FIELD OF THE INVENTION:
This invention relates to an anticancer peptide from Santalum L. and a
process for the isolation and preparation thereof.
BACKGROUND OF THE INVENTION:
Cancer is the second biggest killer of the world after cardiovascular diseases.
Moreover, the incidence of many cancers, including cancers of the skin,
prostate, colon, breast, and cervical, continues to increase .According to the
WHO report cancer killed 7.6 million people in 2005, and by 2015 the
number is expected to rise to 9 million and increase further to 11.5 million
in 2030. Surgery and radiation therapy were most preferred means of
treatment during 20th century. However, over the time it was realized that
neither surgery nor radiation or the two in combination could adequately
control the metastatic cancer and that, for treatment to be effective, therapy
needed to reach every organ of the body. The development of a new class of
anticancer drugs that lack the toxicity of conventional chemotherapeutic
agents and are unaffected by common mechanisms of chemoresistance
would be a major advance in cancer treatment. Drug resistance by tumor
cells is one of the important causes of chemotherapy failure. Therefore, one
of the strategies for seeking anticancer drugs is to identify multi-drug
resistance inhibitors focusing on drugs, biological molecules and immune
mediated therapies. Existing cancer drugs act as cell cycle inhibitors,
apoptosis stimulators, signal transduction inhibitors, anti-inflammatory
compounds, anti-invasive agents, anti-angiogenic compounds and
differentiating agents.
Targeted therapy encompasses a wide variety of direct and indirect
approaches like peptide therapy, ligand targeted therapy, immunotherapy,
apoptosis agonists, metalloproteinase inhibitors and tyrosine kinase
inhibitors. Recently, some attempts have been made for this purpose
including the usage of monoclonal antibodies or small molecules to inhibit
the tumor growth. Most small molecule drugs like peptides are distributed in
large volumes when given intravenously which often leads to narrow
therapeutic index due to a high level of toxicity in normal tissues. It has

been reported that encapsulation of these drugs in a macromolecular carrier,
led to significant increase in concentration of drug in the tumor, resulting in
a decrease in non-specific toxicities. Therefore, identification of peptide
ligands is highly desirable. Peptide targeted therapy via carrier may be able
to allow us to carry higher dosage of drugs to the tumor tissue and help us
overcome some of the obstacles to effective cancer therapy.
Further, the decreasing number of approved drugs contributes to a revival of
interest in peptides as potential therapeutics. This interest has led to the
progress of several peptides based new drug development projects
particularly in identification and targeting specific peptide receptors and
innovative formulation methods for treating infectious diseases.
There has been a significant increase of pathogenic microbial strains that
are resistant to current antimicrobial agents used. This demands multiple
screenings for new classes of antimicrobial agents (Maroti et al., 2011).The
next serious issue is the deadly cancer. Although plant-derived compounds
isolated from the various plant sources (vincristine, Vinblastine,
camptothecin, paclitaxel) have played very crucial role in the field of anti-
cancer drugs but their cytotoxic nature and low specifity has led to search
for alternative anti cancer agents (Islam et al., 2009) The quests for
alternative antimicrobial and anticancer agents have been answered by
emergence of peptides as "the magic bullet". Peptides have attracted huge
attention as a drug candidate owing to possession of certain key advantages
such as their small nature, strong specificity, low cytotoxicity and easy
modification (Janin, 2003; Mader and Hoskin, 2006). Several natural
peptides and their modified derivatives have advanced to clinical trials. The
efficacies of natural peptides are now being increased by new strategies such
as incorporation of D isomers of amino acids into native molecules, use of
peptomimetics and peptide engineering (Bhutia et al., 2008).Now-a-days
much attention is being payed to develop efficient peptide delivery methods
to enhance the potential of peptides (Ali and Manolios, 2002).

