SUPPLEMENTARY MATERIAL
Effects of antioxidant and anticancer activities of methylpheophorbide a, isolated from wheat grass (Triticum aestivum Linn.)
Priyabrata Dasa, Suvra Mandala, Sudeshna Gangopadhyaya, Kalipada Dasb, Arpita Ghosh
Mitrac, Swati Dasguptaa, Soma Mukhopadhyaya, Ashis Mukhopadhyaya, * aNetaji Subhas Chandra Bose Cancer Research Institute, 16 A, Park Lane, Kolkata, West
Bengal, India bBose Institute, 93/1, Acharyya Prafulla Chandra Road, Kolkata, West Bengal, India cMedica Superspeciality Hospital , Mukundapur, Kolkata, West Bengal, India
*Corresponding author: Ashis Mukhopadhyay
Tel: +913322276161; Fax: +913322264704; Email: molecularbiology.ncri@gmail.com

ABSTRACT:
Methylphophorbide a (MPa) has been isolated from wheat grass plant and its antioxidant, hydroxyl radical scavenging activities and reducing capcity have been compared with those furnished by aqueous extract of the plant and is found to show a significant increase in these properties. The compound shows iron binding capacity where the Fe2
+ binds with MPa by two types of binding pattern with dissociation constants 157.17 and 27.89. It has antioxidant and effective cytotoxic effect on HeLa and Hep G2 cells. The percentage of cell survival decreases with increasing concentration of MPa. These findings have provided evidences for the traditional use of wheat grass plant in the treatment of cancers, oxidative stress as well as iron overloaded disorders.
Keyword: wheat grass, methylpheophorbide a, antioxidant activity, Anti-cancer,
Iron binding affinity.
1. Experimental
Chemicals
Thiobarbituricacid, De-oxyribose, 2,2′-Azinobis-3-ethyl-benzothiazoline-6-sulfonic acid
(ABTS
+
), Trichloroethanoic acid, Xylenol orange, Ferrozine, Hydrogen peroxide and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Plant Material and Isolation Procedure
Plant Material
Wheat plant ( Triticum aestivum Linn.) was cultivated in calcareous soil at Darjeeling district,
West Bengal, India. The plant was authenticated by Dr. M.N. Das, former Assistant Director
(Pharmacognosy), National Research Institute for Ayurvedic Drug Development, Kolkata,
India. Specimens (voucher number WG-04) were preserved in our laboratory (Netaji Subhas
Chandra Bose Cancer Research Institute, Kolkata) for further reference.
Purification of active compound:
Wheat ( T. aestivum ) was cultivated in calcareous soil of Darjeeling district, W.B., India, and
7-8 days old wheat grass were collected. 1 kg of fresh leaves with stems of wheat grass was cut into small pieces and soaked in 5 litres of methanol at room temperature. After 72 h, the methanol extract was concentrated by distillation under reduced pressure (0-5 mm of Hg).

