Custard Apple or Anonas (Annona squamosa Linn.) as antibiotic compound in Pasig City in the summer of 2012

A Research

Presented to the Faculty of EnglishOf Pasig City Science High school

Rainforest Park, Pasig City, Philippines

In Partial FulfilmentOf the Requirements

In Research I

FN MI LNRuth Antonette E. RaganitMary Joy C. CamuloJadine Kyzle R. Dalago Yorienhel M. EdmaApple Y. Calion

Chapter IThe Problem and Its Scope

Background of the Study The purpose of the study is to inquire the probability of processing the extract of A. squamosa L. in order to formulate a possible antibiotic. Due to the fact that there has been many literary records and studies in the past up to the present about the effectiveness of the medicinal properties on various fields of ailments and illnesses ranging from mild lice head infestation to something severe as cancer and tumors. It can be said that the plant part of the A. squamosa L. that has the most abundant attributes in making a antibiotic compound, the leaves will be sufficient enough. The A. squamosa L., more locally known as sugar apple or atis, has many chemicals that can be considered as having an antibiotic property such as Annonaine which can be found in the leave, barks, and roots of the fruit. Not only in the means of being an antibiotic, the An. Squamosa L. also has other benifitable medical properties. It can serve as an emmenagogue, an agent that promote menstruational discharge. It can serve as a febrifuge, a type of medicine that alleviates or lowers body temperature. These two attributes of the sugar apple are undoubtly beneficial to the society. While there are properties which can be considered as unhelpful or bad for the society, such as being an abortificient and antiovulatory agent, you cannot overlook the fact that the A. squamosa L. possesses many properties, such as being an antibiotic agent, and therefore making this research highly possible and plausible. Based on all that is written in the above statements, we conclude that it is highly possible to develop a compound that will disinfect or remove any bacterial build-up on any surface especially on a surface as sensitive as a human’s skin.

The antibiotic properties of the A. squamosa L. is due to the A. squamosa yielding various compounds that are used in antibiotic creams like phenols, tannins, alkaloids, saponins, lavanoids, reducing sugars and oil. A. squamosa L., from here on out be referred to as the technical term “sugar apple”, also has methanol extracts that are shown to be quite effective against the bacteria called E. coli. Extracts from the seeds also show that it has a great antifungal property that T. rubum which is one of the most infectious agents in the world. There is also a study that screened the ethanol crude extract of the fruit which show antimicrobial activity against pathogenic organisms. The ethanol crude extractalso shows restrictive properties against S aureus and S pneumoniae, which are common bacteria that can be found in an average household. Sugar apples have also been screened and have been proven to also have insecticidal properties which can also be considered as antibiotic when taking into mind that it can prevents any insects coming into contact with open wounds. It has also been shown to serve as a valuable source of compounds with therapeutic antibiotic potentials. Sugar apples also have dermatological properties in terms of shortening the time spent for a wound to heal. With all these, we conclude that that it is highly possible to develop a compound that will disinfect or remove any bacterial build-up on any surface especially on a surface as sensitive as a human’s skin.

Basing on our thesis proposal, being an antibiotic compound can help in temporary or either permanent curing of various skin diseases and problems, ranging from minor cases to major cases. Skin problems not only cause peer issues for the victim but also internal and bodily issues. If ever a skin problem is left unattended or undetected, it could get larger in scale and cause major injuries to the person inflicted or worse, result in death. It is also necessary to

prove this thesis possible and doable because it can benefit the lower-class society. In this case, people who cannot afford expensive antibiotics and can only rely on herbal medicine. Despite the fact that making a medicinal product out of sugar apples is considered a herbal medicine, this fact is backed up by science because of the many researches done on this fruit and its plant parts. With all these, besides being only a “temporary” cure to common skin problems like burns and cuts, further going into this study will surely make this fruit an all-possible panacea.

