Download POTENT ANTITUMOR-PROMOTING ACTIVITY OF

POTENT ANTITUMOR-PROMOTING ACTIVITY OF HYDROLYZABLE TANNINS
IN TALISAY (Terminalia catappa L.) APPLIED IN
TUMOR CELL LINES
A Research Proposal
Presented to
The Philippine Science High School – Bicol Region Campus
In Partial Fulfillment
of the Requirements in Research 2
by
Espaldon, Ian Estolas
Imperial, Maria Feona Vargas
Roquid, Kenneth Anthony Remot
IV - Electron
SY 2010 – 2011
Chapter I
INTRODUCTION
Background of the Study
As estimated by the National Cancer Institute in January of 2006, approximately 11.4
million Americans have already had cancer. This year, about 569,490 of the populace are
expected to die of cancer, with more than 1,500 people a day. Cancer is the second most
common cause of death in the US, exceeded only by heart disease. In actual fact, cancer accounts
for nearly 1 of every 5 deaths.
Cancer, which currently claims the lives of one out of every five people in the United
States and other developed nations, is a disease of the cell cycle. Cancer cells divide excessively
to form masses called tumors, and if unchecked, they may continue to grow until they kill the
organism. A tumor is defined as an abnormally growing mass of body cells, (Campbell, et al). If
the abnormal cells remain at the original site, the lump is called a benign tumor. Benign tumors
can cause problems if they grow in and disrupt certain organs, but can be completely removed by
surgery. In contrast, a malignant tumor can spread into neighboring tissues and other parts of the
body, displacing normal tissue and interrupting organ function as it goes. Cancer cells may
separate from their original tumor, and enter the blood and lymph vessels of the circulatory
system, where they may proliferate to form new tumors. This spread of cancer cells via the
circulatory system beyond their original site is called metastasis. Thus, an individual with a
malignant tumor is said to have cancer.
Metastasis is a unique feature of malignant neoplasms. It is a major factor in the fatal
course of the disease and is the main restriction to its successful treatment. Given that fact, it is
of paramount clinical importance to diagnose and treat potentially malignant tumors before the
progression to metastasis, whenever possible. In culture, cancer cells are immortal; they can go
on dividing indefinitely, as long as they have a supply of nutrients.
Squamous cell carcinoma is a malignant type of growth that can metastasize either
locally or systemically. It arises both on the skin and in mucous membranes, and may develop
from already present ulcerated area. The lesion may appear rough and scaly and its border wide
with signs of swelling and inflammation. Infections can accompany this lesion particularly if
located in and around an area where the rat is likely to be able to scratch. Common sites for this
type of cancer are the face, jaw, inner ear canal, and near the base of the ear involving Zymbal’s
gland, the auditory sebaceous gland in rats. However it can arise in other areas of epithelial tissue
and mucous membranes.
Many tumors can be successfully treated -- a tumor may be removed surgically,
alleviated by high-energy radiation, which damages DNA in cancer cells much more than it does
to normal cells, apparently because cancer cells have lost the ability to repair such damage.
However, there is sometimes enough damage to normal body cells to produce bad side effects.
To lessen this global enigma, the researchers thought of using medicinal plants to
decrease the utilization of synthetically-produced medicine which are prone to side effects.
Tannin is a polyphenol highly present in Terminalia catappa L., and is proven to possess
antitumor activity. Researchers thought that by extracting tannin from the leaves of Terminalia
catappa L., they can come up with a substance that can control metastasis and growth of tumor
cells.
We, the researchers, had thought that since Talisay trees have high tannic acid content,
which is already known to inhibit tumor initiation, this species may inhibit the multistage process
of carcinogenesis.
Statement of the Problem
This research sought to test the antitumor activity of Talisay (Terminalia catappa L.)
bark. Specifically, it aimed to answer the following questions:
a. Is there a significant difference among the different concentrations of tannin extracted
from Terminalia catappa L. bark applied in tumor cell lines? (Concentrations will be
specified upon planning of methodology)
b. Is there a significant difference in the efficacy of Terminalia catappa L.bark extracts
concentration than that of the commercially produced antitumor drugs in terms of
Tumor Cell Death Index (TCDI)?
c. Is there a significant difference in the efficacy of Terminalia catappa L. bark extracts
concentration than that of the commercially produced antitumor drug Camptotetchin
for not damaging normal cell lines in terms of Cell Death Index (CDI)?