Peptides (12 to 50 AAs) have been identified in virtually all forms of life from
archaea to humans. (Martin et al., 1995; Wang, 2004) Such peptides isolated
from different sources have shown great versatility and their wide spectrum
of action against resistant bacterial strains, viruses, and fungi have been
reported. In addition to this, immunomodulatory, antitumor, and angiogenic
actions have been reported for peptides for example, dermaseptin-B2,
natural peptide, isolated from that frog skin is reported to have both
antimicrobial and antitumor properties.
Peptides from plants
The exploration of active molecules with its function of natural products,
possessing antimicrobial and antitumor activities, is still an intense area of
research. Plant peptides based on their chemical structure, peptides can be
divided into linear and cyclic peptides. Studies have shown that most
peptides isolated from plants are cyclic peptides; so-called cyclopeptides
which is further categorized into cyclopeptides and cyclotides. (Ma et al.,
2006) They exhibit more potent biological activities, possibly due to the
stable configuration provided by their cyclic structure.
The East Indian Sandalwood, Santalum album L., is a tropical, woody,
perennial, essential oil yielding tree and has been praised for centuries for
its medicinal properties as well as it for yellowish heartwood (middle of the
tree). The traditional curative properties of Santalum album, known till date
are anti-inflammatory, anti acne, antiseptic, demulcent (reduces irritation),
and antitumor etc. Phenylpropanoids and terpenoids rich tissue extracts,
from sandalwood revealed antimicrobial, antioxidant, a-amylase inhibition,
anti-tyrosinase and neuroprotective potentials in vitro (Misra, 2010).
OBJECTS OF THE INVENTION:
It is therefore an object of this invention to propose an anticancer peptide
from Santalum album which is cost effective and simple.
A further object of this invention is to propose an anticancer peptide from
Santalum album and a process for the isolation and preparation thereof,
which has antitumor activity.

Another object of this invention is to propose an anticancer peptide from
Santalum album and a process for the isolation and preparation thereof,
which is non-toxic to normal cells.
Yet another object of this invention is to propose an anticancer peptide from
Santalum album and a process for the isolation and preparation thereof,
which is easy to synthesise and scale up for industrial use.
These and other objects of the invention will be apparent from the ensuing
description, when read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS;
Figure 1: Gel filtration Chromatogram of crude fraction
Figure 2 HPLC reversed-phase chromatogram profile (ZORBAX-SB300-C18
column) of crude peptide extract from sandalwood leaves. Diagonal red line
indicates the acetonitrile gradient.
Figure 3: MALDI TOF Mass Spectrum of fraction 4 (SaP4). Spectra were in
positive ion linear mode using Applied Biosystem Voyager DE™ Pro. CHCA
was as matrix.
Figure 4: MALDI -TOF -PSD for peptide sequencing of SaP4
Figure 5: Antiproliferative activities of RP- HPLC purified SaP4 on various
cancer cell lines.
Figure 6: Effect of SaP4 on cellular & nuclear morphology of various cancer
cell lines.
Figure 7: ESI-MS spectrum (positive-ion mode) of synthesized peptide
Figure 8: Flowchart of the steps in producing natural and synthetic cyclic
peptides
Figure 9: Cyclic structure of the octapeptide (SaP4)
Figure 10: Solid phase synthesis of the peptide
DETAILED DESCRIPTION OF THE INVENTION:
According to this invention is provided an anticancer peptide from Santalum
album L, a process for the isolation and preparation thereof.

In accordance with this invention, the leaves of the East Indian Sandalwood,
Santalum album L. have been used for extraction of the peptide. The leaves
are freeze-fried, ground and pre-extracted. The residual extracts are air-dried
and sequestered with ethanol. The ethanol extract is acidified and purified
for removal of tannins. The crude ethanolic extract is analyzed for the
peptides and further subjected to gel filtration. The gel filtration column is
eluted with solvent A (50% ethanol + 2% acetic acid in "solvent"). All the
fractions are collected, monitored, lyophilized and checked for
antiproliferative activity. The active gel elute fraction is further analyzed by
reverse phase-HPLC. The mobile phase is acetonitrile (A) and 0.1% TFA in
water(B). Elution is monitored at 215 nm and 280 and all the peaks of HPLC
chromatogram are collected separately, lyophilized and checked for
antiproliferative activities. Each fraction is then re-chromatographed with
similar mobile phase to test its purity.
The peptide sample is lyophilized and analysed by mass spectrometry.
The purified peptide is hydrolysed in hydrochloric acid and spotted on silica
gel TLC plate alongwith unhydrolysed peptide. Absence of ninhydrin positive
spot (unhydrolyzed) confirmed the presence of a cyclic peptide (stahl 1969),
Arg-Leu-Gly-Asp-Gly-Cys-Thr-Arg (Cyclisation between Arg 1 and Arg 8). The
peptide is a cationic cyclic octapeptide (C-RLGDGTCRL 858Da) and it acts as
a potent anticancer agent, with RLGD motif and unique amino acid
sequence exhibiting potent anticancer activities, possibly due to the stable
configuration provided by its cyclic structure. It is non-toxic to normal cells
and induces apoptotic events.
In accordance with this invention is also provided a process for the synthesis
of the peptide, by solid phase peptide synthesis. The cyclic peptide is
synthesized from Arg-Leu-Gly-Asp-Gly-Cys-Thr-Arg in a series of steps.
(i)Fmoc-Arg(Pbf)-2-chlorotrityl resin is used as the starting material for
coupling amino acid residues thereto to form the straight chain peptide resin
of formula H-Arg(Pbf)-Leu-Gly-Asp(tbu)-Gly-Cys(trt)-Thr-(tbu)-Arg(Pbfj-2-
chlorotrityl resin. The amino acid residue is added by using Arg-Leu-Gly-