The concentrated mass (10.5 g) was chromatographed over silica gel column (E Merck, 60-
100 mesh) using solvents of increasing polarity starting from n-hexane as eluents. n-Hexaneethylacetate (4:1) solvent system yielded five fractions which on concentration afforded an intense black solid material, which was recrystalized and the structure was determined by spectral analysis.
Charecterisation of Compound
The black solid was crystallized from n-Hexane-acetone mixture as a deep greenish black crystalline compound in 0.11% yield. R f
0.33 [n-Hexane-ethylacetate 2:1; silica gel 60 F
254
(E.Merck) TLC plate]. UV, IR, 1H-NMR and Mass spectrometric analyses of the compound
(Fig.S1, S2, S3 and S4) identified it as methyl pheophorbide a. The analytical and spectral data were comparable with those reported previously by Smith et al. 1985 and Boxer et al.
1974. This pure compound (MPa) was used in the experiments (Fig.S5).
Preparation of dried juice used in the experiments
All 7-8 days germinated whole plants were washed and dried by distilled water. Then the plants were crushed in distilled water and got greenish extract. The purified component and whole plant extracts were dried by rotary evaporator (40°C) to get a solid mass.1 mg whole plant extract was dissolved in 1 ml distilled water and prepared the working solution.
Biochemical Analysis
Analysis of antioxidant activity
Antioxidant activity was measured following 2,2′-Azinobis-3-ethyl-benzothiazoline-6sulfonic acid (ABTS
+
) radical cation decolorization assay at 734 nm using the standard protocol( Re et al. 1999)
Hydroxyl radical scavenging activity
A1.2 ml of the reaction mixture was prepared containing 2.8 mM of 112 µl deoxyribose, 25 mM of 150 µl FeSO
4
, 100 µM of 24 µl EDTA, 2.8 mMof 3.8 µl H
2
O
2
in 10 mMof 600 µl phosphate buffer pH 7.4 and different concentration of dried juice and MPa were added to it
(Aruoma&Cuppette 1997, Porter 1984). Finally 100 µM of 24 µl ascorbate was added to initiate the reaction and added required distilled water to make upto 1.2 ml. The reaction mixture was incubated for an hour at 37°C. Next 1ml of 1% TBA in 15mmol NaOH and 1ml of 2.8% TCA were added and the mixture was heated at 90°C for 30 mins. Samples were cooled; the colour produced was extracted in 2ml butanol and absorption was read at 532nm.
Percentage of inhibition was calculated with respect to the values of control. The level of

hydroxyl radical scavenging activity of MPa and extract were also similar to those of the atocopherol positive control.
Analysis of reducing activity
The reducing power of the sample was determined following the known procedure Oyaizu et al., 1986 (Oyaizu M 1986).This assay was performed by using 0.2 M sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide and 1 mg/ml sample. After 20 min of incubation at 50°C, 10% TCA was added in the mixture and then centrifuged. The supernatant was collected and mixed with 0.1% ferric chloride and the absorbance was read at 700 nm. The result was expressed as a percentage of the activity shown by 0.01 mg/ml of ascorbic acid.
Determination of iron dissociation constant
In determination of iron dissociation constant assay, different concentrations (17.98µM,
35.97µM, 89.92µM, 179.85µM, 359.69µM, 719.39µM, 1079.07µM ) of FeSO
4 solution were added to 16.502 µM MPa solution and each volume was made up to 1 ml by adding distilled water. After shaking, the mixture was incubated at 37ºC for 24 h. The spectrum was collected from 200 to 800 nm. Dissociation constant was established on the basis of 400 nm absorbance result using the standard protocol of ΔA=ΔA
max
- K d
ΔA/ [FeSO
4
], ΔA↔ΔA/
[FeSO
4
],(Fersht 1998).
Cell culture
Two human cancer cell lines, such as cervix adenocarcinoma cells (HeLa) and hepatocellular carcinoma cells (HepG2),were used to evaluate the cytotoxic effects of MPa. Cultures were maintained in DMEM (Life Technologies, Inc., Rockville, MD), supplemented with 10%
(v/v) fetal bovine serum (FBS, Life Technologies), 100 iu/ml penicillin (Himedia
Laboratories), 100µg/ml streptomycin (Himedia Laboratories) and kept in an incubator with humidified air with 5% CO
2
at 37°C.
Cell survival for a wide range of concentrations of MPa in 24–72 h was checked by MTT assay. Cell killing efficacy of MPa was found to be dependent upon the concentration and time of treatment. Current study revealed that cytotoxic effects of MPa on normal human cells (PBMC) were significantly different (P < 0.05) than cancer cells.
Initial seeding concentration was calculated in such a way that even after 72 h, cells should be semi-confluent so that the decrease in OD570 value (a measure of the viability)