Hypotheses
General Hypotheses
Ho: Terminalia catappa L. leaf extracts does not have an inhibiting effect against tumor
cells in Tumor Cell Lines.
Ha: Terminalia catappa L. leaf extracts has an inhibiting effect against tumor cells in
Tumor Cell Lines.
Specific Hypotheses
a. Ho: There is no significant difference among the different concentrations of tannin
extracted from Terminalia catappa L. bark applied in tumor cell lines (Concentrations
will be specified upon planning of methodology).
Ha: There is a significant difference among the different concentrations of tannin
extracted from Terminalia catappa L. bark applied in tumor cell lines (Concentrations
will be specified upon planning of methodology).
b. Ho: There is no significant difference in the efficacy of Terminalia catappa L.bark
extracts concentration than that of the commercially produced antitumor drugs in
terms of Tumor Cell Death Index (TCDI).
Ha: There is a significant difference in the efficacy of Terminalia catappa L.bark
extracts concentration than that of the commercially produced antitumor drugs in
terms of Tumor Cell Death Index (TCDI).
c. Ho: There is no significant difference in the efficacy of Terminalia catappa L. bark
extracts concentration than that of the commercially produced antitumor drug
Camptotetchin for not damaging normal cell lines in terms of Cell Death Index (CDI).
Ha: There is no significant difference in the efficacy of Terminalia catappa L. bark
extracts concentration than that of the commercially produced antitumor drug
Camptotetchin for not damaging normal cell lines in terms of Cell Death Index (CDI).
Significance of the Study
Considering the consequences of tumor-related illnesses to mankind, the demand for
potent antitumor and antimetastatic medicines has arisen through decades.
This serves as guide to those concerned and will aid in solving pharmaceutical dilemma
and provide cheaper alternatives available in the market. It is a healthful option as well because
of the natural raw materials used. Talisay tree parts are proven to be used as herbal medicines
through time. Planting also promotes a cleaner and greener environment.
Moreover, the results of this project will open the doors for further innovations on the
end-product, exploring its potentials on curing other illnesses. This indicates that more
merchandise will be available for use. This means further that more medical and economic
activities will be generated by these innovations.
This research is significant to the following beneficiaries:
Philippine Economy – Since the product is proudly Filipino-made, it can generate trade
among countries, with the Philippines as the primary source and general exporter. Thus, this can
provide employment to small and local scale businessmen, as well as in the medical industry,
making the Philippine economy ascend.
Botany - This would be another great discovery for it marks the beginning of the
production of tumor remedies, accordingly, it will lead to the exploration of the potentials of
other plants in the same family. Furthermore, authorities can conduct studies and discover other
plants that can be efficient sources of pharmaceuticals, and other necessities, thus, promoting the
industry.
Health – This will help reduce, or if possible, gradually eradicate the number of tumor
cases in the country as well as in the globe. By joining hands or establishing partnerships
towards research and development, the government can further improve the study, giving
benefits at no cost to the needy.
Pathology – The realization or attainment of the goals of the study will provide the long
yearned for treatment for tumors. This will later serve as a breakthrough in the field of
pathology, provided that the materials utilized were all natural, and limited use of synthetics will
be observed.
Cultivators – This will be very useful to create a new source of income or business so that
the unemployment rate in the Philippines will be trimmed down.
Cancer patients – Medication will no longer be out of reach for the ultimate consumers,
given the fact that the researchers endeavored to pursue the study in their favor. With herbs as
the underpinning, treatment is easier to access – nature will endow raw materials, only with
human’s scientific intervention.
Apparently, the scientific study offers higher chances of
survival to the hopeless.