Asp-Gly-Cys-Thr-Arg. This amino acid is coupled with the chlorotrityl resin
in a solid phase peptide synthesis, using a coupling agent O-Benzotriazole-
N,N,N',N'-tetramethyl-uranium-hexafluoro-phosphate(HBTU), 1 -
hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIEA) in a polar
solvent such as DMF or DCM. Piperidine in DMF, usually 20% piperidine in
DMF is used as a deprotectant alongwith the coupling agent. The
piperidine/DMF removes the N-terminal Fmoc group. The coupling is
effected over a period of about 30 min.
In next step, (ii) the product of step (i) is cleaved with a solution comprising
98% DCM and 2% TFA for about 2 hrs, to give the straight chain peptide of
formula H-Arg(Pbf)-Leu-Gly-Asp(otbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)-OH. In
the following step, (iii) the product of step (ii) is cyclised with PyAOP, HOAT,
DIAT, DCM for about 6 hours to lead to Arg-(Pbf)-Leu-Gly-Asp(otbu)-Gly-
Cys(trt)-Thr(tbu)-Arg(Pbf). In the next step, (iv) the product of step (iii) is
deprotected using 98% DCM and 2% TFA for about 2 hrs and this crude
peptide product is purified by HPLC to a purity of ≥ 99.
The invention will now be explained in greater details with the help of the
following non-limiting examples.
EXAMPLES;
Isolation of peptides from Sandalwood
The extraction of peptides was performed as described (Claeson et al. 1998) with
slight modification. Leaves (45 ± 0.1g) were freeze-dried, ground to a fine powder,
and pre- extracted with dichloromethane. The residual extracts were air-dried and
sequestered with 50% aqueous ethanol. The ethanol extract was then acidified using
2% acetic acid and passed through polyvinylpyrrolidone column for maximum
removal of tannins.
Detection of Peptides: The crude ethanolic extract was analyzed
chromatographically, on silica gel 60 F254 (10x5 cm) and developed with butanol:
acetic acid: water (3:1:1). Ninhydrin reagent was sprayed onto plate in order to
confirm the presence of peptides (Stahl 1969).

