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EFFICACY OF CARICA PAPAYA ENZYME AS ANTIBACTERIAL

HAND SANITIZER

A Research
Presented to
The College of Nursing
Our Lady of Fatima University
Antipolo Campus

In Partial Fulfillment
Of the Requirements for
Microbiology and Parasitology

By:
BSN1Y2-6

April, 2020
ABSTRACT

Every year there is a significant increase in the mortality rate of people due to lack of sanitation,
poor hand-washing practices or simply because of little no access to water. The current water shortage
in the Philippines intensifies this problem. Based on numerous studies, Papaya contains antiseptic and
antibacterial properties. An enzyme from papaya leaves yields antibacterial and antifungal behavior
called papain enzyme which will help in reducing the number of bacteria where it is no longer poses a
threat. The researchers want to formulate an alternative antibacterial sanitizing product that can
provide protection against Staphylococcus aureus at half of the cost. The researchers will make use of
the Dilution method for the extraction wherein they will leave ground papaya leaves in liquid for a
period of 24 to 48 hours. Then apply the Agar Dilution Process for the testing of the antibacterial efficacy
of the aforementioned product.

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INTRODUCTION

Papaya belongs to Caricaceae family under kingdom Plantae. It originates from Mexico and
South America. The botanical name of papaya is Carica papaya. It is a small tropical tree with a
straight stem marked by scars where leaves have fallen from it directly. There are many varieties of
papaya, but the main varieties grown in the United State are Red Lady, Maradol, and various Solo
types. Christopher Columbus (2012) reputedly referred to the tropical fruit papaya as ‘fruit of
angels.’ It is rich in vitamin A, E, K, and B, fiber, calcium, magnesium, phosphorus and zinc, as well as
the essential nutrients lycopene, folate, lutein and enzymes. According to the book Healing Foods by
DK Publishing House, papaya is known to have antibacterial properties and promotes good digestion
and almost every part of the plant can be used.

Carica papaya plants produced natural compounds (Annonaceous acetegenins) in leaf bark and
wing tissues. The leaves are large, usually are 50-70cm in diameter, deeply palmately-lobed, with
seven lobes. Several scientific investigations on the biological activities had been done through the
leaves. It was suggested that a potential lucrative industry based simply on production of plant
biomass could develop for production of anti-cancer drugs, pending Food and Drug Agency
approval, and natural botanical pesticides (McLanghlin, 1992). The high level of natural self-defense
compounds in the tree makes it high resistant to insect and disease infestations. Fresh and green
papaya leaf has a therapeutic value due to its antiseptic quality. It cleans the intestines from
bacteria. In French Guiana, both leaf and root are prepared in combination with other ingredients
for the treatment of malaria fever (Vigneron et al., 2005). Dried and pulverized leaves are sold for
making tea; also, the leaf decoction is administered as purgative for horses and used for the
treatment of genetic urinary system.
Papaya fruits are widely consumed in food industry, while the processing of papaya fruits,
papaya leaf is not consumed in any industry. In order to solve such problem, the study on the
papaya leaf had been done. From the study done by Simmonne (2005), papain enzyme from the
papaya leaf yields the antibacterial behavior. As referring to the research title, production of
antibacterial hand sanitizer by the natural enzyme are important for the bio green product which is
preferable than the chemical made product of hand sanitizer. It is also environmental-friendly and
non-toxic product which solve the garbage formation by papaya tree.

Papain enzyme can be obtained from papaya fruit, latex or roots. The researchers used papaya
leaf extract to obtain papain enzyme which have phenolic compounds such as protocatechuic acid,
p-coumarie acid, dimethoxymarin, caffeic acid, kaempferol, quereetin, chlorogenic acid. These
compounds have antimicrobial activity and have been proven to be able to inhibit the growth of
Rhizupos stolonifer. Papaya leaf has been chosen for this research because it provides the best
potential for further commercial form and most preferable material used for the papain enzyme
extraction. Then, Dilution Method was chosen for the papain extraction to be followed by the Agar
Dilution Method to test its susceptibility against the bacteria Staphylococcus aureus.

