Asian Journal of Chemistry; Vol. 27, No.

7 (2015), 2513-2517

ASIAN JOURNAL OF CHEMISTRY

[Link]

Green Synthesis of Magnesium Oxide Nanoparticles Using Brassica oleracea

and Punica granatum Peels and their Anticancer and Photocatalytic Activity

P. SUGIRTHA1, R. DIVYA1, R. YEDHUKRISHNAN2, K.S. SUGANTHI2, N. ANUSHA1,*, V. PONNUSAMI1 and K.S. RAJAN2
1
School of Chemical & Biotechnology, SASTRA University, Thanjavur-613 401, India
2
Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), SASTRA University, Thanjavur-613 401, India

*Corresponding author: Fax: +91 4362 264120; E-mail: anusha@[Link]

Received: 8 May 2014; Accepted: 25 July 2014; Published online: 30 March 2015; AJC-17069

Experiments were carried out on the synthesis of magnesium oxide nanoparticles using the water extract of cauliflower and the water
extract of pomegranate peels. The primary particles were of sizes 30-45 and 50-65 nm in the powder synthesized using the water extract
of cauliflower and the water extract of pomegranate peels respectively. Polycrystalline powders were obtained when synthesis was carried
out using the water extract of pomegranate peels, white crystalline powders were obtained when using the water extract of cauliflower.
The green-synthesized magnesium oxide nanoparticles exhibited good anticancer activity, with 31.2 µg/mL being the nanoparticles
concentration required for destruction of 50 % of HeLa cells. The green-synthesized magnesium oxide nanoparticles were found to
possess photocatalytic activity both under UV irradiation and sunlight.

Keywords: MgO, Nanoparticles, Green synthesis, Anticancer, Photocatalytic.

INTRODUCTION Green synthesis is an attractive technique for preparation of

nanomaterials predominantly owing to its eco-friendliness.
It is well recognized that the nanoparticles have wide When the green synthesis of nanoparticles is successfully
ranging applications in the field of agriculture, optical fibres, scaled up, they are likely to be cost-effective when compared
antimicrobials, electronics, catalyst, biomedical, bio labelling, to chemical and physical methods12. This cost-effectiveness is
sensitized solar cell, pharmaceuticals, etc.1,2. The performance attributed to fact that the green synthesis is performed at near-
of a nanomaterial can be tuned depending on the size, shape ambient conditions without the requirement of use of high
and crystal structure. Nanomaterials can be prepared through pressure, temperature or heat energy13,14.
green, chemical or microbial synthesis3. Magnesium oxide is Pomegranate (Punica granatum) belongs to the family of
one of the widely used metal oxide for catalytic applications. Lythraceae. It is one of the oldest edible fruit and contains poly-
Magnesium oxide is nontoxic and biodegradable. However, phenols at higher concentrations. The other major components
MgO is toxic to microbes, through its binding to intracellular are flavones, flavan-3-ols and anthocyanidin. The important
protein at low concentration4. Magnesium oxide is also used tannins in the pomegranate are hydrolazable tannins including
in remediation of toxic waste and as superconductor products5. gallo tannins, ellagic tannins and condensed tannins. It may
Gas and humidity sensors have been made with MgO as the be recalled that the tannins are good antioxidants15. The peels
key constituent. Magnesium oxide is also a good adsorbent of pomegranate too contain polyphenols such as ellagic tannins,
for vapours and gases such as acetone, ammonia, carbon gallic acid and ellagic acid. Pomegranate has therapeutical
dioxide, methanol, dimethylamine, trimethyl acetaldehyde, applications, apart from being used in preparation of cosmetics,
benzaldehyde and acetaldehyde6. food recipes, tinctures, etc16.
Chemical methods used for the preparation of MgO nano- Cauliflower (Brassica oleracea) contains a number of
particles include microwave-induced combustion, precipi- phytonutrients such as glucosinolate, ascorbic acids, polyphenols,
tation, hydrothermal, aerosol combustion, flame spray pyrolysis, anthocyanins, sulphoraphane and indole-3-carbinol, which are
chemical vapour deposition, flame metal combustion, well-known antioxidants. Anthocyanins are known to provide
sonication, sol-gel method and lysine nitrate combustion5,7,8. protection against free radicals17. It has been reported that the
Magnesium oxides have been synthesized in different shapes glycosinolate derived product possess anticancer and antioxidant
leading to nanoplates, nanocubes, nanoparticles and nanowires9-11. activity, while indole-3-carbinol inhibit growth of cancer cells18.
2514 Sugirtha et al. Asian J. Chem.

