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th
The 6 International Conference on Natural Products for Health and Beauty (NATPRO6)
January 21-23, 2016
Organized by Khon Kaen University
In vitro-In silico of alpha- beta- gamma-mangostin from
Garcinia mangostana pericarp extract
Songpol Limpisood*a, Pattarawit Rukthongb, Nattawut Sukkhumb
Nawong BoonnakC, Korbtham Sathirakula,d
a
Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 10210
Department of Pharmaceutics, Faculty of Pharmacy, Srinakarinwirot University, Nakornnayok
Thailand 26120
c
Department of Chemistry, Faculty of Science, Thaksin University, Phatthalung, Thailand 93110
d
Drug Discovery and Development center, Thammasart University (Rangsit campus), Pathum Thani,
Thailand
b
*[email protected]
phone +66-864101454
ABSTRACT
The purpose of experiment was to predict pharmacokinetics properties and pharmacodynamic properties of
alpha- beta- gamma -mangostin extracted from Garcinia mangostana L. pericarp. The study on pharmacokinetics
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properties was conducted via ADMET Predictor software. The software was used as a predictive modeling of
absorption, distribution, metabolism, elimination, and toxicity (ADMET) of chemical substances from molecular
structures. ADMET risk rule set, S+Absn_Risk (risk of low absorption from oral dose), TOX_Risk (overall toxicity risk) and
CYP_Risk (metabolic risk) plus two additional rules, high plasma protein binding and high steady-state volume of
distribution were used to determine the comparative relation between efficacy and safety. From overall of ADMET risk
rule set, the results showed that from 24 criteria, the scores of 2.95, 2.65 and 2.17 were for alpha- beta- and gammamangostin respectively. In general, the scores of 0-24 indicates the number of potential ADMET problem. These findings
may be valuable to explain pharmacokinetics data of these compounds. For pharmacodynamic study, the cytotoxicity on
human MCF7 breast carcinoma cells were tested. The average value of half maximal inhibitory concentration (IC50) of
alpha- beta- gamma-mangostin and 5-FU to MCF7 cells were 34.52±4.38, 83.54±0.87, 92.69±0.35 and 64.00±1.52
respectively. According, alpha-mangostin is the most cytotoxic to MCF7 cells. However, alpha-mangostin has highest
ADMET risk score. Thus, further study of mangostins should focus on the optimization of their pharmacokinetic and
pharmacodynamic properties.
Keywords: anticancer activity, Garcinia mangostana, alpha- beta- gamma-mangostin
1. INTRODUCTION
In silico Biopharmaceutics study has found widespread effectiveness as vital tool in drug discovery and
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development [1]. The ADMET Predictor software was used as a predictive modeling of absorption, distribution,
metabolism, elimination, and toxicity (ADMET) of chemical substances from molecular structures. Mangosteen (Garcinia
mangostana L.), a tropical evergreen tree with purple-red fruit, its thick rinds have been used as traditional medicine
treatment for skin wounds, diarrhea and infection [2]. The pericarp crude extract can be isolated to fifty xanthone
compounds, alpha-, beta- and gamma-mangostins, gartanin etc [3]. The main isolated xanthone, yellow color from
pericarp mangostin was name as mangostin [4.]. Several studies have been designed to investigate mechanism of
inducing cell death via mangostins treatment in cancer cells line. [5-7]. Breast cancer is the most frequently diagnosed
cancer among women in worldwide [8].Progression of breast cancer is a multistep process, which involves hormones and
genes. Estrogen is a hormone which activates growing of breast cancer [9]. MCF7 cell line is a general studied model for
hormone-dependant human breast cancer. These cells contain functional estrogen receptors and show a pleotropic
response to estrogen. Estrogen stimulates proliferation of these cells in vitro[10]. Therefore the purposes of this study
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th
The 6 International Conference on Natural Products for Health and Beauty (NATPRO6)
January 21-23, 2016
Organized by Khon Kaen University
are to predict pharmacokinetics properties of mangostins via in silico and to investigate effect of mangostins on MCF7
breast carcinoma cells.