Definition of Terms
Tannic acid – This refers to a specific commercial form of tannin, a polyphenol. It is found in the
seeds, bark, cones, and heartwood, and is a common mordant used in the dyeing process for
cellulose fibers. It is a polyphenol found in Talisay trees that suggests its feasibility of being a
good antitumor agent. In this study, the researchers will use tannic acid as the major element in
screening of antitumor activity in Talisay trees.
Polyphenol - A kind of chemical that may protect against some common health problems and
possibly certain effects of aging. Polyphenols can also block the action of enzymes that cancers
need for growth and they can deactivate substances that promote the growth of cancers.
Talisay - A large deciduous tree reaching a height of 20-25 meters. The branches are horizontally
whorled. Leaves are shiny, obobate, 10-25 cm long, tapering to a narrow and heart-shaped base
with a expanded rounded apex. Leaf stalks are short and stout. Flowers are white, small, and
borne on 6- 18 cm long spikes in the axils of leaves. Fruit is smooth and ellipsoid, 3-6 cm long,
and prominently bi-ridged to the sides. Pericarp is fibrous and fleshy, the endocarp hard.
Tumors - This refers to an abnormal growth of tissue resulting from uncontrolled, progressive
multiplication of cells and serving no physiological function; a neoplasm. In this research study,
the researchers will focus on treating tumors or controlling tumor growth using natural
substances.
Oncogenes – This refers to genes that contribute to the conversion of a normal cell into a
cancerous cell. Oncogenes can derive from cellular genes that undergo mutations that alter their
expression or activity, or from viruses that carry oncogenes within their genome and that are
transferred into the cell by infection.
Benign tumor – This refers to a mild and non-progressive disease, incapable of invading
surrounding tissues and does not metastasize.
Malignant – This refers to a tumor that may grow quite rapidly and can be fatal. This cancer also
sheds cells that travel through the bloodstream, starting new tumor growth at other locations in
the body.
Metastasis – This refers to the transmission of pathogenic microorganisms or cancerous cells
from an original site to one or more sites elsewhere in the body, usually by way of the blood
vessels or lymphatics.
Neoplasm - This refers to an abnormal mass of tissue as a result of neoplasia. Neoplasms may be
benign, pre-malignant or malignant.
Neoplasia – This refers to the abnormal proliferation of cells. The growth of this clone of cells
exceeds, and is uncoordinated with, that of the normal tissues around it. It usually causes a lump
or tumor.
Scope and Delimitation
This research study involves investigation of the inhibiting activities of Terminalia
catappa L. only on squamous cell carcinomas on rats. It does not include, however, other types of
tumors such as pituitary and the like. Furthermore, the study concentrates on the antitumorpromoting activity of Terminalia catappa L., not to the extent of its antimetastatic activity. It
deals with reducing the mass and packed cell volume of the neoplasm. Prevention from
transmission of oncogenes from the source site to other sites or antimetastasis is excluded, but
can be subjected to further improvements.
Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
Related Literature
As described in the Species Profiles for Pacific Island Agroforestry, Tropical almond
(Terminalia catappa) is a large, spreading tree now distributed throughout the tropics in coastal
environments. Commonly referred to in the Philippines as Talisay, the tree is tolerant of strong
winds, salt spray, and moderately high salinity in the root zone. It grows principally in freely
drained, well aerated, sandy soils. Tropical almond has a vast natural distribution in near coastal
areas of the Indian Ocean, through tropical Asia, and into the Pacific Ocean.
The species has traditionally been very important for coastal communities, providing a
wide range of non-wood products and services. It has a spreading, fibrous root system and plays
a vital role in coastline stabilization. It is widely planted throughout the tropics, especially along
sandy seashores, for shade, ornamental purposes, and edible nuts. The timber makes a useful and
decorative general-purpose hardwood and is well suited for conversion into furniture and interior
building timbers. Fruits are produced from about 3 years of age, and the nutritious, tasty seed
kernels may be eaten immediately after extraction. Tropical almond is easily propagated from
seed, and is fast growing and flourishes with minimal maintenance in suitable environments.