In the present work, attempts have been made to utilize amount of the synthesized powder was carefully sprinkled on
the potential of this pomegranate peels and cauliflower for the stub used to place the specimen for imaging. The stub
preparation of magnesium oxide nanoparticles. While the with the synthesized powder was coated with gold before
extracts of cauliflower and pomegranate peels have been used imaging was performed at the acceleration voltage of 3 kV.
to synthesize silver nanoparticles 19,21,22 and gold nano- The crystalline nature of the sample was ascertained using
particles19,20,22, to the best of our knowledge, this is the first a powder X-ray diffractometer (D8 Focus, Bruker, Germany),
report on the synthesis of magnesium oxide nanoparticles using by carrying out scan in the ‘2θ’ range of 25-55. The wavelength
pomegranate peel and cauliflower. The prepared nanomaterial of the radiation used for X-ray diffraction experiment was
has been characterized and its anticancer and photocatalytic 1.54 Å.
activity are being reported. A dispersion of synthesized product was made in water
to enable determination of hydrodynamic size distribution and
EXPERIMENTAL zeta potential. A zetasizer (Nano-ZS, Malvern, UK) was utilized
Magnesium sulphate heptahydrate (MgSO4·7H2O) was for this purpose. A standard operating procedure designed for
obtained from Merck, Mumbai. Fresh Brassica oleracea particle dispersions in water was used for the measurements.
(Cauliflower) and Punica granatum (Pomegranate peels) were Photocatalytic activity: One hundred millilitre of dye
obtained from local market. Double distilled water was utilized solution containing 50 ppm of reactive black dye was taken as
for the preparation of extracts. the feed solution for experiments carried out to ascertain the
Reactive black dye was purchased from Indian dyes and photocatalytic activity of synthesized powders. A photochemical
chemicals, Coimbatore. HeLa cell lines were obtained from reactor fitted with a lamp of 125 W was used as the source for
National Centre for Cell Sciences, Pune (NCCS). Minimum ultraviolet radiation. The wavelength of UV radiation emitted
Essential Medium was purchased from Hi Media Laboratories, by the lamp was 254 nm.
Mumbai. Fetal Bovine Serum (FBS) was purchased from Cistron The dye sample was withdrawn at intervals of 10 min to
Laboratories, Andhra Pradesh. Trypsin, methylthiazolyl determine the absorbance at 580 nm, which is the wavelength
diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide corresponding to the maximum absorption coefficient for the
(DMSO) were procured from Sisco Research Laboratory dye solution. The dye degradation was studied in the presence
Chemicals, Mumbai. of sunlight, without the use of external UV source also. The
Preparation of water extract and nanoparticles: About mass of nanoparticles used for degradation studies was 50 and
500 g of fresh Brassica oleracea (cauliflower) and 24 g dry 100 mg, respectively for degradation using photochemical
powder of Punica granatum (pomegranate peels) was taken reactor and sunlight, respectively.
into separate beaker and double distilled water added to make Anticancer activity: The HeLa cells were maintained as
