ACUTE ORAL TOXICITY STUDY OF AQUILARIA CRASSNA AND α-TOCOPHEROL IN MICE

ACUTE ORAL TOXICITY STUDY OF AQUILARIA CRASSNA AND α-TOCOPHEROL IN MICE

Sheah Yee Ghan¹, Jin Han Chin², Yin Yin Thoo ³, Hip Seng Yim ¹ and Chun Wai Ho^*1

Department of Food Science and Nutrition ¹, Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras,56000 Kuala Lumpur, Malaysia.

Department of Biochemistry ², Faculty of Medicine, MAHSA University, Jalan Universiti Campus, Jalan Universiti, 59100 Kuala Lumpur, Malaysia.

School of Science ³, Monash University Malaysia, Jalan Lagoon Selatan,46150 Bandar Sunway, Selangor, Malaysia.

ABSTRACT: The combination of agarwood (Aquilaria crassna) young leave with α-tocopherol (α-TOH) showed a synergistic effect which helps to protect the food from lipid oxidation. The present study was designed to elucidate the acute toxicity of the mixture of A. crassna young leaves crude ethanolic extract (CE) and α-TOH on Balb/c mice. This acute oral toxicity study was carried out based on OECD 423 guidelines by using female Balb/c mice weighing 15-25 g. First group was served as control group which received dimethyl sulfoxide (vehicle) while second, third and fourth group were orally administered with single dose of 2000 mg/kg of CE, α-TOH and a mixture of CE and α-TOH (1:2), respectively. Throughout 14 days of observation, no behavioural changes were seen in all the treated animals. Body weight for each mouse and the relative organ weights for liver, kidney, lung, heart and spleen showed insignificant different (p>0.05) when compared to the control group. These results indicate the safety of the acute exposure of the A. crassna CE, α-TOH and the mixture of A. crassna CE and α-TOH at dose of 2000 mg/kg in female mice, without causing any adverse effects. The oral lethal dose(LD50)of A. crassna CE, α-TOH and CE/α-TOH mixture were suggested to be greater than 2000 mg/kg body weight in female mice.

Acute oral toxicity; Aquilaria crassna; α-Tocopherol; LD₅₀; Mice

INTRODUCTION: Plants have been extensively used as medicines since a thousand years ago and the use of herbal products should be based on scientific origin in order to make sure the plants are safe to consume. Unfortunately, many people underestimate the toxicity of natural products and use them without further scientifically proven.

Atropa belladonna and Digitalis purpurea are typical example for toxic herbal product which show severe systemic toxicity if taken orally ¹. Therefore, in order to prevent exposure to poisonous plants, the use of an appropriate animal model in toxicity test was conducted in present study. This is a key stage in ensuring the safety of the plant and acute toxicity studies are just one of the toxicity tests that are commonly used ².

Agarwood (Aquilaria crassna) is a kind of dark resinous heartwood which belongs to the Aquilaria genus of the Thymelaeceae family ³. The leaves of A. crassna are commonly consumed in the form of herbal tea for the treatment of high blood pressure, constipation, headache and diabetes ^4-6. In the recent years, the pharmacological properties of A. crassna leaves such as antibacterial, antipyretic and antioxidant have been previously reported ^5-6. The exhibited antioxidant activity is likely attributed to the presence of flavonoids, alkaloids, tannins, saponins, cardiac glycosides, mangiferin and genkwanin in A. crassna leaves ^7-8. α-Tocopherol(α-TOH) is a lipid-soluble antioxidant which readily donate hydrogen from the hydroxyl (-OH) group on ring structure to free radicals and itself becomes unreactive. It has been reported that plant polyphenols can help to reduce α-tocopheroxyl radical by regenerating α-TOH back to its active form, producing synergistic antioxidant effect ^9-10.

Antioxidant synergism has been observed when A. crassna young leaves crude extract (CE) combined with α-TOH at a ratio of 1:2 ¹¹. However, the safety for this plant is not well established. Up to date, no data have been documented on acute toxicity for the crude ethanolic leaves extract of A. crassna. The only data in this regard is the toxicity study for the aqueous leaves extract of A. crassna at 2000 and 15000 mg/kg body weight (bw) using mice ⁶. Scientific evidence for the antioxidant efficacy of A. Crassna young leaves and α-TOH are widely studied but systemic safety studies are still lacking. Therefore, it is essential to determine the potential adverse effects that might be expected after introduction of this CE and α-TOH (CE/α-TOH) mixture into the food system.

The present study was aimed to investigate the possible acute oral toxic effect of CE/α-TOH mixture in female Balb/c mice at a dose of 2000 mg/kg bw and to determine the LD₅₀ value of the mixture in experimental animals. The acute oral toxicity testing was carried out under the Organization for Economic Cooperation and Development (OECD) 423 guideline ¹².

METHODS AND MATERIALS:

Chemicals:

Ethanol and dimethyl sulfoxide (DMSO) were supplied by Merck, Darmstadt from Germany. α-Tocopherol was purchased from Sigma Aldrich, St. Louis, USA.