Selected cultivars of the species warrant wider commercial planting for joint production of
timber and nuts. The tree has a demonstrated potential to naturalize in coastal plant communities,
but not to adversely dominate such communities. The Talisay thrives in places with full sunshine
and moist, well drained soil. It is tolerant to salt and drought, but not to frost and extremely low
temperatures.
For its constituents and characteristics (stuartxchange.com): Seed contains 51.2 percent
fixed oil, Catappa oil, with 54% olein, pamitin, and 46 % stearin. Bark contains tannin. Leaves
are sudorific, antihelminthic. Bark and roots are astringent. Phytochemical analysis yielded
saponin, saponin glycosides, steroid, cardiac glycoside, tannins, volatile oils, phenols and balsam
(gum).
Tannins are polyphenols that are obtained from various parts of different plants belonging
to multiple species. Deriving it name from the technical word ‘tanning’ that meant converting
animal hides to leather through chemical processes; tannin is basically used for this function. It is
found in abundance in the tree bark, wood, fruit, fruit pod, leaves, and roots and also in plant
gall. Since earlier times, people obtained tannin for tanning from plants like wattle (Acacia sp.),
oak (Quercus sp.), eucalyptus (Eucalyptus sp.), birch (Betula sp.), willow (Salix caprea), pine
(Pinus sp.), quebracho (Scinopsis balansae). (Herbs2000.com)
The term tannin defines a very heterogeneous group of phenolic compounds that are
identified, based on certain properties: astringency, bitterness, reaction with ferric chloride, and
the ability to bind with proteins. It was the characteristic interaction with proteins that
traditionally differentiated tannins from other phenols. However, not all phenols that bind with
proteins elicit an astringent response, and tannins are not the only compounds in wines that
cause astringency.
Tannins are found as shapeless yellowish or light brown masses like powder, flakes or
sponge. Interestingly, tannins are found almost in all plants and in all climates all over the world
-- although algae, fungi and mosses do not contain much tannin. The percentage of tannins
present in the plants, however, varies. While they are present in significant proportions in some
plants, many others have too little of them. Tannins are usually found in large quantities in the
bark of trees where they act as a barrier for micro-organisms like bacteria and fungi and protect
the tree.
As tannins often lower the absorption of some materials into the body, tannins are also
often known as anti-nutrients. For example, tannins are found in tea and coffee and consuming
too much of these beverages without milk may lead to calcium and iron deficiency in the body
and often lead to osteoporosis and anemia.
On the contrary, tannin has a high potential of being a good antitumor agent. Previous
researches on the said polyphenol have shown that the tannin-containing fraction of plants
exhibited antitumor activity. In the last years, several immunomodulating antitumor agents have
demonstrated in the nature, particularly from Aloe plant and rice bran. These plants are proven to
have high tannin content in them.
Tannins can be classified into two broad groups - hydrolysable tannins and condensed
tannins. Hydrolysable tannins are basically derived from simple phenolic acids like gallic acid or
ellagic acid and when heated they give away pyrogallol. Pyrogallol is also known as hepatotoxic
and has antiseptic as well as caustic properties. Owing to the hepatotoxic property, plants that
have a concentration of tannins are not appropriate for application on open wounds. When out in
the open air hydrolysable tannins normally change to a brownish color and are accountable for
the brown color of many plant dyes. On the other hand, condensed tannins, also known as nonhydrolysable tannins, do not split easily and hence it is difficult to analyze these. Condensed
tannins are basically flavonoid dyes formed through bio-synthesis of flavins and catechins. When
these non-hydrolysable tannins are heated up in acids they synthesize to yield a red insoluble
substance known as tannin reds or phlobaphenes. Phlobaphenes are flushed precipitates found in
some plants that have reddish tints and this is an indication that these plants have rich
concentration of condensed tannins. When condensed tannins are heated, catechol emerges as the
final product. Unlike hydrolysable tannins, condensed tannins do not possess any trace of
hepatotoxicity or any adverse side effects and hence are favorable for use.
