Health Administrator Vol : XX Number 1&2 : 69-75 Pg.

17 - SCREENING OF NATURAL PRODUCTS FOR

ANTICANCER AND ANTIDIABETIC PROPERTIES
K. Krishnamurthi*
What is Cancer ?

It is a group of diseases caused by loss of cell cycle control.

Cancer is associated with abnormal uncontrolled cell growth.
Carcinogens are substances which cause cancer by mutating DNA.
There are many genes that can mutate to cause loss of control of the cell cycle or cause genomic
instability (DNA damage).

Timeline| Genetic landmarks in cancer research

Cloning of the Identification of the first
Activated oncogenes gene respon- familial breast cancer
in tumour DNA - first sible for familial susceptibility gene,
Boveri demonstration of a BRCA129-30. Mutations
aderomatous
identified the casual genetic event in this gene and its
polyposis
Chromosome associated with close relative BRCA2
(APC) 28-29.
as the unit of cancer. DNA derived Loss of cause familial breast The first
This gene was
heredity and directly from tumours heterozygos- cancer, but, in contrast draft of the
subsequently
proposed that was shown to ity analysis Cloning and to some other high- human
shown to act as
chromosomal transform normal cells was used to indetification of penetrance susceptibil- gerome
Krudson a `gatekeeper.
aberrations when introduced into map the first RB - the first ity genes, they are not sequence
proposed the APC mutations
were the these cells as a high- tumour- tumour commonly mutated in is published
two-hit are also seen in
cause of molecular-weight suppressor suppressor sporadic cancers. (see online
hypothesis 24 sporadic colon
Cancer 3-4 complex 14. gene AB25 gene 21 inks box).
cancers.

1902-1914 1960

Discovery of the
`Philadelphia
1971 1976

Discoveryof
cellular proto-
1979 1983

Identification of
p53 in a
1984 1986

Identification of
the telemeric
1989 1991

Demonstration that
TPSS was a human
1993 1994

Discovery of
microsatellite
1997-1998

Cloning of
f
2001

oncogenes that telemerase

chromosome -a complex with sequence of tumor-suppressor instability in human
are related to (TERT) and
translocationbetween viral Trpanosoma gene, rather than (or tumours and
the transform- demonstration that
chromosomes 9 proteins31-32 bruce36 in addition to) an identification of a
ing genes of telomerase
and 22, leading to oncogene 37. mismatch repair gene
retroviruses 7 expression can
the activation of the Subsequent studies (MSH2) as the first extend the
Abelson leukaemia showed that TP53 gene responsible for lifespan of human
virus (ABL) was the most hereditary non- cells 49-52.
oncogene 6 frequently mutated polyposis colon
gene in human cancer (HNPCC)42-44.
cancers.