Gel filtration chromatography
The tannin free crude ethanolic extract was subjected to Sephadex G-25 (Sigma) gel
filtration chromatography (24 x 1 cm). The column was eluted with solvent A (50%
ethanol + 2% acetic acid in "solvent". A flow rate of 0.5 mL min -1 was maintained
(Claeson et al. 1998). All the fractions collected were monitored at 280 nm,
lyophilized, and checked for antiproliferative activities.
Purification of sandalwood peptides by RP-HPLC
The active gel elute fraction was further analyzed by reverse phase-HPLC (Agilent
1100 series) with a ZORBAX-SB-C18 column (5 μm, 250 x 4.6 mm), at a flow rate of
lml min-1 as described (Conlon 2007). Mobile phase was acetonitrile (A) and
0.1%TFA in water (B). Elution was done with a linear gradient of B (5-35%, v/v) for
30 min. The elution was monitored at 215 nm and 280 nm with a UV-DAD detector.
All peaks of the HPLC chromatogram were collected separately, lyophilized, and then
checked for their antiproliferative activities. Each fraction was then again re-
chromatographed with similar mobile phase to test its purity.
Characterization of peptide using mass spectrometry
Peptide sample was lyophilized and then 50 ul of 10 mM DTT/0.1M NH4HCO3 was
added. The mixture was incubated for 30 min at 56°C. 50 ul of 55mM
iodoacetamide/0.1 mM NH4HCO3 was next added and was further incubated for 15
min. Sample was lyophilized to dry and 50 mM NH4HCO3, 5 mM CaCb, 12.5 ng/μl
of trypsin was added. It was incubated overnight at 37° C followed by purification of
sample with ZipTip C18 from Millipore. The lyophilized dried peptide was
resuspended in 5% (v/v) acetonitrile solution containing 0.01% (v/v) trifluoroacetic
acid. Peptide solution (4 μl) was mixed up with 24 ul of matrix (CHCA, 10 mg ml-1)
and 1.0 μl of this mixture solution was spotted onto the MALDI 100 well stainless
steel sample plate and allowed to air dry prior to the MALDI analysis. To obtain
MALDI mass spectra a Voyager time-of-flight mass spectrometer (Applied
Biosystem, USA), equipped with 337 nm N2 laser was operated in accelerating
voltage 20 kV. The mass spectra were recorded in the positive ion linear mode (Gauri

et al. 2011). The spectra were recorded in the post-source decay (PSD) ion mode as
average of 100 laser shots with a grid voltage of 75%. The reflector voltage was
reduced in 25% steps and guide wire was reduced 0.02-0.01%) with an extraction
delay time 100 ns. Reproducibility of the spectrum was checked 5 times from
separately spotted samples
Hydrolysis of peptides
Purified peptide (12 μl) was hydrolyzed in 6N HCl at 110° C for 24 h. The
hydrolyzed peptide was then spotted on silica gel TLC plate along with unhydrolyzed
peptide. Absence of ninhydrin positive spot (unhydrolyzed) confirmed presence of
cyclic peptides (Stahl 1969).
Synthesis of Arg-Leu-Gly-Asp-Gly-Cys-Thr-Arg
The steps of using i)using Fmoc-Arg(Pbf)-2-chlorotrityl Resin as the starting
material and coupling amino acid residues thereto, using coupling agent in
polar solvent to give the straight chain peptide resin compound of formula
H-Arg(Pbf)-Leu-Gly-Asp(tbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbfj-2-Chlorotrityl
Resin, ii) cleaving the product of step i) with a solution comprising 98% DCM
and 2% TFA, 2hrs, to give straight chain peptide of formula H-Arg (Pbf)-Leu-
Gly-Asp(otbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)-OH iii)Cyclization of the product
of step ii with 7-Azabenzotriazol-l-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyAOP), l-Hydroxy-7-azabenzotriazole (HOAt), DIEA,
DCM,6hrs[Arg(Pbf)-Leu-Gly-Asp(otbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)];iv)
Deprotecting of the product of step iii with 98% DCM and 2% TFA, 2hrs;
v)purifying the crude peptide of step iv)by HPLC to a purity of >99.
One example of quantitative proportion for synthesis


The peptide obtained is studied for its properties:
a) Anti proliferative activity
Cell Lines: MCF-7 (breast cancer), B-16 mouse melanoma, mouse fibroblast (L929),
(NCCS, Pune, India.) were cultured in Dulbecco's Modified Eagle Medium (DMEM),
supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% fetal
bovine serum.
Anti proliferative assay: MCF- 7, B-16, L929, cells were harvested and the cell
concentration was adjusted to 1 x 10 5cells/ml and cells were plated in a 96 well flat
bottom culture plates and incubated with various concentrations of peptides. All
cultures were incubated for 72 h at 37 °C in a humidified incubator, which maintained
a constant 5% CO2. Inhibition of cell proliferation was monitored by MTT assay
(Mosmann, 1983).

