CHAPTER I

INTRODUCTION

Background of the Study
In 2000, according to the World Health Organization, at least 171 million people
worldwide suffer from diabetes, or 2.8% of the population. Its incidence is increasing
rapidly, and it is estimated that by 2030, this number will almost double. Diabetes is
occurring worldwide, however concentrated on Asian and African countries, especially the
developing countries like Philippines.
With the rapid increase of diabetes, Filipinos cannot evade this disorder because most
of us are used to Western-style diets present in American-based fast-food chains and cuisine.
Many diabetic people fall under the category of Class C to D. They cannot afford the
expensive medical treatments and either resort to die or to have the folkloric medicines.
Folkloric medicines are naturally occurring medicines, mostly herbal in nature. Most
of them are foliage or parts of certain plants with almost relatively bitter taste. During the
current century, most people resort to folkloric medicine for it is organic in nature and
cheaper than most commercial drugs.
One such example of these medicines is star apple. Star Apple or Caimito (varies
among places) is a tree characterized with a green upper leaf and golden lower leaf. Star
apple is considered as a traditional form of medicine or folkloric medicine. Different parts
are used for different diseases. The bark is used as antitussive. Infusion of the leaves is long-
2

time used against diabetes and articular rheumatism. The fruit is said to have antioxidant
properties.
Another example is soursop. Soursop is a medicinal tree, recently being identified as
a potent anti-cancer drug. Soursop is mainly used against high blood pressure, acts as a
sedative and as a stimulant of the central nervous system to treat nervous tension, and as an
antibiotic, antiviral and antibacterial agent.
Statement of the Problem
The project aimed to identify the phytochemicals present on the leaves extract as well
as identify its effect on the blood glucose of laboratory mice. Specifically, this research
sought to answer the following questions:
1.What are the phytochemicals present on the leaf extracts of Star Apple and
Soursop?
2. What is the effect of the different treatments in the blood glucose level of
laboratory mice?
3. Are there significant differences between and among the treatment means?
Significance of the Study
This study identified the phytochemicals present on the leaves of star apple and
soursop so that it will be a basis if these folkloric medicines are safe enough to use or they
are useless to use as treatment to diabetes.
3

Aside from the identification of its safety, the phytochemical analysis showed us the
chemicals present on the leaves that can alter the blood chemicals that can lead to the
metabolism or deposition of glucose.
If this study showed the expected result (hypoglycemial condition),it can help the
medical industry in the Philippines for many medical laboratories will start to explore other
folkloric medicines and test their effectivity.
Scope and Delimitation of the Study
The study was conducted to laboratory mice. All procedures must be controlled
wherein application of treatments was done in the morning and in the afternoon, fasting
hours was six hours and 1mL of treatments used for the weight bracket of the mice) to cope
up with their small size. Whatever the results will be is not totally applicable to humans since
we differ a lot from the laboratory mice. Also, statistical tests such as ANOVA cannot be
conducted due to the small number of replicates.
Definition of Terms
Acclimatization It is the process allowing the experimental units (mice) to adapt to the
new environmental condition of the locale of the study.
Blood Glucose Level (BLG) It is the level of glucose present on the blood of the test
subjects.
Blood Chemical Analysis It is a procedure or part of the experiment where the blood of
the mouse is analyzed specially for the level of the glucose present.
4

Crude Extract It is the solution that was obtained after soaking the leaves of C. cainito
and A. muricata for about 48 hours.
Dose It is the amount of extract (1mL of treatment: 30g lab mouse) that were orally taken
by the lab mice. It was proportionate according to the body mass of the experimental units.
Extraction It is a process where the powdered leaves are soaked in ethanol and other
related solvents.
Phytochemical These are organic chemicals found in plants that can affect the homeostasis
of an animal.
Phytochemical Analysis It is a series of process and chemical reactions used to test the
presence of a certain phytochemical on the samples prepared.

5

CHAPTER II
REVIEW OF LITERATURE

Chrysophyllum cainito

Fig. 1. Star Apple and fruits
Chrysophyllum is a genus of about 70-80 species of tropical trees, growing rapidly to
1020 m or more in height. The genus is native to tropical regions throughout the world, with
the greatest number of species in northern South America. One species, C. oliviforme,
extends north to southern Florida (Wikipedia, 2011).
The leaves are oval, 315 cm long, green above, densely golden pubescent below,
from which the genus is named. The flowers are small (38 mm), purplish white and have a
sweet fragrant smell; they are clustered several together, and are hermaphroditic (self fertile).
The fruit is edible; round, usually purple skinned (sometimes greenish-white), often green
6

