VCO as a Novel Radioprotector – 1
INTRODUCTION
Statement of the problem
Does virgin coconut oil reduce chromosomal damage induced by gamma ray exposure
in Mus musculus Linn.?
Background of the Study
Virgin coconut oil (VCO) has emerged as a very popular food supplement with the
advent of surges in research and marketing efforts of both local and international groups. Virgin
coconut oil has earned the label “drugstore in a bottle” (Dayrit, 2005) due to its diverse benefits ranging from being a dietary, anti-hypertensive, anti-diabetic, anti-cancer and anti-microbial
supplement. Recent studies have also shown that the use of VCO has anti-mutagenic (Buenafe
et al,. 2008) and anti-teratogenic benefits (Morales et al., 2009). However, there is still a need to
generate substantial scientific bases for the many health claims of VCO (PCIERD, 2008).
Marina et al. (2009) reported that VCO had significantly high antioxidant content, owing
to the phenolic content of the oil and processing method used. The polyphenols of VCO are
capable of a higher degree of in vitro lipid peroxidation as compared to phenols from other
coconut oils (Nevin & Rajamohan, 2006). Nevin and Rajamohan (2006) also proved that the
antioxidant property of VCO is due to its extraction where there are more biologically active
components retained such as vitamin E and polyphenols as compared to other coconut oils (i.e.
copra oil and groundnut oil). Antioxidants, being able to sequester free radicals that cause
cellular and genetic damages, render possible anti-mutagenic effects. Free radicals may be
generated through exposure to radiation and cause subsequent mutagenetic effects that can
cause genetic material damage and/or cell death.
According to Nair et al. (2001), many plant extracts contain substantial antioxidants that
offer radioprotection – examples of which are Curcmin, Orientin, and Vicinin. Virgin coconut oil,
noted to have high antioxidant property, is very promising in rendering radioprotection. To date,
there is a lack of reported studies on using VCO as a radioprotective agent. This study attempts
to determine the possible radioprotective effect of VCO.
General Objective
This study aims to determine the possible radioprotective potential of VCO against whole-body
gamma irradiation in Mus musculus Linn.
VCO as a Novel Radioprotector – 2
Specific Objectives
1. To compare the effects of whole-body gamma irradiation (4.0 Gy) on the formation of
micronucleus in VCO-treated, amifostine-treated positive control mice, and non VCOtreated control mice;
2. To compare the possible radioprotective potential of two different dosages of VCO, 54µL
VCO/20g mouse (No Adverse Effect Level, NOAEL) and 168µL VCO/20g mouse (based
on normal human dietary usage) as pre-treatment to exposure of ionization radiation
using Micronucleus assay.
Hypotheses
Null hypothesis (Ho) : There is no significant difference in the effect of administering VCO as
prophylaxis in reducing the incidence of micronucleus formation brought about by the wholebody gamma irradiation of Mus musculus Linn.
Alternative hypothesis (Ha) : There is a significant difference in the administration of VCO as
prophylaxis in reducing the incidence of micronucleus formation brought about by the wholebody gamma irradiation of Mus musculus Linn.
Significance of the Study
A person exposed to radiation is at an increased risk of developing radiation-related
conditions such as radiation sickness and several neoplastic processes. This study will be
beneficial because it will identify whether VCO, a substance which is very readily available,
cheap and associated with various other health benefits, can serve as prophylaxis to people
with anticipated exposure to radiation (occupational or radiotherapy) to prevent development of
such radiation-related conditions.
Scope and Delimitation
This study will focus on the radioprotective potential of VCO, which has a known
antioxidant property. Based on the NOAEL dosage, 54µL per 20g mice and the equivalent of
the normal human consumption dosage for 20g mice (168µL) will be used. Amifostine will be
administered via IV push 10-15 minutes prior to irradiation. Forty randomly selected male albino
Swiss-Webster ICR (Institute of Cancer Research) mice will be subjected to irradiation and
grouped into the following: untreated (distilled water), treated with 54µL VCO/20g mouse,
treated with 168µL VCO/20g mouse, and treated with amifostine at 910mg/m2 per estimated
surface area of the mouse. Distilled water and VCO treatments will be administered through oral
VCO as a Novel Radioprotector – 3
gavage. The protective effect of VCO treatment prior to exposure of 4.0 Gy gamma radiation on
mice will be investigated. Treatment with VCO post-radiation will not be performed. Extraneous
effects and other histological analysis will not be performed. The study will only consider the
induction of micronucleus formation in the mice bone marrow. The exact mechanism of action
by which VCO exerts its possible radioprotective effects will not be included in the scope of the
study.
VCO as a Novel Radioprotector – 4
REVIEW OF RELATED LITERATURE
Radiation
The process of energy emission in the form of waves and particles is known as
radiation. A substance is said to be radioactive when it undergoes spontaneous decay
accompanied by the emission of radiation. The three major kinds of radioactive decay are alpha
(α), beta (β), and gamma (gamma) radiation (Brown LeMay and Bursten, 2000).
Ionizing and non-ionizing radiation are the two types of radiation that occur in nature
(Brown LeMay and Bursten, 2000). Examples of non-ionizing radiation include microwaves,
infrared waves and radiowaves. These are not considered as harmful as ionizing radiation
(Jagetia and Baliga, 2003; Naoghare, Kwon, Song, 2009). Examples of ionizing radiation
include x-rays and gamma radiation (Jagetia and Baliga, 2003). Ionizing radiation is commonly
used in radiotherapy and imaging modalities in the field of medicine, however, in excess these
may cause genetic damages (11th Report on Carcinogens, 2004).
Harmful Effects of Radiation
Ionizing radiation is that which has sufficient energy to remove electrons from atoms.
Ionization produces negatively charged free electrons and positively charged ionized atoms
(11th Report on Carcinogens, 2004). Damage to living tissues through a series of molecular
events is brought about by ionizing radiation. Human tissues contain about 80% water; thus, the
major radiation damage is caused by aqueous free radicals which are generated by the action
of such radiation on water. The interaction of these free radicals with cellular macromolecules
results in cellular dysfunction and mortality (Nair and Nomura, 2001).
Ionizing radiation, being a classical mutagen, induces a wide range of detrimental effects
- the most important of which is the damage it produces on cellular DNA. The DNA of exposed
cells undergoes breaks in its strand as well as damage to its bases. These effects eventually
cause chromosomal aberrations, and the fate of the exposed cells depends on the severity of
the damage. The cells may be killed and eliminated through apoptosis, or they may incorporate
the damage and proliferate, leading to carcinogenesis (Jagetia, Ventakesh and Baliga, 2003).
