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 English Of Pasig City Science High school Rainforest Park, Pasig City, Philippines

In Partial Fulfilment Of the Requirements In Research I

FN Ruth Antonette Mary Joy Jadine Kyzle Yorienhel Apple

MI E. C. R. M. Y.

LN Raganit Camulo Dalago Edma Calion

Chapter I The 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 humans 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 extract also 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 humans 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 allpossible panacea. There have been numerous researches and studies that attempted and succeeded in providing useful information about the sugar apples medicinal properties. It has been proved every part of the sugar apples 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 gramflour 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 overthe-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 woundcleaner. 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 Problem Generally, 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 processing of the sugar apple (A. squamosa into an antibiotic compound sets forth the hypothesis which is that this study is successful in producing the product that hoped to achieve. and L.) null not was

drawn: The sugar apple (A. squamosa L.) extract cannot be used to make an antibiotic compound.

From the stated problem, the hypothesis is

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 II Theoretical and Conceptual Framework Theoretical Framework

Literatures of many research works prove that every part of A. squamosa various (sugar apple) medical at hand possesses medicinal properties. The sugar apple plants parts and contain the helpful attributes and useful. Almost all of the sugar apple plants parts can be used to The cure bark for is various known to ailments. be a For example, roots are employed internally in spinal diseases. powerful astringent. Fruits are considered to be a good tonic; enriches blood, used as an expectorant, increases burning sedative muscular to heart strength: and and tendency relieves cooling to lessens Ripe sensation biliousness; medical attributes

range from the common to the extremely complex

vomiting.

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 leaves with

in

prolapsus make a

of

children. to

Bruised induce

salt

cataplasm

suppuration which are applied in the extraction of guinea-worms. Studies show that there are

Leaves

contain

4-(2-nitroethyl)-1-((6-O-B-DBenzyltentrahydro-

xyropyanosyl-B-D-glucopyranosyl)oxy)benzene,Anonaine, isoquinoline, Borneol, Camphene, Camphor, car-3ene, Carvone, B-Caryphyllene, eugenol, Farnesol, Geraniol, Higenamine, 16-Hentriacontanone, Isocorydine, Hexacontanol, Linalool, pBLinomine,

Linalool acetate, Menthone, Menthylanthranilate, Methysalycilate, hydro)isoquinoline, Pinene, of the Rutin, leaves Methylheptenone, n-Octacosanol, Stigmasterol, curing property -Pinene, (hydroxybenzyl)-6,7-(2-hydroxy,4B-Sitosterol, in curing

Thymol, and n-Triacontanol. Due to the uniqueness 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 aureus negative The zone

because Two and Bacillus

of Gram

their positive subtilis) coli showed

antibacterial (Staphylococcus and two Gram and Pseudomonas the in highest methanol by Aeruginosa of some

properties.

(Escherichia results Ps. extract

aeruginosa) bacteria was selected for screening. screening of that inhibition against ether was observed against

extract petroleum study

Aeruginosa

followed Ps.

and methanol extract against E. coli. The present demonstrates the presence Borneol, the strains coli) 96), and was phytochemicals antibacterial screening, Escherichia Staphylococcus aeruginosa test (Linalool, activity. standard (E. 741), aerus coli (MTCC In Eugenol,

Farsenol, and Geraniol) in extracts which provide antimicrobial used (MTCC were 443), subtilis by pour the

(MTCC

Pseudomonas

Bacillus inoculated

(MTCC 441). A 1% of the standard inoculum of the bacterial strain 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 37 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 regression generated

parameters and

were

left

at

default were

settings. The chromatograms were integrated and analysis using statistical evaluation data WinCATS 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, analysis presence Reprostar of of the five III. Anonna known Present squamosa phytochemical displayed the (Linalool,

compounds

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 any at other 0.68 and there in was no overlap with component analyzed in

sample at 200mn-450mn. In the extraction results, percentage yield was found to be increased 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 extract against against S. S. aereus; chloroform extract aereus; methanol

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 this was plant studied P. activity. were for its Volatile compounds for activity But was of its by in not also studied

antibacterial activity. Petroleum ether extract antibacterial and S. S. [13]; and conclude its high efficacy against E. coli, aeruginosa, Patels aereus. aereus Jayshree study,

affected by petroleum ether extract. Conceptual Framework Due the to the fact about that the on there have been of many the of literary records and studies in the past up to present effectiveness various medicinal properties fields

ailments and

and

illnesses we

ranging that

from it

mild is

lice

head infestation to something severe as cancer tumors, conclude 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 humans 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, Hibiscus, Calendula, Holy Basil, Catuaba, Lemon Chapparal, Myrtle, Chrysanthemum, Cinnamon, Coconut oil, Cranberry, Grass,

Periwinkle, Calamus

Parsley,

Pine,

Sage,

Sarsaparilla, bud oil, All

Thyme, Wormwood, Ajwain Seed, Alfalfa, Bee Balm, root, Uva Garlic pepper will Burdock Ursi, root, Yarrow, Clove Coptis, Couch, Elecampane, Grindelia, Kava-kava, Schisandra, Rosemary, Cayenne Sandalwood Sassafrass, Cohosh berries, the mix to Spice oil, Andrographis, Barberry root, Bayberry, extract, also be Blue root, and to extract, Juniper added

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 antiseptic cream

To create a topical antibacterial 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 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 A chemical substance obtained

from plants that is biologically active but not

Antiseptic tissues Inoculum produce disease or

substance

that

destroys

micro-

organisms that carry disease without harming body

substance

(a

virus to

or a

toxin

or

immune serum) that is introduced into the body to increase immunity particular

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 on a cloth and

dressing to

consisting skin to

of a treat

soft heated mass of meal or clay that is spread applied the inflamed areas or improve circulation etc. Guinea-worms contaminated with A painful Guinea worm and debilitating that can

infestation contracted by drinking stagnant water larvae 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) by dangerous and to health;

characterized

progressive

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 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 pathogenic; can be a threat to food

Annona Reticulata - Small tropical American tree bearing fruit Pseudomonas bacteria Bark - Tough protective covering of the woody aeruginosa A species of aerobic a bristly heart-shaped acid tropical

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 it used is mainly in in electrical ores applications; found several

including wolframite and scheelite