around the calyx, with a star pattern in the pulp; the flattened seeds are light brown and hard.
The fruit skin is chewy like gum, and contrary to some reports, is edible (Wikipedia, 2011).
Star apple is originally used as an ornamental plant. Many of the people that include
star apple in their gardens do not recognize the healing wonders of star apple tree.
According to traditional medicine of the Filipinos, star apple leaves are infused in a tea
solution and make the patients drink it. However, this practice is not yet proven
(Stuarexchange.org).
A research in Kenya studies the components of a relative of C. cainito, C. albidium.
The researchers investigated the effect of oral administration of the leaf extract of
Chrysophyllum albidium G. on biochemical and hematological parameters in albino rats for
16 days. The researchers write: In conclusion, the study has demonstrated that the leaf
extract of Chrysophyllum albidium may not cause any adverse effect on the biochemical and
hematological indices of toxicity. Moreover, the extract was found to possess anti-platelet
and hypoglycemic (lowers blood sugar) properties and might be employed in the
management of myocardial infarction (heart attack) and diabetes mellitus, respectively.
Further investigation is needed to establish the anti-platelet property of the extract.
(come2health.blogspot.com)




7

Annona muricata

Fig. 2. Soursop and fruit
Soursop or soursop in English is a small tree about 5 to 7 meters in height. The
leaves are alternate, oval in shape, pointed at both ends, smooth and shinning, 7 to 20
centimeters long and with petioles about 5 millimeters long. The flowers are large, yellowish
or greenish yellow and solitary. There are six large, freshy or leathery petals in two series.
They are heart-shaped, with pointed tip, and up to 5 centimeters in length and 3centimeters in
breadth. In the center of the flower is a cone-shaped mass of many carpels which will form
the fruit, and below this are very numerous stamens (Department of Agriculture,
Philippines).
Several studies have described the purposes of Annona muricata and have outlined
the social history of the plants use (Ayemuricata (Linn.) (Family, Annonaceae) called
Soursop is a small, upright evergreen tree growing 5 to 6 meters in height. Young
branchlets are rusty-hairy, the malodorous leaves, norm evergreen, are alternate, smooth,
glossy on the upper surface, lighter beneath; oblong, ell or narrow-obovate, pointed at both
ends, 6 - long and 2 6 cm wide. The flowers are borne singly, may emerge anywhere on the
8

trunk, branches or twigs. They are short stalked, 4 - 5 cm plump, and triangular-conical; the 3
fleshy spreading, outer petals yellow-green, with 3 close set inner petals pale-yellow (de Feo,
1992; 1990).
Alcoholic extract of soursop leaves yielded as essential oil. The extract also yielded a
dark-green resin containing: myricyl alcohol, sitosterol, fatty acids (oleic, linoleic, and
stearic) and anonol. Flesh of fruit contains saccharose 2.53%, dextrose 5.05 %, and levulose
0.04%. Bark contained an alkaloid found to be poisonous. Seed contains a non-poisonous
alkaloid. Recent studies isolated three acetogenins: annonacin, annonacin A and annomuricin
A (Stuartxchange.org).
Phytochemicals
Phytochemicals are non-nutritive plant chemicals that have protective or disease
preventive properties. They are nonessential nutrients, meaning that they are not required by
the human body for sustaining life. It is well-known that plant produce these chemicals to
protect themselves but recent research demonstrate that they can also protect humans against
diseases. Most phytochemicals are classified under tannins, flavonoids, saponins,
anthraquinones, reducing sugars, alkaloids, terpenoids, and the rest are classified under other
minor phytochemicals (phytochemicals.info, retrieved on March 8, 2013).
9


Fig. 3. An example of tannin
Tannins are organic compounds mostly produced by the phenolic metabolism of a
plant. According to Bate-Smith and Swain(1962), tannins are water soluble phenolic
compounds having molecular weight between 500 to 3000 giving the usial phenolic
reactions and possesses special properties such as the ability to precipitate alkaloids, gelatin
and other proteins.
Tannins have diverse effects on biological systems because they are potential metal
ion chelators, protein precipitating agents, and biological antioxidants. Because tannins can
play such varied biological roles, and because of the enormous structural variation among
tannins, it has been difficult to develop models which allow accurate prediction of the effects
of tannins in any system. An important goal of the future work on the biological activities of
tannins is the development of structure/activity relationships so that biological activites can
be predicted (Hagerman 2002).


