1.
INTRODUCTION
Globally, skin cancer is the most common form of cancer due to level on
exposition of toxicity agents, UV lights and microorganisms that lead to skin carcinogenesis
which initiation, promotion, and progression of cancer (Ng C. Y., Yen H. and Su S. C.,
2018). Cancer remains to be one of the leading cause of deaths of mankind and can be treated
with chemotherapy and radiotherapy which would later on cause side effects; in some cases,
these treatments would be less effective.
The term “chemoprevention” first created by Michael Sporn in year 1976, that widely
use as reference in the use of pharmacologic or natural medium to impede the stimulation of
carcinogenesis formation process. Studies shows, presence of phytochemical in herb plants
are active compounds as in biologically properties that may possess the capability on fitness
utility, particularly in chemoprevention of carcinoma and had been used throughout history of
medicinal purpose. It is believed that phytochemicals may have a distinctive role in skin
cancer. Several promising phytochemicals have been found in basil leaves, Ocimum sanctum
Linn or Ocimum Tenuiflorum Lin or Holy Basil (Bhattacharyya P. and Bishayee A., 2013).
Holy basil plants are composed of complex chemical composition and manifest
massive range of phytochemicals. The existence of the elements in basil herb relied on the
germinating process and storage state. The traits scent of O. sanctum influenced by the
existences of essential oil that mainly concentrated in the leaf with approximate of 70%
eugenol and 20% methyl eugenol. Scientific studies that carried out in the last several
decades, according to the standard protocols have actually shown that O. Sanctum own anti-
neoplastic, chemopreventive and radioprotective consequences. Although, there were certain
current studies that provides an overview of the numerous pharmacological features of O.
sanctum on the utilization of this herb for either chemoprevention or therapy of oncologic
diseases, but they have not discussed the details in the published writing. Therefore, this
research experiment might be an attempt to completely evaluate the effectiveness of
extraction basil leaves against skin cancer.
2. PROBLEM STATEMENT
There are more than 100 types of cancer diseases, skin cancer is one of the typical
formation of cancer and it also can occur on surface areas of skin which is not or rarely
exposed to sunlight. There are few early treatments and prevention were suggested to
overcome it, such as immunotherapy, surgery, chemical peel, targeted therapy,
chemotherapy, radiation therapy, other drug therapy and avoid exposes to UV radiation and
substance that trigged skin cancer. Yet, the treatments caused side effects and needs follow-
up test for quit long periods to make sure the cells are stop spreading and prevent from
reformation of cancer cell for the time being. Therefore, cancer is more likely to be
potentially cured with treatment at an early stage (Davis C. P., 2018).
Cancer is a disease cause by uncontrolled growth of abnormal cells during cells
separation in a body which causes of morbidity and mortality. According to Medical News
Today (The MNT, 30 March 2016) stated; WHO estimated there were 8.2 million deaths that
were associated with carcinoma and 14 million current cancer cases in 2012 in their most
recent data. They also provide general information about cancer worldwide; cause of cancer
death been continue rising about a 70% with an estimation 13.1 million deaths in 2030.
Although treatments are provided, the cost of diagnosis and treatment therapy are
expensive at private medical care while the related equipment and the materials are
insufficient in government hospitals. In contrast, basil leave possess natural chemical
compositions; phytochemical such as volatile oil (Bhattacharyya P., 2013) that might be
effective to cure skin cancer permanently with or without side effects.
3. OBJECTIVE
The objectives of this study are outlined as below:
To determine the potential of extracts of basil leaves towards the growth of skin
cancer cell.
To deduce the composition components of the Ocimum sanctum Linn that have high
potential on anti-cancer effect.
To evaluate the phytochemical characteristic of basil leaves.
4. LITERATURE REVIEW
4.1. Ocimum sanctum Linn
Ocimum sanctum Linn or Ocimum Tenuiflorum Lin is regularly recognized as basil or
'selasih hitam' across Malaysia and it is a highly aromatic herbaceous plant that came from
Labiatae family. In India, it is also known as Holy Basil and recognized as “tulsi which
believed to be the most sacred herb. Tulsi has been significant on its diverse healing
properties that lead on usage in Ayurveda for thousands of years. Different parts of flower,
leaf, shrub, seeds, rhizome and the entire plant of O. Sanctum L. used to make Ayurvedic
medicament for treat normal flu, sore head, inflammation, stomach affliction, insect bite,
heart disorder and different forms of poisoning. O. Sanctum can be consumed in traditional
methods like in the form of herbal tea, dried powder or fresh leaf. Even dried Holy Basil
leaves were used to repel insect by mixed with stored grains which have been practices for
centuries (Bhattacharyya P. and Bishayee A., 2013).
