4) or relative organ weights (Table 1) of all the treated rats. In addition, no death was recorded in both sexes of the treatment groups as well as their respective control group, during or after the course of the experiment. The controls and the treated rats appeared uniformly healthy at the end of the experiment and throughout the 28 days treatment period.
|Data are expressed as mean ± SEM.|
|Data are expressed as mean ± SEM.|
3.3.3. Blood Analyses
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 2). Meanwhile, analyses of biochemical parameters (alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), creatinine, bilirubin, urea, sodium, potassium, and chlorine) also showed no significant differences in any of the parameters tested between the control and the treated groups of both sexes (Table 3).
|Data are expressed as mean ± SEM.|
|Data are expressed as mean ± SEM.|