Related Studies
In a research study presented by Richard Spjut and Richard Marin (2000), World
Botanical Associates in Laurel, Maryland and Temecula, California, plants in Baja California
were screened for antitumor activity. They collected samples from the well known diverse
vegetation of the place, and subjected them to antitumor screening. They have found out three
antitumor active compounds in 400 species, namely colubrinol, holacanthone, and bouvardin.
Extracts from 74 samples representing 56 species that were collected in Baja California plants
were later screened against 60 tumor cell lines. The cell lines then were divided into panels.
Each panel includes one or more assays pertaining to a particular organ such as lung, colon,
ovarian, etc. None of the Baja plant extracts showed specific cytotoxicity—that is activity that is
selective to one panel, as opposed to general cytotoxicity in many tumor panels that is seen more
often. Additionally, extracts of these samples were screened for anti-AIDS activity, and the Baja
plants were being considered for screening of drug resistant antibiotics. The lack of discoveries
thus far should not be interpreted as negative as screening criteria by which plants are being
selected for drug development has become more stringent.
Approximately 100 samples collected during 1986-1990 from Baja California were also
screened by university chemists, Dr. Ching-jer Chang at Purdue University, and Dr. John
Cassady at Ohio State University. They also employed the NCI human cancer cell lines, but on
amore limited scale. Included were five cancer lines in four panels involving lung (1), breast (1),
colon (1) and two of the skin (2). Species that were of interest in these screens are indicated as
active in 1 to 5 cell tumor lines. However, most recollections did not meet activity criteria to
justify further pharmacological study.
A research study concerning antitumor activity of plants was published in the in the
Indian Pharmacological Society, In vitro cytotoxic activity of Lantana camara Linn, by C Raghu
et al,. They utilized Lantana camara Linn., a large evergreen strong-smelling herb, native of
tropical America, but now naturalized in many parts of India. The leaves are used as a bechic,
antitumoral, antibacterial and antihypertensive agent. Several tri-terpenoids, napthaquinones,
flavonoids, alkaloids and glycosides isolated from this plant are known to exert diverse
biological activities including cytotoxic and anticancer properties. The crude methanolic extract
of different parts of this plant was studied for its in vitro cytotoxic potential. Natural products
provide an inexhaustible source of anticancer drugs in terms of both variety and mechanism of
action. Hence, in continuation of their studies to identify potent natural products for antitumor
activities, investigation on the in vitro cytotoxic properties of the crude extracts of different parts
of Lantana camara was taken up against four cancerous cell lines viz. HEp-2, B16F10, A-549
and DLA and a normal NRK-49F cell line using standard procedures.
Bioactivity and drug potential of tannins by Tripathi Yogesh C. and Rathore Mala NonWood Forest Products Division, Arid Forest Research Institute, Jodhpur-342005 (India). The
binding of tannins with proteins participates in the antidiarrhoeic activity of tannins by protecting
the digestive organs from injurious attack. It also participates in the antihaemorrhagic effects of
tannin rich drugs. The detoxification of snake venoms and bacterial toxins by persimmon tannin
is another activity most probably based on the strong binding activity of this tannin (Asquith &
Butler, 1986: Murdiati & McSweeney, 1987). The natural occurrence of tannins, in pasture
species, in addition to its potential of controlling bloat, could have a direct nutritional benefit by
protecting leaf protein from degradation in the rumen of animals (Mangan, 1988). Tannins at
relatively high concentrations usually inhibit the activity of enzymes, but at low concentrations
they often stimulate enzyme activity (Maxson et al., 1973). The inhibition of mutagenecity of
several carcinogens by tannins has been partly attributed to the binding of tannins with these
compounds. Mechanism of inhibition rather depends on the type of the mutagen. Strong
inhibition of tumor promotion is exhibited by several tannins and has been demonstrated
experimentally on two stage carcinogenesis. Several oligomeric hydrolysable tannins and related
compounds revealed strong host mediated antitumor activity which may be due to the
enhancement of immune response of the host animals through the actions on tumor cells and
some immunocytes (Okuda et al., 1991).