10


Fig. 4. An example of saponin
Saponins, group of naturally occurring oily glycosides that foam freely when shaken
with water. They occur in a wide variety of plants, including acacia, soapwort, soaproot,
California pigweed, and many others. Saponins have been, and sometimes still are, used as
cleaning agents and as foam producers, notably in fire-extinguishing fluids. They have a
bitter taste and when ingested orally are practically non-poisonous to warm-blooded animals.
When injected directly into the bloodstream, however, they are dangerous and quickly
dissolve red blood cells. Hydrolysis of saponins, brought about by acids or by enzymes, gives
a sugar (often, but not necessarily, glucose) and a sapogenin, the latter being either a
triterpene or a steroid. Some of the sugars and saponins are useful as raw materials for
synthesis of steroid hormones (Microsoft Encarta 2009).
Saponins have many health benefits. Studies have illustrated the beneficial effects on
blood cholesterol levels, cancer, bone health and stimulation of the immune system. Most
scientific studies investigate the effect of saponins from specific plant sources and the results
cannot be applied to other saponins (www.phytochemicals.info, retrieved on March 8, 2013).
Saponins are complex compounds that are composed of a saccharide attached to a
steroid or triterpene. Saponins are found in a number of plants. In the animal kingdom
11

saponins are found in most sea cucumbers and starfish. Saponins have hemolytic,
expectorative, anti-inflammatory and immune-stimulating activity. Beyond that, saponins
demonstrate antimicrobial properties particularly against fungi and additionally against
bacteria and protozoa (www.raysahelian.com/saponin.html, retrieved on March 17, 2013).

Fig. 5. Examples of alkaloids
Alkaloids, group of mildly alkaline compounds, mostly of plant origin and of
moderate molecular complexity. Even in very small amounts, the alkaloids produce strong
physiological effects on the body. All contain nitrogen atoms that are structurally related to
those of ammonia (Microsoft Encarta 2009).
Nearly 3000 alkaloids have been recorded; the first to be prepared synthetically
(1886) was one of the simplest, called coniine, or 2-propyl piperidine, C
5
H
10
NC
3
H
7
. It is
highly poisonous; less than 0.2 g (0.007 oz.) is fatal. Coniine, obtained from seeds of the
hemlock, was the poison used in the execution of Socrates. Some 30 of the known alkaloids
are used in medicine. For example, atropine, obtained from belladonna, causes dilation of the
pupils; morphine is a painkiller; quinine is a specific remedy for malaria; nicotine is a potent
insecticide; and reserpine is a valuable tranquilizer (Microsoft Encarta 2009).
12

Some alkaloids, such as nicotine, are used in pesticides, and others are used as
chemical reagents. The primary use of alkaloids, however, is in medicine, because they can
act quickly on specific areas of the nervous system. Alkaloids are the active components of
many anesthetics, sedatives, stimulants, relaxants, and tranquilizers. They are taken by mouth
and administered by injection. Except under a physician's supervision, use of alkaloids is
dangerous, because most are habit-forming (for example, almost all narcotics are alkaloids)
and large doses can be poisonous.
Strychnine, used in small doses as a stimulant and a tonic, is highly poisonous.
Quinine, used in treating malaria, can cause dizziness if taken in large doses. Morphine and
cocaine are among the most effective drugs known for temporarily relieving pain without
causing loss of consciousness. However, these two alkaloids are habit-forming and can be
harmful if their use is continued. Curare, used as a muscle-relaxing drug and in arrow
poisons used by South American Indians, is a mixture of various alkaloids
(science.howstuffworks.com/alkaloid-info.htm, retrieved on March 17, 2013).

13


Fig. 6. An example of flavonoid
Flavonoids are water soluble polyphenolic molecules containing 15 carbon atoms.
Flavonoids belong to the polyphenol family. Flavanoids can be visualized as two benzene
rings which are joined together with a short three carbon chain. One of the carbons of the
short chain is always connected to a carbon of one of the benzene rings, either directly or
through an oxygen bridge, thereby forming a third middle ring, which can be five or six-
membered. The flavonoids consist of 6 major subgroups: chalcone, flavone, flavonol,
flavanone, anthocyanins and isoflavonoids. Together with carotenes, flavanoids are also
responsible for the coloring of fruits, vegetables and herbs (www.phytochemicals.info,
March 2013).
Flavonoids are polyphenolic compounds that are ubiquitous in nature and are
categorized, according to chemical structure, into flavonols, flavones, flavanones,
isoflavones, catechins, anthocyanidins and chalcones. Over 4,000 flavonoids have been
14