There have been numerous researches and studies that attempted and succeeded in providing useful information about the sugar apple’s medicinal properties. It has been proved every part of the sugar apple’s plant possesses useful medicinal properties. For example, roots are employed internally in spinal diseases. The bark is known to be a powerful astringent. Fruits are considered to be a good tonic; enriches blood, used as an expectorant, increases muscular strength: cooling, lessens burning sensation and tendency to biliousness; sedative to heart and relieves vomiting. Ripe fruit is a maturant and the mixture along with salt is used against malignant tumors to hasten suppuration. Dried unripe fruit is powdered and mixed with gram-flour to destroy vermin and to act as insecticide. The seeds are said to be abortifacient and good to destroy lice in hair in Yunani medicine. Seed yields oil and resin which acts as detergent and their powder, mixed with gram-flour, is good hair wash. Seeds are powerful irritant of conjunctiva and produce ulcers in eyes. Leaves are used as poultice over boils and ulcers and also to kill lice. Leaf infusion is efficacious in prolapsus of children. Bruised leaves with salt make a cataplasm to induce suppuration which are applied in the extraction of guinea-worms. Therefore, the chances of extracting an antibiotic compound from the A.

squamosa L. (sugar apple) is high or in fact, very possible.

From this research, we expect to produce an antibiotic product from the extract of the A. squamosa L. (sugar apple) that will benefit both the civil community and also the medical community. With the help of this research, we can be sure that the A. squamosa L. (sugar apple)’s demand will greatly inflate because of its use aa an antibiotic compound. For example, if this research were to be proven true and possible, if could serve as a substitute for the usual over-the-counter antibiotics which are rather costly and hard to find. It will also shed some use for all the sugar apple trees that are left unused in the backyard of many citizens’ homes. In the medical community, it can also help in substituting common antibiotics and antimicrobials found in hospitals if ever a shortage in supplies occurs because sugar apple trees are very common for a Filipino citizen to have in his own backyard. Also, as our investigation on the background concerning this study (A. squamosa L.), we have found out that not only does this fruit own antibiotic property, but also antiseptic and antibacterial properties. In fact, in the past, indigenous tribes have many uses for the A. squamosa L., like being an expectorant, astringent, and also as a wound-cleaner. So we expect to widen the knowledge that a common citizen knows about sugar apples, not only serving as food but also as possible medicine.

Statement of the ProblemGenerally, this research seeks to determine the relationship between the possible antibiotic product from the extract of A. squamosa L. and the probability of the product to aid in making the process of healing common skin problems faster.

Specifically, it seeks to answer the following:

1. What are the components of the research?2. What are the advantages or disadvantages of

the research?3. Is it possible to introduce the product of

this research to the general product

Hypothesis This study about the extraction and processing of the sugar apple (A. squamosa L.) into an antibiotic compound sets forth the null hypothesis which is that this study is not successful in producing the product that was hoped to achieve.

From the stated problem, the hypothesis is drawn:The sugar apple (A. squamosa L.) extract cannot be used to make an antibiotic compound.

Significance of the Study This study on the extraction and processing of the sugar apple (A. squamosa L.) into an antibiotic compound will contribute greatly in identifying of finding a cheap and easy-to-find substitute for over-the-counter antibiotics.

More specifically the results of this study will be useful to the following:

Researches – This will help helping further improving studies and theses about the subject of the thesis.

Civil Community – The product of this research will act as a cheap alternative to antibiotics.

Medical Community – Like the civil community, it will also serve as an efficient, cheap, and science-proven substitute for antibiotics.

Chapter IITheoretical and Conceptual Framework

Theoretical Framework

Literatures of many research works prove

that every part of A. squamosa (sugar apple)

possesses medicinal properties. The sugar apple

plant’s parts contain various helpful medical

attributes and the medical attributes at hand

range from the common to the extremely complex

and useful.

Almost all of the sugar apple plant’s parts can

be used to cure for various ailments. For

example, roots are employed internally in spinal

diseases. The bark is known to be a powerful

astringent. Fruits are considered to be a good

tonic; enriches blood, used as an expectorant,

increases muscular strength: cooling lessens

burning sensation and tendency to biliousness;

sedative to heart and relieves vomiting. Ripe

fruit is a maturant and the mixture along with

salt is used against malignant tumors to hasten

suppuration. Dried unripe fruit is powdered and

mixed with gram-flour to destroy vermin and to

act as insecticide. The seeds are said to be

abortifacient and good to destroy lice in hair in

Yunani medicine. Seed yields oil and resin which

acts as detergent and their powder, mixed with

gram-flour, is good hair wash. Seeds are powerful

irritant of conjunctiva and produce ulcers in

eyes. Leaves are used as poultice over boils and

ulcers and also to kill lice. Leaf infusion is

efficacious in prolapsus of children. Bruised

leaves with salt make a cataplasm to induce

suppuration which are applied in the extraction

of guinea-worms.

