Results

4.1. The in silico anti-inflammatory activity of selected xanthone ligands against

COX-2:

The concept of docking was utilized to understand the interaction between COX
proteins and xanthones of Mangosteen. The molecular structures of ligands, α-Mangostin
(Figure-12A), β-Mangostin (Figure-12B) and γ-Mangostin (Figure-12C) were generated
and three-dimensionally optimized using ACD/ChemSketch software. Later the files
were converted into SDF or MOL2 format with the help of Mercury software for docking
analysis.

The three-dimensional protein coordinates of COX-1 (PDB_ID: 3N8V) and

COX-2 (PDB_ID: 3NTG) were derived from PDB database. PDB is a repository for the
three-dimensional structural data of large biological molecules, such as proteins. The
active site and the chain with large area of protein pockets and cavity of target enzymes
were identified by CASTp analysis. CASTp includes a graphical user interface, flexible
interactive visualization, as well as on-the-fly calculation for user uploaded structures. It
provides an online resource for locating, delineating and measuring concave surface
regions on three-dimensional structures of proteins. Based on CASTp analysis chain A of
COX-1 (Figure-13A) and COX-2 (Figure-14A) with large area of protein pockets and
cavity was selected and saved in PDB format with the aid of SPdbV for docking.

The xanthone ligands α-Mangostin (Figure-12A), β-Mangostin (Figure-12B) and

γ-Mangostin (Figure-12C) were docked into the active sites of COX-1 (Figure-13B) and
COX-2 (Figure-14B) using GOLD 3.0.1. GOLD is a genetic algorithm based docking
program allows flexible ligand docking. The active site radius was set as 10A˚ with the
centroid as HH atom of Phe220 and Arg170 for COX-1 and COX-2 respectively. The
docking solutions for COX-1 and COX-2 were scored according to the GOLD fitness
function.

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 Results

Figure. 12. Structure of xanthone ligands (Red-Oxygen, Aqua-Carbon, and White-

Hydrogen). (A). α-Mangostin (C24H26O6), (B). β-Mangostin (C25H28O6) and (C). γ-
Mangostin (C23H24O6).

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 Results

Figure. 13. (A). The ribbon diagram shows the three-dimensional structure of chain A of
COX-1 (PDB_ID: 3N8V) and (B). Active site (green) predicted by CASTp.

Figure. 14. (A). The ribbon diagram shows the three-dimensional structure of chain A of
COX-2 (PDB_ID: 3NTG) and (B). Active site (green) predicted by CASTp.

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 Results

Among the three xanthones, α-Mangostin and β-Mangostin showed two hydrogen
bonds with same residues of COX-1 but differ in bond lengths. α-Mangostin showed two
hydrogen bond interactions of bond lengths 2.656A˚ and 1.817A˚ with Arg49 and Leu52
respectively (Figure-15A). β-Mangostin also showed two hydrogen bonds of bond
lengths 2.180A˚ and 1.806A˚ with Arg49 and Leu52 respectively (Figure-15B). But γ-
Mangostin exhibited a different pattern of interaction with COX-1, a single hydrogen
bond of length 2.488A˚ was observed with Val33 of COX-1 (Figure-15C). The atoms
involved in hydrogen bond interactions and the docking energies of Mangosteen ligands
with COX-1 were tabulated (Table-3).

On the other hand, α-Mangostin extended O11 and O13 of its oxygen atoms to
form two hydrogen bonds of length 2.019A˚ and 2.556A˚ with Ala18 of COX-2 (Figure-
16A). Similarly, β-Mangostin exhibited two hydrogen bonds but with Ser23 and Asp38
of COX-2. One bond was observed between Asp38 of COX-2 and H39 of β-Mangostin
with 1.894A˚ bond length; another bond was observed between Ser23 of COX-2 with
O20 of β-Mangostin (Figure-16B). As shown in Figure-4C, γ-Mangostin showed three
hydrogen bond interactions with Ala18, Cys22 and Asp38 of COX-2. The bonds are
between Asp38 of COX-2 and H37 of γ-Mangostin with a bond length of 1.980A˚. Other
interactions are with Cys22 and Ala18 of COX-2 with O21 and O22 of γ-Mangostin
respectively (Figure-16C). The atoms involved in hydrogen bonding, their bond lengths
and docking energies of all the three xanthone ligands were scored based on GOLD
fitness function for COX-2 and were tabulated (Table-4).

The docking results showed that all the three xanthone derivatives of Mangosteen
are active COX inhibitors with a significant preference to COX-2. Among the three
xanthones, based on the hydrogen bond interactions, number of hydrogen bonds and
GOLD docking score α-Mangostin was utilized for further experimental investigations.

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 Results

Figure. 15. Docking of xanthone ligands into the active site of COX-1 (PDB_ID: 3N8V)
using GOLD 3.0.1. Hydrogen bonds are shown as green dashed lines.
(A). α-Mangostin orientation (stick model) in COX-1 active site pocket,
(B). β-Mangostin orientation (stick model) in COX-1 active site pocket and
(C). γ-Mangostin orientation (stick model) in COX-1 active site pocket.

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 Results

Table. 3. Docking score and hydrogen bond interactions of Mangostin’s with COX-1
using GOLD 3.0.1.

No. of Atoms Bond Docking

Molecule Hydrogen length (Aº) score
bonds Protein Molecule (kcal/mol)
Arg49(HH2) O(21) 2.656 14.77
α-Mangostin 2 Leu52(O) H(41) 1.817
Arg49(HH2) O(21) 2.180 10.20
β-Mangostin 2 Leu52(O) H(41) 1.806
γ-Mangostin 1 Val33(O) H(50) 2.488 16.20

Table. 4. Docking score and hydrogen bond interactions of Mangostin’s with COX-2
using GOLD 3.0.1.