up to 250 mL. This mixture was boiled for about 15-20 min. per standard protocol. HeLa cells were plated in 24-well plates
The mixture was then cooled, followed by filtration to separate at the rate of 1 × 105 cells per well and incubated at 37 ºC and
solid matter from the extract. The extract was collected and 5 % CO2 condition. Once the cells reached confluence, the
stored for subsequent use. synthesized powder were added at different concentrations and
About 250 mL of magnesium sulphate heptahydrate incubated for 24 h. The sample was removed from the well
solution of 0.1 M concentration was prepared using double- after incubation and washed with pH 7.4 phosphate-buffered
distilled water. To this solution, 250 mL of water extract of saline or MEM without serum. 100 µL/well, corresponding to
cauliflower and pomegranate peels was added drop by drop 5 mg/mL of 0.5 % 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
with continuous stirring in a magnetic stirrer. The total time tetrazolium bromide (MTT) was added and incubated for 4 h.
consumed for addition of 250 mL of water extract of cauli- Upon completion of incubation, DMSO was added to all the
flower and pomegranate peels was about 12 h. Solid-liquid wells at an amount of 1 mL each. The cell concentration was
dispersion was obtained at the end of the reaction, which was determined by measuring the absorbance at 570 nm using a
then centrifuged at 7000 rpm for about 10 min. While the UV-spectrophotometer. DMSO was used as the blank for these
supernatant was discarded, the pellet was washed, dried and measurements. The concentration of synthesized powder
calcined at 450 ºC for 1 h. The mass of sample was measured investigated for the anticancer activity ranged between 7.8 and
before and after calcination to determine the weight loss during 1000 µg/mL. The studies on anticancer activity were out-
calcination. sourced to Life Teck Research Centre, Chennai.
Characterization of extract and the synthesized product:
RESULTS AND DISCUSSION
The ethanol extract of pomegranate peels was analysed using
(GC-MS) gas chromatograph coupled with mass spectrometer Components present in ethanolic extract of pomegranate
(Clarus 500, PerkinElmer, USA). The water extract of cauli- peels and water extract of cauliflower: Fig. 1 shows the
flower was subjected to LC-MS/MS Spectrometer (MicrO results of GC-MS analysis of the ethanolic extract of pome-
TOF-Q II, Bruker Daltonik GmbH, Germany). granate peels. The important components of the extracts were
Electron microscopy is a valuable tool to determine the found to be n-hexadecanoic acid (256), 2-furancardoxaldehyde
morphological features of materials. In the present work, a 5-(hydroxymethyl)- (126), furfural (96), 4H-pyran-4-one 2,3-
field emission scanning electron microscope (JSM, 6701F, dihydro-3,5- dihydroxy-6-methyl- (144), D-allose (180) etc.,
JEOL, Japan) has been used to determine the morphological The classes of antioxidant compounds identified from LC-
features and the particle size of the prepared samples. A small MS/MS analysis of water extract of cauliflower are 6-mercap-
Vol. 27, No. 7 (2015) Green Synthesis of Magnesium Oxide Nanoparticles Using B. oleracea and P. granatum Peels 2515