identified, many of which occur in fruits, vegetables and beverages (tea, coffee, beer, wine
and fruit drinks). The flavonoids have aroused considerable interest recently because of their
potential beneficial effects on human health-they have been reported to have antiviral, anti-
allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities.
Antioxidants are compounds that protect cells against the damaging effects of reactive
oxygen species, such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and
peroxynitrite. An imbalance between antioxidants and reactive oxygen species results in
oxidative stress, leading to cellular damage. Oxidative stress has been linked to cancer,
aging, atherosclerosis, ischemic injury, inflammation and neurodegenerative diseases
(Parkinson's and Alzheimer's). Flavonoids may help provide protection against these diseases
by contributing, along with antioxidant vitamins and enzymes, to the total antioxidant
defense system of the human body. Epidemiological studies have shown that flavonoid
intake is inversely related to mortality from coronary heart disease and to the incidence of
heart attacks.
The recognized dietary antioxidants are vitamin C, vitamin E, selenium, and carotenoids.
However, recent studies have demonstrated that flavonoids found in fruits and vegetables
may also act as antioxidants. Like alpha-tocopherol (vitamin E), flavonoids contain chemical
structural elements that may be responsible for their antioxidant activities. A recent study by
Dr. van Acker and his colleagues in the Netherlands suggests that flavonoids can replace
vitamin E as chain-breaking anti- oxidants in liver microsomal membranes. The contribution
of flavonoids to the antioxidant defense system may be substantial considering that the total
daily intake of flavonoids can range from 50 to 800 mg. This intake is high compared to the
15

average daily intake of other dietary antioxidants like vitamin C (70 mg), vitamin E (7-10
mg) or carotenoids (2-3 mg). Flavonoid intake depends upon the consumption of fruits,
vegetables, and certain beverages, such as red wine, tea, and beer. The high consumption of
tea and wine may be most influential on total flavonoid intake in certain groups of people.
The oxidation of low-density lipoprotein (LDL) has been recognized to play an important
role in atherosclerosis. Immune system cells called macrophages recognize and engulf
oxidized LDL, a process that leads to the formation of atherosclerotic plaques in the arterial
wall. LDL oxidation can be induced by macrophages and can also be catalyzed by metal ions
like copper. Several studies have shown that certain flavonoids can protect LDL from being
oxidized by these two mechanisms (Buhler and Miranda, 2013).

Fig. 7. An example of anthraquinone
Anthraquinone has been shown in certain studies to help aid in digestion as a laxative,
to reduce inflammation in arthritis patients, and to inhibit the growth of cancer cells. There is
more than one kind of anthraquinone, and those used for medical treatment are often found
16

naturally in plants such as aloe vera. Despite promising results in some situations, patients
should be aware that this compound does carry the risk of side effects and certain
complications.
One of the most common medicinal uses for anthraquinone is as a laxative. It has been
shown effective at speeding up bowel movements in those with constipation. Many times it is
not recommended, however, because it can lead to side effects. One troubling issue is a
discoloration of the intestines. This is not dangerous, but it can be upsetting for some
patients, and confusing for doctors.
Another way in which anthraquinone is used is in the treatment of arthritis. Usually, this
treatment isn't performed using the herb alone, but in the form of aloe vera supplements.
Certain forms of anthraquinone are found naturally in aloe, and it is suspected that it, in
combination with other compounds, could help reduce inflammation and pain associated with
some types of arthritis.
Cancer is another ailment which may be fought with certain plants containing forms of
anthraquinone. One common example is rhubarb, as this is one of the most widely tested in
terms of effectiveness. There are three separate anthraquinones found in rhubarb which could
help kill cancer cells and prevent the growth of new ones. More studies are needed to
determine how effective this treatment could be, but those done so far have been promising.
The fact that some anthraquinones have been shown to prevent or even treat cancer is not the
only connection between these compounds and cancerous cells. Some other studies have
shown that certain anthraquinone forms might have a carcinogenic effect on the body. This
17

may be more likely in those produced synthetically, such as those found in certain dyes and
other commercial products. These are typically different in molecular makeup than ones
found naturally in certain plant species.
Although natural, patients should not attempt to use herbal supplements without first
consulting a doctor. Not only can they have side effects in some patients, but they can cause
interactions with other drugs. It is also important to establish a clear diagnosis before
attempting any kind of treatment (www.wisegeek.com/what-are-the-medicinal-uses-of-
anthraquinone.htm, retrieved March 23, 2013).

Fig. 8. An example of coumarin
Coumarin is a chemical compound which is found naturally in some plants, although
it can be synthetically produced as well. It has a distinctive odor which has led people to use
it as a food additive and ingredient in perfume. Due to concerns about coumarin as a
potential liver and kidney toxin, its use as a food additive is heavily restricted, although it is
perfectly safe to eat foods which naturally contain the compound.
The chemical name for coumarin is benzopyrone. The distinctive sweet odor reminds
many people of freshly cut grass or hay, and it has been used in perfumes since the late
1800s. In a pure form, this compound has a crystalline structure, and it is said to taste faintly
18

like vanilla. When ingested, it acts as a blood thinner, and it also appears to be effective in
treating some tumors. Coumarin has fungicidal properties as well. However, other much
safer substances can be used for all of these purposes, although the compound is sometimes
used in combination with other blood thinners for medical treatment.
One natural source of coumarin is tonka beans, tropical beans which are known by
the French as coumarou. To release their captive coumarin, the beans are soaked in alcohol
and then fermented. The substance also occurs in sweet clover, strawberries, cherries, bison
grass, woodruff, and apricots. Coumarin has traditionally been used as a vanilla substitute in
various foods, especially tobacco, although this usage is restricted in some countries
(www.wisegeek.org/what-is-coumarin.htm, retrieved on March 17, 2013).