No. of Atoms Bond Docking

Molecule Hydrogen length (Aº) score
bonds Protein Molecule (kcal/mol)
Ala18(H2) O(11) 2.019
α-Mangostin 2 Ala18(H1) O(13) 2.556 22.33

Ser23(HG) O(20) 2.122

β-Mangostin 2 Asp38(O) H(39) 1.894 21.34

Ala18(H1) O(22) 1.667

γ-Mangostin 3 Cys22(H) O(21) 2.630 22.91
Asp38(OD1) H(37) 1.980

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 Results

Figure. 16. Docking of xanthone ligands into the active site of COX-2 (PDB_ID: 3NTG)
using GOLD 3.0.1. Hydrogen bonds are shown as green dashed lines.
(A). α-Mangostin orientation (stick model) in COX-2 active pocket,
(B). β-Mangostin orientation (stick model) in COX-2 active pocket and
(C). γ-Mangostin orientation (stick model) in COX-2 active pocket.

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 Results

4.2. In silico ADME properties of α-Mangostin:

The in silico ADME properties of α-Mangostin and Indomethacin were predicted

using PK/DB database based on pharmacokinetic properties (PK) measurements by
Hologram QSAR Technique. Indomethacin, a standard NSAID, was recruited as
comparative control in this analysis. The pharmacokinetic measurements includes human
intestinal absorption (HIA), human oral bioavailability (F), plasma protein binding
(PPB), blood brain barrier (logBB) and water solubility (logS).

α-Mangostin and Indomethacin structures were edited manually using PK/DB

sketcher and converted into SMILES format for the pharmacokinetic analysis. The in
silico predicted pharmacokinetic properties (PK) are HIA was 60.65%, 104.31% and PPB
was 84.80%, 102.46% for α-Mangostin and Indomethacin respectively (Table-5).

Table. 5. Pharmacokinetic properties (PK) of α-Mangostin and Indomethacin.

Human Human Oral Plasma Blood Brain Water

Compound Intestinal Bioavailability Protein Barrier Solubility
Absorption (F, %) Binding (BBB, logBB) (logS)
(HIA, %) (PPB, %)
α-Mangostin 60.65 48.66 84.80 -0.05 -5.97

Indomethacin 104.31 92.28 102.46 -1.05 -5.31

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 Results

4.3. α-Mangostin potently inhibited COX-2 and iNOS analyzed by AutoDock:

To identify the potential anti-inflammatory lead compound between α-Mangostin

and Indomethacin (comparative control), individual docking studies were performed
against pro-inflammatory genes (COX-2 and iNOS). The protein coordinates of COX-2
(PDB_ID:1CX2) and iNOS (PDB_ID:1NSI) used in this study were correspond to their
X-ray structures, which were retrieved from PDB. The active sites of target enzymes
were predicted by CASTp analysis and based on this chain A of COX-2 (Figure-17A)
and chain B of iNOS (Figure-18A) were selected for docking. Hetero atoms were
removed from the receptor file, polar hydrogen atoms were added and partial atomic
charges were added to the corresponding carbon atoms and saved in pdb format for
docking analysis. The α-Mangostin (Figure-12A) and Indomethacin (Figure-19)
structures were generated using ChemSketch software. AutoTors was used to transform
the ligand MOL2 file into a PDBQ file and to merge non-polar hydrogen atoms and to
define torsions. The molecular docking of α-Mangostin and Indomethacin into the active
site of COX-2 (Figure-17B) and iNOS (Figure-18B) was carried out using AutoDock 3.0.
Docking was performed by defining a grid box with dimensions of 48x54x62A˚ for
COX-2 and 52x56x48A˚ for iNOS in order to enclose the active site residues as
mentioned in Table-6.

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 Results

Figure. 17. (A). The ribbon diagram shows the three-dimensional structure of chain A of
COX-2 (PDB_ID:1CX2) and (B). Active site (green) predicted by CASTp.

Figure. 18. (A). The ribbon diagram shows the three-dimensional structure of chain B of
iNOS (PDB_ID:1NSI) and (B). Active site (green) predicted by CASTp.

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 Results

Figure. 19. Structure of Indomethacin (Aqua-Carbon, White-Hydrogen, Red-Oxygen,

Blue-Nitorgen and Lime-chlorine).

Table. 6. Active site residues of COX-2 and iNOS.

Protein Active site residues

COX-2 His90, Leu117, Arg120, Gln192, Val349, Leu352, Ser353, Tyr355,

Leu359, Trp387, Arg513, Ala516, Phe518, Met522, Val523, Gly526,
Ala527, Ser530 and Leu534.

iNOS Trp194, Cys200, Gln263, Trp346, Pro350, Val352, Phe369, Gly371,

Trp372, Tyr373, Met374, Glu377, Asp382, Ile462, Trp463, Tyr489.

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 Results

The binding interactions of α-Mangostin with COX-2 showed two hydrogen

bonds with Ser353 and Ser530 (Figure-20A) with a bond length of 1.724A˚ and 1.927A˚
respectively (Table-7). But the complex of Indomethacin with COX-2 showed hydrogen
bonds with three residues Arg120, Ser353 and Trp387 and these residues lie at the
junction of the anchoring site of the COX-2 active site (Figure-20B) and their bond
lengths were tabulated in Table-7.