100
13.07 23.90 200
4.80 97 73
96
180 From cauliflower
From pomegranate peels
11.20
43
160
18.40
60
140
%
120

Intensity
20.77
73
9.80 12.39 13.66 15.62 17.97 22.88 100
9.10 95 86 68 43
43 18.76 99
6.74 7.43 43 43
2.77 98 43 25.08 80
44 4.04 109
44
0
60
3.39 5.39 7.39 9.39 11.39 13.39 15.39 17.39 19.39 21.39 23.39 25.39 27.39
40
Fig. 1. GC-MS analysis of water extract of pomegranate peels 20
0
25 30 35 40 45 50 55
topurine (152.01), resveratrol (228.07), 7-hydroxyflavone 2θ (°)
(238.06), ipriflavone (280.10), kaempferol (286.04), catachin Fig. 3. Powder X-ray diffraction patterns of MgO nanoparticles using water
(290.07), herperetin (302.07), quercitrin (448.10), glycyrrhe- extracts of cauliflower and pomegranate peels
tinic acid (470.38), naringin (580.17) etc.,
Characteristics of the product: Fig. 2a,b show the Several components of water extract have hydroxyl groups
scanning electron micrograph of the as-synthesized, calcined that may react with MgSO4. H2O leading to the formation of
powders using the two water extracts. It is evident from Fig. 2a Mg(OH)2. The evidence for this pathway stems from the weight
that the product synthesized using water extract of cauliflower loss observed during calcination. While the theoretical weight
contains nearly spherical nanoparticles with diameter ranging loss calculated from stochiometry during the transformation
between 30 and 45 nm. Very few particles of relatively diameter of Mg(OH)2 to MgO is 30 % , the actual weight loss observed
are also seen, but they are relatively less in number upon was 31 and 31 % for intermediates obtained during the reaction
comparison with particles in the size range of 30-45 nm. The between MgSO4·7H2O and water extract of cauliflower and
product synthesized using the water extract of pomegranate between MgSO4·7H2O and water extract of pomegranate peels,
peels contains particles in the size range of 50-65 nm (Fig. 2b). respectively, which are closer to the theoretical value.
The difference in particle size could be attributed to the diffe- Hydrodynamic size distribution: Hydrodynamic size
rence in the nature of water extracts. The powder X-ray diff- refers to the diameter of the particles and the surrounding liquid
raction patterns (Fig. 3) reveal the presence of MgO in the layer formed when particles are dispersed in a liquid. Though
product synthesized using water extract of cauliflower, as the the hydrodynamic diameter depends on the nature of particle-
spectra matches with that of reference spectra for MgO (JCPDS liquid interaction and the presence of ions in the liquid also
No-270759). The broad nature of peaks indicates the presence apart from the primary size of the particles, hydrodynamic
of nanocrystalline MgO. The crystallite size was calculated diameter is widely used to characterize the state of aggregation
using Scherer’s formula and was found to be 24.13 nm for in nanoparticles dispersion23-25. A lower value of hydrodynamic
MgO synthesized using water extract of cauliflower. The MgO diameter denotes lower extent of particle aggregation, while a
nanoparticles synthesized using water extract of pomegranate higher value is indicative of higher degree of aggregation.
peels were found to be polycrystalline. The hydrodynamic size distribution for MgO nanopar-
ticles synthesized using the two extracts in water is shown in
Fig. 4. It is evident that the size distribution is monodisperse,
with the average hydrodynamic diameter of 206 and 350 nm,
respectively for samples prepared using the water extract of
cauliflower and pomegranate peels, respectively. While com-
paring the average hydrodynamic size with the particle size
measured using scanning electron microscopy (30-45 and 50-
65 nm) and the crystallite size determined by Scherer’s formula
(24.13 and 24.64 nm), the ratio of average aggregate size to
primary particle size is about 5.
Fig. 2. Scanning electron micrograph of green-synthesized nanoparticles
The sizes of aggregates are lower when the component of
using [a] water extract of cauliflower; [b] water extract of repulsive forces between the colloidal objects is higher than
pomegranate peels the magnitude of attractive forces. Zeta potential is a measure
of charge strength on the particle surface. Higher magnitude
Role of water extract of cauliflower and pomegranate of zeta potential indicates higher magnitude of repulsive
peels in product synthesis: There are two widely used routes interactions and hence indicates size stability of colloids. The
for synthesis of MgO from MgSO4·7H2O. In one of the routes, a zeta potentials of dispersion of green-synthesized MgO nano-
carbonate such as sodium carbonate is reacted with MgSO4·7H2O particles, prepared using cauliflower and pomegranate peels,
leading to the formation of MgCO3. The calcination of MgCO3 in water was found to be -29.2 ± 1.5 and -30.5 ± 1 mV, respec-
results in the formation of MgO. Another route involves tively indicating the presence of negative charges on the particle
reaction between MgSO4·H2O and a strong base such as NaOH surface when the same was dispersed in water.
or KOH leading to the formation of Mg(OH)2, which upon Anticancer activity: The infleunce of MgO nanoparticle
calcination results in MgO. concentration of viability of HeLa cells was carried in the nano-
2516 Sugirtha et al. Asian J. Chem.
12 49.4
49 (a)
10 48
Using cauliflower 47 512.26
8