Related Studies
1. Effect of aqueous extract of Chrysophyllum cainito leaves on the glycaemia of
diabetic rabbits
N'guessan Koffi1, Amoikon Kouakou Ernest, Tibr Marie-Solange1, Kadja Beugr and Zirihi Gud
Nol1
Abstract
Chrysophyllum cainito is a plant recognized by the traditional healers of Aboud-Mandk, a
village in the Department of Agboville (Cte-dIvoire), as having antidiabetic properties. The
aim of this study is to evaluate, experimentally, the effect of an aqueous decoction of the
plants leaves that we called C. cainito, on rabbits induced with alloxane, a diabetogenic
product. Different graded doses of this herbal medicine were applied on postprandial blood
sugar levels of diabetic rabbits. At doses of 10 g/l, C. cainito does not induce the
19

hypoglycaemic effect. A dose of 20 g/l reduced the hyperglycaemia from 5 g/l to 1.4 g/l. A
dose of 30 g/l of C. cainito produced a graded decrease in hyperglycaemia from 6.3 g/l to 3.2
g/l. After 6 weeks of treatment, the induced diabetic rabbits stopped eating and succumbed
between the 8
th
and the 9
th
weeks of experimentation. It was thus concluded that C. cainito
leaves have glucose lowering effect at doses > 10 g/l and appears toxic and lethal at 30 g/l. C.
cainito produces its hypoglycaemic effect mainly through alkaloids, sterols or triterpens, the
antidiabetic active constituents.

2. Morphological Changes and Hypoglycemic Effects of Annona Muricata Linn.
(Annonaceae) Leaf Aqueous Extract on Pancreatic -Cells of Streptozotocin-Treated
Diabetic Rats
Stephen O. Adewole1* and Ezekiel A. Caxton-Martins2
Abstract
The present study was undertaken to investigate the leaf aqueous extract effects of Annona
muricata Linn. on the morphology of pancreatic -cells and oxidative stress induced by
streptozotocin (STZ)-diabetic rats. Diabetes mellitus was induced in the diabetic animal
groups B and C by intraperitoneal injections of STZ (75 mg/kg body weight), while the
control group received equal volume of citrate buffer (pH 6.3) solution intraperitoneally. The
rats in group C were given A. muricata leaf aqueous extract (AME, 100 mg/kg, p.o.) as from
day 5 post STZ injections, and stopped on the 30th day of the study period. The pancreases
of the rats were excised and randomly processed for histological staining and biochemical
assays for antioxidant enzymes [such as glutathione (GSH), superoxide dismutase (SOD),
glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA) and serum nitric
20

oxide (NO)]. In diabetic state, pancreatic -cells of STZ-treated group B rats histologically
demonstrated marked alterations in the micro-anatomy and cellular integrities. The
morphology of A. muricata-treated rats pancreases showed viable cellularity with distinct -
cell mass. STZ treatment significantly decreased GSH-Px, SOD, GSH, CAT and
pancreatic/serum insulin levels (p<0.05). However, STZ treatment increased blood glucose
concentrations, MDA, and NO. A. muricatatreated rats showed a significant decrease
(p<0.05) in elevated blood glucose, MDA and NO. Furthermore, A. muricata treatment
significantly increased (p<0.05) antioxidant enzymes activities, as well as pancreatic/serum
insulin contents. In conclusion, the findings of the present study indicate that A. muricata
treatment has beneficial effects on pancreatic tissues subjected to STZ-induced oxidative
stress by directly quenching lipid peroxides and indirectly enhancing production of
endogenous antioxidants. Annona muricata protected and preserved pancreatic -cell
integrity.