This plant is found abundantly in several northern and eastern region of Africa, some
of China division, Taiwan, Malaysia, and Australia but they believed it originated from India.
O. Sanctum is propagated with the aid of seeds that easily may grow without need much care
with extensive type of soils and any climatic conditions. Morphologically, basil plant can
reach up 30 to 75 cm tall with shaggy stems, biennial branched and green leaves that has
strong smell conditions. The characteristic of leaves is oblong, ovate, blunt or keen, length up
to 5 cm, and normally has slightly toothed shape at the edge of leaves. While the flowers are
purple in color and tiny in shape, with small fruit and reddish–yellow color seed.
Tulsi plant is a composed of complex chemical composition which represents a wide
number of phytochemicals. Process of growing, harvesting under standard condition
determined the presence of the elements in this plant. The properties scent of O. sanctum
influenced by the existences of essential oil that largely concerted in leaves part with mainly
composed of 70% eugenol and 20% methyl eugenol. Cirsilineol, isothymusin, apigenin and
rosmarinic acid are some examples of phenolic element that similar as significant amounts of
eugenol contain in fresh leaves and stem. There are also some flavonoids compound has been
found when extraction process take place in leave part.
There is also other composition of essential oil such as carvacrol, ursolic acid,
linalool, limatrol, and caryophyllene. O. sanctum is also shown to possess a range of
sesquiterpenes and monoterpenes which consist of αelemene, myrtenal, α- pinenes, β-
pinenes, β–sitosterol and fats. Majority of the chemical substances has potential in health
value and display numerous pharmacological results.
Structure of some important active components of Ocimum sanctum.
Although the effectiveness of O. sanctum in cancer prevention and therapy have
established a lot number of preclinical studies, there is lack of human studies and less
possibilities particularly in clinical drug development. These research required to understand
the protective effects of Holy basil in humans and the underlying mechanism of action. There
are also lack of information on proper method and techniques in conducting the process of
phytochemical composition.
4.2. Skin Cancer
Skin cancer is result of skin carcinogenesis, where these process lead by multistage
processing due to high level exposition of toxic agents, UV attrition and microscopic
organism on skin. UV radiation induces carcinogenesis due to cell undergoes lasting genetic
alteration which allow the cell with the capacity for greater development. Therefore,
regenerative proliferation which is related with reoccurring wound or UV radiation contribute
to skin tumor promotion (Ng C. Y., Yen H. and Su S. C., 2018).
According to several studies on skin cancer articles, there are 3 major variety of skin
cancer which are basal cell carcinoma, squamous cell carcinoma and melanoma. Most of
squamous cell carcinoma occurs when their body parts are exposed to sun and even Homo
sapiens with dark coloured skin are prone to evolving squamous cell carcinoma on parts that
aren't frequently revealed to the sun. While melanoma advances anywhere on the body
include that part of skin hasn't been exposed to the sun.
Some scientific studies are believed; chemoprevention of cancer can be done by using
phytochemicals that derive form herb plants due to its potential health benefits. This is
because, most of phytochemical have polyphenol groups which consist of multiple
hydrophilic hydroxyl that function as chemical agents for free radicals and reactive oxygen
species (ROS) by securing the cells from oxidative damage on DNA, protein, and lipids.
While several phytochemical endeavor anti-inflammatory characteristic by obstructing
cytokine interest or the discharge of inflammatory mediators. Thus, phytochemicals have a
unique role in skin cancer and have been taken into consideration as a way to boost most
cancers chemoprevention and therapy via a couple of procedures (Ng C. Y., Yen H. and Su S.
C., 2018).
Yet the studies did not specify the herb plant that have content of phytochemical
which very effective and they discuss randomly of some characteristic of phytochemical
which contain on certain plants. There also were lacking information on cultivation and
studies on properties of cancerous cell throughout the studies of articles and journals.
