AIP Conference Proceedings 2092, 030024 (2019); https://doi.org/10.1063/1.5096728 2092, 030024

© 2019 Author(s).

Evaluation of encapsulated microparticles ofGarcinia mangostana L. extracts on markerSGOT, SGPT, BUN and creatinine serum ofBALB/c miceCite as: AIP Conference Proceedings 2092, 030024 (2019); https://doi.org/10.1063/1.5096728Published Online: 09 April 2019

Amirah Deandra Diba, Trienty Batari G. Purba, M. Wien Wienarno, Elsa A. Krisanti, Kamarza Mulia, and SitiFarida

Evaluation of Encapsulated Microparticles of Garcinia

mangostana L. Extracts on Marker SGOT, SGPT, BUN and

Creatinine Serum of BALB/c Mice

Amirah Deandra Diba1, Trienty Batari G Purba1, M Wien Wienarno2, Elsa A

Krisanti3, Kamarza Mulia3, and Siti Farida4,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, West

Java, 16424 Indonesia 4Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central

Jakarta 10430 Indonesia

f)Corresponding author: siti.farida@ui.ac.id

Abstract. Garcinia mangostana L. (mangosteen) pericarp containing α-mangostin is predicted to have potential as an

anticancer because it can induce apoptosis and has high antioxidant content. Moreover, the preliminary study showed that

this extract has high antioxidant activity and cytotoxicity based on the brine shrimp lethality test. This indicates that

mangosteen has potential as chemotherapeutic agents or additional therapy in cancers such as colorectal cancer, which the

available management is not as effective. To improve its efficacy and bioavailability in the colon as a targeted area, ethyl

acetate fraction of mangosteen extract was then formulated into a controlled release encapsulated microparticles by

chitosan-alginate material. It is intended that the active ingredient to be released after reaching the colon. In this study,

toxicity of mangosteen was investigated because there is still no toxicity data of encapsulated microparticles of the ethyl

acetate fraction of mangosteen extract on function of mice's kidneys (BUN/serum creatinine) and liver (SGOT/SGPT).

Twenty female BALB/c mice were divided into 4 groups. Three groups were given a single dose of 2, 3, and 5 g/kg body

weight (BW) mangosteen via intragastric feeding tube and one control group without mangosteen. The findings show that

marker of BUN, serum creatinine, SGPT, and SGOT are not significantly different from those of the control in the

administration of microparticles ethyl acetate fraction of mangosteen extract at a single dose of 2 and 3 g/kgBW, however,

significant increases of BUN and SGOT levels were found in single dose of 5 g/kgBW. The results indicate that

administration encapsulated of microparticles ethyl acetate fraction of G. mangostana L. extract at single dose of 2 and 3

g/kgBW cause no toxicity on liver and kidneys function, but the single dose of 5 g/kgBW. cause toxicity to the liver and

kidneys. Further studies on histopathology examinations of liver and kidneys are necessary to ensure its safety level.

Keywords: acute toxicity, control released, ethyl acetate fractionation, Garcinia mangostana L., microencapsulation

INTRODUCTION

Colorectal cancer is a cancer that is formed from the tissue of colon which consists of colon and or rectum [1]. In

Indonesia, colorectal cancer is ranked the third cancer with estimated mortality rate 9.5% among other cancer cases

[2-5]. The risk factors of colorectal cancer are genetic, limited activities, obesity, low fiber and high fat diet, low

vitamin D, smoking, alcohol consumption, and drugs [1,6,7]. With the westernization in Indonesia that affect life style

and diet, the incidence of colorectal cancer in Indonesia has increased to 12.8 per 100.000 [6-9]. However, the

3rd Biomedical Engineering’s Recent Progress in Biomaterials, Drugs Development, and Medical DevicesAIP Conf. Proc. 2092, 030024-1–030024-4; https://doi.org/10.1063/1.5096728

Published by AIP Publishing. 978-0-7354-1822-6/$30.00

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treatment of choice, surgery, is usually not affordable and gives many side effects. Other treatment options for

colorectal cancer are chemotherapy and radiotherapy [5]. However, these therapies have some major side effects and

limitations for Indonesia, and the costs are also high.