21

CHAPTER III
METHODOLOGY

Process Flowchart


Procurementof
Commercial
Antidiabetics
Procurementof
LaboratoryMice
AcquisitionofStar
AppleandSoursop
Leaves
Preparationof
Treatments
StatisticalAnalysis
Acclimatizationof
LaboratoryMice
Applicationof
Treatments
BloodChemical
Analysis
Phytochemical
Analysis
22

Acquisition and Preparation of Leaves
The leaves of soursop and star apple were acquired on the month of March from local
trees found in San Ildefonso, Ilocos Sur. The leaves were sun-dried for three days and air-
dried for one week.
Preparation of Test Samples
The leaves were dissolved on ethanol with the proportion of 1 gram is to 10 mL. The
extract was stored in a cool and dark environment for seven days. The extract obtained was
filtered and put to a water bath until the ethanol was removed.
Phytochemical Analysis
The prepared dried extracts were analyzedphytochemically using the appropriate
protocol.
Test for Tannins
The presence of tannins was tested using the Ferric Chloride Test. One milligram (1
mg) of the dried extract/ one milliliter (1 mL) of crude extract was put on a clean test tube. It
was dissolved in 1.5 mL of distilled water. Three drops of dilute ferric chloride (FeCl
3
) were
added. A dark blue color indicates the presence of Gallic tannins and a dark green color
indicates the presence of catechol tannins.
Test for Anthraquinones
The presence of anthraquinones was tested using 25% ammonia. 1 mg of the dried
extract/ 1 mL of crude extract was put on a clean test tube. 2 mL of 25% ammonia (NH
3
) was
added and the solution was shaken. A cherish-red color indicates the presence of
anthraquinones.

23

Test for Saponins
The presence of saponins was tested. 1 mg of the dried extract/ 1 mL of crude extract
was put on a clean test tube. 3 drops of dimethylsulfoxide was added together with 5 mL
distilled water. The solution was shaken and the presence of a persistent foam for about 15
minutes indicates the presence of saponins.
Test for Alkaloids
The presence of alkaloids was test using Meyers test for alkaloids. 1 mg of the dried
extract/ 1 mL of crude extract was put on a clean test tube. 6 drops of 2% hydrochloric acid
(HCl) was added to dissolve the extract. The solution was then divided into 2 aliquots. To the
first aliquot, 2 mL of distilled water was added and will serve as reference. To the second
aliquot, 2 drops of Mayers reagent was added. A yellowish-white precipitate formed on the
second aliquot indicates the presence of alkaloids.
Test for Reducing Sugars
The presence of reducing sugars was tested using Benedicts test for reducing sugar. 1
mg of the dried extract/ 1 mL of crude extract was put on a clean test tube. 5 mL of
Benedicts solution was added. The solution was continually shaken for five (5) minutes on a
hot water bath. A reddish-orange color indicates the presence of reducing sugars.
Test for Coumarins
The presence of coumarins was tested. 1 mg of the dried extract/ 1 mL of crude
extract was put on a clean test tube. It was then dissolved using 2 mL distilled water and
divided into two aliquots. To the first proportion, 0.5 mL of 10% NH
3
was added and shaken.
The second proportion was regarded as reference. The two proportions was then exposed
under UV light. Intense fluorescence under UV light indicates the presence of coumarin.
24

Preparation of Treatments
The leaves were dissolved on 70% ethanol with the proportion of 1 gram is to 10 mL.
The extract was stored in a cool and dark environment for seven days. The extract obtained
was filtered and put to a water bath until the ethanol was removed.
The dried extracts will be dissolved in distilled water with appropriate proportions to
create the treatments.
Inducement of High Blood Glucose Level
A solution of about three (3) molars of sucrose was prepared using 50 mL of distilled
water and 15 pieces of X.O. Butter Caramel Flavor. The pieces of candy were pulverized
using a clean mortar and pestle. The powdered and small crystals of sugar were added to the
distilled water. The solution was heated so that the sugar was dissolved completely.
Application of Treatments
The treatments were applied orally via syringe every morning to each mouse. The
dosage was 1mL so that they will not suffer from tremendous volume of each dose. The
treatments was applied every morning, and afternoon.
Blood Chemical Analysis
The mice fasted for six hours, as a protocol before having the blood chemical
analysis. Each mouse was pricked on the hind foot using a lancelet. The blood was then
analyzed using a glucometer. The results were obtained and analyzed.



25

Statistical Analysis
The data acquired were subjected to Kruskal-Wallis test in order to determine the
p(same) and to determine if there is a significant difference between and among the treatment
means. Kruskal-Wallis test was used due to the small number of replicates per treatment.












26

CHAPTER IV
RESULTS AND DISCUSSIONS

Phytochemical Analysis
The phytochemical analysis of the dried extracts resulted to the presence of tannins
and reducing sugars.