Among various binding poses in the active site, the best pose for both the ligands
and most stable conformation was selected based on docking energy. The α-Mangostin
with COX-2 (Table-7) showed a docking energy of -12.01kcal/mol which was two folds
greater compared to the docking energy -5.96kcal/mol of Indomethacin with COX-2.

The docking energies of α-Mangostin and Indomethacin with iNOS were found to
be -12.20kcal/mol and -2.75kcal/mol respectively (Table-8). However, the hydrogen
bonds were observed at different positions with different residues of iNOS. α-Mangostin
showed a single hydrogen bond of 2.012A˚ with Tyr489 (Figure-21A, Table-8) and
Indomethacin showed two hydrogen bonds of 2.076A˚ with Gln263 and 2.013A˚ with
Trp346 of iNOS (Figure-21B, Table-8).

The docking of α-Mangostin and Indomethacin against pro-inflammatory

enzymes COX-2 and iNOS clearly shows the anti-inflammatory potential of α-Mangostin
compared to Indomethacin.

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 Results

Figure. 20. Docking of ligands into the binding site of COX-2 (PDB_ID:1CX2) using
AutoDock 3.0. The important residues of the enzyme and the inhibitor are depicted by
stick model and Hydrogen bonds are shown as green lines.
(A) α-Mangostin with COX-2 (blue) and
(B) Indomethacin with COX-2 (violet).

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 Results

Table. 7. Docking energies and number of hydrogen bond interactions with bond lengths
of ligands docked against pro-inflammatory enzyme COX-2 using AutoDock 3.0.

No. of Amino acids Docking

Bond
Protein Ligand Hydrogen involved in energy
lengths (Ao)
bonds Hydrogen bonding (kcal/mol)
Ser353 1.724
α-Mangostin 2 -12.01
Ser530 1.927
COX-2
Arg120 3.095
Indomethacin 3 Ser353 2.149 -5.96
Trp387 1.361

Table. 8. Docking energies and number of hydrogen bond interactions with bond lengths
of ligands docked against pro-inflammatory enzyme iNOS using AutoDock 3.0.

No. of Amino acids Docking

Bond
Protein Ligand Hydrogen involved in energy
lengths (Ao)
bonds Hydrogen bonding (kcal/mol)
α-Mangostin 1 Tyr489 2.012 -12.20
iNOS Gln263 2.076
Indomethacin 2 -2.75
Trp346 2.013

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 Results

Figure. 21. Docking of ligands into the binding site of iNOS (PDB_ID:1NSI) using
AutoDock 3.0. The important residues of the enzyme and the inhibitor are depicted by
stick model and Hydrogen bonds are shown as green lines.
(A). α-Mangostin with iNOS (pink) and
(B). Indomethacin with iNOS (yellow).

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 Results

4.4. α-Mangostin docked into the active sites of mPGES-1 and NF-κB showed
significant binding efficiency.

4.4.1. Docking of α-Mangostin into the active site of mPGES-1:

The mPGES-1 is a terminal synthase in the production of PGE2. The protein

coordinate of mPGES-1 (PDB_ID:3DWW) was retrieved from PDB. The docking of α-
Mangostin (Figure-12A) into the active site pocket of mPGES-1 (Figure-22) was
performed using AutoDock 3.0. The active site of mPGES-1 was predicted by CASTp
analysis and chain B was chosen for docking.

The grid box was defined to enclose the active site residues namely Glu77,
Arg110, Tyr117 and Arg126 with dimensions of 54x66x40A˚. The α-Mangostin showed
a single hydrogen bond interaction with Arg180 of mPGES-1 (Figure-23). The docking
energy and bond length were tabulated (Table-9).

4.4.2. Docking of α-Mangostin into the active site of NF-κB:

NF-κB is a pivotal transcription factor that regulates the transcription of genes

involved in inflammation, cancer development and its progression. A deep understanding
of the interaction with this protein will perhaps lead to modulate the pro-inflammatory
genes COX-2 and iNOS. The three-dimensional protein coordinate of NF-κB (PDB_ID:
2O61) was derived from PDB. The active site of NF-κB was predicted by CASTp
analysis and chain A was preferred for docking analysis. The α-Mangostin (Figure-12A)
was docked into the active site pocket of NF-κB (Figure-24) using AutoDock 3.0.

The grid box was defined to enclose the active site residues namely Lys218,
Lys221, Glu222, Arg246 and Gln247 with dimensions of 40x40x40A˚. The docking
results showed two hydrogen bond interactions between α-Mangostin and NF-κB with -
7.81kcal/mol docking energy (Figure-25). One bond was observed between Arg30 of NF-
κB and α-Mangostin with a bond length of 2.067A˚. The other bond was found between
Leu280 of NF-κB and α-Mangostin with a bond length of 2.010 A˚ (Table-10).

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 Results

Figure. 22. α-Mangostin (pink) in the active site pocket of mPGES-1 (PDB_ID:3DWW).

Figure. 23. Docking of α-Mangostin into the active site of mPGES-1 (PDB_ID:3DWW)
using AutoDock 3.0. The important residues of the enzyme and the inhibitor are depicted
by stick model and Hydrogen bonds are shown as green lines.

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 Results

Table. 9. Docking energies and number of hydrogen bond interactions with bond lengths
of α-Mangostin docked against mPGES-1 using AutoDock 3.0.

No. of Amino acids Docking

Bond
Protein Ligand Hydrogen involved in energy
lengths (Ao)
bonds Hydrogen bonding (kcal/mol)

mPGES-1 α-Mangostin 1 Arg180 1.965 -7.21

Table. 10. Docking score and hydrogen bond interactions of α-Mangostin with NF-κB
using AutoDock 3.0.