could be attributed to the compound rather than to the greater number of cells in the wells leading to scarcity of nutrients and area of proliferation. It was also found that after 72 h, there was nearly 95% of cell survival, and the data was normalized taking this as control set.
This standardization was done for all the cells.
Isolation of human peripheral blood mononuclear cells (PBMC)
Human PBMC were isolated by Ficoll Histopaque (Sigma Chemical Co.) density gradient centrifugation technique from heparinized whole blood of healthy donor using standard protocol (Ariffin et.al., 2009). Cultures were maintained in RPMI-1640 (Life Technologies,
Inc., Rockville, MD), supplemented with 10% (v/v) FBS, 100 iu/ml penicillin, 100µg/ml streptomycin and kept in incubator with humidified air with 5% CO
2
at 37°C.
Preparation of test material
Stock solutions were prepared by dissolving required quantity of the compound in DMSO.
All further dilutions were made in DMEM medium. The final concentration of DMSO to which cell cultures were exposed never exceeded 0.25%.
Treatment with chemicals
Attached cells were plated in 10 cm culture dishes (1 × 10 6
cells/dish) in complete DMEM at concentrations determined to yield 60–70% confluent cultures.PBMC (2 × 10
5
cell/well) were cultured in quadruplicate in 96-well U-bottom tissue culture plates in complete RPMI-
1640 to a final volume of 200 μl/well. All of them were treated with the indicated concentrations of MPa, and harvested at indicated times. As an untreated solvent control, cells were treated with DMSO (Sigma Chemical Co., St. Louis, MO) at a final concentration of <0.1%.
MTT assay for cell viability
The mitochondrial dehydrogenase activity of the cells after exposure to MPa was determined by colorimetric assay (Qubaisi et.al. 2011). For the experiment, all the cultures were treated with or without the compound after 24 h after seeding. MTT assay was carried out after stipulated time interval using the standard protocol and the optical density was measured at
570 nm using a UV/Vis spectrophotometer (Shimadzu model 2450). The control value corresponding to untreated cells was taken as 100% and the viability of the treated samples were expressed as a percentage of the control. The LD50 values were determined as the concentration that reduced cell viability by 50%.
Morphological Study with Fluorescence Microscope

The AO/EB staining (Acridine orange/Ethidium bromide) method was used to observe the morphology of apoptotic cells (Qubaisi et.al. 2011). Cells were suspended at a final concentration of 1x10
5 cells/well and cultured above 35 mm coverslip in 6-well flat-bottomed plates and allowed to adhere for 24 h before MPa treatment. Cells were exposed to the compound at different concentrations for 72 h. They were washed thrice in PBS at room temperature and then AO/EB mixture [25 μL, containing 100 μg/mL AO and 100 μg/mL EB in PBS (pH 7.4)] (Sigma Chemical Co.) was added to the culture plates for 30 min prior to observation under a fluorescence microscope (Carl Zeiss).
Statistical Analysis
All experiments were done in triplicate. Statistical calculation has been done accordingly and all the values were expressed as mean ± SEM (Standard Error of Mean). Statistical evaluation was calculated by student t- test. For all comparisons, p< 0.05 was considered as significant.
References
Ariffin SHZ, Omar WHHW, Ariffin ZZ, Safian MF, Senafi S, Wahab RMA. 2009. Intrinsic anticarcinogenic effects of Piper sarmentosumethanolic extract on a human hepatoma cell line. Can Cell Int.9:6.
Aruoma OI, Cuppette SL. 1997. Antioxidant methodology. In vivo and in vitro concepts. IL:
New York, AOCS Press.
Boxer SG, Closs GL, Katz JJ. 1974. The effect of Magnesium coordination on the
13
C and
15 N magnetic resonance spectra of chlorophyll a. The relative energies of nitrogen nπ * states as deduced from a complete assignment of chemical shifts. J Am Chem Soc. 96: 7058-7066.
Fersht A. 1998. Structure and mechanism in protein science. W.H. Freeman company, New
York.
Oyaizu M. 1986. Studies on products of browning reaction.Antioxidative activities of products of browning reaction prepared from glucosamine. Japanese J Nutri Diet. 44: 307–
314.
Porter NA. 1984. Chemistry of lipid peroxidation. In: Packer L, editor. Methods in
Enzymology. Vol. 105, New York, Academic Press; p 273-282.