Extraction of Plant Materials:

61. crassna young leaves (first five leaves from the leaf buds) were collected from a plantation in Klang, Selangor, Malaysia and oven dried for 22 h at 50^oC in a convention oven (Model UNB 400, Memmert, Germany). The dried young leaves were grounded into powder form and extracted with 61.01% ethanol at room temperature ¹³. All the extracts were concentrated using multivapor (Multivapor P-6, Buchi, Switzerland) at 40^oC, followed by freeze drying (Alpha 1-4 LD Plus, Christ, Germany) at -50^oC for 24 h. The resulting crude extract (CE) was stored at -18^oC prior to further use.

Selection of Experimental Animals:

The present toxicity study was carried out in accordance to OECD 423 Test Guidelines with a single dose exposure ¹². A total of 20 healthy 8-12 weeks old female Balb/c mice, weighing about 15-25 g, were obtained from Sinar Scientific, Seri Kembangan, Selangor, Malaysia.

Assignment of Animals:

Female Balb/c mice were randomly divided into 4 groups comprising of 5 animals each (n=5). All the animals were identified by marking with black stain on the tail to ease observation. The animals were housed 5 in each polypropylene cage under standard animal housing conditions with controlled lighting (12 h dark-light cycles), temperature (25 ± 2^oC) and relative humidity (75 ± 5%).During the entire experimental period, the animals were provided with food and water ad libitum, except for the short fasting period where the drinking water was still in free access but no food supply was provided. The animals were allowed to acclimatise for 5 days before the experiment started. The experiment procedures were conducted after the approval by faculty ethical research committee of UCSI University (Proj-FAS-EC-14-038) and were in strict accordance with animal ethical committee guidelines for the care and use of laboratory animals.

Test Substance Administration:

The test substances were administered in a single dose by using oral gavage needle for mice and the animals were fasted 3 h prior to dosing. Following the period of fasting, animals were weighed and orally treated with a single dose of test substances at 2000 mg/kg bw. The composition of experimental treatments is listed in Table 1. First group was served as a control group which received dimethyl sulfoxide (DMSO) as the vehicle. The treatment groups received CE (Group 2), α-TOH (Group 3) and a mixture of CE/α-TOH which combined at a ratio of 1:2(Group 4), dissolved in 100%DMSO respectively. The administration volume was 10 mL/kg bw of the animal. The quantity of the test substance was calculated based on the body weight of the animal and prepared in DMSO before administered directly to the mice. After the administration of test substance, the animals were fasted for 2 h (only food was withheld but not water).

TABLE 1: EXPERIMENTAL TREATMENTS OF RESPECTIVE GROUPS

Observation Period:

All the animals were closely observed via cage-side observation during the first 30 min after treatment, followed by observation during the first 4 h for mortality, moribund or ill health, observed periodically for the next 24 h and once daily for the next 14 days ¹⁴. The animals were observed daily for the purpose of recording any signs of intoxication including changes in the skin and fur and behavioural changes.

Body Weight:

Body weight (g) of the female mice was individually weighed and recorded on day-0, day-3, day-7 and day-14 by using Mettler weighing balance (Mettler Toledo Type BD6000, Mettler-Toledo GmbH, Greifensee, Switzerland).

Organ Weight:

At the end of the experiment, the animals were sacrificed to obtain the relative organ weights. At day-14, all the animals were overnight fasting for 16 h. At day-15, the animals were sacrificed by cervical dislocation and measured the organ weights (g) namely liver, kidney, heart, spleen and lungs. The organs obtained from each treated mouse were grossly examined to see the changes compared to the control group and the relative organ weight (ROW) of each organ was then calculated.

Statistical Analysis:

The results were expressed as mean ± standard deviation (SD). Comparisons were made between the treatment groups with the control group using the one-way analysis of variance (ANOVA) followed by Dunnett’s test using Minitab 17 software (Minitab Inc., USA). In all cases, a p-value of < 0.05 was considered as significant difference when compared to the respective control group.

RESULTS: Based on the cage-side observation, all the female mice in treatment and control groups did not show noticeable toxic signs which included changes on skin and fur, abnormal behaviour pattern and coma post 24 h of treatment as well as during 14 days observation duration. No mortality was observed in any of the mice during this duration. In additional to that, there were gradual increases in body weight of treated and control mice on 0-14 days, as shown in Table 2. The changes in body weights and the relative organ weights of the treated mice (CE, α-TOH and CE/α-TOH mixture) were not significant difference (p>0.05) as compared to the control mice (Table 2 and 3). Based on macroscopic observation, gross examination of all the organs from the 3 treatment groups did not reveal any abnormalities or changes in the colour of the internal organs compared to the control group.

TABLE 2: ACUTE EFFECT OF AQUILARIA CRASSNA LEAVES CRUDE EXTRACT (CE), α-TOCOPHEROL (α-TOH) AND CE/α-TOH MIXTURE ON BODY WEIGHT CHANGES IN FEMALE MICE

Note. CE/α-TOH= mixture of A. crassna leaves crude extract and α-Tocopherol (1:2).