*Senior Scientist, Environmental Biotechnology Division, NEERI, Nagpur

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same mutations.
Transplantable
Dedifferentiated: cells lose their specialized identity
Different appearance: reflects dedifferentiation
Lack contact inhibition: will divide in a crowd of cells
and pile on top of each other
Induce angiogenesis (local blood vessel formation)
Increased mutation rate
Invasive: squeeze into any space available
Metastasize: cells move to new location in the body
Past Scenario:
An individuals genes, environment, and diet
have long been acknowledged contributors to
cancer; however, the underlying mechanisms have
been unclear and black boxed in the past.
Present Scenario:
Advancements in scientific research, however,
have enabled researchers to begin characterizing
and understanding the mechanisms which cause
cancer. Finally, the black box has been opened
and scientists can begin formulating a true
understanding of the underlying causes of cancer.
What causes cancer?
Cancer begins with damage (mutations) in
your DNA. Your DNA is like a set of instructions for
your cells, telling them how to grow and divide.
Normal cells often develop mutations in their DNA,
but they have the ability to repair most of these
mutations. Or, if they cant make the repairs, the
cells often die. However, certain mutations arent
repaired, causing the cells to grow and become
cancerous. Mutations also cause cancer cells to
live beyond a normal cell life span. This causes
the cancerous cells to accumulate.
Environmental cancer:
Decades long work has resulted in the widely
accepted estimate that 80 to 90 percent of human
cancer is due to environmental factors. The
gold standard for distinguishing genetic from
environmental traits has been the study of twins
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(Robert N. Hoover, M.D., National Cancer
Institute NEJM-2000)
Environmental Factors:
Environmental factors which, from a scientists
standpoint, include smoking, diet, and infectious
diseases as well as chemicals and radiation in our
homes and workplaces.
Among these environmental factors, tobacco use,
unhealthy diet, and not enough physical activity are
more likely to affect your personal cancer risk than
trace levels of pollutants in food, drinking water, and
the air.
However, the degree of risk from those
pollutants depends on the concentration, intensity,
and exposure. Substantial increases in cancer risk
have been shown in settings where workers have
been exposed to high levels of ionizing radiation,
certain chemicals, metals, and other substances.
Even exposures at low doses, which pose only
small risk to individuals, can still cause substantial
ill health across the whole population. For example,
passive tobacco smoke increases risk in the large
numbers of people who do not smoke but are
exposed to the smoke of others.
Natural Products:
Medicinal plants are the most exclusive source
of life saving drugs for the majority of the worlds
population. Medicinal herbs have been widely used
for treatment of diseases in traditional way for
several generations. An interaction between
traditional medicine and modern biotechnological
tools is to be established towards New Drug
development. The interface between cell biology,
in vitro assays and structural chemistry will be the
best way forward to obtain valuable leads.
There is considerable scientific evidence to
suggest that nutritive and nonnutritive plant-based
dietary factors can inhibit the process of
carcinogenesis effectively. Cancer
chemoprevention involves pharmacologic
intervention with synthetic or naturally occurring
chemicals to prevent, inhibit or reverse
carcinogenesis or prevent the development of
invasive cancer.
 Out of an estimated 250 000 higher plants,
less than 1% have been screened
pharmacologically. In recent years, focus on plant
research has increased all over the world. The
present study is focused to screen traditionally used
medicinal plants like neem, tulsi, thymol, garlic for
anticancer and antidiabetic effect.
What are Natural Products?
The natural world has been medicines most
effective arsenal, providing life-saving antibiotics
and our most potent anti-cancer drugs. The lush
tropical rainforests and colorful coral reefs of our
planet have long been a source of promise in the