Mechanism by which Radiation becomes Harmful
According to Nair et al. (2001), the radiation damage to a cell is potentiated or mitigated
depending on several factors such as the presence of oxygen, sulfhydryl compounds and other
VCO as a Novel Radioprotector – 5
molecules in the cellular milieu. Hydrated electrons and H atoms react with molecular oxygen to
produce radicals. In the presence of oxygen, there is an increase in the sensitivity of cells to
ionizing radiation, and this is called the oxygen effect. The oxidative damage to cellular genetic
material is involved in both mutagenesis and carcinogenesis. The DNA aberrations are caused
by highly reactive oxygen radicals, which eventually lead to mutations and cell death.
The lethal damage caused by ionizing radiation is produced through heterologous
double strand breaks in the DNA. This is identified as the most common type of lesion that leads
to cell death especially in mammals. Such lesions in the DNA occur through direct interaction
with x-rays, or with reactive oxygen intermediates inside the cell, generated by exposure to
radiation. Although mammalian cells have the capacity to repair most double strand breaks in
the DNA, not all lesions can be repaired. Unrepaired DNA lesions lead to post-mitotic cell death,
which is linked to chromosomal abnormalities and DNA dysfunction (Haimovitz-Friedman et al.,
1994).
Brown et al. (2000) stated that the cells which are severely damaged by exposure to
radiation are those capable of reproducing at a rapid rate. Such category includes bone marrow
and blood-forming cells.
Impact of Radioprotection on the Effects of Radiation
Ionizing radiation (x-ray and gamma) causes damage to biological systems, which
proves to be significant especially in the control of growth of abnormal cells such as cancer cells
(Jagetia and Baliga, 2003; Naoghare, Kwon, Song, 2009; Nair, 2001). The need to provide
appropriate protection from unwanted effects of ionizing radiation therapy is thus essential
(Naoghare, Kwon and Song, 2009).
The prevention of damage in the exposed cells may possibly reduce chromosomal
instability, mutagenesis and carcinogenesis (Jagetia et al., 2003). According to Lee et al.
(2005), radioprotectors are chemical compounds which are capable of reducing the adverse
effects (lethality, mutagenicity, and carcinogenicity) produced by ionizing radiation on cells.
Since radiotherapy is the most common modality utilized for the treatment of human
cancers, a balance between the total dose of radiotherapy administered and the threshold limit
of normal critical tissues must be maintained. Therefore, the role of radioprotective substances
is really important in clinical radiotherapy (Nair et al., 2001).
An ideal radioprotector is defined as a substance that is relatively non-toxic to normal
cells. It should not degrade performance nor compromise the therapeutic effects of radiation
VCO as a Novel Radioprotector – 6
treatment. The ease of administration is also one of the criteria that should be fulfilled in order
for a substance to be considered as an ideal radioprotector (Lee et al., 2005).
Recently, the radioprotective action of plant extracts, herbal preparations and
phytochemicals in in vivo and in vitro studies have been reported. The ability of such
substances to provide protection against radiation is attributed to their antioxidant and free
radical scavenging properties. Phytomedicine, otherwise known as herbal medicine, is now
considered a well-established form of complementary medicine (Lee et al., 2005 and Nair et al.,
2001).
Radiation Dose
Maurya et al. (2007) and Nair and Salvi (2007), reported clinically significant exposure to
whole-body radiation of mice at 4.0 Gy gamma-radiation. Exposure levels of at least 0.4 Gy
were able to demonstrate significant cellular damage (Sofyan et al., 2005). According to several
studies testing the radioprotective potential of compounds, 4.0 Gy was employed (Maurya et al.,
2007; Nair and Salvi, 2007). It will also be adopted in this study.
Radioprotective Agents
Nair et al., (2001) have listed three categories of available radioprotective agents.
Radioprotectors consist of antioxidants and sulfhydryl compounds that limit damage to the
intestines and central nervous system. Adaptogens are usually derived from plants and animals
and present relatively minimal toxicity, and serve to activate the natural defenses of an
organism against radiation, such as the nonspecific immune system. Absorbants protect from
internal radiation by limiting the integration of radionuclides into cellular components.
Several mechanisms of radioprotection have been identified given the extensive
literature available on the subject.
The most commonly cited are free-radical scavenging
properties that are found in antioxidants, DNA-binding ligands, and plant extracts.
Other
mechanisms include immune stimulation, toxicity reduction via cellular protection, protease
inhibition, and prostaglandin synthesis, among others (Nair et al., 2001).
Chemical Agents
Among the most studied of radioprotective sulfhydryl compounds is WR 2721 which was
found to selectively conserve the integrity of normal cells in patients undergoing radiation (Nair
et al., 2001).
Prophylaxis with this compound showed decreased adverse effects on the
kidneys, nerves, blood, pelvic organs, and mucous membranes. It also reduced the levels of
VCO as a Novel Radioprotector – 7
cisplatin toxicity in patients undergoing treatment for lung and metastatic breast cancer.
Furthermore, there was no cumulative toxicity observed in patients with fractionated
radiotherapy.
In 1994, Blickenstaff, Reddy, Witt, and Lipkowitz studied the radioprotective possibility of
12 Schiff bases prepared using siacyladehyde. Results after irradiation with 6 mV photons were
variable among the different bases but the best results were obtained from mixtures of paminopropiophenone and its corresponding Schiff base, or of the former with the 1-(paminophenyl)-1-propanol Schiff base.
Lidocaine, amifostine, and pilocarpin were studied for their protective effect on the
parotid gland following X-ray irradiation. No significant difference of salivary ejection fraction
was noted relative to the control group. However, lidocaine (alone or with pilocarpin) was found
to reduce adverse effects on the gland’s smooth muscle actin and tenascin-C levels. It also
offered greater protection of gross structure compared to the control group, a result also
observed with amifostine (Hakim et al., 2005).
Jensen and Meister (1983) found that lymphoid cells treated with glutathione after being
supplied exogenously did not experience decreased viability after irradiation from a cobalt-60
source. They also found, however, that the intracellular glutathione as well the enzyme yglutamyl transpeptidase had to be present in order for the repletion and protection to occur.
Organic Agents
Many studies have showed that organic extracts can serve as viable and effective
radioprotective agents. There has been a recent increase in their use as such because of their
decreased side effects upon administration (Veerapur et al., 2007; Nair et al., 2001).
Phyllanthus amarus extract was given to mice as prophylaxis and again a month after
whole-body radiation (Kumar and Kuttan, 2004). The extract showed extensive radioprotective
potential by increasing leukocyte and bone marrow cellularity, glutathione levels, and the activity
of various antioxidant enzymes. It also reduced serum and tissue lipid peroxidation secondary
to irradiation from a cobalt-60 source (Kumar and Kuttan, 2004).
Jagetia and Baliga (2005) found that prophylaxis of mangiferin from Mangifera indica
increased protection from gamma-radiation-induced gastrointestinal and bone marrow damage.