Knowing the limitations of these treatments, alternative treatment that costs less and has effective effects for

colorectal cancer is needed. One of the potential substances is from a fruit plant, Garcinia mangostana L. Many studies

found that the extract of G. mangostana L. is beneficial as an anticancer agent for cancers, including colorectal cancer

[10-15]. As colorectal cancer is located in the gastrointestinal tract, the medicine should be encapsulated with polymer

substances so the drugs can be slowly released in the colon. Chitosan-alginate encapsulation microparticles are proven

to be the basic formulation for controlled drug release [11].

In this research, the extract of G. mangostana L. which is encapsulated by chitosan-alginate microparticles was

given to 4 groups of BALB/c mice to find the oral acute toxicity of the drug with different doses on marker function

of mice’s kidneys using BUN (Blood Urea Nitrogen) and serum creatinine and liver using SGOT (serum glutamic

oxaloacetic transaminase) and SGPT (serum glutamic pyruvate transaminase). The objectives are to evaluate the effect

of this drug to SGOT, SGPT, BUN, and creatinine serum levels so it can be used as a consideration for a safe anti-

colon cancer agent. Elevation of marker levels of function kidneys and liver indicate toxicity and reduction of their

function.

MATERIALS AND METHODS

Plant material and extract preparation

The extract of chitosan-alginate microparticles encapsulated mangosteen was obtained as a result of a research that

was done by Faculty of Engineering, Universitas Indonesia. The pericarp of mangosteen was obtained in 2014 from

Solo and was labeled as Garcinia mangostana Linn. by Herbarium Bogoriense, Research Center for Biotechnology-

Indonesian Institute of Sciences (LIPI). Standard α-mangostin (98%) was from China and the chitosan (medical grade;

deacetilation degree of 93.6%; viscosity of 23.3 cp) was obtained from Biotech Surindo, Indonesia. Other materials

such as calcium chloride and sodium alginate were obtained from Merck. While Sodium tripolyphosphate (food grade)

was obtained from Brataco Chemical, Indonesia. The extract that was already within ethyl acetate fraction was then

suspended in Department of Medical Pharmacy, Faculty of Medicine Universitas Indonesia.

The dry pericarp of mangosteen was extracted using a modified procedure stated by Jung, et al [12]. Subsequently,

the extract was macerated using ethanol 96% for seven days applying mangosteen powder to ethanol ratio of 1:3 (m/v)

and periodic stirring was done to the mixture. The macerated mixture was then filtrated and evaporated under a reduced

pressure to be crude ethanolic extract. Afterwards, the mixture was fractionated using a mixture of ethyl acetate and

water with 1:1 volume ratio.The evaporation of this ethyl acetate fraction under reduced pressure generated mangostin

powder/paste [11]. The mangostin powder was then emulsified using gum arabic to form a more soluble drug to be

administrated.

Experimental Animals

Female BALB/c mice were obtained from Central of Health Research Development, Ministry of Health of the

Republic of Indonesia and acclimatized to the laboratory conditions. Twenty 6-8 weeks, nulliparous and not pregnant

mice, 25-30 g of body weight with 20% maximum of weight distribution were prepared. All mice were housed 5 per

cage in a room maintained at 25°C, 50 ± 10% relative humidity and a 12-hour-light/12-hour-dark cycle throughout

the experiment. All mice had free access to water and food before the beginning the experiment. All of the procedures

in this study have been approved by the Health Research Ethic Committee Faculty of Medicine, Universitas Indonesia

(Approval no. 0430/UN2F1/ETIK/2018).