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REVIEW OF RELATED LITERATURE

2.1 Antimicrobial Activity of Carica Papaya (Pawpaw Leaf) on Some Pathogenic Organisms of Clinical
Origin from South-Western Nigeria
Some studies proved that the bioactive compound of leaf and root extracts of Carica papaya
using water and organic solvents were investigated for antibacterial activity against some human
pathogenic bacteria using the agar diffusion method. The aqueous extracts of the root extracts did
not show significant activity, but the organic extracts had significant activity with the methanol
extracts demonstrating the highest activity against the test bacteria. The root extracts demonstrated
higher activities against all the gram-positive bacteria than the gram-negative bacteria tested, with
the highest activity demonstrated against Pseudomonas aeruginosa while the aqueous leaf extract
showed pronounced inhibition demonstrating higher activities against the test bacteria than the
organic solvents. The extracts demonstrated higher activities against all the gram-positive bacteria
than the gram-negative bacteria tested, with the highest activity demonstrated against P.
aeruginosa. Increase in temperature enhanced the activity of the extracts, while alkaline pH
decreased the activity. C. papaya may be used for the treatment of gastroenteritis, uretritis, and
otitis media and wound infections. (Anibijuwon and Udeze, 2009)
Papain enzyme can be obtained from papaya fruit, latex or roots. The researchers used papaya
leaf extract to obtain papain enzyme which have phenolic compounds such as protocatechuic acid,
p-coumarie acid, dimethoxymarin, caffeic acid, kaempferol, quereetin, chlorogenic acid. These
compounds have antimicrobial activity and have been proven to be able to inhibit the growth of
Rhizupos stolonifer. Papaya leaf has been chosen for this research because it provides the best
potential for further commercial form and most preferable material used for the papain enzyme
extraction. Then, Maceration Method was chosen for the papain extraction to be followed by the
Agar Dilution Method to test its susceptibility against the bacteria Staphylococcus aureus.

2.2 Morphology of Staphylococcus aureus


Staphylococcus aureus is a facultative anaerobic, Gram-positive coccus, which appears as grape-
like clusters when viewed through a microscope, and has round, usually golden-yellow colonies,
often with hemolysis, when grown on blood agar plates. Some strains of S. aureus are capable of
producing staphyloxanthin - a golden colored carotenoid pigment. This pigment acts as a virulence
factor, primarily by being a bacterial antioxidant which helps the microbe evade the reactive oxygen
species which the host immune system uses to kill pathogens.
This kind of bacteria is highly vulnerable to destruction by heat treatment and nearly all
sanitizing agents. Thus, the presence of this bacterium or its enterotoxins in processed foods or on
food processing equipment is generally an indication of poor sanitation. S. aureus can cause severe
food poisoning. It has been identified as the causative agent in many food poisoning outbreaks and
is probably responsible for even more cases in individuals and family groups. ( Tallent, Hait, Bennett
and Lancette, 2001). The presence of a large number of S. aureus organisms in a food may indicate
poor handling or sanitation. Also, Gordon L. Archer stated from his book (Clinical Infectious Diseases)
that S. aureus is a virulent pathogen that is currently the most common cause of infections in

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hospitalized patients. S. aureus infection can involve any organ system. The success of S. aureus as a
pathogen and its ability to cause such a wide range of infections are the result of its extensive
virulence factors. The increase in the resistance of this virulent pathogen to antibacterial agents,
coupled with its increasing prevalence as a nosocomial pathogen, is of major concern.

2.3 Effectiveness of Electrolyzed Water as a Sanitizer for Treating Different Surfaces


Some researchers tried different ways or methods how to kill S. aureus. Hoon Park, Yen-Con
Hung and Chyer Kim-student researchers of University of Georgia from Griffin, Georgia evaluated the
effectiveness of electrolyzed (EO) water at killing S.aureus in pure culture. One milliliter of the
bacterium was subjected to EO water or control water at room temperature for 30 s. Inactivation of
pathogens occurred within 30 s after exposure to EO water. The effectiveness of EO water in
reducing S. aureus on different surfaces (glass, stainless steel, glazed ceramic tile, unglazed ceramic
tile, and vitreous china) was also evaluated. No viable cells of either strain were observed in the EO
water after treatment, regardless of agitation. However, large populations of both pathogens were
recovered from control wash solution after treatment.

