1
INTRODUCTION
Background of the study
According to the World Health Organization (WHO) in 2017, medicinal plants are used
throughout the world, and the regulations defining their proper use, such as purity and
concentration of the required chemical compounds, are widely recognized. The use of herbal
medicine products has increased tremendously over the past three decades with not less than
80% of people worldwide relying on them for some part of primary health care (Martins Ekor,
2013). It is becoming more mainstream as improvement analysis and quality along with
advantage in clinical research show the value of herbal medicine in treating and preventing
disease (K. P. Sampath Kumar, Debjit Bhowmik, 2010).
Ethanol ingestion with alcoholic intoxication is one of the commonest emergencies
followed by “alcohol withdrawal syndrome” in chronic alcoholics presenting to the department
with medical conditions (Meera Ekka, Praveen Aggarwal, 2015). Ethanol produces intoxication
because of its depressive effects on various areas of the brain causing physical and mental
impairments in a progressive order as the person’s alcohol level increases (Jerry R. Balentine,
Steven Doerr, MD, 2018).
In connection to this, Aloe Vera (Aloe barbadensis) is the oldest medicinal plant ever
known and the most applied medicinal plant worldwide. Its leaves are full of a gel-like substance
that contains numerous beneficial compounds like vitamins, minerals, anti-oxidant and amino
acids (Joe Leech, 2017). A related study investigated the hepatoprotective of aloe vera
polyssacharides against chronic alcohol-induced hepatotoxicity in mice. This study shows that
AVGP (A. vera polysaccharides) exerts a potent protective effect against chronic alcohol-
2
induced liver injury. Its hepaprotective effect appears to associated with its antioxidant capacity
and its ability to accelerate lipolysis and inhibit inflammatory response. The results of this study
indicate that AVGP could be considered as potent food supplement in the prevention of AVGP
(Yan Cui et al, 2013). Another valuable medicinal plant from cucurbitaceae family is “bitter
melon” (Momordica charantia), which has gained an enormous attention in recent years as an
alternative medicine in developed countries. Most commonly used as a folk medicine, bitter
melon has been subjected to having antiviral, anti-leukemia, anthelmintic, antidiabetic and
anticancer properties. One study, the wild bitter gourd against alcoholic fatty liver in mice by
attenuating oxidative stress and inflammatory responses, investigated whether the wild bitter
gourd possessed protective effect against chronic alcohol-induced liver injury in mice. The mice
were fed an alcohol containing liquid diet for 4 weeks to induce alcoholic fatty liver. The mice
with alcoholic fatty liver were treated with ethanol extracts from four different wild bitter gourd
cultivars: Hualien no.1; Hualien no. 2, Hualien no. 3 and Hualien no. 4. This study shows that
daily administration of 500mg kg body weight of a Hualien no.3 extract (H3E) or Hualien no. 4
extract (H4E) markedly reduced the steatotic alternation of liver histopathology (Kuan-Hung Liu
et al, 2014).
Several studies had been conducted using Caenorhabditis elegans or C.elegans because it
involves vast majority of molecular signaling pathways common to human development and cell
behaviour, thus Caenorhabditis elegans offers great potential for developmental toxicity testing,
(Markaki, M. & Tavernarakis, N., 2010).With this note, the researchers investigated aloe vera
(Aloe barbadensis) and bitter melon (Momordica charantia)on Caenorhabditis elegans to see
their therapeutic effect on alcoholic addiction. This study will be beneficial especially to those
who are alcohol addicted or had been intoxicated by alcohol for it is a baseline study for
3
development of medications on such cases. This will also benefit to Department of Health
(DOH) for developing new medicine and assessing the potential therapy for alcohol addiction
carried by aloe vera (Aloe barbadensis) and bitter melon (Momordica charantia) on
Caenorhabditis elegans.
