SYNERGISTIC EFFECT OF TOCOTRIENOLS AND CURCUMIN
FIELD OF INVENTION
The present invention relates to a transdermal fluid wherein the main ingredient is
tocotrienol; and curcumin is the supporting ingredient.
BACKGROUND ART
Breast cancer worldwide affects nearly one million women per year and although current
treatments do help many patients, more than 350,000 die from the disease. In Malaysia,
breast cancer is the most common cancer where the statistic shows that 18.0% of the total
cancer case reported is from breast cancer. It is documented that 11,952 new breast cancer
cases were reported to the National Cancer Registry within year 2003 - 2005, which
comprised 3 1.3% of all cancers in women.
The established risk factors for breast cancer include a family history of breast cancer, early
menarche, late age at first childbirth, late age at menopause and history of benign breast
disease. With the exception of the genetic predisposition to the disease the rest of the risk
factors point to the life time exposure of women to estrogen. Estrogen does not cause the
disease but is involved in the progression and development of breast cancer. Anti-estrogens
are therefore used as therapy in the control of breast cancer progression.
Scientific investigations have been undertaken to associate possible functional properties,
antioxidant or otherwise in the diet, which could be efficient in preventing diseases like
cancer. One such antioxidant is vitamin E. The tocotrienol (or T3) group together with
tocopherols compose the vitamin E family. Both have four isomers, which are a- , b-, g- , d-
tocopherols and a- , b- , g- , d-Tocotrienols (Machlin ef a/., 1991).
The major structural difference of tocotrienol from tocopherol is through its unsaturated side
chain that has three double bonds in its farnesyl isoprenoid tail. Tocotrienols also display a
variety of functions that are clearly distinct from that of a-tocopherols (Sen et ai, 2006)
Tocopherols are abundant in common vegetables and nuts, while tocotrienols can be found
in rice bran, wheat germ and most abundantly in the fruit of palm (Sundram, et ai., 2002;
Sookwong, er a/., 2007). Crude palm oil extracted from the fruits of oil palm (Elaeis
guineensis) particularly contains a larger concentration of tocotrienols (up to 800 mg kg 1)
than all other natural sources (Theriault et ai., 1999). The tocotrienol-rich fractions (TRF)
composed of 32% a-tocopherol and 68% tocotrienols can be obtained from palm oil after
esterification and following distillation, crystallization and chromatography (Sundram et ai,
1992).
Tocotrienol isomers of vitamin E in palm oil have been reported to contain biological and
physiological properties which include potential blood cholesterol lowering and
cardioprotective effects, efficient antioxidant activity in biological systems, and possible
anticancer and neuroprotective effects (Sen et ai, 2006).
Previous studies showed that tocotrienols are the components of vitamin E responsible for
growth inhibition in human breast cells in vitro as well as in vivo (Nesaretnam et ai, 1998).
The inhibitory effects on cancer cell growth was found to be different in the four isomers of
tocotrienols with studies reported that g- and d-tocotrienols have pronounced inhibitory effect
compared to a- and b-tocotrienol (Yu et ai, 1999). TRF and d- and g-tocotrienol are shown
to inhibit the proliferation of PC-3, a prostate cancer cell whereas a-tocopherol showed no
significant effects (Nesaretnam et ai., 2008). Various findings have demonsrated the
superiority of tocotrienols over tocopherols in terms of their anti-cancer property.
Tocotrienols, but not tocopherols, inhibited the growth of normal mouse mammary epithelial
cells, ZR-75-1, a responsive human breast cancer line and MDA-MD-435 oestrogenreceptor-
negative human breast cancer cells (Mclntyre et ai, 2000; Nesaretnam et ai, 2000;
Nesaretnam et ai, 1995). Noguchi and colleagues (2003) reported that a-tocotrienol
suppresses the expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion
of THP-1 monocytic cells to human umbilical vein endothelial cells (HUVECs). In fact, the
efficacy shown by a-tocotrienol was 10-fold higher than that of a-tocopherol. In addition, atocotrienol
also exhibits neuroprotective activities through its protection against glutamateand
stroke-induced neurodegeneration, a property not seen in a-tocopherol (Khanna et ai,
2005).