Cell morphology observation
MCF-7, B-16 melanoma and L929 (2 x 105) cells mL-1 treated with 100μg mL-1 of
SaP3 at 37 °C for 48 h. After treatment for 48 h, MCF-7 cells were washed three
times with PBS and 6-well plate was viewed under phase contrast microscope (IMT-
2, Olympus Opticals Co. Ltd., Japan) to observe the morphology of the cells
DAPI staining for nuclear morphology observation
DAPI staining for nuclear morphology observations was performed as described
(Uthaisang et al. 2004). MCF-7 and B-16 melanoma (2 x 105) cells mL-1 were treated
with SaP3 at 100μg mL-1 for 48 h. Then, cells were rinsed with phosphate buffer
(PBS) and fixed in pretreatment solution for 5 min. Cells were incubated for 30 min
with 1 mg mL-1 of 4', 6-diamidino-2-phenylindole (DAPI, Sigma) staining solution.
DNA alterations like condensation or fragmentation were observed by fluorescence
microscope (IX 51, Olympus) high-performance CCD camera using Image-Pro
discovery 5.1 software.
Live -Dead assay using FDA/PI
Live-dead assay was carried out as described (Jones and Senft 2007). MCF-7 and B-
16 melanoma cells (2 x 105) treated with 100 μg mL-1 of SaP3 for 48 h were stained
with fluorescein diacetate (lmg mL-1) for 15 min. Then, it was washed twice in PBS
and incubated with propidium iodide (1 mg mL-1) solution for 15 min at room
temperature to stain the dead cells red. It was again washed in PBS and fluorescein
diacetate/propidium iodide (Himedia) stained images were visualized under
fluorescence microscope. The cells were photographed with a fluorescence
microscope (IX 51, Olympus) high-performance CCD camera using Image-Pro
discovery 5.1 software.
Apoptosis double staining using Hoechst 33258/PI
MCF-7 cells (2 x 105 ellsmL-1) treated with various concentrations of SaP3 for 48
h were rinsed two times with PBS, and resuspended in 500 μl PBS containing

Hoechst 33258, Sigma (1 mg mL-1) and Propidium Iodide (1 mg mL-1) for 15 min at
room temperature in dark (Zang et al. 2005). After washing with PBS, the cells
were visually examined under fluorescence microscope (IX 51, Olympus) high-
performance CCD camera using Image-Pro discovery 5.1 software.

WE CLAIM:
1. A cyclic peptide obtained from East Indian Sandalwood, Santalum
album L, having anticancer activity, said peptide having the structure
Arg-Leu-Gly-Asp-Gly-Cys-Thr-Arg (cyclization between Arg1 and Arg8)
2. A process for the isolation of the peptide as claimed in claim 1,
comprising subjecting the leaves of Santalum album L., to freeze-
drying, grinding to the powder and pre-extracting with a solvent,
followed by drying the extracts and sequestration with ethanol,
acidification of the ethanol extract and passing the same through a
polyvinylpyrrolidone column for removal of tannins and purification,
to obtain the peptide.
3. A process for the synthesis of the cyclic peptide as claimed in claim 1
comprising the steps of using i)using Fmoc-Arg(Pbf)-2-chlorotrityl
Resin as the starting material and coupling amino acid residues
thereto, using coupling agent in polar solvent to give the straight
chain peptide resin compound of formula H-Arg(Pbf)-Leu-Gly-
Asp(tbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)-2-Chlorotrityl Resin, ii)
cleaving the product of step i) with a solution comprising 98% DCM
and 2% TFA, 2hrs, to give straight chain peptide of formula H-Arg
(Pbf)-Leu-Gly-Asp(otbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)-OH
iii)Cyclization of the product of step ii with PyAOP, HOAT, DIEA, DCM,
6hrs[Arg(Pbf)-Leu-Gly-Asp(otbu)-Gly-Cys(trt)-Thr(tbu)-Arg(Pbf)];iv)
Deprotecting of the product of step iii with 98% DCM and 2% TFA,
2hrs; v)purifying the crude peptide of step iv)by HPLC to a purity of
≥99.
4. The process as claimed in claim 3, wherein the amino acid residue
used for coupling is Arg-Leu-Gly-Asp-GlyOCys-Thr-Arg.
5. The process as claimed in claim 3, wherein the coupling agent used is
HBTU, HBOT.

6. The process as claimed in claim 3, wherein the deprotectant used in
20% piperidine DMF.
7. The process as claimed in claim 3, wherein the polar solvent used is
DMF or DCM.

ABSTRACT

This invention relates to a cyclic peptide obtained from East Indian
Sandalwood, Santalum album L, having anticancer activity, said peptide
having the structure Arg-Leu-Gly-Asp-Gly-Cys-Thr-Arg (cyclization
between Arg1 and Arg8)