2.4 Investigation on Antibacterial Activity of Carica Papaya Leaf Extracts against Wound Infection-
Causing Bacteria
Due to increase in the thrust for the production of plant-based antimicrobials, many studies
tested the properties of Carica papaya leaves. The leaf extract was prepared by using acetone,
methanol, and water. The antimicrobial nature of the extract was studied by agar well diffusion
method against wound infection-causing pathogens viz., Escherichia coli, Staphylococcus aureus,
Proteus vulgaris, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The acetone leaf extracts
exerted pronounced antibacterial effect on gram negative bacteria especially Pseudomonas sp. The
study revealed that papaya leaves could contain active antimicrobial compounds which may hinder
the growth of wound infection causing pathogens in in-vitro conditions. The results obviously
justified the importance of topical application of papaya leaf extracts to treat the wound infection as
a traditional practice.( Aruljothi, Uma, Bhuvaneswari, 2014).

2.5 The In-vitro Assessment of Antibacterial Effect of Papaya Seed Extract Against Bacterial Pathogens
Isolated From Urine, Wound and Stool

Carica papaya is used in traditional medicine for variety of purposes in treating infectious
and noninfectious diseases. The objective of the study was to assess the antibacterial effect of
papaya seed extract against bacterial pathogens isolated from wound, urine and stool. This
analytical experimental study was conducted in Jimma University, School of Medical Laboratory
Technology, Microbiology laboratory between February to March 2005 by Yismaw G, Tessema B,
Mulu A, Tiruneh M. The antibacterial activity of methanol extract of papaya seed was
investigated against specific pathogenic bacteria isolated from wound, urine and stool by an
agar dilution technique and the crude preparation was assessed by an agar diffusion technique.
The growth or inhibition of control strains of Escherichia coli, Staphylococcus aureus, Salmonella
typhi, and Pseudomonas aeruginosa as well as the clinical isolates of these bacteria were
determined in growth media. Based on experimental procedure of this study, Papaya seed could
be used as an effective antibacterial agent for the tested organisms. Nevertheless, preclinical

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studies including in vivo animal models and clinical trial on the effect of the seed are essential
before advocating large-scale therapy.

2.6 Effect of extraction conditions on total phenolic compounds and antioxidant activities
of Carica papaya leaf aqueous extracts

A study from Journal of Herbal Medicine includes some research about the effect of extraction
conditions of Carica papaya. The study aimed to optimize extraction conditions and determine the
effect of aqueous extraction on the yield of polyphenols from papaya leaves. The efficiency of water
extraction was compared to the organic solvents acetone, ethanol and methanol. A method to
prepare crude powder from the leaves was developed, with its composition and antioxidant
properties also examined. The researchers show that temperature, extraction time and water-to-
leaf ratio had significant effects on the extracted polyphenol yield as well as the scavenging and
total antioxidant activities. A simple and scalable method was developed to obtain approximately
190 g of powder from 1 kg of dried papaya leaves. The crude powder contained 6.3% polyphenols,
and when compared to butylated hydroxytoluene (BHT), Vitamins C and E and epigallocatechin
gallate (EGCG) had lower scavenging and total antioxidant activity. However, as this method used
water for extraction, it is considered safe and offers great potential for further purification and
application in future studies.

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METHODOLOGY

3.1 Procedure
The papaya leaves used in this research were 20-25 cm in length. It was washed, dried,
and extracted, 70g of papaya leaves produced 60mL of pure extract. The extract was then
divided in four test samples: 100% pure extract, 75% extract with 25% water, 50% extract with
50% water, and 25% extract with 75% water. After that dilution procedure is done, it was tested
to determine its effectiveness against pathogenic bacteria: Staphylococcus aureus.
3.2 Preparation of the sample
· Collect fresh leaves of the plant sample.
· Remove the extraneous matter by washing the sample with water.
· Wipe and dry the leaves with cloth.
· Extract the leaves using mortar and pestle, while pounding the leaves, ethanol was added.
· Divide the extract in four separate test tubes: 100% pure extract, 75% extract with 25% water,
50% extract with 50% water, and 25% extract with 75% water.