This effect was maximal at a dose of 2 mg/kg and declined at higher doses. The extract also
showed the ability to decrease the symptoms of extreme radiation sickness such as lethargy,
anorexia, diarrhea, weight loss, and facial edema, among others (Jagetia and Baliga, 2005).
VCO as a Novel Radioprotector – 8
Veerapur et al. demonstrated that ethanol extract of Ficus racemosa, given as
pretreatment an hour before gamma-irradiation, decreased the amount of post-radiation DNA
lesions after micronuclear assay of lung fibroblast (V79) cells in Chinese hamsters (2007). They
also found that administration of the extract does not alter the cell cycle, thus opening
possibilities for the radioprotective effects of F. racemosa (Veerapur et al., 2007). A similar
study conducted by Rao (2007) on Coleus aromaticus extract revealed that it significantly
decreased micronuclei percentages in V79 cells if given as pretreatment. However, only a
moderate suppression of lipid peroxidation was observed.
Jagetia, Venkatesh, and Baliga (2003) studied radioprotection of human blood
lymphocytes using the hydroalcoholic extract of Aegle marmelos. Treatment with the extract
significantly reduced the percentage of micronuclei in radiation-exposed cells by inhibiting free
radicals formed. Pretreatment with Tinospora cordifolia extract also significantly reduced
micronuclei in gamma-irradiated mice, as well as increasing spleen CFU counts and S-phase
cell levels (Goel et al., 2004). Total lymphocyte cell counts were found to be restored to normal
15 days after radiation and 30-day survival had increased 76.3% relative to the control (Goel et
al., 2004).
Amifostine
Amifostine is a thiol/synthetic radioprotective drug. It has the structural formula of
H2N(CH2)3NH(CH2)2S-PO3H2 (RxList, 2008).
Figure 1. Amifostine structural formula
It is indicated as a cytoprotective agent in chemotherapy, and is used specifically to prevent the
occurrence of xerostomia in patients undergoing head and neck radiation therapy (MIMS, 2009).
Amifostine is usually given intravenously, 15-30 minutes before chemotherapy, at a dose
of 910mg/m2 if it is to be used as a cytoprotective, or at a dose of 200mg/m2 if it is to be used for
prevention of xerostomia. (MIMS, 2009). It can also be given subcutaneously, in order to lessen
the adverse effects of this drug (Hosseinimehr, 2007).
Side effects include hypotension and nausea (Hosseinimehr, 2007). The radioprotective
effect of amifostine—and thiol radioprotectors in general—have been proposed to be due to free
VCO as a Novel Radioprotector – 9
radical scavenging, hydrogen transfer, inducing hypoxia, and stabilizing DNA through direct
binding (Hosseinimer, 2007).
The cost of this drug in the Philippines is P9,450 per 10mL vial (Philippine
Pharmaceutical Directory, 2004).
Components of Virgin Coconut Oil
Virgin coconut oil is described as the oil extract from the mature kernel of a fresh
coconut. It differs from plain coconut oil in that it does not go through chemical refining,
bleaching, or deodorizing and that the coconuts used in the latter are dried in the kiln or under
the sun (Philippine Herbal Medicine, 2009). Extraction of VCO often goes through the process
of wet-milling, or the mechanical shredding of the meat of the coconut. The meat is basically
cold-pressed for the coconut milk and is then allowed to ferment for a day or two. It is in this
fermentation process where the oil is produced. The oil is then finally separated from the milk
either by filtration or centrifuge.
Virgin coconut oil is mainly composed of saturated fats (92%) and is rich in lauric acid
(43-56%) (Buenafe et al., 2008). Majority of the saturated fats are made of medium long chain
fatty acids (MLCFA), which do not elevate Low Density Lipoproteins (LDL) or the “bad
cholesterol” in the body. This is in comparison to the other polyunsaturated vegetable oils such
as canola and sunflower oil (Philippine Herbal Medicine, 2009). Below are the other
components of VCO according to the study done by the Bureau of Food and Drugs (BFAD),
Philippine National Standard (PNS and Asian and Pacific Coconut Community (APCC) as cited
in Buenafe et al. (2008):
Table 1. Component comparison of virgin coconut oil
Fatty Acid
BFAD
PNS
APCC
Caproic (C 6:0)
---
ND – 0.7
0.4 – 0.6
Caprylic (C 8:0)
4.6 – 10.0
4.6 – 10.0
5.0 – 10.0
Capric (C 10:0)
5.0 – 8.0
5.0 – 8.0
4.5 – 8.0
Composition
Lauric (C 12:0)
Myristic (C 14:0)
45.0
– 45.1
– 43.0
56.0
53.2
53.0
16.8
– 16.8
– 16.0
21.0
21.0
21.0
–
–
VCO as a Novel Radioprotector – 10
Palmitic (C 16:0)
7.5 – 11.0
7.5 – 10.2
7.5 – 10.0
Stearic (C 18:0)
2.0 – 4.0
2.0 – 4.0
2.0 – 4.0
Oleic (C 18:1)
5.0 – 10.0
5.0 – 10.0
5.0 – 10.0
Linoleic (C 18:2)
1.0 – 2.5
1.0 – 2.5
1.0 – 2.5
Virgin coconut oil was found to have a lethal dose of (LD50) of 36.7g/kg in mice in the
study of Pekson (2007, as cited in Buenafe et al., 2008). The same study reports that no
adverse effects were observed below 2.3 g/kg in mice. Based on the recommended dose of
VCO in humans, 40.5-54 g/70 kg or 3-4 tablespoons per day, the LD50 of mice in Pekson’s study
is seven times lower than that of the normal dose for humans. These differences should be
considered in the formulation of dosing schedules for the current study so as to prevent the
subjects from overdosing.
Antioxidants as Components of Virgin Coconut Oil
Antioxidants have been reported to offer a degree of radioprotection to cells exposed to
ionizing radiation due to their ability to scavenge free radicals (Jagetia and Baliga, 2003; Nair et
al., 2001). Virgin coconut oil was reported to exhibit antioxidant property and thus holds
potential as radioprotective agent. Other components of VCO such as phenols and some acids
have also been reported to be beneficial in biological systems exposed to radiation.
Furthermore, results from the research of Buenafe et al. (2008) reporting on anti-mutagenicity,
could be suggestive of the radioprotective potential of VCO. The ability of VCO to deter mutation
in the genetic material of viable cells may be also effective in protection against ionizing
radiation.