fight against cancer and other diseases. Natural
products, especially those from plants, have been
a valuable source of new cancer drugs for many
decades.
Over 60 percent of the current anticancer
drugs are derived in one way or another from natural
sources, and, thus far, natural products chemistry
has proved superior to that of de novo combinatorial
chemistry. New drugs are needed desperately to
replace and improve existing medicines, and to
provide new avenues for treating cancers that resist
treatment with current drugs.
NEEM:
sNeem (Azadirachta indica Juss), abundantly
prevalent in the tropical countries of the world.
sMultidirectional therapeutic uses of neem
have been known in India since the Vedic times.
sAlmost all parts of the tree have been in use
as traditional medicine for house-hold remedies
against various human ailments from antiquity.
sAlthough there is lot of research work been
available in neem.
sMolecular mechanism studies like apoptosis
induction,cell cycle analysis are lacking. s
THYMOL:
sThymol, the major constituent of Ajowan oil, is a
powerful antiseptic and has an agreeable odor
sThymol, a plant derived phenol occurring in the
volatile oils of Thymus vulgaris and Carum
copticum , has molecular weight of 149.66 having
molecular formula C10H14O
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sIts a large, colorless, translucent crystal of
hexagonal system with aromatic thyme like odor
and a pungent aromatic taste
sThymol is also used in toothpaste and perfumery.
It is used in a steeped liquid form against diarrhea
and flatulence
sThymol possesses strong antibacterial and
antimicrobial activity and it is used to delay or inhibit
the growth of pathogenic microorganisms
TULSI:
Ocimum sanctum Linn (OS), is a small erect
herb belonging to the family Labiatae (Lamiaceae.
Commonly known as sacred Tulsi, it is a fragrant
bushy plant found in semi tropical and tropical parts
of India.
The leaves of OS are commonly used to treat
fever, vomiting, cholera, constipation, postnatal
disorders, cough, edema, hydrosarca, septicemia,
dog bite and many other ailments
Although many active components have been
isolated from the leaves of this plant, eugenol is
identified as the major active constituent by means
of liquid chromatography of the essential oil from
the leaves.
Other components identified include eugenol
methyl ester, caryophyllene, terpinene-4-ol,
decyladehyde, -selinene, -pinene, -pinene,
camphor and cervacrol. It was also revealed that
among the different parts of the plant, leaves contain
the highest percentage of essential oil
GARLIC:
Garlic is the edible bulb from a plant in the lily
family. Garlic, onions, leeks, scallions, shallots
and chives are classified as members of the
Allium genus. Thus, they are commonly
described as Allium vegetables.
Garlic is rich in natural sulfur and iodine that
have favorable effects on body system and has
enzymes that are capable of neutralizing the activity
of carcinogenic substances. It is known that garlic
contains numerous sulfur compounds and
glutathione precursors, which acts as antioxidant
and also exhibit anticarcinogenic properties.
Garlic contains allyl sulfur and other compounds HPLC system:- Pump: Water LC 1525 pump;
that slow or prevent the growth of tumor cells. Allyl Detector: UV-Visible detector 2487;
sulfur compounds, which occur naturally in Column: C-18 (5mm), 3.9mm (id) X 300mm;
garlic and onions, make cells vulnerable to the recorder:
stress created by products of cell division.
1cm/min; mobile phase: methanol : water.
Drug Design Approach: Apigenin and Urosolic acid was found in tulsi extract
by LC-MS analysis
sIn vitro model and test
Apoptosis studies showed DNA fragmentation in
sBinding assays:
agarose gel electrophoresis
sAnnexin-V FITC/PI
sCell Cycle Analysis Cytotoxicity:
sCaspases Cytotoxicity of distillate of OS leaf extract
Cellular Experiments: was determined by trypan blue dye
sInhibition experiments exclusion assay.
Approximately 1x106 human
sCytotoxicity polymorphonuclear leukocyte cells,
sLipid Peroxidase suspended in phosphate buffer saline
sCatalase Incubated with different concentrations such
sDNA Fragmentation as 1l/ml, 10l/ml, 25l/ml, 50l/ml, 100l/ml, 200l/
sCOMET Assay ml and 500l/ml of distillate of OS leaf extract for
different time interval of up to twelve hours
sFADU
sGenomics (DNA Microarray) Apoptosis:

