AIP Conference Proceedings 2092, 030020 (2019); https://doi.org/10.1063/1.5096724 2092, 030020
© 2019 Author(s).
Liver and kidney function analysis on mice given ethyl acetate fraction of Garcinia
mangostana as potential colorectal cancer therapeutic agent
Cite as: AIP Conference Proceedings 2092, 030020 (2019); https://doi.org/10.1063/1.5096724 Published Online: 09 April 2019
Andika Trya, M. Wien Wienarno, Kamarza Mulia, M. Idzhar Arridzal, Erni Hernawati Purwaningsih, and Siti Farida
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Liver and kidney function analysis on mice given ethyl acetate fraction of Garcinia mangostana as potential
colorectal cancer therapeutic agent
Andika Trya
1, M Wien Wienarno
2, Kamarza Mulia
3,5, M Idzhar Arridzal
1, Erni Hernawati Purwaningsih
4, and Siti Farida
4,a)1Undergraduate student, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2National Institute of Health Research and Development, Indonesian Ministry of Health, Jl. Percetakan Negara No.29, Central Jakarta 10560, Indonesia
3Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok 16424, West-Java, Indonesia
4Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
5Research Center for Biomedical Engineering, Faculty of Engineering Universitas Indonesia, Kampus UI Depok 16424, West-Java, Indonesia
.
Corresponding author: a)[email protected]
Abstract. The incidence of colorectal cancer in Indonesia reaches 12.8 per 100,000 adult populations with mortality rate reaching 9.5% of all cancer cases. Current chemotherapy widely used has not been satisfactory, quite expensive, and may produce untoward side effect. Therefore, it is necessary to develop an effective and safety alternative agent from herbs to eradicate colorectal cancer. Previous studies has shown that α-mangostin contained in mangosteen peel extracts and fraction (Garcinia mangostana) has high antioxidant content and activities as anti-inflammatory, antibacterial, and antivirus. To get a higher level of α-mangostin content, the fraction used in this study is acquired by ethyl acetate fraction, and so the fraction yields 25% α-mangostin. The preliminary study done by our research team showed that the fraction has high cytotoxicity based on brine shrimp lethality test and high antioxidant activity. These results indicate the potential of ethyl acetate mangosteen fraction as an anticancer agent. In this study, we investigate acute toxicity of ethyl acetate mangosteen fraction on liver and kidney function of mice. Twenty of female BALB/c mice are divided into 4 groups. Three groups are given intra gastric administration of a single dose of 2, 4, and 6 g/kg body weight (BW) of ethyl acetate mangosteen fraction and another group as a control. The findings show that the functional marker of kidney – blood urea nitrogen (BUN) and creatinine serum, and liver – serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT), do not differ from those of the control at a single dose of 2 g/kg BW of ethyl acetate fraction. However, an increase of serum creatinine occurs at the administration of dose 6 g/kg BW compared with the control. The results of this study indicate that a single dose of 2 and 4 g/kg BW of ethyl acetate fraction of Garcinia mangostana do not cause toxicity on liver and kidney function of mice. To ensure the safety level of the fraction, further histopathological examination is needed.
Keywords: ethyl acetate fraction, fraction, Garcinia mangostana L, kidney function analysis, liver function analysis,
INTRODUCTION
Colorectal cancer is the third highest cause of morbidity and mortality throughout the world [1].Colorectal cancer represents 9.4% of all incidences of cancer in males and 10.1% in women in the world [2]. The predicted prevalence of colorectal cancer in Indonesia is 12.8 per 100.000 adults [3]. Mortality rate of colorectal cancer in Indonesia reaches
9.5% from all cancer incidences [4]. Such high rates of morbidity and mortality of colorectal cancer are caused by high metastatic rates, difficulty on applying early detection, poor prognosis, and ineffective therapies of choice for colorectal cancer [5,6].
Chemotherapy, radiotherapy and surgery remain as therapies used in clinical settings for treating colorectal cancer.
The low efficacy, such high cost, and wide possibilities for side effects and potential complications of current therapies also remain as challenges for clinicians to successfully eradicate colorectal cancer in patients. Such challenges require adequate solutions, one which may be provided by using natural products derived from plants as a source of drugs to establish high efficacy and low complications on future patients.
One of the plants which has such potential to be the solution, as a chemopreventive and chemotherapeutic agent, is Garcinia mangostana Linn. A specific component found in the pericarp of Garcinia mangostana Linn. called α- mangosteen, according to studies, has other pharmacological effects other than chemopreventive and chemotherapeutic effects, such as antioxidant, anti-tumor, anti-allergy, anti-inflammation, antibacterial, antifungal, and antivirus effects [7-9].
The administration of chemotherapeutic agent to treat colorectal cancer has an extensive period of time. As so, toxicity tests to evaluate Garcinia mangostana Linn. (and at the same time, α-mangosteen) is required. An acute toxicity test done by Kosem8 using crude methanolic extract has shown that at 250mg/kg BW or higher dose, dose- related toxicity signs and symptoms had been observed. The toxicity signs and symptoms observed are significant weight loss and elevations of SGOT, SGPT, BUN levels, indicating general toxicity and loss of kidney and renal function.
Different from other experiments, our experiment used ethyl acetate fraction to acquire 25% α-mangosteen in the extract, a much higher concentration than using hydroethanolic extract [9].There is no further available data found on the toxicity and efficacy of ethyl acetate fraction of Garcinia mangostana Linn. Considering the remarkable potential of this natural agent as a chemotherapeutic agent with low cost, low side effects, and high efficacy, we wished to obtain new knowledge of this potential agent by analysing the liver and kidney function of mice given the fraction.