Transmittance (%)
Intensity (%)

Using pomegranate
46
peels
6 45 2851.99
1095.27
4 44
2922.47
43 3416.42
1643.11
2
42

0 41
75
175
275

375
475
575

675
775
875

975
1075
1175

1275
1375
1475

1575
40.0
Size (nm) 4000 3000 2000 1500 1000 400
Fig. 4. Influence of source of water extract on hydrodynamic size Wavelength (cm )
–1

distribution of dispersions of MgO nanoparticles in water

40.3
40
(b)
particle concentration range of 7.8-1000 µg/mL. When com- 39
pared to the control, the cell viability decreased significantly 38
with the addition of nanoparticles (Fig. 5). At about a nano-

Transmittance (%)
37 520.41
particle concentration of 31.2 µg/mL, the cell viability was
36 3398.80
reduced to about 50 %. The fact that the cell viability was found 2920.83

to be independent of green reagent, indicates that both the 35

green sources (cauliflower and pomegranate peels) contain a 34 1616.52
1102.41
number of components with anticancer activity, which could 33
be adsorbed on the particle surface. 32

120 31
30.0
100 4000 3000 2000 1500 1000 400
–1
From cauliflower Wavelength (cm )
From pomegranate
Cell viability (%)

80 Fig. 6. FTIR spectra of MgO nanoparticles synthesized using [a] water

extract of cauliflower; [b] water extract of pomegranate peels
60

40

20

0
0 7.8 15.8 31.2 62.5 125 250 500 1000
Nanoparticle concentration (µg/mL)
Fig. 5. Influence of MgO nanoparticle concentration and the green source
on cell viability of HeLa cells

The FTIR spectra of the synthesized nanoparticles (Fig. 6)

shows absorption bands characteristics of several functional
groups associated with organic compounds. For instance, peaks
around 3400, 2900 and 1600 cm-1 may be attributed to the
presence of phenols and alcohols, alkanes and amides func-
tional groups. The anticancer activity is also confirmed through
microscopy (Fig. 7a,b), in which the extensive damage to
cellular morphology is evident for nanoparticle concentrations
of 31.2 µg/mL and above. All these testify the anticancer
activity of green synthesized MgO nanoparticles. Fig. 7. Microscopic features of HeLa cells (control) and cells treated with
Photocatalytic activity: The photocatalytic activity of various concentrations of MgO nanoparticles prepared using [a]
MgO nanoparticles synthesized using the water extract of water extract of cauliflower; [b] water extract of pomegranate peels
cauliflower and pomegranate peels, for degradation of dye
under ultraviolet irradiation is shown in Fig. 8. It is evident water extract of pomegranate peels (82 % dye degradation
from Fig. 8 that MgO nanoparticles synthesized from both after 50 min). The photocatalytic activity of MgO nanoparticles
the green sources possess photocatalytic activity. However, synthesized using water extract of cauliflower (Fig. 9), under
the MgO nanoparticles synthesized using water extract of sunlight (30 % dye degradation after 1 h) is better than that of
cauliflower have shown better photocatalytic activity (93 % MgO prepared using the water extract of pomegranate peels
dye degradation after 50 min) than those prepared using the (26 % dye degradation after 1 h). The surface area of particles
Vol. 27, No. 7 (2015) Green Synthesis of Magnesium Oxide Nanoparticles Using B. oleracea and P. granatum Peels 2517
100 and scalable method for preparation of MgO nanoparticles
MgO from cauliflower
90
MgO from pomegranate peels
for a variety of applications.
80
ACKNOWLEDGEMENTS
Dye degradation (%)

70
60 This work was supported by (i) PG teaching grant no:
50 SR/NM/PG-16/2007 of Nano Mission Council, Department of
40 Science & Technology (DST), India; (ii) SASTRA University
30 for Infrastructural support.
20
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