sProteomics HL-60 (human acute myeloid leukemia), and

(HPNLs) were grown in DMEM media while Jurkat
sIn vivo model and test
E-6 cells were grown in RPMI-1640 medium with a
sKnock-out models density of 1x106 cells/ml.
sInduced cancer
After growing the cells for 24 hour, cell viability
sToxicity immune response studies studies were carried out with different
concentrations (50l/ml, 100l/ml and 250l/ml) of
EXTRACTION OF NATURAL PRODUCTS
distillate of OS leaf extract for a time period of 48
Leaves (Tulsi, Neem,Thymol and Garlic (pods) hours.
were washed with distilled water about 50 grams On the basis of viability studies, the toxic
of leave of were kept in soxhlet apparatus and concentration of distillate of OS leaf extract
extraction was done for 16h in 45 oC. Further was determined.
concentrated by rotary evaporator. Stored in at -
20 0 C until use. Known quantity dissolved in
autoclaved water filtered through 0.45 filter
HPLC with Electro spray ionization mass
spectrometry (ESI LC-MS) Identification and
quantification of compounds in distillate of OS
leaf extract
The individual active compounds present in
the distillate of OS leaf extract (OSLE) were
identified and quantified using HPLC analysis.

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IDENTIFICATION OF COMPOUNDS IN DISTILLATE OF OS LEAF EXTRACT HPLC
DETECTION
Absorbance(269mm)

Absorbance(234mm)
Neem induced apoptosis
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0
C 1 5 10
/ml. extract

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 Cntrl NEEM 24 hrs HL60.008
File: cntrl NEEM 24 hrs HL60.008 Patient ID:
104

Sample ID: cntrl NEEM 24 hrs HL60 Panel: Apop Annexin

Propidium Iodide Control

Tube: tube # 1 Gate: No Gate

103

Acquisition Date: 18-Oct-06 Total Events: 5000

Gated Events: 5000 Y Parameter: FL2-H Prop
102

X Parameter: FL1-H Annexin V FITC control (Log)

Quad Location: 10, 10
101

Log Data Units: Linear Values

100

100 101 102 103 104

Annexin Y FITC

Quad Events % Gated % Total X Mean X Geo Mean Y Mean Y Geo Mean
UL 1 0.02 0.02 5.67 5.67 10.27 10.27
UR 4 0.08 0.08 128.26 62.22 11.25 11.24
LL 4781 95.62 95.62 1.30 1.19 1.12 1.09
LR 214 4.28 4.28 38.34 29.78 2.98 2.09

1ul trtd NEEM 24 hrs HL60.008

File: 1ul trtd NEEM 24 hrs HL60.003 Log Data Units: Linear Values
104

Sample ID: 1ul trtd NEEM 24 hrs HL60 Patient ID:

Panel: Apop Annexin Acquisition Date: 18-Oct-06
103
Propidium Iodide

Gate: No gate Gated Events: 5000

Total Events: 5000 X Parameter: FL1-H Annexin V
102

Y Parameter: FL2-H Propidium Iodide (Log) Quad Location: 10, 10

101 100

100 101 102 103 104

Annexin Y FITC

Quad Events % Gated % Total X Mean X Geo Mean Y Mean Y Geo Mean
UL 1 0.02 0.02 1.37 1.37 10.18 10.18
UR 7 0.14 0.14 227.46 114.59 11.27 11.24
LL 4493 89.86 89.86 1.46 1.28 1.14 1.09
LR 499 9.98 9.98 41.74 30.50 2.53 1.73

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 5ul trtd NEEM 24 hrs NEEM 24 hrs HL60.008
104

Log Data Units: Linear Values

103

File: 5ul trtd NEEM 24 hrs HL60.004

Sample ID: 5ul trtd NEEM 24 hrs HL60 Patient ID:
Panel: Apop Annexin Acquisition Date: 18-Oct-06
102

Gate: No Gate Gated Events: 5000

Total Events: 5000 X Paraneter: FL1-H Annexin V
101

Y Parameter: FL2-H Propidium Iodide (Log) Quad Location: 10, 10

Propidium Iodide 0
10

100 101 102 103 104

Annexin Y FITC
Quad Events % Gated % Total X Mean X Geo Mean Y Mean Y Geo Mean
UL 0 0.00 0.00 *** *** *** ***
UR 1 0.02 0.02 9.91 9.91 10.75 10.75
LL 4628 92.56 92.56 1.42 1.27 1.17 1.14
LR 371 7.42 7.42 77.49 50.60 1.69 1.34

5ul trtd NEEM 24 hrs HL60.004

104

Log Data Units: Linear Values

103

File: 10ul trtd NEEM 24 hrs HL60.007

Sample ID: 10ul trtd NEEM 24 hrs HL60 Patient ID:
Panel: Apop Annexin Acquisition Date: 18-Oct-06
102

Gate: No Gate Gated Events: 5000

Total Events: 5000 X Parameter: FL1-H Annexin V
101

Y Parameter: FL2-H Propidium Iodide (Log) Quad Location: 10, 10

100

100 101 102 103 104

Annexin Y FITC
Propidium Iodide

Quad Events % Gated % Total X Mean X Geo Mean Y Mean Y Geo Mean
UL 1 0.02 0.02 9.39 9.39 10.75 10.75
UR 3 0.06 0.06 55.18 48.70 10.98 10.97
LL 4664 93.28 93.28 1.35 1.21 1.09 1.07
LR 332 6.64 6.64 37.75 29.44 2.09 1.55

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