No. of Amino acids Docking

Bond
Protein Ligand Hydrogen involved in energy
lengths (Ao)
bonds Hydrogen bonding (kcal/mol)
Arg30 2.067
NF-κB α-Mangostin 2 -7.81
Leu280 2.010

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 Results

Figure. 24. α-Mangostin (aqua) in the active site pocket of NF-κB (PDB_ID: 2O61).

Figure. 25. Docking of α-Mangostin into the active site of NF-κB (PDB_ID: 2O61) using
AutoDock 3.0. The important residues of the enzyme and the inhibitor are depicted by
stick model and Hydrogen bonds are shown as green lines.

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 Results

4.5. The in vivo anti-inflammatory and in vitro antioxidant potential of α-Mangostin.

4.5.1. Acute toxicity test:

The oral doses of α-Mangostin up to 1500mg/kg did not produce any evident sign
of toxicity and mortality in rats when observed up to 14 days since administration. Thus,
the median lethal dose (LD50) was determined to be higher than the maximum
(1500mg/kg) dose tested.

4.5.2. In vivo anti-inflammatory activity of α-Mangostin.

4.5.2.1. Carrageenan induced acute inflammatory analysis in rats:

In the present study, the acute inflammation in rats was experimentally induced
by carrageenan to determine the anti-inflammatory activity of α-Mangostin. After 1h of
oral treatment with α-Mangostin (0.5, 5 and 10mg/kg, b.w, p.o), Indomethacin (10mg/kg,
p.o) and normal saline (control), the acute inflammation was produced by subplantar
administration of % carrageenan in the left hind paw of rats. After 3h of carrageenan
injection the rat paw oedema volume was measured and the inhibition of paw oedema
was clearly observed in α-Mangostin (10mg/kg) treated group compared to control group
(Figure-26). α-Mangostin significantly (p<0.001) inhibited the carrageenan induced paw
oedema in dose dependent manner by restricting its volume to 0.883±0.012ml at 10mg/kg
compared to standard Indomethacin (10mg/kg) group (Table-11).

α-Mangostin at the doses of 0.5, 5 and 10mg/kg showed an inhibition of 21.66%,

25.72% and 40.21% respectively against acute paw oedema induced by carrageenan in
comparison with Indomethacin (Figure-27).

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 Results

4.5.2.2. Cotton pellet induced chronic inflammatory analysis in rats:

The chronic inflammation in rats was experimentally induced by cotton pellet to

confirm the anti-inflammatory activity of α-Mangostin. The rats were treated with α-
Mangostin (0.5, 5 and 10mg/kg, b.w, p.o), Indomethacin (10mg/kg, p.o) and normal
saline (control) were administered orally for seven consecutive days from the day of
cotton pellet implantation. The animals were anesthetized on the 8th day and the
formation of granuloma around subcutaneously implanted cotton pellets in the groin
region of rats was observed in control group whereas it was not observed in 10mg/kg of
α-Mangostin treated group (Figure-28).

The cotton pellets were removed surgically, freed of extraneous tissues and dried
for 24h at 60ºC. The dry weight of cotton pellets were taken as the measure of granuloma
formation. α-Mangostin significantly (p<0.001) reduced the formation of granuloma at
10mg/kg as the dry weight of cotton pellet was 23.3±0.15mg (Table-12) compared to
standard group. The maximum inhibition of 66.71% was observed at 10mg/kg of α-
Mangostin but Indomethacin showed 50.57% inhibition of granuloma formation at
10mg/kg (Figure-29). The α-Mangostin showed significant in vivo anti-inflammatory
activity both in acute and chronic phases of inflammation compared to Indomethacin.

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Figure. 26. The anti-inflammatory activity of α-Mangostin was evaluated in acute phase
of inflammation in rats. The acute inflammation was experimentally induced by
carrageenan showing paw oedema in control group (a) and inhibition of paw oedema in
10mg/kg b.w of α-Mangostin treated group (b) after 3h of induction.

Figure. 27. The inhibition of paw oedema by α-Mangostin was evaluated by carrageenan
induced acute inflammation in rats.

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 Results

Table. 11. Effect of α-Mangostin on carrageenan induced paw oedema in rats.

Group / Treatment Dose Mean paw oedema

(mg/kg,p.o) volume (ml) ± SEM
Group 1 / Control -- 1.477±0.117
Group 2 / Indomethacin 10 0.910±0.070*
Group 3 / α-Mangostin 0.5 1.157±0.042*
Group 4 / α-Mangostin 5 1.097±0.038*
Group 5 / α-Mangostin 10 0.883±0.012*

All values are expressed as mean ± SEM of six individual observations and
*
p<0.001 when compared to control group.

Table. 12. Effect of α-Mangostin on cotton pellet induced granuloma in rats.

Group / Treatment Dose Dry weight of cotton

(mg/kg,p.o) pellet (mg) ± SEM
Group 1 / Control -- 70.0±0.13
Group 2 / Indomethacin 10 34.6±0.03*
Group 3 / α-Mangostin 0.5 55.3±0.09*
Group 4 / α-Mangostin 5 42.0±0.05*
Group 5 / α-Mangostin 10 23.3±0.15*

All values are expressed as mean ± SEM of six individual observations and
*
p<0.001 when compared to control group.

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Figure. 28. The anti-inflammatory activity of α-Mangostin was evaluated in chronic

phase of inflammation in rats. The chronic inflammation was induced by cotton pellet
showing formation of granuloma around subcutaneously implanted cotton pellets in the
groin region of rats in control group (a) but not in 10mg/kg b.w of α-Mangostin treated
group (b) after 7 days of induction.