2. MATERIALS AND METHODS
Prediction pharmacokinetic porperties of mangosteen pericarp extract
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MedChem Designer sofeware version 2.0.0.34, Simulations Plus, Inc. was used to create illustration of
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xanthones’s IUPAC chemical structure in specific file type for ADMET Predictor . ADMET Predictor software version
6.0.0007, Simulations Plus, Inc. was used to predict pharmacokinetic properties of mangostins shown in Table 1.
Table 1. Summary of ADMET risk 24 scores via ADMET Predictor™ modules
ADMET risk
S+Absn_Risk (risk of low absorption from oral dose)
1. Too large molecule(Sz)
2. Too flexible molecule(Rb)
3. Too many H-bond donors(HD)
4. Too many H-bond acceptors(HA)
5. Excessive partial atomic charge(ch)
6. Too lipophilic(ow)
7. Low permeability(Pf)
8. Low solubility(Sw)
TOX_Risk (overall toxicity risk)
9. hERG liability(hE)
10. Acute toxicity in rats(ra)
11. Carcinogenicity in chronic rat studies(Xr)
12. Carcinogenicity in chronic mouse studies(Xm)
13. Human liver hepatotoxicity(Hp)
14. SGOT and SGPT elevation in blood (severe hepatic injury)(SG)
15. Mutagenicity (following five strains of Salmonella typhimurium,
each with and without microsomal activation)
CYP_Risk (metabolic risk)
16. High metabolic clearance by human CYP P450 1A2(1A)
17. High metabolic clearance by human CYP P450 2C19(19)
18. High metabolic clearance by human CYP P450 2C9(C9)
19. High metabolic clearance by human CYP P450 2D6(D6)
20. High metabolic clearance by human CYP P450 3A4(3A)
21. Inhibition of human CYPP450 3A4 with midazolam as substrate(mi)
22. Inhibition of human CYPP450 3A4 with testosterone as substrate(ti)
23. Plasma protein binding(fu)
24. Steady-state volume of distribution(Vd)
Criteria
MWt. > 500, Number atom > 35
Number of free lotate bond > 10
H-bond Donor Protons > 4
H-bond acceptors > 9
NPA partial atomic charges > 21
logP > 4.5 or logD >3.5
jejunal < 0.25 or MDCK<30
Solubility in water <= 0.010
TOX_hERG > 6
TOX_RAT < 320
TOX_BRM_Rat
TOX_BRM_Mouse < 25
TOX_MUT_Risk > 2
Is substrate and CLint in each
CYP > 30
PrUnbnd < 3.5%
Vd > 5.5
Primary cell culture
Human MCF7 breast carcinoma cells were grown in T-25 flask at 37 °C in an atmosphere of 5% CO2. The
adherent cells were cultured in Dulbecco's Modified Eagle media(DMEM) with 10% Fetal bovine serum and 1% Pen strep.
The medium was replaced regularly three times a week until the flask reach 80% confluence. The cells were moved from
the flasks by incubating the monolayers with 0.5% trypsin for 2-3 min at 37 °C. The cells were collected into centrifuge
tubes, and then centrifuged at 1,000 rpm for 4 minutes and the pellets were resuspended in DMEM. Cells used for this
study were in third passage in late subconfluency.
Cytotoxicity effect of mangostin to MCF7 cell
In vitro cytotoxicity studies Cytotoxicity of alpha- beta- and gamma-mangostin was evaluated for MCF7 cells
4
using the MTT assay. MCF7 cells were cultured in 96-well plates for 24 h at a seeding density of 1.0×10 cells/well before
the addition of test samples. Mangostin were diluted with medium as culture medium to different concentrations.
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th
The 6 International Conference on Natural Products for Health and Beauty (NATPRO6)
January 21-23, 2016
Organized by Khon Kaen University
Control test samples were medium, 5-FU in medium (positive control) and medium (negative control). Experiments were
initiated by replacing the culture medium in each well with 100 μl of sample solutions at 37 C in the CO2 incubator. After
24 h of incubation, 10 μl of MTT reagent (1 mg/ml) in the phosphate buffer saline was added to each well. The plates
were then incubated at 37 C for another 24 h. At the end of the incubation period, the intracellular formazan was
solubilized with 50 μl DMSO and quantified by reading the absorbance at 590 nm on a micro-plate multi-detection
instrument, SpectraMax M2. Percentage of cell viability was calculated based on the absorbance measured relative to the
absorbance of cells exposed to the negative control. The percentage of cell viability was calculated as follow: % cell
viability=(OD of treated cells)/(OD of untreated cells )×100. Half maximal inhibitory concentration (IC50) was calculated
via Graphpad prism version 6.