4
Statement of the Problems
This study aimed to assess the potential therapeutic effect of aloe vera (Aloe barbadensis)
extract and bitter melon (Momordica charantia) extract for alcohol addiction using
Caenorhabditis elegans assay.
Specifically, it sought answers to the following questions:
1. How may the aloe vera (Aloe barbadensis) and bitter melon (Momordica charantia)
extracts be described in terms of their phytochemicals?
2. Is there a significant difference between the aloe vera (Aloe barbadensis) extract and
bitter melon (Momordica charantia) extract and the controlled variable in terms of
2.1 preference index of C.elegans that exhibit reduced ethanol addiction?
Hypotheses
Ho: There is no significant difference between the aloe vera (Aloe barbadensis) extract and bitter
melon (Momordica charantia) extract and the controlled variable in terms of:
1. preference index of C.elegans that exhibit reduced ethanol addiction?
Ha: There is a significant difference between the aloe vera (Aloe barbadensis) extract and bitter
melon (Momordica charantia) extract and the controlled variable in terms of:
1. preference index of C.elegans that exhibit reduced ethanol addiction?
5
Research Paradigm
IV
DV
Therapuetic effect of aloe vera
(Aloe barbadensis) leaf extract
and bitter melon (Momordica
charantia) fruit extract for
alcohol addiction in terms of:
1. preference index of C.elegans
that exhibit reduced ethanol
addiction?
Aloe Vera (Aloe barbadensis)
leaf extract and Bitter melon
(Momordica charantia) fruit
extract
CV
Caenorhabditis elegans without
treatment
Figure 1.0: Research Paradigm
6
Definition of Terms
Assay. This refers to an investigative (analytic) procedure in laboratory medicine, pharmacology,
environmental biology and molecular biology for qualitatively assessing or quantitatively
measuring the presence, amount, or functional activity of a target entity (the analyte).
Alcohol Addiction. ‘Addiction’ means having a dependence on a substance or activity.
Ethanol. This refers to a chemical compound that is a type of alcohol.
Caenorhabditis elegans. This refers to microscopic, soil-dwelling roundworm that has been
powerfully used as a model organism also known as “C.Elegans”.
Preference Index. This refers to the frequency of approaches of C.elegans spends in proximity
to each item of the investigated research during a multiple-choice trial.
Phytochemical. This refers to any of various biologically active found in plant compounds.
Scope and Delimitation
This study in only limited to the assessment of the therapeutic effect of aloe vera (Aloe
barbadensis) leaf extract and bitter melon (Momordica charantia) extract for alcohol addiction
on Caenorhabditis elegans assay. The aloe vera and bitter melon were gathered in Olongapo
City. The extracts of aloe vera and bitter melon underwent phytochemical analysis in Department
of Science and Technology (DOST). The Caenorhabditis elegans were cultured at UP – Manila,
College of Medicine with proper approval form from Dr. Paul Medina, Director of the said
college. The testing was conducted in the laboratory of Olongapo City National High School
(OCNHS) with the supervision of the researchers’ adult sponsor and qualified scientist.
7
Significance of the Study
This study will be beneficial to the following:
Researchers. This study could provide a significant contribution to other researchers that
are in the same field as this study. They could use this as a reference for their own investigatory
project.
Department of Health (DOH). The result of this study may also benefit DOH for
developing medicine on treatments that can help people and in determining the potential therapy
for alcohol addiction carried by aloe vera (Aloe barbadensis) and bitter melon (Momordica
charantia) on Caenorhabditis elegans.
8
Review of Related Literature
Aloe Vera
Aloe vera, commonly known as Barbados is a popular herbal
medicine that has been known and utilized for centuries for its
wellbeing, magnificence, therapeutic and skin care properties use by
a variety of cultures. Concerted effort by the scientific research
community, through in vivo (real life) and in vitro (within an
Figure 1.2
https://plantshop.ae/product/aloe-vera/
artificial environment) studies (human and animal) has shed new
light on Aloe vera and its abilities. It has brought Aloe vera out of
the realm of folk medicine, providing it with a solid medical and scientific foundation as a
healer. Aloe vera’s numerous and varied constituent have provided this botanical with multifaceted abilities, uses and applications. There are over 300 species of aloe, which develops
basically within the dry districts of Africa, Asia, Europe and America. It could be a characteristic
item that is presently a day regularly utilized within the field of cosmetology (Oliver
Grundmann, 2012).
