AIP Conference Proceedings 2230, 020011 (2020); https://doi.org/10.1063/5.0007461 2230, 020011
© 2020 Author(s).
Subacute oral toxicity test of chitosan- alginate coated microparticle of Garcinia mangostana Linn extract
Cite as: AIP Conference Proceedings 2230, 020011 (2020); https://doi.org/10.1063/5.0007461 Published Online: 04 May 2020
Gede Bagus Yoga Satriadinatha, Siti Farida, Kamarza Mulia, and Desak Gede Budi Krisnamurti
ARTICLES YOU MAY BE INTERESTED IN
The combined process of coagulation-flocculation and membrane separations to treat wastewater from tofu industry
AIP Conference Proceedings 2230, 030010 (2020); https://doi.org/10.1063/5.0003027 Application of combination of coagulation-flocculation and membrane separation processes for tofu industrial wastewater treatment
AIP Conference Proceedings 2230, 030007 (2020); https://doi.org/10.1063/5.0003025 Sensitivity analysis of gas distribution pipeline risk assessment methodology in Indonesia AIP Conference Proceedings 2230, 030006 (2020); https://doi.org/10.1063/5.0002612
Subacute Oral Toxicity Test of Chitosan-alginate Coated Microparticle of Garcinia mangostana Linn Extract
Gede Bagus Yoga Satriadinatha
1, Siti Farida
2, Kamarza Mulia
3, Desak Gede Budi Krisnamurti
2, a)1Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
3Department of Chemical Engineering, Faculty of Engineeringe, Universitas Indonesia, Kampus UI Depok, West Java, 16424 Indonesia
Corresponding author: a) desak.gede@ui.ac.id
Abstract. Chitosan-alginate coated microparticle of Garcinia mangostana Linn extract promises an alternative treatment of colon cancer patients, as the component of Garcinia mangostana Linn extract has anticancer activity in colon cancer cell lines, and chitosan-alginate could deliver compound faster to colon and accelerate its absorption. However, there have not been any studies evaluating sub-acutely the toxicity effects of this compound on the liver (SGOT and SGPT) and clinical signs of toxicity. This study was conducted to evaluate the subacute toxicity effect of Chitosan-alginate coated microparticle of Garcinia mangostana Linn extract’s oral administration. Fifty BALB/c mice were divided into five groups, including doses of 0.5 gram/kg, 1.0 gram/kg, 2.0 gram/kg, negative control, and normal group. Each rat was given an extract by force-feeding via intragastric tube for 14 days (subacute). The results showed that differences in SGOT level (p = 0.061), SGPT level (p = 0.82), and weight gain (p = 0.076) were not significant between the treatment and control groups, accompanied by the absence of clinical signs during the study. These indicate that subacute administration of chitosan-alginate coated microparticle of Garcinia mangostana Linn extract is safe until a dose of 2.0 gram/kgBW. Further studies are needed to evaluate the toxicity effects of this extract on other parameters and in a longer duration of exposure.
INTRODUCTION
The mangosteen (Garcinia mangostana Linn) pericarp has been used extensively in traditional health practices.
The bioactive compounds contained in the mangosteen pericarp are believed to reduce abdominal pain, dysentery, cholera, infectious wounds, chronic ulcers, and other diseases in traditional Southeast Asian medical practices [1].
These bioactive compounds have also been shown to have chemopreventive effects,[2] through reactive oxygen species (ROS) stabilization, stimulation of cellular defense, and inhibition of lipid peroxidation events [3].
Xanthones, secondary metabolites isolated from mangosteen pericarp, are active compounds that contribute to the pharmacological effects of this fruit [4]. One of the most common Xanthones derivatives found in mangosteen SHULFDUSLVĮ-mangostin [5]. 3UHYLRXVVWXGLHVKDYHUHSRUWHGWKDWWKHFRQFHQWUDWLRQRIĮ-mangostin (1,3,6-trihydroxy- 7-methoxy-2,8-bis (3-methyl-2-butenyl)-9H-xanthen-9-one) correlates with the pharmacological activity of mangosteen pericarp extract [2].