Statistical analysis
Statistical data analysis was done using statistical package for GraphPad Instat version 6. The values are
expressed as mean. Hypothesis testing method included one-way analysis of variance (ANOVA) followed by post hoc
testing performed with least significant difference test. For all comparisons, differences were considered statistically
significant at p<0.01.
3. RESULTS
These results were characterized to ADMET risk score, the scores of 2.95, 2.65 and 2.17 were for alpha- betaand gamma-mangostin, respectively. In general, the scores of 0-24 indicate the number of potential ADMET problem.
These findings may be valuable to explain pharmacokinetics data of these compounds. Lower score can determine to low
ADMET problem and has high possibility to be developed to new drug. All mangostins had closely ADMET risk score and
were too lipophlilic(LogP>4.5 or LogD>3.5). Gamma-mangostin had lowest score(2.17). Nevertheless, gamma-mangostin
had ADMET problem in low permeability (jejunal < 0.25 or MDCK<30) and high plasma protein binding (PrUnbnd < 3.5%).
ADMET risk score and potential ADMET problems of mangostins shown in Table 2.
Table 2. ADMET risk score and ADMET_Code of alpha-beta-gamma-mangostin via ADMET Predictor
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software.
2.95
ow,Sw,fu,ra,C9
4.497
4.377
0.01
40.814
4.801
betamangostin
2.65
ow,Sw,fu
4.811
4.772
0.004
89.332
6.812
gammamangostin
2.17
ow,Pf,fu
4.213
4.089
0.015
22.456
3.165
304.327
1.139
99.146
334.06
1.274
nonsubstrate
331.008
1.357
nonsubstrate
Name
alpha-mangostin
ADMET Risk
ADMET_Code
ow: Logarithm of partition coefficient
Logarithm of distribution coefficient
Sw: Native water solubility
Pf: Apparent MDCK COS permeability
Human jejunal effective permeability
ra: Acute toxicity in rats
fu: Percentage of Plasma protein non-binding
C9: CLint by human CYP P450 2C9
Criteria
> 4.5
> 3.5
<= 0.010
< 30
< 0.25
< 320
< 3.5%
> 30
The cytotoxicity of mangostins in MCF7 cells was evaluated by MTT assay. Cells were treated with various doses
of mangostins (0.1μM to 100μM) for 24h. The average value of half maximal inhibitory concentration(IC50) of alphabeta- gamma-mangostin and 5-FU to MCF7 cells were 38.42±4.38, 83.54±0.87, 92.69±0.35 and 64.00±1.52 respectively.
The significant values represent mean ± SD of three separate experiments(n=3). 5-FU was significant difference with
alpha- beta- and gamma-mangostin(p<0.01). According, alpha-mangostin is the most cytotoxic to MCF7 cells that shown
in Figure 1.
297
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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6)
January 21-23, 2016
Organized by Khon Kaen University
100
P < 0.01
80
Mean IC50 (microM)
60
40
20
0
alpha-mangostin
beta-mangostin
gamma--mangostin
5-FU
Figure 1. Cytotoxicity effect of various concentrations of alpha- beta- gamma-mangostin and 5-FU on MCF7 cells.
4. CONCLUSIONS
From the result alpha-mangostin is the most cytotoxic to MCF7 cells. However, alpha-mangostin has highest
ADMET risk score. Thus, further study of mangostins should focus on the optimization of their pharmacokinetic and
pharmacodynamic properties and should be more studied via in vivo or other in vitro models
ACKNOWLEDGEMENTS
Foremost, I would like express gratitude to Assoc. Prof. Dr. Korbtham Sathirakul, my advisor for kindness
supports to my research, immense knowledge and advices. I also benefited by outstanding works from Mr. Pattarawit
Rukthong’s help with his particular skill in handling precisely delicate equipment and technique. Special thanks to Mr.
Nawong Boonnak for perfect material and funding from Srinakarinwirot University and my family, for everything I have
until this day.
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