PROJECT PROPOSAL

ON

EFFICACY AND HYPERTOXICITY OF GUAVA

MISTLETOE ON CANCER

BY

SALIHU, HAJARAT OYIZA

FPN/SAS/2016/2017/HSLT/BC/191

SUPERVISED BY

MR. M. T. ALOYSIUS

SUBMITTED TO

THE DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY

SCHOOL OF APPLIED SCIENCE

THE FEDERAL POLYTECHNIC NASARAWA, NASARAWA STATE

APRIL, 2017

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 ABSTRACT
Various studies on mistletoe did not intensify toxicity. Toxicological evaluation of a
species of mistletoe growing on guava plant in Nigeria will be carry out with the
observation of rats hematological and serum biochemical changes for a period of thirty-
day administration. Acute toxicity and sub chronic toxicity tests will be carry out using
standard methods. Rats of average weight 150g will be use. In acute toxicity, rats will be
administer orally with methanolic extract of mistletoe using 10, 20, 30 and 50mg/kg
doses respectively, while observations will be made over a period of twenty four hours
to seven days. Serum biochemical parameters to analyze will be total protein, albumin,
urea, creatinine, aspartate transaminase, Alanine transaminase, Alanine phosphate,
triglyceride, glucose, billirubin, cholesterol, high density lipoprotein and low density
lipoprotein. Statistical analysis will be carry out on the results of these parameters to
check for their significance.

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INTRODUCTION

Chemotherapy kills tumor cells and thereby slows down the progression of cancer.
However, as you know, most conventional chemotherapy has significant toxicity leading
to severe side effects. In many countries, doctors question the efficacy of chemotherapy
for solid tumors of the colon, lung, pancreas and breast. It has been observe that for these
types of tumors, the usual chemotherapy regimens are much less effective than for blood
or lymph types (Alison, 2000). An alternative and nontoxic therapy now widely used in
Europe Iscador, a medicine made from the lacto-fermented extract of fresh sap of the
plant known as mistletoe (Viscum album). Mistletoe has been use in folk medicine for
hundreds of years, especially by the Druids. Its first use as a cancer medicine came from
the indications of Rudolf Steiner in the 1920s. Steiner pointed out the remarkable
similarity between the life and growth patterns of mistletoe and cancer. It proliferates in
an undifferentiated way, growing in a ball bigger and bigger, always with the same cell
and tissue types without any regard to its host. The Druids were aware by the fact that the
plant was so liberated from normal earthly rhythms that it flowered and fruited at
Christmas, the time of the winter solstice. They saw this as a sign that this plant was a gift
to mankind from the heavens (Bocci, 1999).

Mistletoe is a saprophytic plant meaning that it derives nourishment from its host just as
cancer derives its nourishment from the host, even eventually directing the blood supply
into feeding the tumors. Viscum preparations made from certain species of host trees are
therefore utilize to treat specific types of cancers. The most commonly used variants of
Iscador are Viscum mali from apple trees which is used for cancer in female patients,
Viscum quercus from the oak tree for cancers in men, and Viscum pini from the pine tree
which is given a mixed use, but is most famous for breast cancer (Bown, 1995).

The common route of administration is by injection of the Viscum just under the skin. As
each day of therapy progresses, a more concentrated version is administered. After
reaching the highest concentration of Iscador, the injections are often continued for a
week or longer, depending upon the clinical situation. The side effects of Iscador therapy

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can include low-grade fever, and redness and irritation at the injection sites (Deeni,
2002).

STATEMENT OF THE PROBLEM

Today, it is known that Cancer has the highest incidence (25.9 %) and mortality (17.1 %)
of all types of ailment. Cancer requires multimodal therapy with primary surgery
followed by adjuvant radio-, chemo- and/or hormonal therapy depending on individual
criteria relating to tumor status, lymph node disease, menopause and hormone receptor
status (adjuvant therapy) (Ofem et al., 2009). In view of the frequent side effects of
adjuvant therapy and the risk of a substantial impairment in quality of life,
complementary treatment to limit or prevent the symptoms associated with the disease or
treatment is becoming increasingly important. This is despite the fact that there is still no
convincing evidence of its effectiveness in reducing the progression of the disease and
prolonging survival. Of the complementary treatments used by cancer patients, European
mistletoe extracts (Viscum album L.) are the most common in Europe and Africa (Ofem
et al., 2009).

Cancer drugs are made from synthetic chemicals. Patient takes it for long period of time
and therefore become addicted to it and not all the drugs are used for all types of cancer
like leukemia, breast, brain (tumor) cancers. Most of the drugs use today in treating of
cancer causes many side effects and not all people can afford the drugs, and it was
observed that not all people have the knowledge and the information on how to use those
drugs.