Inhibition of the autoxidation of co-existing substances by tannins is regarded as being
due to polyphenolic structures that can produce stable free radicals. The use of alkylgallates as
antioxidants of fatty oil and the use of gallic acid as a preservative of fresh fish are examples of
practical applications of this effect. The long lasting inhibitory effects of tannins (Feeny, 1969),
may be due to the presence of many phenolic hydroxyl groups which produce stable free radicals
one after another. The formation and solubilization of precipitates with several metal ions is
accompanied by reduction of the metal ions. This property of tannins may induce a reduction in
the toxicity of several metallic ions under mild conditions. Tannins have been shown to inhibit
the growth of HIV and herpes simplex virus (Okuda et al., 1991)
Antitumor-promoting activities of hydrolyzable tannins in mouse skin by Gali et al.
Ellagic acid and gallic acid and its derivatives, applied topically to female CF-1 mice 20 min
before each 12-O-tetra-decanoylphorbol-13-acetate (TPA) treatment inhibit the inductions of
epidermal ornithine decarboxylase activity, hydroperoxide production and DNA synthesis caused
by this potent tumor promoter in relation with their abilities to inhibit the promotion of skin
papillomas and carcinomas in the two-step initiation-promotion protocol. Because of its potency
against TPA promotion, tannic acid, which is already known to inhibit tumor initiation, may
inhibit the multistage process of carcinogenesis.
Antitumor Activity and in vivo Antioxidant Status of Mucuna pruriens Seeds against
Ehrlich Ascites Carcinoma in Swiss Albino Mice by Rajeshwar et al, aimed to evaluate the
antitumor effect and antioxidant role of Mucuna pruriens against EAC bearing Swiss albino
mice. The effect of methanol extract of Mucuna pruriens (MEMP) on tumor growth and host's
survival time was studied by the following parameters: tumor volume, packed cell volume,
viable and non-viable cell count and life span of the host. MEMP was administered at a 125 and
250mg/kg b.w. once a day for 14 days, after 24 h of tumor inoculation. Decrease in tumor
volume, packed cell volume, and viable cell count were observed in MEMP treated animals
when compared to EAC treated animals. Treatment with MEMP at a dose of 125 and 250mg/kg
increased the mean survival time to 29.5 ± 0.55 and 34 ± 0.2 days respectively. The extract also
decreased the body weight of the EAC tumor bearing mice. Hematological studies reveal that the
Hb content was decreased in EAC treated mouse, whereas restoration to near normal levels was
observed in extract treated animals. There was a significant decrease in RBC count and increase
in WBC counts in extract treated animals when compared to EAC treated animals. The study was
also extended to estimate the liver biochemical parameters such as LPO, GSH, and antioxidant
enzymes like SOD, CAT etc. Treatment with MEMP decreased the levels of lipid peroxidation
and increased the levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT).
The results suggest that the methanol extract of Mucuna pruriens seeds exhibits significant
antitumor and antioxidant effects in EAC bearing mice.
In-vitro Antimicrobial and Antitumor Activities of Stevia Rebaudiana (Asteraceae) Leaf
Extracts by Jayaraman et al, published in the Tropical Journal of Pharmaceutical Research in
December 2008, Four solvent extracts (ethyl acetate, acetone, chloroform and water) of Stevia
rebaudiana leaves were investigated against Staphylococcus aureus, Salmonella typhi,
Escherichia coli, Bacillus subtilis, Aeromonas hydrophila and Vibrio cholerae by using agar well
diffusion method. Candida albicans, Cryptococcus neoformans, Trichophyton mentagrophytes
and Epidermophyton species were used to test anti-yeast and antifungal activity. The cytotoxic
effects of the extracts on Vero and HEp2 cells were assayed using 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide [MTT]. Among the four extracts tested, acetone extract had
effective antibacterial potential, followed by ethyl acetate extract. The acetone extract showed
greater activity against Gram-positive than against Gram-negative organisms. All the extracts
were active against Epidermophyton species and Candida albicans. The 1:8 dilution of the
acetone extract was non-toxic to normal cells and also had both anticancer and anti-proliferative
activities against cancerous cells. The study confirms the antimicrobial and antitumor activities
of Stevia rebaudiana leaves extracted using various solvents, and is therefore, a potential drug
that requires further studies and development.