Value = mean ± standard deviation; n=5. Analysed using Dunnett’s test

TABLE 3: ACUTE EFFECT OF AQUILARIA CRASSNA LEAVES CRUDE EXTRACT (CE), α-TOCOPHEROL (α-TOH) AND CE/α-TOH MIXTURE ON RELATIVE ORGAN WEIGHTS IN FEMALE MICE

Note. CE/α-TOH= mixture of A. crassna leaves crude extract and α-Tocopherol (1:2).

Value = mean ± standard deviation; n=5. Analysed using Dunnett’s test

DISCUSSION: In present study, acute toxicity of CE, α-TOH and CE/α-TOH mixture were evaluated in female Balb/c mice. Female mice are commonly used in toxicity test because they are small, easy to handle and relatively economical to obtain ¹⁵. Additionally, female is more sensitive to see the effect of treatment compared to male ¹⁶. Prior to dosing, the animals were fasted for 3 h to prevent food and other chemicals in the digestive tracts in affecting the absorption and reaction of the compound.

Generally, the changes in body weight gain and internal organ weights of mice would reflect the toxicity after exposure to the toxic substances ¹⁷. The relative organ weight is used as one of the parameters to examine the severity of toxicants on the targeted organs. The heart, liver, kidney, spleen and lungs are the primary organs affected by drugs and chemicals ingested through the oral route ^17-18. Normally when the test substance is toxic, in acute response, it will damage liver and kidney, causing the relative organ weight for liver and kidney to increase due to swelling ¹⁹. Based on the result obtained, an increase in the body weight of the animal and insignificant effect (p>0.05) on the relative organ weights indicate that the administration of CE, α-TOH and CE/α-TOH mixture has negligible level of toxicity on the growth of the animals. This finding is in agreement with the result reported by Kamonwannasit et al. who demonstrated that no sign of toxicity or death was observed in mice treated with aqueous extract of A. crassna leaves at the doses of 2000 and 15000 mg/kg ⁶. Additionally, α-TOH was reported to have LD₅₀ greater than 2000 mg/kg bw in mouse ²⁰.

Lethal concentration (LD₅₀) is normally shown in acute toxicity study as the dose which kills 50% of the animals. The higher the LD₅₀ value, the lower the toxicity. According to the OECD 423 guideline method, a high starting dose of 2000 mg/kg bw was suggested in order to determine the maximum dose which is most likely to produce mortality in some of the treated animals. At this dose, no deaths, hazardous signs of toxicity or organ damages were recorded in these animals during the 14 days period of observation after acute treatment through the oral administration. This clearly indicates that the CE/α-TOH mixture does not cause any acute toxicity effect in mice and the same effect is likely to be demonstrated in human as well. As a result, it is concluded that the LD₅₀ of the CE/α-TOH mixture was greater than 2000 mg/kg in mice.

Due to the high price of naturally derived antioxidants, a new food additives trend where blending of antioxidants to cut costs has emerged. Young leaves are discovered to have higher antioxidant activity than mature leaves. The synergistic antioxidant interaction between A. crassna young leaves CE and α-TOH allows suppliers to partially replace the natural antioxidants with A. crassna young leaves to minimise the cost. With no sign of acute oral toxicity, CE/α-TOH mixture is valuable to the food industry as natural antioxidants and can potentially be incorporated into novel functional foods or beverages with optimum health benefits. However, acute toxicity studies have limitation in detecting the toxic effects on vital functions of cardiovascular, central nervous and respiratory systems which should be evaluated prior to human exposure²¹. Hence, further studies are recommended to assess the long-term safety of CE/α-TOH mixture using repeated dose-toxicity and safety pharmacology studies.

CONCLUSION: The normal behaviour of the treated animals during a period of 14 days suggests the non-toxic nature of the CE, α-TOH, and CE/α-TOH mixture since all the animals grow healthy and normal without being affected by the treatments. The single oral dose of the CE, α-TOH and CE/α-TOH mixture did not produce mortality or significant (p>0.05) changes in the body weight and relative organ weight of the animals. Hence, CE/α-TOH mixture at 2000 mg/kg bw exhibits no acute toxic effects in female Balb/c mice. Since acute toxicity study only provides preliminary data relevant to single exposure or over-dosage in human, further study on its chronic toxicity is essential for further support of this antioxidants mixture.

ACKNOWLEDGEMENTS: Financial support given by the University: CERVIE research grant scheme (Proj-In-FAS 007) is gratefully acknowledge. The authors would like to thank UCSI University for the laboratory facilities.

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    How to cite this article:

    Ghan SY, Chin JH, Thoo YY, Yim HS and Ho CW: Acute Oral Toxicity Study of Aquilaria Crassna and α-Tocopherol In Mice. Int J Pharm Sci Res 2016; 7(4): 1456-61.doi: 10.13040/IJPSR.0975-8232.7(4).1456-61.

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