5. METHODOLOGY
5.1. Equipments
The equipment that are needed is, tubes, racks of reaction tubes, petri dish, Bunsen burners,
microscopes, scalpel, glass objects, cover glass, blender, hot plate, paper discs, a
micropipette, pipette drops, mixer, Erlenmeyer flask, measuring cup, haemocytometer, digital
scales and rotary vacuum evaporator.
5.2. Materials
The substance that are required is a sample of skin cancer cell, basil leaves (Ocimum sanctum
L.), aquades, ethanol 96%, Formalin, Dimethyl sulfoxide (DMSO) 10%, DMEM, MTT assay
and MSA.
5.3. Media Preparation
The Erlenmeyer flask is placed in an autoclave along with petri dish at a temperature of
121 ˚ C 1 ATM pressure for 15 minutes. The SDA materials in liquid state included as
much as 20 ml in each Petri dish.
5.4. Solvent extracts of basil leaves
Basil leaves is diluted with the solvent Dimethyl sulfoxide (DMSO). Then, mash basil leaves
into powder after drying it under the sunlight for 3 days. Necessary amount of basil powder is
macerate using ethanol over 3 x 24 hours at room temperature. The obtained solution is then
filtered using a filter paper and then it was evaporated using the rotary evaporator vacuum.
The final extract is then diluted with the concentration needed.
5.5. Culturing and identification of cancer cell
5.5.1. Primary Culture
Sample of cancer cells can be obtained from any resources that are available and reproduced
by culturing on media (DSA) with 10% (v/v) fetal calf serum, 100 units/ml penicillin and 100
g/ml streptomycin sulphate. The cultured cells were preserved at 37°C in a humidified
atmosphere with 5% CO2.
5.6. Feeding and Maintenance
These cell cultured should be inspected systematically and day-to-day to evaluate the
existence of contaminants such as fungus and bacteria. The colour, turbidity and pH of the
medium should be supervised, media (plus serum and other additives) should be altered
frequently and are not permitted to be diminished, the cells must have enough required
nutrients and the cells should not become acidic.
5.6.1. Subculture of cell
Subculture is required to reduce the cell density and not to wear out the medium of nutrients
too quickly. Proteolytic enzymes such as trypsin use to breaks the cell-cell and cell-substrate
links and creates a single-cell suspension.
5.6.2. Cloning
This individual cell is isolates by cloning process so that it can develop into progeny. Thus,
the yield culture consists of genetically homogeneous. Then, the media incubate at
temperature of 25 C for two to seven days for obtain pure isolation.
5.6.3. Cell counting
The hemocytometer use for cell count and trypan blue use to test cell viability after done the
trypsinization process
5.7. MTT Cell Viability Assay(cytotoxicity test)
The sample treat with different concentration of basil extract in 96-well tissue culture plate
and incubate at 37°C with 5% CO2 for 48 and 72 hrs, optimal treatment time of the extracts
of the cell line the result. Then MTT is add to each sample and incubate again at 37 °C with
5% CO2 for 4hrs. Next, 100µL of acidified isopropanol and 100 µL of distilled water were
added into every well. Using ELISA reader at 550 nm, the cell viability will detected.
Example of basil leaves extract
6. EXPECTED OUTCOME
Successful research will prove:
• Tendency of Ocimum sanctum Linn reduce the growth skin cancer cell
• The composition components of the Ocimum sanctum Linn that have high potential on
anti-cancer effect has been deduced
• The evaluation of phytochemical composition
7. PROJECT MILESTONE
Appendix Thesis 1 Thesis 2
Year 2019 2019
Month/Project JAN FEB MAR APR MAY JUN JULY
Activities 2019 2019 2019 2019 2019 2019 2019
Proposal & Literature
Review
Submission of
research proposal &
proposal presentation
Sample Preparation
Preparation of Media
Isolation and
Preliminary Screening
Cytotoxicity test
(ELISA)
Thesis Writing
Viva presentation
This project will be conduct and run for 2 semesters. The research milestone is designed
based on the objectives of the project.
Table 1: Gantt Chart
8. EXPECTED BUDGET
The total cost of this study or project is calculated to be RM 4980.45 approximately.
The budget is needed to purchase chemical/materials and consumables needed for this
project. The budget of this project is tabulated below.