There is a lack of reliable hepatotherapeutic drugs in modern medicine in the
management of alcohol/drug-induced liver damage. According to the other study, where they
evaluate the hepatotherapeutic activity of aqueous extract of aloe vera gel in rat. Aloe vera
treatment maintained hepatic architecture similar to that seen in the control. This study shows
that aqueous extract of aloe vera gel is hepatotherapeutic and thus lends credence to the use of
the plant in folklore medicine in the management of alcohol-induced hepatic dysfunction (Saka
WA, et al. Pak J Biol Sci. 2011).
9
Bitter melon
Momordica charantia commonly referred to as Bitter
melon is widely cultivated and eaten across Southeast Asia,
Africa, China, Japan, Oceania and even in the Caribbean. It has
been used as traditional medicine for treating various ailments
Figure 1.3
https://seedsnpots.com/product/karela/
and containing bioactive properties like alkaloids, vitamins and
minerals. It is also used as an antibacterial agent, antioxidant, and an immune system modulator.
It also contains notably phytonutrient, plant insulin known to lower blood sugar levels. Also, it
composes hypoglycemic agent called Charantin which increases glucose uptake and glycogen
synthesis inside the cells of the liver, muscle, and fatty (adipose) tissue. According to studies,
bitter melon contains p-insulin, which can significantly lower blood glucose levels on diabetic
patients by injection.
Bitter melon enhances the antioxidant and reduces pro-inflammation cytokines (Lu KH,
et al. Food Function). Fatty liver occurs when too much builds up in liver cells. Although it is
normal to have a tiny amount of fat in these cells, the liver is considered fatty if more than 5% of
it is fat. Hepatoprotective effect of bitter melon extracts is mainly attributed to its antioxidant
capacity to scavenge free radicals and reduced inflammation in liver due to noxious stimuli. The
plant extract at a dose of 5ml/kg also produced significant protection of liver damage due to high
dose of acetaminophen administration in rabbits. A recent study also suggests that bitter melon
supplementation a meliorates oxidative stress in liver of fructose fed offspring of rats by
improving the antioxidant enzymes activity such as GPx, SOD and catalase (Hiroki Kinoshita,
Yasuyuki Ogata, 2018).
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In addition, several studies were able to investigate whether wild bitter gourd
possessed protective effects against chronic alcohol-induced liver injury in mice. Results
demonstrated that Bitter melon have beneficial effects against alcoholic fatty liver, in which they
attenuate oxidative stress and inflammatory responses.(Kuan Hang Lu, Hui-Chun Tseng et al.,
2013).
Caenorhabditis elegans
C. elegans is a non-hazardous, non-infectious, nonpathogenic, non-parasitic organism. It is small, growing to about
1 mm in length, and lives in the soil – especially rotting
vegetation – in many parts of the world, where it survives by
Figure 1.4
https://seedsnpots.com/product/karela/
feeding on microbes such as bacteria. C. elegans is about as
primitive an organism that exists which nonetheless shares
many of the essential biological characteristics that are central problems of human biology. The
worm is conceived as a single cell which undergoes a complex process of development, starting
with embryonic cleavage, proceeding through morphogenesis and growth to the adult. It has a
nervous system with a ‘brain’ (the circumpharyngeal nerve ring). It exhibits behavior and is even
capable of rudimentary learning.
Caenorhabditis elegans senses multiple environmental stimuli through sensory systems
and rapidly changes its behaviors for survival. With a simple and well-characterized nervous
system, C. elegans is a suitable animal model for studying behavioral plasticity.