In recent years, the medicinal properties of turmeric and its bioactive compound curcumin
have increasingly been recognized. Curcumin, a bioactive constituent derived from the
rhizomes of Curcuma longa is one of the major yellow pigments found in turmeric and has
over many years of history in traditional medicinal uses. There are many evidences for its
cytotoxic, antiproliferative, and / or proapoptotic activity toward neoplastic cells in vitro, and
suppression of tumorigenesis in rodent models (Sharma et al., 2005; Duvoiz et al., 2005;
Aggarwal et al., 2005). These findings further give ways for curcumin's translation into
therapeutic modalities to combat cancer. Previous study has shown curcumin potentiates the
growth inhibitory effect of celecoxib by shifting the dose-response curve to the left. The
synergistic growth inhibitory effect was mediated through a mechanism that probably
involves inhibition of the COX-2 pathway and may involve other non COX-2 pathways (Lev-
Ari et al., 2005). Extracellularly, curcumin acts as a strong antioxidant (Subramaniam e al.,
1994; Mukundan et al., 1993) an anti-inflammatory agent and reduces free radical
production (Huang et al., 1991). Curcumin is a small, lipophilic molecule that can pass
through the cell membranes and exert intracellular effects as well. Curcumin's most
observed property is its pronounced anti-proliferative action, described in several cell types,
including colon (Ramsewak et al., 2000) and microglial (Lim et al., 2001) cells as well as its
ability to induce apoptosis in cancer cells (Ruby et al., 1995). Curcumin also known to
disrupts the conformation of the p53 protein required for its serine phosphorylation, its
binding to DNA, its transactivation of p53-responsive genes and p53-mediated cell cycle
arrest (Moss et al., 2004). Menon et al (1995) reported that curcumin-induced inhibition of
B16F-10 melanoma lung metastasis in mice. Oral administration of curcumin at
concentrations of 200 nmol/kg body weight reduced the number of lung tumor nodules by
80%. The life span of the animals treated with curcumin was increased by 143.85% (Menon
et ai, 1995).
SUMMARY OF INVENTION
In one embodiment of the present invention is a composition containing an active amount of
tocotrienol or derivative thereof as the main ingredient; and an addition of curcumin, for
prevention or inhibition of a cancer and/or a tumour in a mammal, preferably breast cancer,
wherein curcumin is added to the composition to enhance the anti-cancer effect in
tocotrienol.
Further, there is also provided a composition containing an active amount of tocotrienol or
derivative thereof as the main ingredient; and an addition of curcumin, for prevention or
treatment of an inflammatory disorder in a mammal.
The present invention consists of several novel features and a combination of parts
hereinafter fully described and illustrated in the accompanying drawings, it being understood
that various changes in the details may be made without departing from the scope of the
invention or sacrificing any of the advantages of the present invention.
It is to be noted that tocotrienols have potent anti-cancer effects and the addition of curcumin
enhances the anti-cancer effect in tocotrienols.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
To further clarify various aspects of some embodiments of the present invention, a more
particular description of the invention will be rendered by references to specific embodiments
thereof, which are illustrated, in the appended drawings. It is appreciated that these
drawings depict only typical embodiments of the invention and are therefore not to be
considered limiting of its scope. The invention will be described and explained with additional
specificity and detail through the accompanying drawings in which:
FIG. 1 shows inhibitory effects of TRF, Curcumin, Curcumin + 1mM TRF, TRF, and TRF +
1mM Curcumin on MCF-7 human breast cancer cells.
FIG. 2 shows the percentage of inhibition wherein the composition is composed only of TRF
whose concentration is varied from 1 mM to 100 mM
FIG. 3 shows the percentage of inhibition wherein the composition is composed of TRF
whose concentration is varied from 1 mM to 100 mM and the concentration of Curcumin is 1
mM
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to a transdermal fluid wherein the main ingredient is
tocotrienol; and curcumin is the supporting ingredient. Hereinafter, this specification will
describe the present invention according to the preferred embodiments of the present
invention. However, it is to be understood that limiting the description to the preferred
embodiments of the invention is merely to facilitate discussion of the present invention and it
is envisioned that those skilled in the art may devise various modifications and equivalents
without departing from the scope of the appended

claims.
In the present invention, the efficiency of curcumin when added to a composition with
tocotrienol as the active ingredient is claimed.