7KHĮ-mangostin has been widely studied in various in vitro and in vivo studies. A study examining the anti- carcinogenesis property UHSRUWHGWKDWĮ-mangostin has a cytotoxic effect on oral epidermoid carcinoma (IC50 = 2.8 ȝJP/[6]. Į-mangostin also reported to have an inhibitory effect on several human colorectal adenocarcinoma cell line proliferation, including COLO 205 and MIP-101 [7]. Į-mangostin has been shown to have an inhibitory effect on the activity of enzyme DNA polymerase and topoisomerase [8]. 7KH IDFW WKDW Į-mangostin has an
LQKLELWRU\HIIHFWRQWKHSUROLIHUDWLRQRIFRORUHFWDODGHQRFDUFLQRPDLQYDULRXVFHOOOLQHVVKRZVWKHSRWHQWLDOIRUĮ- mangostin as a pharmacotherapy agent in cancer cases colorectal.
An oral acute toxicity test RI Į-mangostin compound extracted with ethyl acetate fraction was carried out by involving Sprague-Dawley rats as samples. In this study, it was reported that the LD50 was reached at a dose
>15.480 mg/kg BW. There were no clinical signs related to toxicity evaluated during 14 days of study, indicates that Į-mangostin extracted with ethyl acetate fraction is safe to use within that period and has a wide margin of safety [9].
,Q RUGHU WR LPSURYH WKH HIILFDF\ RI Į-mangostin, encapsulation of this extract was carried out with chitosan- alginate microparticles. This encapsulation has been demonstrated by Kamarza et al [10]. This technique was chosen because chitosan-alginate capsules have mucoadhesive properties in the colon epithelium, so that they can reduce the side effects caused by the administration of this drug within a certain time period [11]. An acute toxicity study has also been conducted to assess the toxicity of these preparations. In the study, administration of this preparation did not have a toxic effect on the liver (parameters in the form of SGOT and SGPT) and kidney (parameter in the form of creatinine BUN) at a single dose of 2 and 3 grams/kg BW [12].
Despite the promising pharmacological effects of the compounds Į-mangostin, preclinical testing of these compounds to be used extensively in the medical practice is still inadequate. Further testing is needed to evaluate the effectivity and toxicity of these preparations, which include subacute to chronic toxicity tests, and moreover, clinical trials before finally being used widely. For these reasons, we propose this study, which will evaluate the subacute toxicity effects of oral administration of Garcinia mangostana Linn extract, using ethyl acetate fraction and coated within chitosan-alginate capsules in mice. This toxicity effect was evaluated by observing liver function, which is one of the parameters of drug toxicity according to PerKBPOM RI nomor 7 Tahun 2014 Tentang Pedoman Uji Toksisitas Nonklinik secara In Vivo, as well as evaluating clinical signs that appear during treatment.
MATERIALS AND METHODS
Chitosan-alginate-Coated Garcinia mangostana Linn Extract Preparation
Extraction of mangosteen pericarp conducted by using the method demonstrated by Jung et al. [13]. Mangosteen was separated between its endocarp and pericarp. The mangosteen pericarp was cleaned and dried for five days.
Dried pericarp was milled to powder, and then macerated in ethyl-alcohol (96%) solution for seven days. The ratio of pericarp powder to Ethanol was 1:3 (w/v). During the maceration period, the mixture was stirred periodically.
After the maceration period, the mixture was then filtrated and ethyl-alcohol was evaporated with a rotary evaporator in order to get a thick extract [13]. The extract was then fractionated with ethyl acetate solvents to separate the active compounds from the extract obtained. This method was reported by Kamarza et al [10]. F001 thick extract was mixed with distilled water and ethyl acetate with a ratio of 1:1 (v/v). The ethyl acetate fraction was then separated, concentrated, and dried to obtain mangosteen extract [13].
Once Garcinia mangostana Linn extract is obtained, this extract is weighed according to the prescribed dose.
Solvents (GOM) 2% (w/w) and distilled water are used to liquefy the extract before administrated to mice. The volume given to mice is a maximum of 0.7 mL per 30 grams of body weight.