Previous studies have demonstrated acute neurodepressive effects of ethanol on multiple
behaviors of C. elegans similar to ethanol’s effect on other organisms. C. elegans also develops
ethanol tolerance during continuous exposure to ethanol. In mammals, chronic ethanol exposure
11
leads to ethanol tolerance as well as increase ethanol consumption. Ethanol preference is
associated in development of tolerance and may lead to the development of ethanol dependence.
Caenorhabditis elegans can rapidly modify its behaviors based on experience. One well-studied
behavior is olfactory adaptation. Olfactory preferences can be changed by adaptation caused by
pre-exposure to a volatile odorant or by food signals (Colbert & Bargmann 1997; Nuttley et al.,
2002; Chao et al., 2004). Despite a simple nervous system of 302 neurons, C. elegans also shows
relatively complicated associative learning based on responses to chemosensory and
thermosensory cues (Gomez et al., 2001; Saeki et al., 2001; Mori et al., 2008). For instance,
worms modify their olfactory preferences after exposure to pathogenic bacteria to avoid toxic
bacteria (Zhang et al, 2005).
According to the other researchers where they identify mechanisms responsible for
intoxication, they screened for Caenorhabditis elegans mutants with altered behavioral responses
to ethanol. C. elegans behaviors are affected by a short continuous exposure to ethanol (Andrew
G. Davies et al., 2003).
12
METHODOLOGY
Methodology Flowchart
Gathering and Preparation
of Plant Materials
Powderization of Aloe Vera
and Bitter Melon
Extraction of
Aloe Vera and Bitter Melon
Phytochemical Screening
Caenorhabditis elegans
Bioassay
Microscopic Investigation
2.2 time
Comparison of Aloe Vera
and Bitter Melon as Therapy
for Alcohol Addiction
Preference Assay
Collection of Data
Analysis and Interpretation
DETAILED METHODOLOGY
Figure 1.5 Flowchart showing the Methodology of the Study
13
1. Gathering and Preparation of Plant Materials
Aloe vera (Aloe barbadensis) plants and Bitter melon (Momordica charantia) for
extraction was obtained and gathered from the local area from any available supermarket in
Olongapo City (Philippines). Test materials that were used were obtained from the school
laboratory of Olongapo City National High School. The researchers washed the aloe vera to
remove the unnecessary dirt, the aloe vera leaf was put upside down to drain the yellow sap,
which is toxic then the researchers cut the aloe vera in the middle and scoped the jell out with
spoon next the outer leaf was placed in a tray to dry it under the sun or placed it inside a
microwave for 5 minutes. For the extraction of bitter melon, the researchers washed the bitter
melon thoroughly to remove all unnecessary dirt second. The bitter melon was cut into half and
scoped out the seeds then cut it into pieces then placed it under the sun or placed it inside the
microwave for 10 minutes.
Figure 1.6 Cleaning and Drying of the Samples
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2. Powderization of Aloe vera and Bitter melon
For the powderization of dried aloe vera leaf and bitter melon, the researchers used a
blender to powderize dried aloe vera leaf and bitter melon.
Figure 1.7 Powderization of the Samples
2.1Extraction of Aloe Vera and Bitter Melon
250 grams of powdered bitter melon is submerged to ethanol for soxhlet extraction while
the 250 grams of powdered aloe vera is soaked under 2500 ml of 100% ethanol for 3 days and
filtered to get the liquid solution after this it was subjected on a hot bath for 4 hours with
continuous external ventilation.
Figure 1.8 Ethanolic Extraction of the Samples
15
2.2 Phytochemical Screening
For the phytochemical screening, the researchers went to the Department of Science and
Technology (DOST) in Pampanga to see the presence of various phytoconstituents.
Phytoconstituents are the raw ingredients for pharmacological activities. Phytochemicals was
screened through standard procedures. These were carefully identified by characteristic color
changes using standard procedures.