3.3 Materials:
 Weighing scale  Filtering paper  2 Beaker
 Graduated cylinder  Funnel  Inoculating needle
 Distilled Water  Mortar and pestle  Dropper
 4 test tubes  Ethanol  Test tube brush

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Figure No. 1 Figure No. 2 Figure No. 3 Figure No. 4 Figure No. 5

Figure No. 8 Figure No. 9


Figure No. 6 Figure No. 7

Figure No. 10 Figure No. 11 Figure No. 12

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3.4 Extraction Procedure:
1. On a weighing scale measure about 10 grams of ground papaya leaves and mince using a
mortar and pestle.

Figure No. 11 Figure No. 12

2. Add three to four drops of ethanol in each 10 grams of minced papaya leaves.

Figure No. 13

3. Gently squeeze the minced papaya leaves allowing the extract to be collected to a beaker.
Measure the extract by observing the amount collected from 10 grams of minced papaya
leaves shown in the beaker’s indicator.

Figure No. 14

4. Strain the extracts off into a separate container using a coffee filter
placed in a funnel. Make sure the substance is clean and clear from solid materials or else it
will rot over time.

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Figure No. 15

5. Put the extract collected in four separately labeled test tubes placed in a
test tube rack.

Figure No. 16

6. Tightly seal the tubes and allow it to stand for 10 minutes. Note the
amount of alcohol volume used. Occasionally shake the jars to prevent molds from
forming. Label each of the test tubes.

3.5 Dilution
In the dilution process the researcher’s initial step is to place a total of 15 ml in each tube from a 60
ml pure papain extract. The first test tube is labeled as 15 ml pure extract. The second test tube is
labeled as 75% extract with 25% ml distilled water. The third test tube is labeled as 50% extract and 50%
distilled water. The fourth test tube was labeled 25% extract and 75% distilled water. To complete the
dilution process each test tube is place in a test tube rack at a temperature of 37°C.

3.6 Agar Dilution Process


The aim of agar dilution is to determine the lowest concentration of the assayed antimicrobial
agent, minimal inhibitory concentration (MIC) that under defined test conditions inhibits the visible
growth of the bacterium being investigated. MIC values are used to determine susceptibilities of
bacteria to drugs and also to evaluate the activity of new antimicrobial agents.
Furthermore, it involves the incorporation of different concentrations of the antimicrobial
substance into nutrient agar medium followed by the application of a standardized number of cells to
the surface of agar plate.
1. To prepare antimicrobial plates, a 2-ml aliquot of antimicrobial dilution of each concentration to
be tested is added to 18 ml of molten agar and allowed to equilibrate in a water bath to 48 to
52°C. Mix by inverting, taking care to avoid the formation of bubbles. Pour all 20 ml of each
agar/antimicrobial combination into a 100 × 15-mm Petri dish, and allow to solidify overnight on
a level surface covered to protect from light. Allow the plates to dry completely in an inverted
position. Label plates with the type of medium, drug name and concentration, and date
prepared. Agar plates without antimicrobial agents for growth controls are prepared in the
same manner except that 2 ml of ultrapure water replaces the 2 ml of drug.