Marina et al. (2009) established from researches that phenolic compounds contained in
food exhibit antioxidative properties. Foods containing high phenolic levels are gaining
popularity like olive oil and virgin coconut oil (Nevin and Rajamohan, 2006). In a study, upon
exposure to oxidative stress, the levels of superoxide dismutase and glutathione peroxidase
were significantly unchanged in subjects given VCO (Nevin and Rajamohan, 2006). In the same
study, intake of VCO also increased the level of antioxidant enzymes in the body (Nevin and
Rajamohan, 2006). The phenolic compounds found in VCO sequester the reactive oxygen
species or the free radicals and thus rendering them incapable of causing further cellular
damage (Nevin and Rajamohan, 2004). There is a direct relationship between the amount of
VCO as a Novel Radioprotector – 11
reactove oxygen species sequestered. The phenolic acids identified in VCO were
protocatechuic, vanillic, caffeic, syringic, ferulic and p-coumaric acids (Marina et al., 2009).
Due to the low activation energy of phenols, they are able to easily donate hydrogen to
the free radicals in the system (Nair et al., 2001). The resulting antioxidant free radical is stable
because of the delocalization conferred by the ring structure of phenols (Nair et al., 2001).
Furthermore, the reaction between other stable free radicals form complexes that are also
stable enough to remain bonded. The mechanism of antioxidants sequestering free radicals are
shown as below (Nair et al., 2001):
RH  Ro + H
after free radicals are created, due to high energy such as radiation,
Ro + AH  RH + A·
an antioxidant, may donate its hydrogen atom and thus stabilizing the free radical.
Ao + Ao  A-A
Antioxidants may easily form complexes with oxidized antioxidants in the system.
Virgin Coconut Oil with Radioprotective Potential
It has been identified that extracts of Cocos nucifera L. contains fatty acids. The study of
Castelluccio et al. (1995 and 1996), Gardner et al. (2000), and Seneviratne and Dissanayake
(2008), further described coconut oil as having phenolic content of caffeic acid, p-coumaric acid,
ferulic acid, (+/-) catechins, along with a substantial amount of unidentified phenolic acids and
flavonoids. It should be noted from their study that the relative quantities of these components
varied according to the method of extraction and that the known methods used to manufacture
what is accepted as VCO was not investigated.
Polyphenols are abundant in plants, are commonly used in folkloric medicine due to its
putative anti-inflammatory, anti-viral, anti-atherosclerosis, and anti-bacterial effects. The
possible underlying mechanism of these medicinal effects may be due to the antioxidant
properties of polyphenols, as demonstrated.
Caffeic acid is also believed to have antioxidant properties. It has been demonstrated
that it inhibits lipid peroxidation at a dose dependent manner (Gülçin, 2006). Nardini (1997) et
al., demonstrated that antioxidant effects of caffeic acids may occur in vivo and can be
supplementary to the antioxidant defense system in rats, and exerts a sparing effect on αtocopherol. It has been demonstrated that caffeic acid is a radical-scavenger by reacting with –
OH radicals (Bors et al., 2004), and a derivative, caffeic acid phenethyl ester (CAPE) exhibited
VCO as a Novel Radioprotector – 12
protection against free radical mediated oxidative renal impairment in rats induced by mobilephone radiation (Özgüner et al., 2005). Caffeic acids have been demonstrated to be antimutagenic (Karekar et al., 2000; Birosová et al., 2005). It is also anticarcinogenic but the
mechanisms are still unknown.
It has been suggested that p-coumaric acid possesses antioxidant effects (Castellucio et
al., 1996; Castellucio et al., 1995; Gardner et al. (2000) and Laranjinha et al., 1995). It has been
widely investigated that its antioxidant properties are influential in lipid metabolism. Lun-Yi et al.
(2000) reported experimental data suggestive that the mechanism p-coumaric acid involves
directed scavenging of reactive oxygen species (ROS) such as –OH.
Ferulic acid is known to have anti-cancer activity, based on its ability to counter reactive
oxygen species which result in oxidative insult (to DNA, protein, and lipids), reduce proliferation,
and induce apoptosis (Castelluccio et al. (1996) ; Roy et al., 2003; Vermeulen et al., 2003; as
cited in Srinivasan et al., 2007 ). It has established radioprotective effects, via antioxidation of
ionizing radiation-induced oxidative stresses, or via activation of intrinsic antioxidant
mechanisms (Dean et al., 1995; Han et al., 2001, as cited in Srinivasan et al., 2007; Toda et al.,
1991;). Other antioxidant properties were demonstrated in its ability to protect against oxidative
stress induced neurodegeneration (Kanskia, 2002) and diabetes (Hideko et al., 2004).
Mechanism of Action of VCO as a Radioprotective Agent
It has been believed that ionizing radiation (Low-LET and High-LET) induced genomic
instability occurs by direct damage of the particles to living tissues such as DNA. Such damage
leads to mutagenicity as a result of point mutations and deletions. Other biologic effects include
chromosomal aberrations, karyotype abnormalities, sister chromatid exchanges, micronuclei
formation, gene mutation and amplification, change in colony size, delayed apoptosis, and
transformation (Morgan et al., as cited in Limoli et al., 2000).
This theory has been augmented in the past decades by results of various experiments
demonstrating that even unirradiated living cells develop mutagenicity in succeeding
generations (e.g. Limoli et al., 2000; Pampfer and Streffer, 1989; Prise et al., 1998; Chunlin et
al., 2003; reviewed in Little, 2000; Mothershill and Seymour, 2006). The detrimental effects of
ionizing radiation to living tissues through direct ionization are supplemented by other
mechanisms such as what occurs in bystander effects (abscopal effects). The bystander effect
occurs when radiation induces directly hit cells to release signals which are medium soluble that
may be transmitted to nonirradiated adjacent cells via diffusion. Such factors (Chunlin, 2003)
include short-lived reactive oxygen species, nitric oxide, and TGF-β 1. Bystander effects via gap
VCO as a Novel Radioprotector – 13
junctional intercellular communication may also occur resulting in modulation of p53 and p21
gene expression. These responses are ideally protective, but excesses cause DNA mutations,
impairing p53 and tumor suppressor gene (when reaction is independent of p53) expression
and function, resulting to tumorigenicity.
A particular pathway that might be of significance in this study is the genotoxicity to
directly irradiated and neighboring unirradiated cells, that may be the result of oxidative stress
(ROS) induced by irradiation (Gamma ray or Low-LET/Low-Linear Energy Transfer). A
biological end marker, micronuclei formation, also known to be a possible result of irradiation
induced reactive oxygen species, will be utilized in the present study.
Kyung-Mi et al. (2007) demonstrated micronuclei formation along with increased ROS
levels, with attenuation of micronuclei formation in the presence of catalase (degrades hydrogen
peroxide). Specifics on irradiation induced oxidative stress were also found to consist of
irradiation induced mitochondrial production of ROS; Nox1 (member of the Nitric oxide family of
NADPH oxidase) sourcing of ROS; and JNK mediated ROS production. These were
demonstrated by inhibition challenge using Rotenone (inhibitor of the mitochondrial respiratory
chain), RNA interference (disrupts Nox1), and SP600125 (JNK inhibitor).