MATERIALS AND METHODS Plant Material and Extract Preparation
Mangosteen pericarp used in this study is acquired from Solo, Central Java, Indonesia. Sodium tripolyphosphate as the food grade is acquired from Brataco Chemical, Indonesia. Sodium alginate and calcium chloride are acquired from Merck. α-mangostin (98%) as the standard compound is acquired from Aktin Chemicals, China. Chitosan (medical grade) is acquired from Biotech Surindo, Indonesia. α-Amylase enzyme is bought from Claricem Indonesia.
β-glucosidase is bought from Sigma-Aldrich.
The extraction of mangosteen pericarp is done following the procedure reported by Jung et al with several modifications. The maceration is executed using ethanol 96% for 7 days with periodic stirring of the mixture. The ratio of mangosteen powder to ethanol ratio is 1:3 (w/v). The mixture is filtrated and evaporated, accompanied by reduction of the pressure. The fraction is then produced by using a mixture of ethyl acetate and water in 1:1 volume ratio. The fraction, evaporated under reduced pressure, produced mangosteen powder/paste [10].
Animals
Female BALB/c mice are acquired and acclimatized to the laboratory conditions. A total of 20 healthy, nullipara mice of 6-8 weeks old are chosen for the present study. They are provided with water and food to acclimatize them before starting the experiment. The rats are acclimatized for 3-7 days so that their weight would be ideal for the experiment (25-30 g). Experimental protocols are approved by the institutional ethical committee.
Dose Administration
The mice are divided into 3 experimental groups and 1 control group, with each group contains five mice. The extract in doses of 2, 4 and 6 g/kg body weight (BW) respectively dissolved in 1ml distilled water is administered intra gastric in a single oral dose by gavages using a feeding needle. The control group received an equal volume of distilled water. The number of survivors and the toxic symptoms shown is noted after 24 hours and these animals are maintained for further 14 days with daily observations.
Observation and Examination Methods
14 days after the extract administration, all survivors are anesthetized for blood collection. Blood samples are collected for analysing liver and kidney function by using blood chemistry values parameters.
Blood Chemistry Values Parameters
The blood is used for liver and kidney function test which is determined by blood urea nitrogen (BUN), creatinine, SGOT, and SGPT concentration level. SGOT and SGPT concentration level is used to determine the liver function, while urea and creatinine concentration level is used to determine the kidney function.
Statistical Analysis
The statistical significance of the data is analysed using one-way analysis of variance (ANOVA). Each of the dependent variables will be furtherly analysed using Fisher’s Least Significant Difference (LSD) post hoc. P value less than 0.05 is considered significant
RESULTS AND DISCUSSION
TABLE 1. Mean values in mice treated with the mangosteen extract after 14 days of observation
Parameter Dose administered (g/kg BW)
0 2 4 6
SGOT (U/L) 9.38±2.73 5.59±2.09 7.85±1.77 8.24±3.68
SGPT (U/L) 4.65±0.62 4.85±0.16 5.14±0.33 5.43±0.60
BUN (U/L) 26.31±8.75 23.39±2.31 21.71±1.53 24.39±13.68
Creatinine (U/L) 21.57±0.51 21.88±0.86 21.73±0.23 23.06±0.59*
In this study, the toxicity of ethyl acetate fraction of Garcinia mangostana extracts is done to determine the overall safety and the applicable dose for further animal experimentation using the same extract. From Table 1, it is shown that all mean values of SGOT and BUN did not show any significant dose-related effects. All mice groups had higher SGPT levels than the control group, yet the increase is not significant. There is, however, a significant increase of creatinine serum levels found in the high dose group compared to the control group and the 4 g/kg BW group, as shown in Figure 1. This finding indicated that the administration of high dose fraction caused a significance renal function loss observed by the increase of the creatinine serum levels.
**
*
In another experiment done by Kosem et al [9], the repeated administration of crude methanolic extract during a 14-days study showed renal and hepatic cell injury, resulting a significant increase of BUN in ≥500 mg/kg BW dose, SGPT in ≥250 mg/kg BW dose, and SGOT in ≥200 mg/kg BW dose. All mice given 500 mg/kg BW dose had also died before the end of the experiment. A study done by Towatana [10],however, has shown that the mice given 2000 and 5000 mg/kg BW of hydroethanolic extract of mangosteen pericarp did not show any signs of liver and kidney disfunction with no dose dependent increases and decreases of SGOT, SGPT, BUN, and creatinine levels on all mice groups.
CONCLUSION
Our study has the opportunity to use much higher dose of mangosteen fraction to as high as 6 g/kg BW using ethyl acetate fraction. The total of α-mangosteen held in the fraction is higher than the ones used in other studies, contains as much as 25% of α-mangosteen. Yet, we have managed to conclude that acute toxicity at doses lower than 6 g/kg BW of mangosteen fraction did not produce any significant dose-related increase of serum biochemistry parameters, thus no liver and kidney function loss had been observed. There is, however, a significant increase of serum creatinine levels shown at 6 g/kg BW dose, indicating renal function loss of the mice. Further histopathologic study must be conducted to confirm the safety level of the fraction used in this study.
ACKNOWLEDGMENTS
The authors would like to thank Research and Community Engagement Directorate of Universitas Indonesia through the PITTA Grant (Contract No. 2130/UN2.R3.1/HKR.05.00/2018) for the financial support, and Department of Medical Pharmacy, Universitas Indonesia for the technical support throughout the study.
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Control
2 g/kg BW
4 g/kg BW
6 g/kg BW
1 5 0 1 7 5 2 0 0 2 2 5 2 5 0
D o s e g r o u p
Serum creatinine value (g/dl)
FIGURE 1.
*p <0,05 vs. control group (0 g/kg BW), **p <0,05 vs. 4 g/kg BW group
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