Figure. 29. The inhibition of cotton pellet induced granuloma by α-Mangostin was
evaluated by chronic inflammation in rats.

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4.5.3. In vitro antioxidant property of α-Mangostin.

4.5.3.1. DPPH radical scavenging by α-Mangostin:

The DPPH was widely used as a model system to investigate the scavenging
activities of natural compounds. DPPH radical is scavenged by antioxidants through the
donation of proton by forming the reduced DPPH. The color changes from purple to
yellow after reduction was quantified at 517nm. α-Mangostin at different concentrations
(62.5 to 1000µg/ml) was tested for the DPPH free radical scavenging ability. As shown in
Table-13, α-Mangostin showed 95% inhibition at 1000µg/ml and 40% inhibition at
62.5µg/ml concentration. The IC50 for DPPH scavenging was found to be at 100µg/ml of
α-Mangostin. This suggests that α-Mangostin has the proton-donating ability and could
serve as a free radical scavenger.

4.5.3.2. Nitric oxide scavenging assay:

Sodium nitroprusside in an aqueous solution at physiological pH, spontaneously

generates nitric oxide which interacts with oxygen to produce nitrite ions that were
measured at 546nm using Griess reagent. Nitric oxide scavenging effect of α-Mangostin
was found to be 12% and 49% at 62.5µg/ml and 1000µg/ml respectively (Table-14). The
IC50 was found to be at 1000µg/ml of α-Mangostin. The reduction in nitrite production
indicates the antioxidant potential of α-Mangostin, which competes with oxygen to react
with nitric oxide there by inhibiting the generation of nitrite.

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Table. 13. DPPH scavenging activity of α-Mangostin and * represents results of triplicate
determinants.

α-Mangostin Percentage of
(µg/ml) inhibition*
62.5 40
125 64
250 68
500 70
1000 95

Table. 14. Scavenging effect of α-Mangostin on nitric oxide radical and * represents data
of triplicate determinants.

α-Mangostin Percentage of
(µg/ml) inhibition*
62.5 12
125 15
250 20
500 30
1000 49

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4.5.3.3. Scavenging of superoxide radical by α-Mangostin:

The scavenging activity towards the superoxide radical (O2-) was measured in
terms of inhibition of generation of O2- using alkaline dimethyl sulphoxide (DMSO)
method. In the present study, superoxide radical reduces nitroblue tetrazolium to a blue
colored formosan that was measured at 560nm. The result shows that α-Mangostin has a
potent scavenging activity with increasing percentage of inhibition i.e., 27% at 62.5µg/ml
and 89% at 1000µg/ml (Table-15). The IC50 was found to be at 250µg/ml of α-
Mangostin. The possible mechanism of scavenging superoxide anions by α-Mangostin
was may be due to its inhibitory effect towards generation of superoxides in the in vitro
reaction mixture.

4.5.3.4. Lipid peroxidation assay:

In vitro induction of lipid peroxidation is a tool for measuring antioxidant

potential of α-Mangostin. Dose-dependent inhibition of lipid peroxidation (Table-16)
exhibiting the IC50 at 900µg/ml of α-Mangostin. This activity may be related to the H+
ion donating capability of the α-Mangostin which can scavenge the peroxyl radical. It
was observed that free radicals were scavenged by α-Mangostin in a concentration
dependent manner. From the above results it was inferred that α-Mangostin has potent
antioxidant activity.

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Table. 15. Scavenging activity of α-Mangostin on superoxide radical and * represents

results of triplicate determinants.

α-Mangostin Percentage of
(µg/ml) inhibition*

62.5 27
125 39
250 54
500 59
1000 89

Table. 16. Inhibition of lipid peroxidation by α-Mangostin and * represents data of

triplicate determinants.

α-Mangostin Percentage of
(µg/ml) inhibition*
62.5 12
125 19
250 26
500 32
1000 54

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4.6. The effect of α-Mangostin on cell viability and apoptosis of MDA-MB-231

human breast cancer cells:

To test the effect of α-Mangostin on the cell viability and apoptosis, a well
characterized human breast cancer cell line MDA-MB-231 was selected. MDA-MB-231
cells in DMEM medium were seeded in culture plates, kept overnight for attachment and
treated with different concentrations of α-Mangostin for desired time periods. The stock
solution of α-Mangostin was prepared in DMSO and diluted with DMEM to get desired
concentrations. The maximum concentration of DMSO was within permissible limits of
toxicity (≤0.1%). Appropriate concentration of DMSO used (0.1% v/v) was taken as
control in all the experiments.

To set α-Mangostin dosage and exposure time, MDA-MB-231 cells were

incubated with different concentrations (1, 2.5, 5, 10, 15 and 20µM) of α-Mangostin for
24h and observed for morphological changes under optical inverse microscope. Cells
treated with α-Mangostin (1 to 20µM) showed significant morphological alterations like
cell to cell contact inhibition and detaching of adherent cells compared to control (0.1%
DMSO) cells (Figure-30).

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Figure. 30. Photomicrograph in optical inverse microscopy shows the effect of α-

Mangostin on MDA-MB-231 human breast cancer cells. From ‘a’ to ‘g’ are 0.1% DMSO
treated control cells to 1, 2.5, 5, 10, 15 and 20µM of α-Mangostin treated cells. Exposure
of cells to α-Mangostin for 24h showed morphological alterations like retraction,
rounding and some sensitive cells were detached from the surface in d, e, f and g.