Acute Oral Toxicity Study and Dose Administration

The mice were randomly divided into 4 groups (n=5) i.e. three experimental groups and one control group, with

each group contains five mice [16]. The extract in doses of 2, 3 and 5 g/kgBW. Mice are fasted for 3-4 hours (except

water) before treatment. The administration of mangosteen extract dissolved in a solvent (distilled water and gummi

arabicum), and it was administered through intragastric in a single oral dose by gavages using a feeding needle with

maximum volume 1 ml/100 g mice. The control group received an equal volume of solvent. The animals were then

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weighed and given a single dose of mangosteen extract based on their body weight. In the circumstance where it is

not possible to administer a dose with a single feeding, the extract of mangosteen can be administered several times

within 24 hours. The feeds may be given 1-2 hours after treatment. The mice were maintained within 14 days

observations. After administration, the mice were observed during the first day and each of the following 14 days to

look for any signs and symptoms of toxicity and death. After it was done, all of the remaining mice were sacrificed to

see the clinical biochemistry in their blood specimen.

Biochemical Analysis

After the 14 days observations, the remining mice were anesthetized for blood collection. Blood samples were

centrifuged and the supernatant were stored. Examination of clinical biochemistry including SGOT and SGPT levels

are used to determine the liver function, while BUN and serum creatinine concentration levels are used to determine

the kidney function. The blood samples was examined using Spectrophotometry.

Statistical analysis

All data was processed using Stat view 5.0 software. Statistical significance of the difference of clinical

biochemistry in the four groups was tested using one-way analysis of variance (ANOVA). Significance of difference

was considered at p <0.05. The difference among groups was analyzed using Fisher’s Paired Least Significant

Difference (PLSD) post hoc. If the P value is less than 0.05, the result will be considered significant.

RESULTS AND DISCUSSION

Table 1 shows that all mice groups given single dose of mangosteen extract had higher BUN levels than the control

group, but a significant increase occurred at the administration of 5 g/kgBW. Moreover, a significant increase of

SGOT levels was found in the 5 g/kgBW group. On the other hand, serum cretainine and SGPT did not show any

significant dose-related effects. This finding indicates that the administration of single dose encapsulated

microparticles of the ethyl acetate fraction of mangosteen extract at 2 and 3 g/kgBW did not affect the marker of

kidneys and liver function, but a single dose of 5 g/kgBW caused toxicity and reduction of the function of the liver

and kidneys of mice. These findings were different from study by Kosem who conducted repeated administration of

crude methanolic extract during a 14-day 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.

CONCLUSION

From this research, it can be concluded that G. mangostana L. extract with ethyl acetate fraction encapsulated by

chitosan-alginate microparticles is safe seen from kidneys and liver function at a dose until 3 g/kg BW. Therefore, this

extract is potential to be a safe anti-colon cancer drug. However, further research on histopathological examination

needs to be done to ensure the safety of this drug.

Dose administered

(g/kgBW)

Marker

BUN

Serum

creatinine SGPT SGOT

Control 13.03 ± 1.88 22.36 ± 0.58 4.60 ± 2.74 7.22 ± 1.14

2 15.84 ± 2.17 21.88 ± 0.21 5.29 ± 0.24 8.73 ± 1.78

3 15.44 ± 3.79 21.80 ± 0.20 5.24 ± 0.71 8.73 ± 1.81

5 19.45 ± 5.79* 21.92 ± 0.71 4.42 ± 1.66 12.22 ± 1.43*

TABLE 1. The marker level of function of kidneys and liver after administration of the G. mangostana L. extract

*p < 0.05 vs. control group (0 g/kg BW)

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ACKNOWLEDGMENTS

The research was funded by Hibah Publikasi Terindeks International untuk Tugas Akhir Mahasiswa UI (PITTA)

2018. This study, which is an experimental study using mice, has been approved by the ethical commissary of Faculty

of Medicine of University of Indonesia with serial number of 0430/UN2.F1/ETIK/2018. The authors would like to

appreciate also dr. Siti Farida, M.Kes for the support, assistance, and advices during this research. The funding of this

research was supported by the PITTA Grand from Universitas Indonesia with number of contract

2130/UN2.R3.1/HKP.05.00/2018.

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