Table 1. Phytochemical Analysis Results

Phytochemical C. cainito A. muricata
Gallic tannin + +
Catechol tannin + +
Anthraquinone - -
Saponin - -
Alkaloid - -
Coumarin - -
Reducing Sugar + +


The presence of the reducing sugar (e.g. glucose) indicates the oxidation process that
is happening in the organism. The presence of reducing sugars is quite expected because the
source of the extracts was leaves of star apple and soursop.
The presence of the gallic tannin indicates the antioxidant properties of the leaves
extracts. Basing on the metabolic activity of gallic tannins to organism, it acts not only as
antioxidant but can also act against diabetes therefore proves the efficacy of the leaves.
The presence of catechol tannin indicates the antioxidant properties of the leaves
extracts. Basing on the metabolic activities of catechol, it is being utilized by the body to
produce catecholamines, which are released by the sympathetic nervous system in times of
distress, which triggers the release of insulin.
27

With the presence of the gallic and catechol tannins indicates the activity of the leaves
extract against the high blood glucose levels which can therefore treat diabetes.
Blood Glucose Level




Fig. 9. Average Blood Glucose Levels of Lab Mice in mg/dL as affected by Star Apple
The figure shows the graph of the blood glucose level of mice affected by Star Apple
extracts. The peak point of the graph (about 125 mg/dl) shows that the induction of the blood
glucose was successful. After that point, the blood glucose level lowered, seeing the effect of
the star apple extracts on the blood glucose level of laboratory mice.
60
70
80
90
100
110
120
130
B
l
o
o
d

G
l
u
c
o
s
e

L
e
v
e
l

i
n

m
g
/
d
L

Date of Blood Test
Treatment 1
Treatment 2
Treatment 3
Treatment 0-
Treatment 0+
28


Fig. 10. Average Blood Glucose Levels of Lab Mice in mg/dl as affected by Soursop
The figure shows the graph of the blood glucoselevel of mice affected by
Soursop extracts. The peak point of the graph (about 125 mg/dl) shows that the induction of
the blood glucose was successful. After that point, the blood glucose level lowered, seeing
the effect of the soursop extracts on the blood glucose level of laboratory mice.
Discussions
The different graphs show a single result wherein the blood glucose level of
laboratory mice lowered after the treatments were applied. It is evident that the
phytochemicals present on the leaves acted in the blood of the mice, therefore rendering its
efficacy against diabetes.
60
70
80
90
100
110
120
130
B
l
o
o
d

G
l
u
c
o
s
e

L
e
v
e
l

i
n

m
g
/
d
L

Date of Blood Test
Treatment 4
Treatment 5
Treatment 6
Treatment 0-
Treatment 0+
29

Even though with the presence of reducing sugars, the extracts acted and lowered the
blood glucose of the mice, therefore it is safe to assume that the tannins have higher
concentration when compared to reducing sugars.


30

CHAPTER V
SUMMARY CONCLUSION AND RECOMMENDATIONS

Summary
After the phytochemical analysis which resulted to the presence of catechol and gallic
tannins as well as reducing sugar, the research continued to find effects on the blood glucose
level of laboratory mice.
The experimentation included the acclimatization period wherein the mice were being
trained into the environment and procedures, induction period wherein their blood glucose
levels were altered to higher levels, and application period wherein the treatments were
applied to the experimental units.
After the application period, the blood glucose went back to normal, indicating the
treatments effect against diabetes.
Conclusion
1. The phytochemicals present on the leaves extract of star apple are gallic and
catechol tannin and reducing sugars. The phytochemicals present on the
leaves extract of soursop are gallic and catechol tannin and reducing sugars.
2. All experimental treatments reduced the blood glucose level of the laboratory
mice back to normal.
3. According to Kruskal-Wallis test, there is significant difference between and
among the treatments, although the difference does not mean that the
31

treatments dont have the effect on the blood, rather their effect is different
from one another.

Recommendation
The researchers would like to recommend other methods of extraction to efficiently
extract more phytochemicals that could act against diabetes. This may include distillation
process, decoction and more processes other than maceration.
Also the researchers would like to recommend further studies on the effect of the
leaves extract, specifically on a longer span of time, to see its efficacy, side-effects and other
therapeutic claims. Also, the researchers would like to recommend further studies of the
effect of the extracts on other animals.