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2. Agar plates should be stored in sealed plastic bags at 2 to 8°C for no more than three days.
Plates should be removed from the refrigerator and allowed to equilibrate to room temperature
before use. Ensure that the agar surface is dry before inoculating with bacterial suspensions. If
necessary, plates can be placed in an incubator in an inverted position for approximately 30
minutes to hasten drying and temperature equilibration.
3. Remove frozen stock strains of clinical isolates or QC strains from a −80°C freezer and thaw on
the day assay is to be performed. Dilute to 104 –105 CFU/ml in the appropriate broth medium
according to CFU values determined in initial stock 2 concentration. A volume of 10 ml is
sufficient to test several drugs. Discard unused frozen stock.
4. Before testing, make 1:10 and 1:100 dilutions of working stock of the clinical isolates and QC
strains to be tested in carefully labeled sterile tubes to yield volumes of 1 ml each. Incubate
dilutions at 37°C in ambient air for two hours. Test samples will include aliquots of the post-
incubated working stock and of each 1:10 and 1:100 dilution.
5. Once the final organism suspensions are prepared for all clinical isolates and QC strains that are
to be tested, arrange tubes containing suspensions in order in a rack and transfer a small
volume (0.3-0.5 ml) of each inoculum into the appropriate well of the replicator seed block. If a
replicator is not available, the tubes can be sampled directly with a micropipettor or with
disposable calibrated loops.
6. It is helpful to prepare a template or pattern in order to keep track of the organisms and their
respective dilutions. Agar plates should be marked for orientation of the inoculum spots. Plates
must be inoculated on a level surface.
7. Using the replicator, pipettor, or calibrated loop, deliver 1 to 10 μl, depending on device used, of
each of the three dilutions (1:10, 1:100, and 1:1000) of each strain or QC organism to agar plates
containing the appropriate antimicrobial concentrations and antimicrobial-free control pates.
The goal is to obtain 30–300 CFUs per spot of inoculum on the growth control plate of at least
one of the three dilutions tested.
8. Apply the inoculum directly to the surface of the plate, taking care to avoid splashing. Inoculate
the plates containing antimicrobials from the lowest 3 concentration to the highest. Inoculate a
drug-free control plate between each series of drug-containing plates.
9. Inoculate antimicrobial-free plates with spots of each dilution of each clinical isolate being
tested as a growth control. Incubate an uninoculated plate as a media sterility control. Prepare a
solvent control plate by incorporating 2 ml of the highest concentration (1:10 dilution) of solvent
used to dissolve the antimicrobial being tested in the agar instead of the diluted antimicrobial.
This is necessary for some macrolides because they are dissolved in alcohol/methanol or DMSO
rather than water. If water is used as the solvent this step is not required.
10. Allow the inoculated plates to stand at room temperature until the moisture in the inoculum
spots has been absorbed into the agar, but not for more than 30 minutes. Invert the plates
when placing in the incubator.
11. Incubate inoculated plates and controls at 37°C in a humidified incubator with 5% CO2. High
humidity is required to reduce the drying of media during extended incubations and to enhance
the growth of the organism.

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12. Record the MIC as the lowest concentration of the antimicrobial agent that prevents colony
formation when read (with the aid of a stereomicroscope) at the same time the antimicrobial-
free control plate demonstrates growth. All three dilutions are examined. A faint haze or growth
of only a single colony is read as negative. Use the dilution yielding 30–300 colonies for
reporting the MIC and disregard the others.
13. The media (sterility) control plates should show no evidence of growth. 4
14. The solvent control plate for each drug should show approximately the same CFU as the growth
control plates.
15. A colony count of 30–300 colonies on at least one dilution of the growth control plates is
required in order for the assay to be valid.

RESULTS

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DISCUSSION

The primary purpose of this study was to determine the efficacy of Carica Papaya
enzyme as antibacterial hand sanitizer against Staphylococcus aureus bacteria. Earlier research
shows that papain enzyme obtained from the papaya leaf extract have phenolic compounds
such as protocatechuic acid, p-coumarie acid, dimethoxymarin, caffeic acid, kaempferol,
quereetin, chlorogenic acid. These compounds have antimicrobial activity and have been proven
to be able to inhibit the growth of Rhizupos stolonifer.
Our results revealed _________________
These results clearly (shows o contradict o consistent????) some of the earlier research
that claimed the capability of papain enzyme to have antimicrobial activity. (if may maiinsert ka
na study ditto from RRL base sa result, please do so)