Given the antioxidant properties of the components of VCO, it would be of interest in this
study if the phenolic acid contents of Cocos nucifera L. oil will be quantitatively sufficient to
render radioprotective effects in mice. The antioxidant potential of VCO may oppose the free
radicals or reactive oxygen species produced secondary to irradiation, thus reducing the risk of
DNA mutations and micronuclei formation by attenuating one of the pathways to tumorigenicity
and bystander effects described previously.
Micronucleus Assay in Measuring Degree of Radioprotective Effects
Different studies on potential radioprotective agents have made use of various methods
to quantify the radioprotective potential of a given agent. One of the most common method for in
vivo analysis of genetic material analyses is the Micronucleus (MN) Assay. This will be used by
the current study so as to examine the radioprotective potential of VCO.
The micronucleus assay, also called nuclear anomaly assay is based on the presence of
micronuclei, which are whole or partial pieces of chromosomes that have not been incorporated
into the genome of daughter nuclei during cell division. These appear as small round dark
stained structures, very similar to a cell nucleus. The MN assay is also used to segregate
genotoxic compounds from non-genotoxic compounds in the process of selecting the latter for
potential drug development. The MN assay has also been reported to be more sensitive than
VCO as a Novel Radioprotector – 14
other tests, such as Comet Assay, in describing the extent of chromosome abnormalities
(Hartmann et al., 2001).
In the study done by Sofyan, et al. (2005), the MN assay was used to measure the
extent of chromosomal damage in terms of the presence or formation of micronuclei in
polychromatic erythrocytes (MNPCE) as a result of gamma-irradiation. Micronuclei in
polychromatic erythrocytes frequencies were observed to be significantly higher in irradiated
mice as compared to the non-irradiated mice. These differences in MNPCE were observed at
radiation doses of 0.4 Gy and above whereas no significant differences in MNPCE were
observed in mice treated with 0.2 Gy and below. Upon performing correlation analysis, it was
concluded that levels of MNPCE are positively correlated [r=0.846) with irradiation dose.
VCO as a Novel Radioprotector – 15
MATERIALS AND METHODS
Research Design
This study is a double blind posttest group trial.
Sample Size Determination
Sample size calculations are based on an estimated mean difference of 4.9 and a within
group standard deviation of 2.73 (Dela Paz and Tan, 2008). The test of equality of means will be
carried out at the 0.05 level of significance. A sample size of 7 per group gives a probability of
0.869 of rejecting the null hypothesis of equal means if the alternative holds (Pc-Size:
Consultant version 1.00). Three mice per subgroup will be added to account for possible deaths
in the experiment process. Subgroups are: untreated irradiated, treated with 54µL VCO/20g
mouse, treated with 168µL VCO/20g mouse, and treated with amifostine, as positive control.
Acclimatization
Forty male Swiss-Webster ICR mice will be randomly selected and purchased from the
Research Institute of Tropical Medicine (Alabang, Muntinlupa City, Philippines) and housed at
the Department of Pharmacology Animal House, College of Medicine, University of the
Philippines, Manila. The mice will then be housed in a facility with a 12:12-hour light-dark cycle,
at standard room temperature of around 25 to 28°c, for seven days. Standard chow pellets and
distilled water will be provided during this period.
Source of VCO
The source of VCO for the study is called Oleum ®, which will be purchased from the
pharmacy section of the Victor R. Potenciano Medical Center. This specific brand has been
used in previous local studies such as Buenafe, et al (2009)
Treatment
The mice will be randomly assigned temporary numbers on their tails and using a
random number generator, will be grouped into four subgroups, as previously stated. The
normal human dietary consumption of VCO is 40.5 – 54 g/70 kg or 3-4 tbsp/day, which is
equivalent to 168 microliters µL of VCO per 20g mouse given the same density of VCO and a
VCO as a Novel Radioprotector – 16
Basal Metabolic Rate multiplier factor of 10 from the conversion of humans to mice (Buenafe et
al. 2008). The VCO-treated groups will therefore be given the NOAEL dosage of 54 µL VCO per
20g mouse, the equivalent normal human dietary consumption dose of 168 µL VCO per 20g
mouse, and as positive control, amifostine. Placebo and VCO treatment regimens will be
delivered via by oral gavage and given for 8 days, while amifostine will be administered 15-30
minutes prior to radiation exposure. Placebo group will simultaneously receive the same amount
of water as a control.
Irradiation
Two hours after the last VCO treatment and 15-30 minutes after the administration of
amifostine, whole body irradiation will be performed with Gammacell-220 cobalt-60 gammaradiation at the Philippine General Hospital, Manila, Philippines (Tan and Dela Paz, 2008). Mice
will be exposed to 0.5 Gy/min at 38cm for a total dosage of 4 Gy. Each group of mice, control
and treatment groups, will be divided into smaller groups of 5, placed in a well ventilated tote
box, and irradiated simultaneously.
The Micronucleus Assay
Method for MN assay will be based on the protocol used by Schmid (1975) and Sofyan
et al. (2005). Femurs of the freshly sacrificed mice, devoid of muscles, will be taken out and the
end will be removed. With a needle inserted a few millimetres into the proximal portion of the
bone, the marrow will be sucked into a 1 mL syringe and will be dispensed into a 5 mL
centrifuge tube containing 2.5 mL fetal calf serum. The sample will be centrifuged at 1000 rpm
for 5 minutes. The supernatant will be pippetted out while the pellet will be re-suspended. A
small drop of the resuspension will be placed on a microslide. Sample will be spread using a
cover glass held 45 degrees from horizontal and will be air-dried overnight at room temperature.
Staining will be as follows:
a. 3 minutes in undiluted May-Grünwald solution. Sample will be washed in distilled water
for 1 – 2 times.
b. 2 minutes in 50% May-Grünwald in distilled water. Sample will be washed in distilled
water for 1–2 times.
c. 10 minutes in 15% Giemsa stain solution in distilled water. Then, it will be washed in
distilled water for 1 – 2 times.
VCO as a Novel Radioprotector – 17
Sample will then be dried and will be covered with a cover glass. The MN Assay will be
conducted at the Biochemistry Laboratory, at the Institute of Chemistry, University of the
Philippines. (Diliman, Quezon City).
Triplicate slides for each mouse will be prepared. The slides will be coded so as to
eliminate bias and thus a blinded analysis. With the help of a histopathologist, 1000 cells per
slide will be observed for the total number of polychromatic erythrocytes (PCEs) and for
frequency of appearance of micronucleated polychromatic erythrocytes (MPCEs).