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4.6.1. Cell viability examined by Trypan blue dye exclusion assay:

The dye exclusion test is used to determine the number of viable cells present in a
cell suspension. It is based on the principle that live cells possess intact cell membranes
that exclude certain dyes like trypan blue, whereas dead cells do not. In this test, the
MDA-MB-231 cells were incubated with different concentrations (1 to 20µM) of α-
Mangostin for 24h. The cell suspension was mixed with Trypan blue and loaded on to a
haemocytometer, then visually examined to determine whether cells take up or exclude
dye under microscope. The viable cells showed clear cytoplasm by excluding Trypan
blue and cells with blue cytoplasm were counted as non-viable. The percentage of viable
cell number was consistently decreased with increasing concentrations of α-Mangostin.
As shown in Figure-31 marginal decrease in the cell viability was observed up to 2.5µM
of α-Mangostin. The treatment with 10µM of α-Mangostin significantly decreased the
viability of MDA-MB-231 cells to 48.6% at 24h and to 2.4% with 20µM concentration of
α-Mangostin (Figure-31).

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Figure. 31. The effect of α-Mangostin on MDA-MB-231 cells viability determined by

Trypan blue dye exclusion method. The inhibition of cells viability from 0.1% DMSO
(C) to 1, 2.5, 5, 10, 15 and 20µM of α-Mangostin exposure after 24h. Each bar represents
the data of triplicate determinants and * indicates significant difference in comparison to
control (p < 0.01).

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4.6.2. Cytotoxicity of α-Mangostin on MDA-MB-231 breast cancer cells:

Neutral red assay is one of the most widely used cytotoxicity tests with many
biomedical and environmental applications. Neutral red assay is based on the ability of
viable cells to incorporate and bind the supravital dye called Neutral red. It is a weak
cationic dye that readily penetrates cell membranes by non-ionic diffusion and
accumulates intracellularly in lysosomes. Alterations of the cell surface or the sensitive
lysosomal membrane lead to lysosomal fragility result in a decreased uptake and binding
of NR. It is thus possible to distinguish between viable, damaged, or dead cells by this
assay. In the present study MDA-MB-231 cells incubated with different concentrations (1
to 20µM) of α-Mangostin for 24h and stained with Neutral red. As shown in the Figure-
32, in the presence of α-Mangostin (5 to 20µM) less amount of Neutral red dye was bind
to cells. The MDA-MB-231 cells survival was 49.2% with 10µM and 4.7% with 20µM
of α-Mangostin (Figure-33).

The cell viability in vehicle-treated control (0.1% DMSO) was >95% as

determined by Trypan blue and Neutral red assay and the data obtained from both assays
showed that α-Mangostin has an IC50 value of 10µM. The IC50 concentration was defined
as the concentration of α-Mangostin inhibiting cell viability / cell survival by 50%,
compared with the vehicle-treated control (0.1% DMSO). Thus, α-Mangostin displayed a
strong cytotoxic effect on MDA-MB-231 breast cancer cells.

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Figure. 32. Photomicrograph in optical inverse microscopy shows the effect of α-

Mangostin on MDA-MB-231 cells survival by Neutral red uptake assay. From ‘a’ to ‘g’
are 0.1% DMSO treated control cells to 1, 2.5, 5, 10, 15 and 20µM of α-Mangostin
treated cells after 24h.

Figure. 33. The inhibition of MDA-MB-231 cells survival from 0.1% DMSO (C) to 1,
2.5, 5, 10, 15 and 20µM of α-Mangostin treatment determined by Neutral red assay after
24h. Each bar represents the results of triplicate determinants and * indicates significant
difference in comparison to control (p < 0.01).

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4.6.3. Anti-proliferative property of α-Mangostin assessed by MTT assay:

Mitochondrial respiration, an indicator of cell viability, was assayed by the

mitochondrial-dependent reduction of MTT to formazan. MTT a yellow tetrazole is
reduced to purple formazan in living cells. MDA-MB-231 cells were incubated with
different concentrations (1 to 20µM) of α-Mangostin for 24h and assessed by MTT assay
to determine the anti-proliferative activity of α-Mangostin. We observed that more
formazan crystals were formed by control (0.1% DMSO) cells compared to 10µM of α-
Mangostin treated cells. The formazan crystals produced by the mitochondrial
dehydrogenase activity were dissolved in DMSO and the amount of dye released was
read at 570nm for measuring cell proliferation percent. A significant decrease in cell
proliferation with increasing concentrations of α-Mangostin was found at 24h (Figure-
34).

The percentage of MDA-MB-231 cells proliferation was reduced to 47.6% and

3.4% with 10µM and 20µM of α-Mangostin respectively (Figure-35). Thus, α-Mangostin
displayed a strong anti-proliferative activity on MDA-MB-231 breast cancer cells.

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Figure. 34. Photomicrograph in optical inverse microscopy shows the effect of α-

Mangostin on MDA-MB-231 cells proliferation by MTT assay. From ‘a’ to ‘g’ are 0.1%
DMSO treated control cells to 1, 2.5, 5, 10, 15 and 20µM of α-Mangostin treated cells
after 24h.

Figure. 35. The inhibition of MDA-MB-231 cells proliferation from 0.1% DMSO (C) to
1, 2.5, 5, 10, 15 and 20µM of α-Mangostin treatment determined by MTT assay after 24h.
Each bar represents the data from triplicate determinants and * indicates significant
difference in comparison to control (p < 0.01).