Preparation of Animals for Experiment
Fifty mice are enrolled to this study, which was then grouped into 0.5 gram/kgBW (Group M1), 1.0 gram/kgBW (Group M2), 2.0 gram/kgBW (Group M3), negative control group (GOM only), and normal group (Aquadest only).
Based on PerKBPOM RI nomor 7 Tahun 2014 Tentang Pedoman Uji Toksisitas Nonklinik secara In Vivo, experimental animals must be 6-8 weeks old with variation in body weight is less than 20%. The temperature of the experimental room is made 22oC ± 3oC with 30-70% relative humidity, and with 12 hours of light and dark cycles.
The mice were fed with standardized mice food. During the treatment, the mice had access to water [14].
This study was conducted in accordance with ethical approval issued by Komite Etik Penelitian Kesehatan, Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo Hospital in 2018 with the registration number of 0430/UN2.F1/ETIK/2018.
Treatment of Animals
Extracts are administered by force-feeding every day for 14 days. The animals are fasted for 2 hours before being fed. The dose given to the mice was 0.5 gram/kgBW (Group M1); 1.0 gram/kgBW (Group M2); and 2.0 gram/kgBW (Group M3). There is also negative control (GOM only) as well as normal (Aquadest only) group.
During the administration of the treatment (extract), clinical signs related to toxicity are observed, including: (1) Skin changes; (2) Change of fur; (3) Change of eye; (4) Changes in mucous membranes; (5) Changes in secretions;
(6) Change in excretion; (7) Changes in the way of roads; (8) Strange behavior (walking backwards); (9) Seizures;
SGOTand (10) Others. On day 15, mice were terminated and blood was taken to determine SGOT and SGPT levels.
Measurement of Subacute Toxicity Effect (Liver Enzyme)
Blood serums collected from centrifugation were assessed to determine the level of SGOT and SGPT. The measurement of these liver enzymes took place in the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia. SGOT and SGPT levels of animals’ serum were reported in the mean ± standard error of mean U/mL serum.
Statistical Analysis
SGOT and SGPT levels are represented in numerical form. Data processing was done by comparing the values of SGOT and SGPT between the treatment and control groups, to find out if there is a significant difference in SGOT and SGPT levels between the treatment groups and control. In accordance with the design of this study, the statistical test method can be an Oneway ANOVA test or a Kruskal-Wallis test, according to the normality of data distribution.
The normality of data distribution was tested by the Shapiro-Wilk normality test, according to the sample size. If the data obtained is normally distributed, the Oneway ANOVA test will be used. If the data is distributed abnormally, data transformation will be carried out. However, if the data obtained are still not normally distributed, then the Kruskal-Wallis test is conducted to see if there is a significant difference between the treatment and control groups.
RESULTS AND DISCUSSION
Level of SGOT and SGPT, in mice terminated at the end of the treatment, showed the abnormal distribution in both the Shapiro-Wilk and Kolmogorov-Smirnov distribution test. Thus, the data will be presented in the form of a boxplot, which has the median, minimum, and maximum values of each dependent factor tested. The results of observations of body weight will also be presented in the form of the median value.
FIGURE 1. Changes in body mass were observed during treatment. Normal group (distilled water only) was found as a group with the highest weight of mice at the end of treatment, with a median weight of 30 grams (26; 36 grams).
Body mass is one of the factors that represent toxicity conditions. In this study, the group Normal (Aquadest) only was the group with the best trend of increasing body mass, as presented in Figure 1. All groups, other than M3 (dose 2.0 gram/kg BW), showed an increase in body mass at the end treatment compared to day 0. The M3 group (dose 2.0 gram/kg BW) was the only group that experienced a decrease in body mass at the end of treatment compared. Even though there were differences between the five groups, these differences were not significant.
Based on the Kruskal-Wallis nonparametric comparative test, which compared changes in mice body mass from day 0 to day 14, it was found that p = 0.076 (95% CI).