Studies show that there are

Leaves contain 4-(2-nitroethyl)-1-((6-O-B-D-

xyropyanosyl-B-D-glucopyranosyl)-

oxy)benzene,Anonaine, Benzyltentrahydro-

isoquinoline, Borneol, Camphene, Camphor, car-3-

ene, Carvone, B-Caryphyllene, eugenol, Farnesol,

Geraniol, 16-Hentriacontanone, Hexacontanol,

Higenamine, Isocorydine, Linomine, Linalool,

Linalool acetate, Menthone, Menthylanthranilate,

Methysalycilate, Methylheptenone, p-

(hydroxybenzyl)-6,7-(2-hydroxy,4-

hydro)isoquinoline, n-Octacosanol, Á-Pinene, B-

Pinene, Rutin, Stigmasterol, B-Sitosterol,

Thymol, and n-Triacontanol. Due to the uniqueness

of the leaves’ curing property in curing

different ailments, the leaves were selected for

the study of Jayshree Patel. In the phytochemical

analysis and antimicrobial screening conducted by

Jayshree Patel and Vipin Kumar in the Sadbhav

SRISTI Sadsohan Laboratory in Orissa, India, four

different solvent extracts of leaves of Custard

apple (Annona squamosa L.) were chosen to be

studied because of their antibacterial

properties. Two Gram positive (Staphylococcus

aureus and Bacillus subtilis) and two Gram

negative (Escherichia coli and Pseudomonas

aeruginosa) bacteria was selected for screening.

The screening results showed that the highest

zone of inhibition was observed in methanol

extract against Ps. Aeruginosa followed by

petroleum ether extract against Ps. Aeruginosa

and methanol extract against E. coli. The present

study demonstrates the presence of some

phytochemicals (Linalool, Borneol, Eugenol,

Farsenol, and Geraniol) in extracts which provide

antibacterial activity. In the antimicrobial

screening, the standard strains used were

Escherichia coli (E. coli) (MTCC 443),

Staphylococcus aerus (MTCC 96), Pseudomonas

aeruginosa (MTCC 741), and Bacillus subtilis

(MTCC 441). A 1% of the standard inoculum of the

test bacterial strain was inoculated by pour

plate method on Mueller Hinton Agar (MHA). Then,

6 mm were bored in the MHA. Plant extracts were

introduced into each well and allowed to stand

for 1 h to diffuse and incubated at 37֯ C for 24

h. The Inhibition Zone Diameter (IZD) was

measured by antibiotic zone reader to nearest mm

[5]. The MIC was determined by the cylinder agar

method diffusion as described by Fyhrquist et al

[6]. In the phytochemical analysis, the

quantitive and qualitive analysis were performed

with the help of HPTLC instrument. The HPTLC

system (Camag, Mutenz, Switzerland) consists of

(1) TLC scanner connected to a pc running WinCATS

software under Windows NT; (2) Linomat V sample

applicator; (3) Photo documentation system Camag,

Reprostar III. The chromatographic extraction was

performed by streaking the extract in the form of

narrow bands of precoated silica gel 60 F254

aluminum TLC plate (5 cm x 10cm), at a constant

application rate of 150 ul/s and gas flow 10 s/uL

was employed with the help of Camag 100 ul

syringe connected to a Nitrogen tank; using a

Camag Linomat V (Camag, Mutenz, Switzerland). The

space between three bands was kept 15 mm. 15 ul

of 1% concentration solution from three extracts

(Methanol, Chloroform, and Petroleum ether) was

placed as a spot. After spotting the plate, it is

subjected to linear ascending development up to a

distance of about 90 mm in a solvent system at

room temperature just 10 minutes prior to

development. TLC plate was dried in flowing air

at room temperature. Densitometric scanning was

carried out using Camag TTLC Scanner III (Camag,

Mutenz, Switzerland) between wavelength 200-450

nm with a slit dimension of 6.00 x 0.30 mm, with

a scanning speed of 20 mm/s, and data resolution

was at 100 m/step. The source lamps for radiation

were deuterium and tungsten lamps. All remaining

measurements parameters were left at default

settings. The chromatograms were integrated and

regression analysis and statistical data were

generated using WinCATS evaluation software

(Version 1.4.2.8121). After the scanning, images

of the place were taken by using three different

wavelength of lights (254 nm by UV lamps, 366 nm

by mercuric lamp, and 400-800 nm by white lamp)