For the purpose of this project work, which will be aimed at determining the efficacy of
the extracts (guava mistletoe) in treating/curing cancer without causing any side effect to
patients and also, to provide a new means through which people who can afford the
normal cancer drugs can get help using extracts in the control of cancer.

JUSTIFICATION OF THE PROBLEM

A considerable percentage of patients with cancer include treatments from

complementary and alternative medicine (CAM) in their therapeutic approach along with

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oncological therapies. In order to bridge the gap between traditional use and current
research evidence of mistletoe preparations, thought it was necessary to systematically
review efficacy and hyper-toxicity and safety of mistletoe on cancer, to give a short
overview on the possible mechanisms of action and to outline useful prerequisites for
future research while taking into account the patients perspective.

AIM OF THE STUDY

The aim of this study will be to carry out the phyto-chemical, efficacy and hyper-toxicity
screening of mistletoe from Guava plant hosts and also determine the hyper toxicity
activities of mistletoe from Guava plant hosts.

OBJECTIVES OF THE STUDY

The purpose of the study will be to:
1) Evaluate the therapeutic efficacy of long-term complementary therapy in cancer
patients with a standardized mistletoe extract.
2) To examine the hyper-toxicity of mistletoe from Guava plant hosts.
3) To examine the efficacy of mistletoe extract on cancer from Guava plant hosts

MATERIALS AND METHODLOGY

MATERIALS

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Materials to be use for this project work are as follows:
Plant extract (Guava mistletoe)
Weighing balance
Cage
Mortar and pestle
Syringe and needle
Cotton wool

EQUIPMENT
Equipment to be use for this project work are as follows:
Centrifuge
Spectrophotometer
Stirrer
Beakers/Funnel/Filter
REAGENTS
The reagents to be use are:
Arscenic (a cancer chemical)
Normal saline
Hydrochloric acid
Bismult

METHODOLOGY
PLANT MATERIAL AND PREPARATION OF PLANT EXTRACTS
Fresh mistletoe leaves will be obtained from a guava host plant in AMAC Local
Government Area of FCT, Abuja, Nigeria. The plants were identified by a nutritionist in
the Department of Science Laboratory Technology, School of Applied Science, Federal

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Polytechnic Nasarawa, Nasarawa State, Nigeria. The mistletoe leaves will be rinsed in
clean water to remove debris. They will be air dried at a temperature of 25 oC and below.
The dried leaves are be grounded to powder using mortar and pestle. 100g of the dry
sample were to be mix with 500ml distilled water and allowed to stand for 24 hours. The
mixture will then be filtered with Whatmans filter paper (size1). The filtrates were to be
dried at 45 using a laboratory oven. The pasty filtrate obtained after drying will be
weigh using an analytical electronic balance. The stock solutions of the extract are to be
prepared by dissolving 15g of extract in 10ml of distilled water to give a concentration of
1500mg/ml. The stock solution will be appropriately label and refrigerated at 4 until
required for use.

PHYTO-CHEMICAL SCREENING
Phyto-chemical screening of the leaf extracts will be carried out by employing standard
procedures and tests (Trease and Evans, 1989; Sofowora, 1993), to reveal the presence
of chemical constituents such as alkaloids, flavonoids, tannins, terpenes, saponins,
reducing sugars and cardiac glycosides among others.

ANIMAL PREPARATION AND PROTOCOL

Twenty-(20) albino wistar Rats weighing 180 to 220g will be randomly assign one of five
(5) groups, such that each group contained four (4) animals. The groups are to be labeled
as follows: Group 1 (Control, C1), Group 2 (Cancer untreated group), Group 3 (Cancer
group) and Group 4 (Cancer C2 group, treated with mistletoe leaf extract C2+M), and
Group 5 (Control group, treated with mistletoe leaf extract (C1+M).

INDUCTION OF CANCER
The animals will be fasted overnight and Cancer will be induce by a single intra-
peritoneal injection of Arsenic (As), (150 mg/kg body weight) in ice cold 0.9% NaCl
saline solution. Control Rats will be inject with normal saline alone. After 72 hours, to
allow for the development of Cancer, Rats with moderate Cancer will be consider and
used for the drug treatment. The leaf extracts of the plant from the hosts in aqueous
solution will be administer to Rats orally through a gavage method at a concentration of
100mg/kg body weight/Rat/day for 14 days.