Qubaisi, MA, Rozita R, Yeap SK, Omar AR, Ali AM, Alitheen NB. 2011. Selective
Cytotoxicity of Goniothalamin against Hepatoblastoma HepG2 Cells. Molecules.16: 2944-
2959.
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Figures
Fig. S1.UV spectrum of Methylpheophorbide a in ethanol shows the absorbtion at
400nm.

Fig. S2. ESI-Mass spectrometry of Methylpheophorbide a

Fig. S3. IR spectrophotometry of Methylpheophorbide a in PotassiumBromide.

Fig. S4. 300 MHZ 1H-NMR spectrum of MMethylpheophorbide a in cdcl3

Methylpheophorbide a 3D model of Methyl Pheophorbide a
Fig. S5. Structure of Methylpheophorbide a and 3 D model

a b
Fig S6.(a) Showed the spectrums of MPa, MPa with FeSO4 and only FeSO
4
, Where MPa concentration is constant. The spectrum (A) wasshowed for only MPa, (B, C, D, E, F, G,
H)spectrums were showedfor different concentrations (17.984µM, 35.969µM, 89.9225µM,
179.845µM, 359.69µM, 719.39µM, 1079.07µM) of FeSO
4 with of MPa and H1, G1, F1, C1,
B1 spectrums has presented for only 1079.07µM, 719.39µM, 359.69µM, 35.969µM
17.984µM concentration FeSO
4
. (b).showed two types of binding pattern. A was found for rapid binding pattern, where the dissociation constant is 157.166 and the B was for slow binding pattern, Where the dissociation constant is 27.8922.

100
90
80
70
60
50
0 0,005 0,01 0,015
Concentration (uM)
0,02 0,025
Fig. S7. Comparison of cytotoxic effect of MPa on normal and cancerous cell lines, as assessed by MTT assay. Cells were seeded into 24-well plates and allowed to overnight incubation at 37°C overnight. These cells were treated with stipulated concentrations of MPa and incubated for 48 h. The results were expressed as percentage viability of cells (Y-axis) against varying concentrations (X-axis). Mean ± S.D. were given.
healthy donor, HeLa cells, HepG2 cells
PBMC from

Fig. S8. Morphology of HepG2 cells using AO/EtBr staining/Detection by fluorescent microscopy of MPa induced apoptosis in HepG2 cells. Cell were grown for 24 h and then treated with MPa for 72 h followed by AO/EtBr staining. (a) Untreated cells were viable and uniformly green with intact structure. (b, c, d) Cells treated with 0.005 µM (b), 0.01 µM (c) and 0.015 µM MPa (d) displayed a mixed population of viable and early apoptotic cells characterized by loss of membrane integrity and chromatin condensation as bright-green to yellow spots. (e) Cells treated with 0.025 µM MPa showed appearance of late-apoptotic cells as dense orange areas of chromatin condensation with orange intact nucleus depicting secondary necrosis, indicating mixed population of viable, early apoptotic, late apoptotic and necrotic cells. Magnification ×400. Triplicate samples were used for each concentration, and in each sample, a minimum of 200 cells were analyzed.

Table S1: Reducing power was expressed as a percentage of the activity shown by 0.01 mg/mL of ascorbic acid.Trolox was used as a positive control for ABTS and α-tocopherol was used for hydroxyl radical scavenging assay, respectively.
Compound (mg/ml)
Wheat Grass Juice
Antioxidant status
(µM trolox equivalent)
Reducing Power
( % mg/ml)
0.62 ± 0.03
Methylpheophorbide a 1.30 ± 0.02
87.87 ± 0.77
97.78 ± 0.13
Hydroxy
( % mg/ml)
12.20 ± 1.46
47.64 ± 1.16
Radical
Scavenging Activity