with the help of photo documentation system of

Camag, Reprostar III. Present phytochemical

analysis of the Anonna squamosa displayed the

presence of five known compounds (Linalool,

Borneol, Eugenol, Farnesol, and Geraniol on the

basis of their Rf value. The Rf value of Linalool

is visible at Rf value ~0.25, Borneol at 0.22,

Carnevone at 0.47, Eugenol at 0.47, Farnesol at

0.35, and Geraniol at 0.68 and there was no

overlap with any other component in analyzed

sample at 200mn-450mn. In the extraction results,

percentage yield was found to be increased in

accordance with the increasing polarity of the

solvents. Highest yield in methanol, chloroform

and petroleum ether extracts. Phytochemical

studies showed that Linalool, Borneol, Eugenol,

Farnesol, and Geranisol are present in extract

which may be in combination and separately

responsible for antibacterial activity. Two gram

positive and two gram negative were chosen to be

studied. The results of antibacterial screening

indicates the highest inhibition was observed in

methanol extract against P. aeruginosa followed

by petroleum ether extract against Ps. Aeruginosa

and methanol extract against E. coli. Amikacin,

tetracycline, and cefepime were used as positive

controls; whereas DMSO was used in black controls

in experiments. It was observed that petroleum

ether extract against S. aereus; chloroform

extract against S. aereus; methanol extract

against B. subtilis; and water extract against S.

aereus, Ps. Aeruginosa and B. subtilis have not

shown any signs of antibacterial activity. Kotkar

HM and his colleagues [11] had studied the

biochemical of this plant and mentioned that A.

squamosa contains flavonoids which expose strong

antibacterial activity. Volatile compounds of

this plant were also studied for its

antibacterial activity. Petroleum ether extract

was studied for its antibacterial activity by

[13]; and conclude its high efficacy against E.

coli, P. aeruginosa, and S. aereus. But in

Jayshree Patel’s study, S. aereus was not

affected by petroleum ether extract.

Conceptual Framework

Due to the fact that there have been many

literary records and studies in the past up to

the present about the effectiveness of the

medicinal properties on various fields of

ailments and illnesses ranging from mild lice

head infestation to something severe as cancer

and tumors, we conclude that it is highly

possible to develop a cream that will disinfect

or remove any bacterial build-up on any surface

especially on a surface as sensitive as a human’s

skin. If we were to choose what part of the A.

squamosa L. to use in the making of the

antibacterial cream, we would choose the leaves

because of its many components and properties as

mentioned above, that can be said as a component

for being antibacterial. In making the cream, we

have decided to add antiseptic and antibiotic

components to the cream to increase its fullest

potential as an all-round bacteria-killing creams

that the study visualizes. The process in which

we follow in making the antibacterial cream is as

follows: first, the leaves would be grounded up

to pulpy mix and then mix it with pure lavender

oil. The resulting mixture is then boiled in a

separate pot with one and one-half ounces of

water adding another tablespoon of lavender oil.

The mixture will then be strained and be mixed

with a heated oil base and then be stirred to

blend. Tea tree, Lemon, Eucalyptus, Aloe vera,

Arhuja root, Bergamot oil, Blood root, Boneset,

Buchu, Calendula, Catuaba, Chapparal,

Chrysanthemum, Cinnamon, Coconut oil, Cranberry,

Hibiscus, Holy Basil, Lemon Grass, Myrtle,

Periwinkle, Parsley, Pine, Sage, Sarsaparilla,

Thyme, Wormwood, Ajwain Seed, Alfalfa, Bee Balm,

Calamus root, Burdock root, Clove bud oil,

Coptis, Couch, Elecampane, Grindelia, Kava-kava,

Schisandra, Uva Ursi, Yarrow, Sassafrass, All

Spice oil, Andrographis, Barberry root, Bayberry,

Rosemary, Garlic extract, Blue Cohosh root,

Cayenne pepper extract, Juniper berries, and

Sandalwood will also be added to the mix to

increase not only its antibacterial property, but

also its antibiotic and antiseptic attributes.