Clinical signs in the form of: (1) Skin changes; (2) Change of fur; (3) Change of eye; (4) Changes in mucous membranes; (5) Changes in secretions; (6) Change in excretion; (7) Changes in the way of roads; (8) Strange behavior (walking backwards); (9) Seizures; and (10) Others, are also not typically found in certain groups. Death before termination was found in the M3 group (2.0 gram/kg BW). One of the mice was found dead on day 10 of treatment. After surgery, the mice lungs showed infiltration of fluid, which then was known to be the extract.
Therefore, the death of mice is thought not to be caused by toxicity but due to aspiration (faulty administration of extracts). This is confirmed by the absence of clinical signs of toxicity found in mice days before death.
Table 1. Clinical signs evaluated during treatment (a) Toxicity signs were evaluated during treatment. One mouse died on day 10 due to aspiration during force-feeding. (b) Weight gain was also evaluated. Group M2 shows a decrease in median body
mass of -1 gram.
Toxicity
sign M1 M2 M3 Negative Normal
Skin changes Fur changes Orbital changes Mucosal changes Secretion changes Excretion changes Walking pathway changes Bizarre behavior Seizure
Mortality
ض
(1)(a)
Group Day 0 (gram)
Day 7 (gram)
Day 14 (gram)
Weight gain (gram)
M1 26
(25;29)
28 (25;29)
28 (23;32)
2 (-2;7)
M2 26
(25;28)
27.5 (24;30)
26.5 (18;33)
-1 (-8;8)
M3 27
(25;28)
27 (25;29)
26 (17;33)
0 (-10;7) Negative
control 26
(25;29) 28
(25;31) 29.5
(25;33) 3 (-1;6)
Normal 26
(25;28)
29 (25;33)
30 (26;36)
4.5 (0;10) (b)
Assessing toxicity based on clinical signs and changes in body mass is certainly not adequate in concluding the lethal dose of a substance to an organism. Therefore, researchers also evaluated signs of toxicity in the form of liver enzyme levels, SGOT and SGPT. The M1 group showed the highest SGOT value among the other groups, with a median of 104.76 (32.0; 261.9, 95% CI). The Aquadest only (normal) group was the group with the lowest median SGOT level, which was 62,565 (34.9; 174.6, 95% CI). The M1 group showed the highest SGPT value, with a median of 34.92 (11.64; 116.4; 95% CI). The lowest SGPT value was found in the GOM only group, with the SGPT level of 1.75 U/L, and the median SGPT level for the GOM only group was 12,513 (1.75; 55.29, 95% CI). The SGOT and SGPT results are described in the boxplot graph in Figure 2.
The difference in SGOT levels in each group was not significantly different based on the Kruskal-Wallis test (p=0.061; 95% CI). However, when each treatment group was compared individually with the control group, the difference was significant in the group M1, M2, and M3, with p values of 0.017, 0.017, and 0.030 respectively.
SGPT levels in serum in each group did not differ significantly, with a p-value of 0.82 (95% CI). Individual comparisons were also made, where the M1, M2, and M3 groups compared independently to the control group using the Mann-Whitney comparative test. However, all treatment groups differ insignificantly with both control groups.
The insignificant difference between the control and the treatment groups means that chitosan-alginate coated microparticle of Garcinia mangostana Linn extract did not have a significant effect on liver toxicity at all of treatment doses.
(a) (b)
FIGURE 2. Liver enzyme level of mice after treatment (a) SGOT (p=0.061, 95% CI) (b) SGPT (p=0.082, 95% CI). No significant difference was evaluated between treatment and control groups.
SGOT and SGPT are important parameters in assessing toxicity or even a disease course [15]. SGPT (Glutamic Pyruvic Transaminase Serum) or ALT (Alanine Amino Transferase) is an enzyme found in the kidneys, heart, muscles, and liver, with greater concentrations in the liver compared to other body tissues. Normal levels of SGPT in serum are 7–56 U/L. SGPT levels in serum are regulated at a certain level based on individual physiological or pathological conditions. The highest concentration of SGOT (Serum Glutamic Oxaloacetic Transaminase) or AST (Aspartate Amino Transferase) can be found in the heart, compared to the other tissue and/or organ in human body.