Figure 1.9 Submission of the Sample at DOST for Phytochemical Test
3. Caenorhabditis elegans Bioassay
The researchers bought the Caenorhabditis elegans at UP Manila, College of Medicine
Physician-Scientist Association. They were trained by Dr. Raphael Manalo and Dr. Paul
Medinna on how to take care of the C. elegans and perform the assay. The researcher attained
INTEL ISEF Form Certification for using Biohazards for microorganisms.
16
3.1 Microscopic Investigation
The researchers were supervised by a qualified scientist to observe the effects of Aloe
Vera and Bitter Melon extracts as therapy for alcohol intoxication.
Figure 1.10 Microscopic Investigation of the C. elegans Assay
4. Comparison of Aloe Vera and Bitter Melon as Therapy for Alcohol Addiction
The researchers compared the aloe vera extract and bitter melon extract to see which is
most effective therapy for alcohol addiction.
4.1 Preference Assay
C. Elegans were incubated on a control or ethanol plate with OP50 for defined time
intervals and then placed at the origin of an assay plate .The animals remain on the assay plate
for 30 minutes before scoring. EtOH pretreated animals preferentially accumulate in the ethanol
quadrants (A, B). Control animals accumulate primarily to the quadrants without ethanol (C, D).
17
A Preference Index (PI) was calculated as {[the number of animals at A and B] - [the number of
animals at C and D]} / Total number of animals.
5. Collection of Data
The data was collected in a tabular manner, so that uniformity and variability of the
results can be easily noted.
6. Analysis and Interpretation
Data comparison of aloe vera and bitter melon as therapy for alcohol intoxication
was compared to confirm the variability of the data and validity of results.
18
RESULTS AND DISCUSSION
1. Phytochemical Test
1.1 Bitter Melon
As shown in the figure below Alkaloids, Anthraquinones, Flavanoids, and Saponins are
present in the fresh bitter melon. Flavonoids are important for human health because of their
antioxidant, antibacterial, antiviral, and anti-inflammatory activities. They act as free radical
scavengers as they are potential reducing agents that protect from oxidative damage, according to
Saul Ruiz-Cruz et al.
Table 1.1: Phytochemical Screening Test Result for Bitter Melon
Test Parameter
Method
Result
Alkaloids
Meyer/Mayer Test
Alkaloids Detected
Anthraquinones
Bornträger Test
Anthraquinones Detected
Cardenolides and
Keller-Kiliani Test
Bufadienolides
Flavonoids
Tannis and Polyphenolic
Bufadienolides Not Detected
Bate-Smith and Metcalf Test
Ferric chloride Test
Compounds
Saponins
Cardenolides and
Flavonoids Detected
Tannis and Polyphenolic
Compounds Not Detected
Froth Test
Saponins Detected
1.2 Aloe Vera
Phytochemical analysis on Table 1.2 revealed the presence of alkaloid, carbohydrate,
tannin, andtriterpenoid. The proposed roles of alkaloids in plant metabolism, plant catabolism, or
plant physiology are (1) end products of metabolism or waste products, (2) storage reservoirs of
nitrogen, (3) protective agents for the plant against attack by predators, (4) growth regulators.
19
Carbohydrates are body's main source of energy: They help fuel brain kidneys heart muscles and
central nervous system.
Table 1.2: Phytochemical Screening Test Result for Aloe Vera
Test Parameter
Method
Result
Alkaloids
Meyer/Mayer Test
Alkaloids Detected
Carbohydrate
Lead acetate Test
Carbohydrate Detected
Tannin
Borntrager’s Test
Tannin Detected
Triterpenoid
Liebermann-Burchard Test
Triterpenoid Detected
Flavonoids
Bate-Smith and Metcalf Test
Flavonoids Detected
Tannins have been reported to exert other physiological effects, such as to accelerate
blood clotting, reduce blood pressure, decrease the serum lipid level, produce liver necrosis, and
modulate immune-responses. Triterpenoids are widely found in plants. It is found that
triterpenoids have a variety of pharmacological activities including anti-inflammatory, antidiabetic, anti-cancer, regulation of immune function, etc. Flavonoids, like other antioxidants, do
their work in the body by corralling cell-damaging free radicals and metallic ions.