Conceptual Paradigm

GOAL 1 GOAL 2 GOAL 3

To create a topical

antibacterial cream

To create a topical

antiseptic cream

To create a topical

antibiotic cream

The result of this study

SOCIETY

Definition of Terms

Astringent – A drug that causes contraction of

body tissues and canals

Tonic – A medicine that strengthens and

invigorates

Expectorant – A medicine promoting the discharge

of phlegm or sputum from the lungs and out of

the mouth

Phytochemical – A chemical substance obtained

from plants that is biologically active but not

nutritive

Antimicrobial – An agent (as heat, radiation or a

chemical) that destroys microorganisms that might

carry disease

Antibacterial - Any drug that destroys bacteria

or inhibits their growth

Antibiotic - A chemical substance derivable from

a mould or bacterium that can kill microorganisms

and cure bacterial infections

Antiseptic - A substance that destroys micro-

organisms that carry disease without harming body

tissues

Inoculum - A substance (a virus or toxin or

immune serum) that is introduced into the body to

produce or increase immunity to a particular

disease

Sedative – A drug that reduces excitability and

calms a person

Maturant – An agent that helps in developing or

reaching the maturity of something

Suppuration – (medicine) the formation of

morbific matter in an abscess or a vesicle and

the discharge of pus

Abortifacient – A drug (or other chemical agent)

that causes abortion

Irritant – Something that causes irritation and

annoyance

Ulcer – A circumscribed inflammatory and often

suppurating lesion on the skin or an internal

mucous surface resulting in necrosis of tissue

Poultice – A medical dressing consisting of a

soft heated mass of meal or clay that is spread

on a cloth and applied to the skin to treat

inflamed areas or improve circulation etc.

Guinea-worms – A painful and debilitating

infestation contracted by drinking stagnant water

contaminated with Guinea worm larvae that can

mature inside a human's abdomen until the worm

emerges through a painful blister in the person's

skin

Cataplasm – A medical dressing consisting of a

soft heated mass of meal or clay that is spread

on a cloth and applied to the skin to treat

inflamed areas or improve circulation etc.

Prolapsus – The slipping or falling out of place

of an organ (as the uterus)

Conjunctiva - A transparent lubricating mucous

membrane that covers the eyeball and the under

surface of the eyelid

Biliousness - Gastric distress caused by a

disorder of the liver or gall bladder

Rf Value - A radioactive transuranic element

which has been synthesized

Insecticide - A chemical used to kill insects

Tumor - An abnormal new mass of tissue that

serves no purpose

Malignant - (pathology) dangerous to health;

characterized by progressive and uncontrolled

growth (especially of a tumour)

Staphyloccocus aureus - A bacterium that causes

illnesses ranging from minor skin infections and

abscesses, to life-threatening diseases such as

pneumonia, meningitis and septicaemia

E. Coli - A species of bacterium normally present

in intestinal tract of humans and other animals;

sometimes pathogenic; can be a threat to food

safety

Bacillus Subtillis - A species of bacillus found

in soil and decomposing organic matter; some

strains produce antibiotics

Annona Squamosa - Tropical American tree bearing

sweet pulpy fruit with thick scaly rind and shiny

black seeds

Annona Reticulata - Small tropical American tree

bearing a bristly heart-shaped acid tropical

fruit

Pseudomonas aeruginosa - A species of aerobic

bacteria

Bark - Tough protective covering of the woody

stems and roots of trees and other woody plants

Solvent - A liquid substance capable of

dissolving other substances

Boil - A painful sore with a hard core filled

with pus

Inhibition – the quality of being inhibited

Strain - (biology) a group of organisms within a

species that differ in trivial ways from similar

groups

Deuterium - An isotope of hydrogen which has one

neutron (as opposed to zero neutrons in hydrogen)

Tungsten - A heavy grey-white metallic element;

the pure form is used mainly in electrical

applications; it is found in several ores

including wolframite and scheelite