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4.6.4. DNA fragmentation induced by α-Mangostin in MDA-MB-231 breast cancer

cells:

In order to delineate the mechanism of cell death mediated by α-Mangostin, DNA

fragmentation analysis was performed, which is the hallmark of apoptosis. MDA-MB-
231 cells treated with different concentrations (2.5, 5, 10, 15 and 20µM) of α-Mangostin
for 24h indicated the generation of oligonucleosomal fragments of DNA on 1.8% agarose
gel. The degree of nuclear DNA fragmentation was directly proportional to the increasing
concentrations of α-Mangostin (Figure-36).

4.6.5. α-Mangostin caused double strand breaks in DNA of MDA-MB-231 cells:

To assess the DNA damage caused by α-Mangostin, comet assay was performed
in MDA-MB-231 cells. As shown in Figure-37, comet tails of damaged DNA were
observed in the presence of α-Mangostin.

4.6.6. α-Mangostin induced apoptosis in MDA-MB-231 cells assessed by fluorescent

nuclear staining:

The anti-proliferative activity shown by α-Mangostin could be possibly due to the

induction of apoptosis. A distinguishing feature of apoptosis is the condensation and
fragmentation of nuclear chromatin which can be assessed by nuclear staining with
Hoechst fluorescent stain. The MDA-MB-231 cells treated with 10µM of α-Mangostin
after 24h showed fragmentation and condensation of nuclear chromatin whereas
chromatin with distinct nuclei was found in control cells (Figure-38).

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Figure. 36. DNA fragmentation analysis of α-Mangostin treated MDA-MB-231 cells.

After 24h treatment with or without different concentrations of α-Mangostin, DNA was
isolated and analysed on 1.8% agarose gel. Lane M: Φ DNA marker, Lane C: 0.1%
DMSO treated control cells, Lane ‘1’ to ‘5’ are 2.5, 5, 10, 15 and 20µM α-Mangostin
treated cells.

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Figure. 37. Comet assay of α-Mangostin treated MDA-MB-231 cells showing detectable
comet tails, indicative of DNA damage. From ‘a’ to ‘f’ are 0.1% DMSO treated control
cells to 2.5, 5, 10, 15 and 20µM of α-Mangostin treated cells.

Figure. 38. Hoechst nuclear staining of α-Mangostin treated MDA-MB-231 cells.

a) Control cells (0.1% DMSO) and b) α-Mangostin (10µM) treated cells after 24h.
Arrows shows the α-Mangostin treated cells morphological changes like cell shrinkage,
formation of membrane blebs, fragmentation and condensation of nuclear chromatin.

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4.6.7. Effect of α-Mangostin on MDA-MB-231 cell cycle determined by Flow

cytometric analysis:

The induction of apoptosis in cells treated with α-Mangostin was further

confirmed by Flow cytometric analysis. The DNA fluorescence histogram of control cells
shows prominent G1 phase, followed by S and G2/M phases (Figure-39A). The control
cells showed only 1.85% of hypodiploid DNA and the percentage was increased to
13.70% on exposure of cells to 10µM of α-Mangostin (Figure-39). Thus, MDA-MB-231
cells in the presence of α-Mangostin showed significant apoptotic peak to the left of
G1/G0 peak as compared to control cells (Figure-39B).

4.6.8. α-Mangostin activates caspase-3 mediated apoptosis in MDA-MB-231 cells:

Caspase-3 is a critical regulator of cell death during execution phase of cells

undergoing apoptosis. It is necessary for the cleavage of a large number of proteins leads
to morphological changes in cells such as DNA fragmentation and nuclear collapse
during apoptosis. MDA-MB-231 cells treated with 10µM of α-Mangostin for 24h and
fixed with 4% paraformaldehyde. Immunocytochemical staining for caspase-3 protein
expression was done and developed using DAB substrate kit. Then cells were
counterstained with hematoxylin to show nuclear staining (blue). The results showed the
expression of caspase-3 in α-Mangostin (10µM) treated cells, as a marker of apoptosis
(Figure-40).

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Figure. 39. Flow cytometric analysis was performed to determine the apoptotic effect of
α-Mangostin on MDA-MB-231 cells. The cells were exposued to α-Mangostin (10µM)
and 0.1% DMSO for 24h. Then cells were harvested, fixed and stained with propidium
iodide, and the DNA content was analyzed by Flow cytometry. The DNA histogram of
(A) Control cells (0.1% DMSO) and (B) 10µM α-Mangostin treated cells after 24h.

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Figure. 40. Immunocytochemical staining for detecting caspase-3 expression in MDA-

MB-231 cells. a) Control cells (0.1% DMSO) and b) 10µM α-Mangostin treated cells
after 24h. Arrows shows the expression of caspase-3 as a marker of apoptosis in α-
Mangostin treated cells. Pictures were taken with a 40x objective under light microscope.

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4.7. α-Mangostin suppressed the expression of COX-2 and iNOS analyzed by

Immunocytochemistry:

The MDA-MB-231 cells were exposed to α-Mangostin (10µM), stimulated with

LPS (1µg/ml) and analyzed by Immunocytochemistry for the detection of COX-2 and
iNOS expression. The over-expression of COX-2 was observed at 2h of LPS stimulation
in control (0.1% DMSO) cells (Figure - 41a) compared to α-Mangostin (10µM) treated
cells (Figure-41b). The over-expression of iNOS was seen at 12h of LPS stimulation in
control cells (Figure - 42a) compared to α-Mangostin (10µM) treated cells (Figure - 42b).

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Figure. 41. Immunocytochemical analysis for detecting COX-2 expression in LPS

stimulated MDA-MB-231 cells. a) Control cells and b) 10µM of α-Mangostin treated
Pictures were taken with a 10x objective under light microscope.