Table 1.3 Preference Index that Exhibit Reduced Ethanolic Addiction
Quadrant
C. elegans with Aloe
Vera extract
C. elegans with
C. elegans
Bitter Melon
without
extract
treatment
3
5
10
A (with ethanol)
7
6
1
B (with water)
8
8
2
C (with water)
2
1
7
D (with ethanol)
Total Number of C.elegans
20
20
20
The table above shows the preference index of the C.elegans with aloe vera extract is 0.5
or 50% of the population preferred quadrant with water while the preference index of C.elegans
20
with bitter melon extract is 0.4 or 40%. On the other hand the preference index of C.elegans
without treatment is 0.7 or 70%. This means that the Bitter melon has the lesser preference index
compared to the other variables which suggests that bitter melon has a higher therapeutic effect
on ethanol addiction.
Preference Index (PI) = (A+D) – (B+C) / A+B+C+D
21
CONCLUSION
With the conducted experiment as basis, the group therefore concludes that aloe vera leaf
extract and bitter melon fruit extract has therapeutic effect for alcohol intoxication. Therefore,
there is a significant difference between the aloe vera (Aloe barbadensis) extract and bitter melon
(Momordica charantia) extract and the controlled variable in terms of percentages of the
population C. elegans that exhibit reduced ethanol behavior and time duration of the population
that exhibit reduced alcoholic behavior. The results show that the bitter melon is more effective
than aloe vera by 25% in terms of reaction of c. elegans. The results of this work agree with
recent studies in which bitter melon and aloe vera has shown therapeutic effect on alcohol
intoxication.
RECOMMENDATIONS
After careful analysis and experimentation, the group is recommending this study to be
continued and used for further researches. This study is very convenient and efficient if it will be
used for treatment for alcohol intoxication, thus it is also organic and botanical.
Further studies on this project can possibly explore the used of aloe vera and bitter melon
as treatment for alcohol intoxication. Thus, the number patients with alcohol intoxication may be
reduced. This study can be a base-line study for medications of alcohol intoxication.
22
BIBLIOGRAPHY
1. Andrew G. Davis et al., (2003). A Central Role of the BK Potassium Channel in Behavioral
Responses
to
Ethanol
in
C.
elegans.
Retrieved
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https://www.sciencedirect.com/science/article/pii/S0092867403009796
2. Hiroki Kinoshita, Yasuyuki Ogata, (2018). Effect of Bitter Melon Extracts. Retrieved from
https://www.hindawi.com/journals/ecam/2018/4915784/
3.Joe Leech, (2017). Benefits that aloe vera gives Retrieved July 10, 2019
from https://www.medicalnewstoday.com/articles/318591.php.
4. Jungsoo Lee, Changhoon Jee, and Steven L. McIntire, (2009). Retrieved from https://
www.ncbi.nlm.nih.gov/pmc/articles/PMC2880621/?fbclid=IwAR2Nt8E9cn4T2y
kE4GnemJFd-saJd5A6MKClzW2AuvjUm6o35XOtEmH-yl
5. K. P. Sampath Kumar, Debjit Bhowmik, (2010). Traditional medicinal uses and therapeutic
benefits
of
Momordica
charantia.
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July
10,
2019
from
https://www.researchgate.net/publication/292256546_Traditional_medicinal_uses
_and_therapeutic_benefits_of_Momordica_charantia_Linn
6. Kuan-Hung Lu, Hui-Chun Tseng et al., (2013). Wild bitter gourd protects against alcoholic
Fatty
liver
in
mice.