Statistical analysis
Statistical analysis of the Micronucleus assay will be done through a One-way analysis
of variance (ANOVA), using a Completely Randomized Design (CRD) at a 0.05 level of
significance to compare the micronucleated cell frequencies of the sample groups.
VCO as a Novel Radioprotector – 18
Dummy Tables
Table 2. Micronucleated Polychromatic Erythrocyte Count per Group
Group
Mean MPCE Count ± SD
Placebo + Irradiation
54µL VCO/20g mouse + Irradiation
168µL VCO/20g mouse+ Irradiation
Amifostine + Irradiation
Table 3. Micronucleated Polychromatic Erythrocyte Frequency of Mus
musculus Linn. determined through Statistical analysis (ANOVA).
Comparison
Between
Groups
Within Groups
Total
Sum of
Squares
df
Mean
Square
F
Sig.
VCO as a Novel Radioprotector – 19
REFERENCES
Bayliss, M. (1936). Effect of the chemical constitution of soaps upon their germicidal properties.
J. Bacteriol. 31:489-504.
Benkovic, V., Knezevic, A. H., Dikic, D., Lisicic, D., Orsolic, N., Basic, I., Kosalec, I. and Kopjar,
N. (2008). Radioprotective effects of propolis and quercetin in [gamma]-irradiated mice
evaluated by the alkaline comet assay. Biological and Pharmaceutical Bulletin, 31(1),
167-172.
Bergsson, G., Arnfinusson, J., Steingrimsson, O., Thormar, H. (2001). In vitro killing of Candida
albicans by fatty acids and monoglycerides. Antimicrob Agent Chemother. 45:3209-12.
Birosová, L., Mikulásová, M., and Vaverková, S. (2005). Antimutagenic effect of phenolic acids.
Biomedical papers of the Medical Faculty of the University Palacký, Olomouc,
Czechoslovakia, 149(2), 489-491.
Bors, W., Michel, C., Sttetmaier, K., Yinrong, L., and Foo, L. (2004). Antioxidant mechanisms of
polyphenolic caffeic acid oligomers, constituents of Salvia officinalis. Biological
Research, 37 (2), 301-311.
Brown, T., LeMay, H., and Bursten, B. (2000). Chemistry: The central science. 8th ed. Upper
Saddle River, New Jersey: Prentice Hall, Inc.
Buenafe, F.M.A., Cabrera, N., Calderon, J., Campos, E.M., Canoy, I.C., Capili, C., Carasco,
M.A.A., Cielo, P.M., Co, M.L., Collantes, P.A., Concepcion, F.A., Concha, J.S., De La
Cruz, R.A., and Delgado, G. (2009). Virgin Coconut Oil is Non-Mutagenic and AntiMutagenic: Results of Ames Test and Comet Assay. [Thesis]. Manila: University of the
Philippines.
Castelluccio, C., Bolwell, G., Gerrish, C., and Rice-Evans, C. (1996). Differential distribution of
ferulic acid to the major plasma constituents in relation to its potential as an antioxidant.
Biochemical Journal, 316, 691-694.
Castelluccio, C., Paganga G., Melikian N., Bolwell G., Pridham J., Sampson J., and Rice-Evans
C. (1995). Antioxidant potential of intermediates in phenylpropanoid metabolism in
higher plants. FEBS Letters, 368, 188-192.
Kyung-Mi, C., Chang-mo, K., Eun Sook, C., Seong Man., K., Seung Bum., L. & Hong-Duck., U.
(2007). Ionizing radiation induced micronucleus formation is mediated by reactive oxigen
species that are produced in a manner dependent on mitrochondria, Nox1, and JNK.
Oncology Reports, 17, 1183-1188.
Chunlin S., Furusawa, Y., Kobayashi, Y., Funayama, and Wada, S. (2003). Bystander effect
induced by high-LET particles in confluent human fibroblasts: A mechanistic study.
FASEB Journal, 17, 1422-1427.
Collins AR. (2004). The comet assay for DNA damage and repair: principles, applications, and
limitations. Mol Biotechnol 26(3):249-61.
VCO as a Novel Radioprotector – 20
Conley, A. J., and J. J. Kabara. (1973). Antimicrobial action of esters of polyhydric alcohols.
Antimicrob. Agents Chemother. 4:501-506.
Dayrit, C.S. (2005). Thetruthabout coconutoil: The drugstore in a bottle. Ph. Anvil Pub.
Dayrit C. S. (2000). Coconut oil in health and disease: Its and monolaurin’s potential as cure for
HIV/AIDS. In: Proceedings of the XXXVII Coco Tech Meeting/ICC 2000 (“Sustainable
Coconut Industry in the 21st Century”). Retrieved September 19, 2009 from
http://www.apccsec.org/document/Dayrit.PDF.
MIMS. (2009). Amifostine. Retried September 21, 2009 from:
http://www.mims.com/Page.aspx?menuid=mng&name=amifostine&brief=false#Dosage
RxList: The Internet Drug Index. (2008). Ethyol (amifostine for injection). Retrieved September
21, 2009 from: http://www.rxlist.com/ethyol-drug.htm
Philippine Pharmaceutical Directory, 11th ed. (2004). Ethyol. Pasig City: Medicomm Pacific, Inc.
Dean, J., Devarenne, T, Iks, L., and Orlofsky, E. (1995). Properties of a maize glutathione Stransferase that conjugates coumaric acid and other phenyl propanoids. Plant
Physiology, 108, 985–994.
Gardner H., Xianglin, S., Castranova, V., and Vallyathan, V. (2000). Effect of antioxidant
protection by p-coumaric acid on low-density lipoprotein cholesterol oxidation. American
Journal of Physiology – Cell Physiology, 279, 954-960.
Glassman, H. N. (1949). Surface active agents and their application in bacteriology. Bacteriol.
Rev. 12:105-148.
Goel, H., Prasad, J., Singh, S., Sagar, R., Agrawala, P., Bala, M., Sinha, A., and Dogra, R.
(2004). Radioprotective potential of an herbal extract of Tinospora cordifolia. J. Radiat.
Res. 45(1):61-68.
Gülçin, I. (2006). Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology,
217 (2-3), 213-220.
Haimovitz-friedman, A., Kan, C., Ehleiter, D., Persaud, R., McLoughlin, M., Fuks, Z., and
Kolesnick, R. (1994). Ionizing radiation acts on cellular membranes to generate
ceramide and initiate apoptosis. Department of Radiation Oncology and the Laboratory
of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York.
Hakim, S. G., Kosmehl, H., Lauer, I., Nadrowitz, R, Wedel, T., and Sieg, P. (2005). A
comparative study on the protection profile of lidocaine, amifostine, and pilocarpin on the
parotid gland during radiotherapy. Cancer Research, 65, 10486-10493.