Figure. 42. Immunocytochemical analysis for detecting iNOS expression in LPS

stimulated MDA-MB-231 cells. a) Control cells and b) 10µM of α-Mangostin treated
cells. Pictures were taken with a 10x objective under light microscope.

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4.8. Effect of α-Mangostin on the expression of COX-2 and iNOS genes in LPS
stimulated MDA-MB-231 cells:

The effect of α-Mangostin on the transcriptional regulation of pro-inflammatory

genes, COX-2 and iNOS in LPS stimulated MDA-MB-231 cells was analyzed by RT-
PCR. The MDA-MB-231 cells were pre-treated with different concentrations (2.5 to
20µM) of α-Mangostin and with 0.1% DMSO (Control). Then cells were stimulated with
LPS (1µg/ml) for 2h and 12h for the expression of COX-2 and iNOS mRNA
respectively. The total RNA was extracted and cDNA was synthesized by using relevant
primers. The amplified PCR products were electrophoresed on 1.5% agarose gels and
visualized by ethidium bromide staining. As shown in Figure-43, the levels of COX-2
and iNOS cDNA significantly decreased in a dose-dependent manner compared to cDNA
of GAPDH in α-Mangostin treated MDA-MB-231 cells at 12h of LPS stimulation.

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Figure. 43. The effect of α-Mangostin on the expression of COX-2 and iNOS genes in
LPS stimulated MDA-MB-231 cells determined by RT-PCR. Cells were pre-treated with
the different concentrations (2.5 to 20µM) of α-Mangostin and with 0.1% DMSO (C).
Then stimulated with LPS (1µg/ml) for 2h and 12h for the expression of COX-2 and
iNOS respectively and total RNA from MDA-MB-231 cells was used as a template for
cDNA synthesis and then subjected to PCR. The amplified PCR products were
electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. The
cDNA of COX-2 (305bp) and iNOS (370bp) were detected and found that the expression
levels were decreased with increasing concentrations of α-Mangostin. The GAPDH was
used as the loading control. Data shown are representative expression patterns from
triplicate independents.

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4.9. Effect of α-Mangostin on COX-1 and COX-2 protein expression in LPS

stimulated MDA-MB-231 cells:

The impact of α-Mangostin on the expression of COX-1 and COX-2 proteins in

LPS stimulated MDA-MB-231 cells was investigated by Western blot analysis. The
analysis was performed with whole cell lysate from cells pre-treated with different
concentrations (2.5 to 20µM) of α-Mangostin and 0.1% DMSO (Control), stimulated for
2h with LPS (1µg/ml) for COX expression.

As per the results presented in Figure- 44A, no change in COX-1 protein levels
was observed whereas α-Mangostin at 10-20µM showed remarked changes in COX-2
protein levels. GAPDH was used as loading control and the concentration-dependent
inhibition of COX-2 protein expression was observed at 10-20µM of α-Mangostin
compared to GAPDH expression (Figure- 44A, B).

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Figure. 44. (A) The effect of α-Mangostin on the expression of COX-1 and COX-2
proteins in LPS stimulated MDA-MB-231 cells assessed by Western blotting. After pre-
treatment with the indicated concentrations of α-Mangostin and with 0.1% DMSO (C),
cells were stimulated with LPS (1µg/ml) for 2h. Equal loading was confirmed by
stripping the blot and reprobing it for GAPDH. Data shown are representative expression
patterns from triplicate independents.
(B) The densitometric data of COX-2 protein expression representing the relative density
of the Western blot bands normalized to GAPDH.

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4.10. Inhibition of nitric oxide production in α-Mangostin treated MDA-MB-231

cells:

The nitrite concentration, an indicator of nitric oxide production was estimated by

Griess assay in α-Mangostin pre-treated, LPS (1µg/ml) stimulated MDA-MB-231 cells

culture supernatant. As shown in Figure- 45, the levels of nitrite concentration reduced

dose-dependently by α-Mangostin on comparison with control.

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Figure. 45. The nitrite concentration in LPS stimulated MDA-MB-231 cells was
measured by Griess assay. The cells were pre-treated with different concentrations (2.5,
5, 10, 15 and 20µM) of α-Mangostin and with 0.1% DMSO (C), then stimulated with
LPS (1µg/ml) for 12h. The nitrite levels were expressed as mean ± SEM from triplicate
determinants.

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4.11. Effect of α-Mangostin on iNOS protein expression in LPS stimulated MDA-

MB-231 cells:

The iNOS protein expression in LPS stimulated MDA-MB-231 cells was assessed

by Western blot analysis. The analysis was carried with whole cell lysate from cells pre-

treated with different concentrations (2.5 to 20µM) of α-Mangostin and with 0.1%

DMSO (Control), stimulated with LPS (1µg/ml) for 12h for iNOS expression. The results

showed dose-dependent inhibition of iNOS protein expression at 10-20µM compared to

GAPDH expression (Figure- 46A, B).

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Figure. 46. (A) The effect of α-Mangostin on the expression of iNOS in LPS stimulated
MDA-MB-231 cells assessed by Western blotting. After pre-treatment with the indicated
concentrations of α-Mangostin and with 0.1% DMSO (C), cells were stimulated with
LPS (1µg/ml) for 12h. Equal loading was confirmed by stripping the blot and reprobing it
for GAPDH. Data shown are representative expression patterns from triplicate
independents.
(B) The densitometric data of iNOS protein expression representing the relative density
of the Western blot bands normalized to GAPDH.

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