Budget Price
Consumables RM 120.60
I. Gloves
II. Inoculation loop
III. Agar plates
IV. Centrifuge tube
Chemicals/Materials RM 4859.85
I. DMEM
II. Basil plants
III. Thypsin
IV. DMSO
V. Ethanol
VI. Sample cancer cell
Total RM 4980.45
9. ACKNOWLEDGEMENT
I would like to thank Madam Roshani Binti Othaman, lecturer BBS 123 Scientific
Communication of UNISEL for guidance in doing proposal as well as the authors and
publishers who published related articles and journals for references.
10. REFERENCE
Agarwal, P., Nagesh, L., & Murlikrishnan. (2010). Evaluation of the antimicrobial activity of
various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus
mutans: an in vitro study. Indian Journal of Dental Research : Official Publication of
Indian Society for Dental Research, 21(3), 357–359. https://doi.org/10.4103/0970-
9290.70800
Anti-probiotic effect of sweet basil (Ocimum basilicum L.) leaf extract. (n.d.). Retrieved from
https://www.researchgate.net/publication/314895214
Aburigal, Y. A. A., Mirghani, M. E. S., Elmogtaba, Sirible, Hamza, N. B., & Hussein.
(2017). Total phenolic content and antioxidant capacity of basil (Ocimum basilicum L.)
leaves from different locations. International Food Research Journal (Vol. 24).
Retrieved from http://www.ifrj.upm.edu.my/24 (07) 2017 supplementary/(18) R1.pdf
Baliga, M. S., Jimmy, R., Thilakchand, K. R., Sunitha, V., Bhat, N. R., Saldanha, E., …
Palatty, P. L. (2013). Ocimum Sanctum L (Holy Basil or Tulsi) and its phytochemicals
in the prevention and treatment of cancer. In Nutrition and Cancer (Vol. 65, pp. 26–35).
https://doi.org/10.1080/01635581.2013.785010
Bhattacharyya, P., & Bishayee, A. (2013). Ocimum sanctum Linn. (Tulsi). Anti-Cancer
Drugs, 24(7), 659–666. https://doi.org/10.1097/CAD.0b013e328361aca1
Baliga, M. S., Rao, S., Rai, M. P., & D’souza, P. (2016). Radio protective effects of the
Ayurvedic medicinal plant Ocimum sanctum Linn. (Holy Basil): A memoir. Journal of
Cancer Research and Therapeutics, 12(1), 20–27. https://doi.org/10.4103/0973-
1482.151422
Charles Patrick Davis. (2018). Cancer Causes, Types, Treatment, Symptoms & Signs.
Retrieved August 19, 2018, from
https://www.medicinenet.com/cancer_causes/article.htm#what_is_cancer
Chandrappa, P. M., Dupper, A., Tripathi, P., Arroju, R., Sharma, P., & Sulochana, K. (2015).
Antimicrobial activity of herbal medicines (tulsi extract, neem extract) and
chlorhexidine against Enterococcus faecalis in Endodontics: An in vitro study. Journal
of International Society of Preventive & Community Dentistry, 5(Suppl 2), S89-92.
https://doi.org/10.4103/2231-0762.172952
EMA Mahmoud, H., Bashir Supervisor, N. H., Assad, Y. O., & Bashir, N. H. (2017). Effect
of basil (Ocimum basilicum) Leaves Powder and Ethanolic-Extract on the 3 rd Larval
Instar of Anopheles arabiensis (Patton, 1905) (Culicidae: Diptera). ~ 52 ~ International
Journal of Mosquito Research, 4(2), 52–56. Retrieved from
http://www.dipterajournal.com/pdf/2017/vol4issue2/PartA/4-1-9-803.pdf
Hosadurga, R. R., Rao, S. N., Edavanputhalath, R., Jose, J., Rompicharla, N. C., Shakil, M.,
& Raju, S. (2015). Evaluation of the efficacy of 2% Ocimum sanctum gel in the
treatment of experimental periodontitis. International Journal of Pharmaceutical
Investigation, 5(1), 35–42. https://doi.org/10.4103/2230-973X.147231
Jamshidi, N., & Cohen, M. M. (2017). The Clinical Efficacy and Safety of Tulsi in Humans:
A Systematic Review of the Literature. Evidence-Based Complementary and Alternative
Medicine, 2017, 1–13. https://doi.org/10.1155/2017/9217567
Martins Güez, C., Oliveira de Souza, R., Fischer, P., Fernanda de Moura Leão, M., Apollo
Duarte, J., Augusti Boligon, A., … Mansur Machado, M. (2017). Evaluation of basil
extract (Ocimum basilicum L.) on oxidative, anti-genotoxic and anti-inflammatory
effects in human leukocytes cell cultures exposed to challenging agents. J. Pharm. Sci,
53(1), 15098. https://doi.org/10.1590/s2175-97902017000115098
Mallikarjun, S., Rao, A., Rajesh, G., Shenoy, R., & Pai, M. (2016). Antimicrobial efficacy of
Tulsi leaf (Ocimum sanctum) extract on periodontal pathogens: An in vitro study.