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from
https://www.ncbi.nlm.nih.gov/pubmed/24664243
Mark Edgley and the Riddle lab, (2015). What is C. elegans. Retrieved from
(https://cbs.umn.edu/cgc/what-c-elegans)
7. Martins Ekor, (2013). The growing use of herbal medicines: issues relating to adverse reactions
and challenges in monitoring safety. Retrieved July 11, 2019
https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3887317/
23
8. Oliver Grundmann, (2012). Aloe Vera Gel Research Review: An overview of its clinical uses
and proposed mechanisms of action. Retrieved from
(https://www.naturalmedicinejournal.com/journal/2012-09/aloe-vera-gelresearch-review)
9. Ozlem Sultan Aslanturk, (2017). In vitro cytotoxicity and cell viability assays: principles,
advantages,
and
disadvantages.
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from
https://www.intechopen.com/books/genotoxicity-a-predictable-risk-to-our-actualworld/in-vitro-cytotoxicity-and-cell-viability-assays-principles-advantages-anddisadvantages
10. Rebecca A. Richmond, Quan V. Vuong and Christopher J. Scarlett, (2017). Cytotoxic effect
of bitter melon (momordica charantia l.) Ethanol extract and its fractions on
pancreatic
cancer
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vitro.
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25,
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from
https://pdfs.semanticscholar.org/baea/f1b23f68e0449a4cb1bd839a04ea1ad4fb83.
pdf
11. Siroshini Thiagarajan et al, (2019). Momordica charantia (Indian and Chinese Bitter Melon)
Extracts Inducing Apoptosis in Human Lung Cancer Cell Line A549 via ROS
Mediated Mitochondria Injury. Retrieved July 10, 2019 from
https://www.hindawi.com/journals/ecam/2019/2821597/
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Related Diseases. Retrieved from
24
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517695/)
14. Saka WA, Akhigbe RE, Ishola OS, Ashamu EA, Olayemi OT, Adeleke GE, (2011).
Hepatotherapeutic effect of Aloe vera in alcohol-induced hepatic damage.
Retrieved from (https://www.ncbi.nlm.nih.gov/pubmed/22308658)
15. Wadie I. Najm, (2012). An Overview on Nutraceuticals and Herbal Supplements for
Diabetes and Metabolic Syndrome. Retrieved from
(https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/bittergourd).
16. Yamada, Muneo (2012). A study about Ethanol-Induced Hepatic Lipid Accumulation by
Suppressing the Expression of Lipogenic Genes in Mice. Retrieved from
(https://tandfonline.com/doi/abs/10.1271/bbb.120393)
25
ACKNOWLEDGEMENT
The researchers wholeheartedly acknowledge all those who contributed in making this
study possible, most especially to the following:
The Lord Almighty God, the creator, the Life-giver, the ultimate source of strength and
wisdom. Honor and gracious thanks for His Divine providence and for providing the courage and
light to everything that they do in completion of this day.
Ms. Camille Rose M. Tacazon, Research Adviser of Grade 10 sections 3-4, for the
significant information rendered to the researchers, for this unfavorable understanding, assistance
and encouragement to finish this research.
Mr. Rogelio Escobar and Mr. Rafael Manalo for guiding, assisting, and helping us on
how to execute the testing of the product.
To the researchers’ parents and family, who gave unconditional love and financial
support to accomplish this study.
Friends, for their encouragement, moral support and prayer. And who wholeheartedly
extend their helping hands and given significant contribution in this research.
To Olongapo City National High School, for the quality of education that imparted to
the researchers, for the knowledge, wisdom and values that the researchers need in forming right
decision and precise solution to their problems.
26
APPENDICES
27
APPENDIX A – PHYTOCHEMICAL TEST RESULTS
28
APPENDIX B – PLANT IDENTIFICATION/CERTIFICATION
29
30
APPENDIX C – TRAVEL ORDER
31
APPENDIX D – CERTIFICATION FROM UP MANILA
32
INTEL ISEF FORMS