Woodfordin C, a Macro-ring Hydrolyzable Tannin Dimer with Antitumor Activity, and
Accompanying Dimers from Woodfordia fruticosa Flowers (Takashi et al.). Three new dimeric
hydrolyzable tannins, woodfordins A, B and C, along with seven known hydrolyzable tannins,
including oenothein B, a dimer exhibiting marked host-mediated antitumor activity, were isolted
from in Indonesian curde drug, Sidowayah [dried flowers of Woodfordia fruticosa (L.) Kurz
(Lythraceae)]. The structures of the new tannins were elucidated based on chemical and spectral
evidence. Woodfordin C, having a macro-ring structure, was also found to exhibit a significant
antitumor activity.
Chapter III
METHODOLOGY
This chapter discusses the variables, materials, procedure, and the treatment that will be
employed in the present study.
Research Design
FIRST EXPERIMENT
In the first experiment, the researchers shall culture tumor cells then apply different
antitumor agents in a petri-dish. They will use the Posttest only Control Group.
X1
R1
X2
O1
X3
X4
They will only have one group. This group will be applied four treatments, this will be
the tumor cell lines contained in a petri-dish. The first treatment will be the tannin extracted from
Talisay bark is in a 25%-75% ratio with water. The second treatment will be applied on the same
petri-dish but the tannin extracted from Talisay bark is in a 50%-50% ratio with water. The third
treatment will be applied on the same petri-dish but the tannin extracted from Talisay is in a
75%-25% ratio with water. The fourth treatment will be applied on the same petri-dish but tannin
is not to be used, the commercially produced antitumor drug will be the treatment which will be
in a 50%-50% with water. After applying all the treatments in a petri-dish, they will have an
observation to test the effectivity of each of the antitumor agents applied.
SECOND EXPERIMENT
In the second experiment, the researchers shall culture tumor normal cells then
apply different antitumor agents in a petri-dish. They will use the Posttest only Control Group.
X1
R1
X2
O1
X3
X4
They will only have one group. This group will be applied four treatments, this will be
the normal cell lines contained in a petri-dish. The first treatment will be the tannin extracted
from Talisay bark is in a 25%-75% ratio with water. The second treatment will be applied on the
same petri-dish but the tannin extracted from Talisay bark is in a 50%-50% ratio with water. The
third treatment will be applied on the same petri-dish but the tannin extracted from Talisay is in a
75%-25% ratio with water. The fourth treatment will be applied on the same petri-dish but tannin
is not to be used, the commercially produced antitumor drug will be the treatment which will be
in a 50%-50% with water. After applying all the treatments in a petri-dish, they will have an
observation to test the applying it to different parts of the body as an antitumor agent if it does
not damage the normal cells.
Variables
(Linking Variable)
Time
Type of Treatment Used
Tumor Cell Death Index
(Independent Variable)
(Dependent Variable)
Environment, temperature, Pressure, Discrepancies in equipment
(Extraneous Variable)
In the experiment, the independent variable is the type of treatment used. In the
experimental groups, different treatments were assigned, yielding different results. The linking
variable in the experiment is the time in which the experiment took place. The result of the
different kinds of treatment used is the Tumor Cell Death Index (TCDI). Though many measures
are done in order to get accurate results, one can never stop the occurrences of extraneous
variables, like environment, temperature, pressure and discrepancies in equipment.
Materials
Talisay Bark
Tumor cell lines
50 mL Pyrex-glass extraction vials
Incubator
Water
Normal cell lines
Methanol
Paper Disks
Large Diameter Glass Marbles
Petri-dish
Refrigerated Centrifuge
Camptotetchin
Volumetric Flasks
Procedure
Extraction of Tannin
Aliquots of 400mg samples will be weighed into 50ml Pyrex-glass extraction vials. Ten ml 50
percent aqueous methanol will be added and large-diameter glass marbles will be placed on top
of each tube. Initial extraction is performed by placing the tubes in a room-temperature,
ultrasonic water bath for 20 minutes, then in a dry-heating block at 92-95°C for 15 minutes.