Method
Tocotrienols and Curcumin preparation
Stock solution of TRF and curcumin was prepared in dimethyl sulfoxide (DMSO) at a
concentration of 10 mg/mL. For cell growth experiments (as described below), treatments of
TRF and curcumin were diluted in phenol red-free RPMI 1640 medium supplemented with
5% dextran-charcoal-treated FCS (DCFCS) to final concentrations of 1 - 100mM in test
medium.
Cell Viability and Toxicity Assessment: Cell Growth Experiment
MCF-7 cells were suspended from T75 flask by treatment of trypsin to an equal volume of
phenol red free RPMI 1640 medium supplemented with 5% dextran-charcoal treated FCS
(DCFCS), 1% penicillin-streptomycin, 1% L-glutamine and counted on a haemocytometer.
Cells were added to the required volume of phenol red free RPMI medium supplemented
with 5% dextran-charcoal treated FCS (DCFCS), 1% penicillin-streptomycin, 1% Lglutamine.
0.5x1 0 5 MCF-7 cells were seeded in each well of 24-well tissue culture dishes.
After 24 hours, the medium was changed to various concentrations of test compounds (1-
100mM) prepared in phenol red free RPMI medium supplemented with 5% dextran-charcoal
treated FCS (DCFCS), 1% penicillin-streptomycin, 1% L-glutamine, 10 M estradiol. To
examine the synergistic effects of curcumin and tocotrienols, a one to one combination of
these compounds were incorporated into the test medium and tested on MCF-7 cells. For
the cell viability count, cells were washed with 0.9% NaCI to wash off non-adherent dead
cells, and were then lysed in 0.5 ml 0.01 M HEPES buffer/1 .5 mM MgCI2 plus 2 drops of zapoglobin
solution for 15 minutes. Cell proliferation was measured by using an automated
particle counter, Beckman Coulter Particle Counter. Cell counts were taken by diluting
500m of cell solution sample in 10 mL Isoton. The nuclei released were counted in isoton on
a Coulter particle counter. All cell counts were carried out in triplicate. Viable cell counts
were taken after 72 hours.
Determination of IC50 using DPIot Software
IC50 curve for TRF, Individual Tocotrienols and a-Tocopherol was plotted using the DPIot
graphing software. Based on the intersection value at 50%, accurate value was generated
using the interpolate data feature of the DPIot software. The interpolate value was adjusted
to 50 to obtain the IC50 value and this generated for each replicates. The IC50 value was
calculated as an average of three generated value of each replicates.
Statistical Analysis
Comparisons between means of six groups were assessed for significance. In all cases,
statistical significance was set at P < 0.05, and data in the text, tables and figures are
presented as means ± SD.
Table 1: Inhibitory effects of TRF, Curcumin, Curcumin + 1 M TRF, TRF, and TRF + 1mM
Curcumin on MCF-7 human breast cancer cells
Curcumin synergistically enhanced the anti-proliferative effect of tocotrienols on estrogenpositive
MCF-7 human breast cancer cells in vitro.
CLAIMS
1. A composition containing an active amount of tocotrienol or a derivative thereof and
curcumin.
2. The composition according to claim 1 wherein it comprises a-tocotrienol, and/or b-
tocotrienol, and/or g -tocotrienol, and/or d-tocotrienol, preferably d-tocotrienol, or
derivatives of these, or combination of one or more of these.
3. The composition of claim 1 wherein the concentration of tocotrienol is in the range of
1mM to 100mM.
4. The composition of claim 1 wherein the concentration of curcumin is in the range of
0.5mM to 100mM, preferably 1mM.
5. Use of an effective amount of composition of claims 1 - 4 in the manufacture of a
medicament for a prevention or treatment of cancer or a tumour and/or inflammatory
disorder in a mammal, preferably breast cancer.
6 . The use of claim 5 wherein the composition is to be applied topically or orally or
subcutaneously.
7. The composition of claims 1 - 6 wherein the composition is used either in
combination with known medication or used exclusively.
8. A process of manufacturing a composition of claim 1 comprises the following steps:
(a) preparing a stock solution of TRF and curcumin in DMSO
(b) treating the stock solution of (a) by diluting in phenol red-free RPMI 1640 medium
supplemented with 5% dextran-charcoal-treated FCS (DCFCS)