Han, B., Park, C., Takasuka, N., Naito, A., Sekine, E., Nomura, H., Taniguchi, T., Tsuno, H.,
and Tsuda, A. (2001). Ferulic acid derivative ethyl 3-(4' genranyloxy-3-methoxy phenyl)2-proleonyl as a new candidate chemopreventive agent for colon carcinogen in rat.
Japanese Journal of Cancer Research, 92, 404–409.
VCO as a Novel Radioprotector – 21
Hazell, S. L., and D. Y. Graham. (1990). Unsaturated fatty acids and viability of Helicobacter
(Campylobacter) pylori. J. Clin. Microbiol. 28:1060-1061.
Hideko, M., Motoyo, O., Hisatsugu, T., Eisaku, N., Asao, H.., Takuo, T., Satomi, T., and
Hideyuki S. (2004). Antioxidant activity and hypoglycemic effect of ferulic acid in STZinduced diabetic mice and KK-AY mice. Bulletin of the Faculty of Education, Wakayama
University, 54, 43-52.
Hosseinimehr, S. J. (2007) Foundation review: Trends in the development of radioprotective
agents. Drug Discovery Today, 12(19-20) :794-805.
Jagetia, G. C., and M.S. Baliga. (2005). Radioprotection by mangiferin in DBAxC57BL mice: A
preliminary study. Phytomedicine, 20, 209-215.
Jagetia, G., Venkatesh, P., and Baliga, M. (2003). Evaluation of the radioprotective effect of
Aegele marmelos (L.) Correa in cultured human peripheral blood lymphocytes exposed
to different doses of gamma-radiation: a micronucleus study. Mutagenesis vol. 18 no. 4
pp. 387-393.
Jensen, G. J., and A. Meister. (1983). Radioprotection of human lymphoid cells by exogenously
supplied glutathione is mediated by y-glutamyl transpeptidase. Proceedings of the
National Academy of Sciences, 80, 4714-4717.
Kabara, J. J. (1978). Fatty acids and derivatives as antimicrobial agents. A review, p. 1-14. In J.
J. Kabara (ed.), The pharmacological effects of lipids. American Oil Chemist Society,
Champaign, Ill.
Kabara, J. J., Swieczkowski, D.M., Conley, A.J. and Truant, J.P. (1972). Fatty acids and
derivatives as antimicrobial agents. Antimicrob. Agents Chemother. 2:23-28.
Kanskia, J., Aksenovaa, M., Stoyanovaa, A., and Butterfield, D. (2002). Ferulic acid antioxidant
protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal
cell culture systems in vitro: Structure activity studies. Journal of Nutritional
Biochemistry, 13(5), 273-281.
Karekar, V., Joshi, S., and Shinde, S. (2000). Antimutagenic profile of three antioxidants in the
Ames assay and the Drosophila wing spot test. Mutation Research, 468(2), 183-194.
Kato, N., and I. Shibasaki. (1975). Comparison of antimicrobial activities of fatty acids and their
esters. J. Ferment. Technol. 53:793-801.
Knapp, H. R., and M. A. Melly. (1986). Bactericidal effects of polyunsaturated fatty acids. J.
Infect. Dis. 154:84-94.
Kumar, K. B. H., and R. Kuttan, R. (2004). Protective effect of an extract of Phyllanthus amorus
against radiation-induced damage in mice. Journal of Radiation Research, 45, 133-139.
Laranjinha, J., Vieira, O., Madeira, V., and Almeida, L. (1995). Two related phenolic antioxidants
with opposite effects on vitamin E content in low density lipoproteins oxidized by
VCO as a Novel Radioprotector – 22
ferrylmyoglobin: consumption vs regeneration. Archives of Biochemisty and Biophysics,
323, 373-381.
Limoli, C., Ponnaiya, B., Corcoran, J., Giedzinski, E., Kaplan, M., Hartmann, A., and Morgan, W.
(2000). Genomic instability induced by high and low LET ionizing radiation. Advances in
Space Research, 25 (10), 2107-2117.
Lee, T., Johnke, R., Allison, R., O’Brien, K., and Dobbs, L. (2005). Radioprotective potential of
ginseng. Mutagenesis vol. 20 no. 4 pp. 237-243.
Little, J. (2000). Radiation carcinogenesis. Carcinogenesis, 21(3), 397-404.
Manu, K.A., Leyon, P.V. and Kuttan, G. (2007). Studies on the protective effects of Boerhaavia
diffusa L. against gamma radiation: Induced damage in mice integrative cancer
therapies, 6(4), 381-388.
Marina, A.M., Che man, Y.B., Nazimah, A.H., and Amin, I. (2009). Antioxidant capacity and
phenolic acids of virgin coconut oil. [Electronic version]. International Journal of Food
Sciences and Nutrition, 60(S2), 114-123.
Morales, P.I.B., Nantes, M.J.Z., Nepomuceno, M.J., Nera, E.J.R., Ngo, R.E.A., Noroña, B.D.C.,
Omar III, A.T., Ong, F.R.C., Ong, M.M.S., Oreta, P.Y.G., Pabalinas, M.D.A., Padilla,
N.L.G., Paner, I.N., Paragas, J.R., Pascual, T.R.M., & Paulino, P.R.T. (2009). The
Effects of Virgin Coconut Oil in the Embryological Development of ICR Mice (Mus
musculus). [Thesis]. Manila: University of the Philippines.
Morgan W., Day, J., Kaplan, M., McGhee, E., and Limoli, C. (1996). Genomic instability induced
by ionizing radiation. Radiation Research, 146, 247.
Mothershill, C. and C. Seymour. (2006). Dose Response, 4(4), 283-290.
Nair, C.K.K., Parida, D.K., and Nomura, T. (2001). Radioprotectors in radiotherapy. [Electronic
version]. Journal of Radiation Research, 42, 21-37.
Nardini, M., Natella, F., Gentili, V., Di Felice, M., and Scaccini, C. (1997). Effect of caffeic acid
dietary supplementation on the antioxidant defense system in rat: An in vivo study.
Archives of Biochcemistry and Biophysics, 342 (1), 157-160.
Nevin, K.G. and T. Rajamohan. (2006). Virgin coconut oil supplemented diet increases the
antioxidant status in rats. [Electronic version]. Food Chemistry, 99, 260-266.
Nevin, K.G. and Rajamohan, T. (2004). Beneficial effects of virgin coconut oil on lipid
parameters and in vitro LDL oxidation. [Electronic version]. Clinical Biochemistry, 37, 830-835.
Nieman, C. (1954). Influence of trace amounts of fatty acids on the growth of microorganisms.
Bacteriol. Rev. 18:147-163.