Normal SGOT levels in serum are 0 to 35U/L. The increase in these enzymes could be found in hepatitis patients, both due to infection, and alcohol or drug intoxication [16].
When the results of a study did not show significant differences in SGOT and SGPT levels between the treatment and control groups, the administration of those substances was considered as non-toxic substances to the liver. Thus, the administration of Garcinia mangostana Linn extracts microparticle coated chitosan-alginate does not have a toxicity effect on the liver until the extract dose is 2.0 gram/kgBW. This is also strengthened by the absence of clinical signs of toxicity during administration of extracts. The increase in body weight of treatment group mice at the end of the study was also insignificantly different from the control group. The fact that differences in SGOT levels (p = 0.061), SGPT level (p = 0.82), and weight gain (p = 0.076) were not significant between the treatment and control groups, accompanied by no clinical signs during the study, indicating that giving chitosan-alginate coated microparticle of Garcinia mangostana Linn extract is safe until a dose of 2.0 gram/kg BW.
Garcinia mangostana Linn extract is known to contain compounds with antioxidant and anticancer SKDUPDFRORJLFDO DFWLYLWLHV Į-mangostin. This compound has been investigated for its cytotoxic effects on oral epidermoid carcinoma (KB) cells ZLWK,&YDOXHRIȝJP/, breast cancer (BC-1) cell with IC50 value of 3.53 ȝJP/, and small cell lung cancer (NCI-H187) with IC50 value of ȝJP/[17]. Į-mangostin also has an inhibitory effect on human colorectal adenocarcinoma cell proliferation COLO 205 ȝJP/, MIP-101
ȝJP/, and SW 620 ȝJP/ [7]. When the anticancer potency and safety of this extract has been found out, then chitosan-alginate coated microparticle of Garcinia mangostana Linn extract is a promising substance that can be used as an alternative therapy in managing cancer patients. The purpose of coating with chitosan-alginate itself is to facilitate the delivery of this extract to the colon. The delivery efficiency of a substance when wrapped in chitosan-alginate has been proven in several studies [18,19]. Thus, chitosan-alginate coated microparticle of Garcinia mangostana Linn extract promises a new safe and effective alternative management of colon cancer patients.
CONCLUSION
We conclude that chitosan-alginate coated microparticle of Garcinia mangostana Linn extract was safe to a dose of 2.0 gram/kg BW. Thus, this extract promises a new safe and effective treatment of colon cancer patients.
However, further studies are needed to evaluate the toxicity effects of these extracts on other parameters and in a longer duration of exposure.
ACKNOWLEDGMENTS
This study was supported by National Institute of Health Research and Development, Ministry of Health Republic of Indonesia and Faculty of Medicine as well as Faculty of Engineering, Universitas Indonesia. The authors would also like to thank PITTA (Publikasi Internasional Terindeks untuk Tugas Akhir Mahasiswa) Grant for the financial supports in conducting this study (NKB-0532/UN2.R3.1/HKP.05.00/2019). This grant is presented by Direktorat Riset dan Pengabdian Masyarakat, Universitas Indonesia.
REFERENCES
1. M Guo, X Wang, X Lu, H Wang, PE BrodeliusĮ-Mangostin Extraction from the Native Mangosteen
*DUFLQLDPDQJRVWDQD/DQGWKH%LQGLQJ0HFKDQLVPVRIĮ-Mangostin to HSA or TRF. PLoS One 11, (2016).
2. MY Ibrahim, NM Hashim, AA Mariod, S Mohan, MA $EGXOOD$EGHOZDKDE6,HWDOĮ-Mangostin from Garcinia mangostana Linn: An updated review of its pharmacological properties. Arabian Journal of Chemistry 9(3), 317–29 (2016).
3. M Valko, D Leibfritz, J Moncol, MTD Cronin, M Mazur, J Telser. Free radicals and antioxidants in normal physiological functions and human disease. The International Journal of Biochemistry & Cell Biology 39(1), 44–84 (2007).