Journal of Indian Society of Periodontology, 20(2), 145–150.
https://doi.org/10.4103/0972-124X.175177
Luthfi Hakim, M., & Kusdarwati, R. (2018). The Effectiveness of Extracts Basil Leaves
(Ocimum sanctum Linn) against Saprolegnia sp. by in Vitro. Retrieved from
http://iopscience.iop.org/article/10.1088/1755-1315/55/1/012010/pdf
Pranati Eswar, C.G. Devaraj, P. A. (2016). JCDR - Actinobacillus actinomycetemcomitans,
Anti-microbial, Chlorhexidine, Dental plaque, Tulsi extract. Retrieved August 17, 2018,
from http://jcdr.net/article_fulltext.asp?issn=0973-
709x&year=2016&volume=10&issue=3&page=ZC53&issn=0973-709x&id=7468
Pattanayak, P., Behera, P., Das, D., & Panda, S. K. (2010). Ocimum sanctum Linn. A
reservoir plant for therapeutic applications: An overview. Pharmacognosy Reviews,
4(7), 95–105. https://doi.org/10.4103/0973-7847.65323
R.Tambun*, R.R.H. Purba, H. K. G. (2017). Extraction of basil leaves (ocimum cannum)
oleoresin with ethyl acetate solvent by using soxhletation method. Retrieved from
http://iopscience.iop.org/article/10.1088/1757-899X/237/1/012032/pdf
Sakr, S. A., & Al-Amoudi, W. M. (n.d.). Effect of leave extract of Ocimum basilicum on
deltamethrin induced nephrotoxicity and oxidative stress in albino rats. Journal of
Applied Pharmaceutical Science, 2012(05), 22–27.
https://doi.org/10.7324/JAPS.2012.2507
Shraddha J Bennadi, K. K. (2014). Protection of zonisamide induced memory impairment by
tulsi extract and piracetam on mice. https://doi.org/10.4103/2278-344X.138600
Tambun, R., Purba, R. R. H., & Ginting, H. K. (2017). Extraction of basil leaves ( ocimum
canum ) oleoresin with ethyl acetate solvent by using soxhletation method. IOP
Conference Series: Materials Science and Engineering, 237(1), 012032.
https://doi.org/10.1088/1757-899X/237/1/012032
Yates, B. (n.d.). Holy Basil – An Overview of the Research and Clinical Indications.
Retrieved from
https://www.gaiaherbs.com/uploads/1596_HPR_HolyBasil_ResearchPaper-
1371567034.pdf
11. APENDIX
11.1. Appendix 1
Mannitol Salt Agar (MSA) Preparation
Composition:
Ingredients
-Beef extract 1.0g
-Peptone or Polypeptone 10.0g
-Sodium chloride (NaCl) 75.0g
-Mannitol 10.0g
-Phenol Red 0.025g
-Agar 15.0g
Approximately 111g dissolve in 1 litre of distilled water. The media then sterilized by
autoclaving at 121˚C for the 15 minutes.
11.2. Appendix 2
The effectiveness of extracts of basil leaves, Ocimum sanctum Linn against the growth skin
cancer cell.
Sample preparation
Cancer cell Extraction of Basil Leave
Preparation of media Mashing
Cultivation and Subculture
Chemical, DMSO
Isolation and preliminary
MTT Assaay
Cytotoxicity test
BBS 3123 SCIENTIFIC COMMUNICATION
Research proposal
THE EFFECTIVENESS OF EXTRACT OF BASIL
LEAVE AGAINST SKIN CANCER CELL IN-VITRO
NAME: LISHANTHINIE MANIARASU
STUDENT ID: 4171004231
LECTURER: MADAM ROSHANI BINTI OTHAMAN
SUBMISSION DATE: 21ST AUGUST 2018