After cooling to room-temperature, the samples are transferred to a refrigerated centrifuge and
spun in an angle rotor at 9130 × g for 20 minutes. The supernatants from each sample are
combined into 50ml volumetric flasks while the precipitates are re suspended in another 10ml of
50 percent aqueous methanol, and two subsequent extraction-centrifugation cycles are repeated.
The final precipitates are discarded. All supernatants from the same samples are combined in one
volumetric flask, and the final uniform volume of the supernatants is adjusted with aqueous
methanol (up to 25ml) before tannin analysis.
Preparation of Tannin Extracts and Tumor Cell Lines
The tannin extracts should be kept in a cool room and its container must be opaque
because of the reactivity of tannin in sunlight and heat. The tannins should be of proper care in
order to use it efficiently. After the incubation period of the tumor cells, it will be placed in the
the table together with the tannin extracted from Terminalia catappa L. bark, the commercially
produced antitumor drug, Camptotetchin, and the paper discs with a diameter of 2 mm.
Execution of the first Experiment
The aim of the experiment is to test the feasibility of tannin extracted from Talisay as an
antitumor drug. The Tumor Cell Death Index (TCDI) will be used to determine the effectiveness
of the potent antitumor agents. To do so, one petri-dish with the tumor cell lines is needed. In
this case, there should 4 paper disks. One containing 25%-75%, tannin and water ratio
respectively, 50%-50%, 75%-25%, and the other one is 50%-50% Camptotetchin and water
ratio.
All of these paper disks are placed in one petri-dish and is assigned proper location with
proper distances. The paper disks will be dipped in the corresponding agents after 6 hours and
after it will be placed on the petri-dish. After which the paper disks are placed the petri-dishes
will be placed in the incubator for their growth for about 3-4 days and the inhibition each
antitumor agent will produce.
Execution of the second experiment
The aim of the experiment is to test the feasibility of tannin extracted from
Talisay as normal drug which cannot damage normal cells.. The Cell Death Index (TCDI) will be
used to determine its feasibility as a normal drug. To do so, one petri-dish with a normal cell
lines is needed. In this case, there should 4 paper disks. One containing 25%-75%, tannin and
water ratio respectively, 50%-50%, 75%-25%, and the other one is 50%-50% Camptotetchin and
water ratio.
All of these paper disks are placed in one petri-dish and is assigned proper location with
proper distances. The paper disks will be dipped in the corresponding agents after 6 hours and
after it will be placed on the petri-dish. After which the paper disks are placed the petri-dishes
will be placed in the incubator for their growth for about 3-4 days and the inhibition each
antitumor agent will produce.
Gathering of Data in the Experiment
In the first experiment, to determine the effectiveness of the antitumor drug is to measure
the zone of inhibition created by each of them and the Tumor Cell Death Index (TCDI). The
larger the zone of inhibition created by an antitumor antitumor agent the more effective it is to be
an antitumor drug. The zone of inhibition will be measured by a ruler and the longer the radius of
the zone of inhibition the more effective it is.
In the second experiment, to determine the effectiveness of the antitumor drug to not
damage the normal cells is to measure the zone of inhibition created by each of them and the Cell
Death Index (TCDI). The smaller the zone of inhibition created by an antitumor antitumor agent
the more effective it is to be an antitumor drug. The zone of inhibition will be measured by a
ruler and the shorter the radius of the zone of inhibition the more effective it is. For as an
antitumor drug, it should be effective both in killing tumor cells and not creating damage to the
normal cell.
Schematic Diagram
Preparation of
Materials
Extraction of
Tannin
Preparation of
Tumor cell line
Execution of
First experiment
Preparation of
Normal cell line
Execution of
Second
experiment
A
A
Measuring the
zone of inhibition
Measuring the
Tumor Cell Death
Index (TCDI)
Measuring the Cell
Death Index (CDI)
Analysis of Data
Restoration