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CONCLUSION

The present work demonstrates the antimicrobial potential of Carica papaya enzyme
extract by using a solvent. The amount extracted from the leaves was small (10 grams) for both
gram-positive and gram-negative bacteria. The substance was bactericidal while the extracts
have an antibacterial activity that exerts an inhibitory effect on the growth of pathogenic
bacteria S. aureus and E. coli. The results indicate that ethanol is better for the extraction of the
antibacterial properties of the Carica papaya enzyme. The observed inhibition of Gram-positive
bacteria, Staphylococcus aureus suggests that Carica papaya enzymes possess compounds
containing antibacterial properties that can effectively suppress the growth when extracted
using ethanol as the solvent. Comparisons with related data from the literature indicate that
according to the different methodologies of studies on antibacterial activity, the most diverse
outcomes can be obtained. This study provides scientific insight to further determine the
antimicrobial principles and investigate other pharmacological properties of Carica papaya
which is a good ingredient in hand sanitizers on inhibiting the transmission of deadly pathogens
from hands and other parts of the body that can cause various effects on human health. Thus,
the formulation will be in the form of a gel that can be easily dissolved and absorbed by the
hand, good for the children and adults. Based on the present finding, Carica papaya enzymes
possess the capabilities of being a potential candidate in the search for a natural antimicrobial
agent against infections and/or diseases caused by S. aureus.

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RECOMMENDATION

Proper hand washing techniques should be practice and improve to avoid such diseases and
illness, promoting organic cleaning agent like carica papaya antibacterial hand sanitier will help student
have a safer and easier way in cleaning their hands and maintaining healthy body. The researchers
recommend the reads to consider using organic cleaning agents instead of regular chemical cleaning
agents. Also, the researchers would like the future researchers to use this as a basis for their study and
widen their scope to improve this research better.
In general, promoting organic cleaning agent is necessary to gain a better understanding in
proper hand washing techniques for quality contribution and health impacts. This includes more proper
hand washing monitoring programs and additional health risk assessment. Lastly, understanding the
health risk attribute to an individual requires a health risk assessment specific to the individual, and their
effort should be prioritized t provide insight to every individual.

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REFERENCES

Romasi, F. (2011). Antibacterial Activity of Papaya Leaf Extracts Against Pathogenic Bacteria.
Makara Journal of Technology. https://www.neliti.com/publications/151390/antibacterial-
activity-of-papaya-leaf-extracts-against-pathogenic-bacteria
Anibijuwon, I. I. and Udeze, A. O. (2009) Antimicrobial Activity of Carica Papaya (Pawpaw
Leaf)
on Some Pathogenic Organisms of Clinical Origin from South-Western
Nigeria. Ethnobotanical Leaflets. https://opensiuc.lib.siu.edu/ebl/vol2009/iss7/4
Taylor, T. and Unakal, G. (2019). Morphology of Staphylococcus Aureus.
StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK441868/
Perk, H., Hung, Y., Kim, C. (2002). Effectiveness of electrolyzed water as a sanitizer for treating
different surfaces. Department of Food Science and Technology, College of Agricultural
and Environmental Sciences, University of Georgia.
https://www.ncbi.nlm.nih.gov/pubmed/12182480

Aruljothi, S., Uma, C., and Sivagurunathan, M. (2014). Investigation on Antibacterial Activity of
Carica
Papaya Leaf Extracts against Wound Infection-Causing Bacteria. Bhuvaneswari
Division of Microbiology, Annamalai University. International Journal of Research
Studies in Biosciences (IJRSB). https://www.arcjournals.org/pdfs/ijrsb/v2-i11/3.pdf
Yismaw, G., Tessema, B., Mulu, A., Tiruneh, M. (2008). The invitro assessment of antibacterial
effect of papaya seed extract against bacterial pathogens isolated from urine, wound and
stool. Department of Microbiology and Parasitology, College of Medicine and Health
Sciences, The University of Gondar. https://www.ncbi.nlm.nih.gov/pubmed/18711992

Vuong, Q., Roach, P. and Bowyer, M. (2013). Effect of extraction conditions on total phenolic
compounds and antioxidant activities of Carica papaya leaf aqueous extracts. School of
Environmental and Life Sciences, University of Newcastle.
https://www.sciencedirect.com/science/article/abs/pii/S2210803313000432

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