4. F Gutierrez-Orozco, ML Failla. Biological Activities and Bioavailability of Mangosteen Xanthones: A Critical Review of the Current Evidence. Nutrients 5(8), 3163–83 (2013).
5. EB Walker. HPLC analysis of selected xanthones in mangosteen fruit. J Sep Sci. 30(9), 1229–34 (2007).
6. T Suksamran, P Opanasopit, T Rojanarata, T Ngawhirunpat. Development of Alginate/Chitosan Microparticles for Dust Mite Allerge. Tropical Journal of Pharmaceutical Research 10(3), (2011).
7. R Watanapokasin, F Jarinthanan, Y Nakamura, N Sawasjirakij, A Jaratrungtawee, S 6XNVDPUDUQ(IIHFWVRIĮ-mangostin on apoptosis induction of human colon cancer. World J Gastroenterol 17(16), 2086–95 (2011).
8. Y Mizushina, I Kuriyama, T Nakahara, Y Kawashima, H <RVKLGD,QKLELWRU\HIIHFWVRIĮ-mangostin on mammalian DNA polymerase, topoisomerase, and human cancer cell proliferation. Food Chem Toxicol 59, 793–800 (2013).
9. F Rahmayanti, DF Suniarti, ZA Mas`ud, BM Bachtiar, Y Wimardhani, GP Subita. Acute Oral Toxicity Testing of Ethyl Acetate Fraction from Garcinia mangostana Linn Extract in Sprague-Dawley Rats. Res J Med Plants 10, 261–4 (2016).
10. K Mulia, N Halimah, E Krisanti. Effect of alginate composition on profile release and characteristics of chitosan-alginate microparticles loaded with mangosteen extract. AIP Conference Proceedings 1823(1), 020010 (2017).
11. A Philip, B Philip. Colon Targeted Drug Delivery Systems: A Review on Primary and Novel Approaches. Oman Medical Journal 25(2), 70–8 (2010).
12. AD Diba, TBG Purba, MW Wienarno, EA Krisanti, K Mulia, S Farida. Evaluation of encapsulated microparticles of Garcinia mangostana L. extracts on marker SGOT, SGPT, BUN and creatinine serum of BALB/c mice. AIP Conference Proceedings 2092(1), 030024 (2019).
13. H-A Jung, B-N Su, WJ Keller, RG Mehta, AD Kinghorn. Antioxidant Xanthones from the Pericarp of Garcinia mangostana (Mangosteen). J Agric Food Chem. 54(6), 2077–82 (2006).
14. Peraturan Kepala Badan Pengawas Obat dan Makanan Republik Indonesia Nomor 7 Tahun 2014 Tentang Pedoman Uji Toksisitas Nonklinik Secara in vivo [Internet]. HK.04.01.73.04.11.3361 p. 1. Available from: http://jdih.pom.go.id/
showpdf.php?u=b04A9w86hyKB%2Be6W2EBSvqz2wj0x%2FHHrSSRcBG%2F%2Bw QE%3D
15. S Gowda, PB Desai, VV Hull, AAK Math, SN Vernekar, SS Kulkarni. A review on laboratory liver function tests. The Pan African medical journal [Internet]. 2009 [cited 2019 Feb 7];3. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2984286/
16. BR Thapa, A Walia. Liver function tests and their interpretation. Indian J Pediatr. 74(7), 663–71 (2007).
17. S Suksamrarn, O Komutiban, P Ratananukul, N Chimnoi, N Lartpornmatulee, A Suksamrarn. Cytotoxic Prenylated Xanthones from the Young Fruit of Garcinia mangostana. Chem Pharm Bull. 54(3), 301–5 (2006).
18. P He, SS Davis, L Illum. In vitro evaluation of the mucoadhesive properties of chitosan microspheres. International Journal of Pharmaceutics. 166(1), 75–88 (1998).
19. R Hejazi, M Amiji. Chitosan-based gastrointestinal delivery systems. Journal of Controlled Release 89(2), 151–65 (2003).