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EXPERIMENTAL DESIGN
The animals will be treated as follows: Group I: Cancer Rats administered orally with
leaf extract of mistletoe from Guava at a dose of 100mg/kg/day in aqueous solution for
14 days. The Cancer of all the Rats will be record at regular intervals during the
experimental period. For the acute study, the Tumor will be monitor after 1, 3 and 7 hours
of administration of a single dose of the extract, and at the end of 1, 7 and 15 days for the
prolonged treatments. The Tumor will be monitored in the Rats by the tail tipping method
and the readings will be noted (WHO, 1980).

STATISTICAL ANALYSIS
Data will be reported as mean + standard error of the mean (SEM) and will be analyzed
statistically using One way ANOVA followed by Tukey-kramer multiple comparison test
and values and 0.05 will be considered significant.

EXPECTED RESULTS
The results of the phyto-chemical screening of the leaf extracts of V. album from guava
plant hosts showed differential compositions in terms of amount and type of secondary
metabolites present. Mistletoe contains alkaloids, tanins, catechols, saponins, terpenes,
flavonoids, and cardiac glycosides. Alkaloids, tannins, catechol, saponins, flavonoids,
terpenes and cardiac glycosides might likely be present in the leaf extract of V. album
from D. edulis. In the leaf extract of V. album from P. guajava, tannins, saponins,
flavonoids, anthraquinones, cardiac glycosides and terpenes might be found while
alkaloids and catechol, phlobatanins and cyanogenic glycosides might be absent.

EFFICACY TEST
As an anthroposophical medicine, mistletoe is one of the most important herbal drugs and
is potentially effective in cancer is recommended due to its minimal side effects and the
fact that these side effects are not life threatening. A few cases of allergic reactions should
be reported in the Rats.

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ACUTE TOXICITY TESTS
The mistletoe extract will be administer to the rats for acute toxicity tests. Different doses
ranging from 1000 to 5000 mg/kg will be administer to the rats that will be kept under the
same condition-using rat cannula after which observation will be made over a period of
24 hours to seven days. The diluents that will be used isolive oil as the extract is oily and
not easily miscible in water. The weight of extract in milligram per body of rat in
kilogram will be used (mg /kg body weight).

CONCLUSION
At the end of this research work, it would expect that the aqueous extract of leaf will be
compare with the standard drugs and the toxicity level will be safe and the extract will be
able to reduce the mortality a morbidity in cancer thereby be recommended as a cancer
drug in the control of tumor.

REFERENCES
Alison M., Yasser H. A., Abdel W., and Flatt P. R., (2000). The traditional plant

treatments of Sameucus nigra Chpt 130, pg: 15-20

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Bocci B., (1999), Mistletoe (Viscum album) lectins as cytokine inducers and immune

adjuvants in tumour therapy. Journal of Biology and Regulatory Homoeostatic

Agents. Pg 7: 1- 6

Bown D. (1995) The Royal Horticultural Society Encyclopedia of Herbs and Their

Uses. Dorling Kindersley Ltd: London.

Deeni Y Y., Sadiq N. M., (2002). Antimicrobial properties and phytochemical

constituents of leaves of African mistletoe (Tapinanthus dodoneifolius (DC)

Danser) (Loranthaceae): an ethnomedicinal plant of Hausaland, Northern Nigeria.

J. Ethnopharmacol., 83 (3): 235-240.

Harmsma M., Ummelen M., Dignef W., Tusenius K J., Ramaekers F C., (2006). Effects

of mistletoe (Viscum album L.) extract Iscador on cell cycle and survival of tumor

cells. Arzneimittel for schung, 56 (6A): 474-82.

Nwanjo HU. 2007. Free radicals scavenging potential of the aqueous extract of Viscum

album (Mistletoe) leaves in cancer Wistar rats hepatocytes. The Internet Journal

of Nutrition and Wellness, 3: 2.

Nwaegerue E, Nweke IN, Ezeala CC, Unekwe PC. 2007. Glucose lowering effect of leaf

extracts of Viscum album in normal and cancer rats. J. Res. Med. Sc., 12(5): 235-

240.

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Ofem OE, Eno AE, Nku CO, Antai AB. 2009. Viscum album (mistletoe) extract prevents

changes in levels of red blood cells, PCV, Hb, serum proteins and ESR in high

saltfed rats. J. Ethnopharmacol., 126(3): 421-426.

Ogutoye SO, Olatunji GA, Kolawole OM, Enonbun KI. 2008. Phytochemical screening

and antibacterial activity of Viscum album (mistletoe) extracts. Plant Sciences

Research, 1(3): 44 46.

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