Özgüner, F., Oktem, F., Ayata, A., Koyu, A., and Yilmaz, H. (2005). A novel antioxidant agent
caffeic acid phenethyl ester prevents long-term mobile phone exposure-induced renal
VCO as a Novel Radioprotector – 23
impairment in rat. Prognostic value of malondialdehyde, N-acetyl-beta-Dglucosaminidase and nitric oxide determination. Molecular and Cellular Biochemistry,
277 (1-2), 73-78.
Pampfer S., and C. Streffer. (1989). Increased chromosome aberration levels in cells from
mouse fetuses after zygote x-irradiation. International Journal of Radiation Biology, 55,
85.
Petschow, B. W., Batema, R. P., and Ford, L. L. (1996). Susceptibility of Helicobacter pylori to
bactericidal properties of medium-chain monoglycerides and free fatty acids. Antimicrob.
Agents Chemother., p. 302-306 Vol. 40, No. 2
Philippine Council for Industry and Energy Research and Development (PCIERD). (2008).
DOST pursues national program on virgin coconut oil. Retrieved September 12, 2009,
from Department of Science and Technology Sectoral Planning Council, Philippine
Council for Industry and Energy Research and Development Web site:
http://pcierd.dost.gov.ph/index.php?option=com_content&task=view&id=18&Itemid=27
Prabhakar, K.R V.P. Veerapur, Punit Bansal, Vipan Kumar Parihar, Machendar Reddy
Kandadi, P. Bhagath Kumar, K.I. Priyadarsini and M.K. Unnikrishnan. (2007).
Antioxidant and radioprotective effect of the active fraction of Pilea microphylla (L.)
ethanolic extract. Chemico-Biological Interactons 165, 22-32.
Prise, K., Belyakov, O. Folkard, M., and Michael, B. (1998). Studies of bystander effects in
human fibroblasts using a charged particle microbeam. International Journal of Radiation
Biology, 74(6), 793-798.
Rao, E. (2007). Antioxidant, anticlastogenic and radioprotective effect of Coleus aromaticus on
Chinese hamster fibroblast cells (V79) exposed to gamma radiation.
Retsky, K., Freeman, M., and Frei, B. (1993). Ascorbic acid oxidation product(s) protect human
low density lipoprotein against atherogenic modification. Journal of Biologic Chemistry,
268, 1304-1309.
Roy, M., Chakrabarty, S., Sinha, D., Bhattacharya, R., and Siddiqi, M. (2003). Anticlastogenic,
antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol.
Mutation Research, 523, 33–41.
Seneviratne K. and D. Dissanayake. (2008). Variation of phenolic content in coconut oil
extracted by two conventional methods. International Journal of Food Science and
Technology, 43, 597-602.
Sofyan, R., Sumpena, Y., Lukita, M. and Fitrisari, A. (2005). The use of micronucleus assay on
swiss-webster mice (Mus musculus) bone marrow for the mutageneicity test of gamma
irradation. Atom Indonesia, 31(2), 103-110.
Srinivasan, M., Sudheer, A., and Menon, V. (2007). Ferulic acid: Therapeutic potential through
its antioxidant property. Journal of Clinical Biochemistry and Nutrition, 40(2), 92-100.
VCO as a Novel Radioprotector – 24
Toda, S., Kumura, M., and Ohnishi, M. (1991). Effects of phenolic carboxylic acids on
superoxide anion and lipid peroxidation induced by superoxide anion. Plant Medicine,57,
8–10.
Veerapur, V.P., Prabhakar, K.R., Parihar, V.K., Kandadi, M.R., Ramakrishana, S., Mishra, B.,
Satish, Rao, B.S., Srinivasan, K.K., Priyadarsini, K.I., and Unnikrishnan, M.K. (2007).
Ficus racemosa stem bark extract: A potent antioxidant and a probable natural
radioprotector. Evidence-Based Complementary and Alternative Medicine, 6, 317-324.
Vermeulen, K., Van Bockstaele, D., and Bernemanm, Z. (2003). The cell cycle: a review of
regulation deregulation and therapeutic targets in cancer. Cell Proliferation, 36, 131–
149.
Zaidi, K., Patil, M.S., Bhatt, M.B., Bagewadikar, R.S., Subramanian, M., Rajan, R., Kaklij, G.S.,
and Singh, B.B. (2001). Effect of whole body hyperthermia on radiation therapy of
transplanted fibrosarcoma in swiss mice [Abstract]. International Journal of
Hyperthermia, 17(5), 428-438.
11th Report on Carcinogens. (2004). “Ionizing Radiation”. 11th ed. U.S. Department of Health
and Human Services, Public Health Service, National Toxicology Program. Retrieved
September 13, 2009 at http://ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s097zird.pdf.
VCO as a Novel Radioprotector – 25
Appendix A
Proposed Budget
Item
Albino Swiss-Webster ICR Mice
Animal House- Pharmacology
Gavage (1 week)
VCO
Technician
Chow pellets
Distilled water
Irradiation Treatment
Gamma cell- 1 load
Positive control- Amifostine (10ml)
Marrow Extraction
Syringes
Needles
5 ml Centrifuge tubes
Staining
Histo slides (cover slip and slide)
Staining Solutions (SigmaAldrich)
May- Grunwald/ Jenner’s stain
Modified Giemsa
Fetal Calf Serum
Histopathology Reading
Miscellaneous Fees (transportation,
labeling materials, overtime
expenses for animal house,
importation of reagents)
TOTAL
*SGD, Singaporean dollars
Units
40 pcs
Estimated Cost [in Philippine Peso]
1600.00
0.00
1 bottle
8 days
25 kg
20 L
169.00
1000.00
2000.00
1000.00
1 session
2 vials
2500.00 per load (PNRI rate)
18900.00
100 pcs
100 pcs
50 pcs
500.00
200.00
500.00
130 sets
500.00
120 slides
4815.62 (143.29 SGD*)
4237.24 (126.08 SGD)
1157.44 (34.44 SGD)
3000.00
4000.00
46,6079.30
VCO as a Novel Radioprotector – 26
Appendix B
GANTT CHART
Activity
Week
Research Proposal
Search for Research Topic
Literature Search
Draft Preparation
Writing of Final Research Proposal
Research Proposal Defense
Submission of Revised Paper
Pre-experiment
Securing Venue for Experimentation
Acquisition of VCO from Victor R.
Potenciano Medical Center Pharmacy
Purchase of Mice from RITM
Preparation of Treatment and Solutions
Acclimation Period for Mice
Experiment Proper
Administration of Treatment
Irradiation of Mice
Mice Sacrifice
Micronucleus assay
Reading of Slides for Histopathology
Statistical Analysis of Results
Writing of Final Research Paper
September
2009
1
2 3 4
October
2009
1 2 3 4
November
2009
1 2 3 4