32

Bibliography

Journals
E., H. A. (2002). Tannin Chemistry.
Murphy S., X. J. (2012, January 11). Deaths : Preliminary Data for 2010. United States of
America.
Webpages
Anonymous. (n.d.). Retrieved March 8, 2013, from phytochemicals.info:
http://www.phytochemicals.info
Anonymous. (n.d.). "HowStuffWorks" Alkaloids. Retrieved March 17, 2013, from
"HowStuffWorks": http://science.howstuffworks.com/alkaloid-info.htm
Anonymous. (2011, January). Retrieved February 15, 2012, from
come2health.blogspot.com/2011/01/researchers-employ-star-apple-to-treat.html
Anonymous. (n.d.). Caimito - Chrysophyllum cainito. Retrieved October 7, 2012, from In
Rio Dulce: http://www.inriodulce.com/links/Caimito.html
Anonymous. (n.d.). Diabetes mellitus. Retrieved December 2011, from Wikipedia:
http://en.wikipedia.org/wiki/Diabetes_mellitus
Anonymous. (n.d.). GRAVIOLA (Anona muricata). Retrieved October 7, 2012, from Whole
World Botanicals: http://www.wholeworldbotanicals.com/herbal_graviola
Anonymous. (n.d.). Terpenes, terpenoids. Retrieved March 17, 2013, from
www.cyberlipid.org: http://www.cyberlipid.org/simple/simp0004.htm
Anonymous. (n.d.). Terpenoids. Retrieved March 12, 2012, from Wikipedia:
http://en.wikipedia.org/wiki/Terpenoids
Anonymous. (n.d.). What is Coumarin? Retrieved March 17, 2013, from wiseGEEK:
http://www.wisegeek.org/what-is-coumarin.htm
Chua, D. P. (2008, July 28). Diabetes warning, INQUIRER.net Philippine News for
Filipinos. Retrieved October 20, 2012, from Global Nation Inquirer:
http://globalnation.inquirer.net/cebudailynews/opinion/view/20080728-
151202/Diabetes-warning
33

Ray Sahelian, M. (n.d.). Saponins in plants health benefits. Retrieved March 17, 2013, from
www.raysahelian.com: http://www.raysahelian.com/saponin.html


34

APPENDIX A
PLATES


Plate 1. Preparation of Treatments

Plate2. Phytochemical Analysis
35


Plate 3. Procurement of Commercial Antidiabetics
36


Plate 4. Procurement and Acclimatization of Lab Mice

Plate 5. Application of Prepared Treatments

37

APPENDIX B
STATISTICAL TOOLS

Kruskal-Wallis Test

H: 35.27
H
c
: 35.36
p(same): 2.877 x 10
-9


*significantly different
There is a significant difference among the treatments efficacy on the blood glucose levels
38

APPENDIX C
RAW DATA

Table 2. Average Blood Glucose Levels of All Treatments in mg/dL

Day T
1 T
2
T
3
T
4
T
5
T
6
T
0-
T
0+

Oct-7-12 69.33 67.67 70 82.33 72.67 79.33 60.33 74.67
Oct 10-12 84.67 89.33 87.33 74.67 70 82.33 84.33 71.67
Oct-13-12 76 93 83.33 83.33 96.67 97 66.67 79.33
Oct-16-12 96.33 76.33 67.33 75.33 65.67 67.33 85.67 99
Oct-19-12 122 126.33 112.33 85.67 93 113.67 123.33 126
Oct-22-12 71.67 80.67 70 73.67 75.67 90.33 67 74
Oct-25-12 80.33 81.67 75 78 80.33 87.67 74.33 74.67



39

CURRICULUM VITAE

NAME: Clarence Ave Martin L. de Guzman
NICKNAME: Marty
BIRTHDAY: May 31, 1997 (15)
ADDRESS: A. Luna St., Central East,
Bauang, La Union
PRIMARY SCHOOLING: BHC
Educational Institution Inc., 2003-2009
ACHIEVEMENT IN HIGH SCHOOL:
- Best in Microbiology, 2012
- Best in Social Science IV, 2013
- Student Alliance President, S.Y. 2012-2013
- Student Alliance Vice President, S.Y. 2011-2012
- SMT Representative, S.Y. 2010-2011
PRINCIPLE IN LIFE: Make the most out of every opportunity given
AMBITION: To become a Medical Doctor
MESSAGE: Work hard. Finish early. Dont be lazy.

40



NAME: Gwynne Micah S. Ritua
NICKNAME: Gwynne
BIRTHDAY: July 24, 1997 (15)
ADDRESS: Bobonot, Dasol, Pangasinan
PRIMARY SCHOOLING: Shekinah
Grace School, 2003-2009
ACHIEVEMENT IN HIGH SCHOOL:
- Best in Earth Science, 2010
- Best in Biology III, 2013
- Best in Developmental Biology, 2013
- Directors List 2
nd
Quarter S.Y. 2009-2010
- Directors List 2
nd
and 3
rd
Quarter S.Y. 2011-2012
- Directors List 3
rd
and 4
th
Quarter S.Y. 2012-2013
- IRC Debate League Vice Chairperson
PRINCIPLE IN LIFE: I am what I am; I can do all things if I believe in God
AMBITION: To become a world-renowned researcher and a good father
MESSAGE: What has been planned is already planned. Do not procrastinate.