Acta. Chim. Asiana.

, 2024, 7(2), 517-525

e-ISSN/p-ISSN 2550-0503/2550-049x 517
Available online at: [Link]

RESEARCH PAPER

Physical test and irritation test of water guava leaf extract (Syzygium
aqueum) as a natural spray hand sanitizer
,a a
Sri Winarni Sofya* , Lalu Busyairi Muhsin

[a] Department of Pharmacy, Bumigora University, Jl. Ismail Marzuki No.22, Cilinaya, Mataram, Indonesia
*
E-mail: winarni@[Link]

DOI: 10.29303/aca.v7i2.212

Article info: Abstract: This study explores the manufacture of water guava
(Syzygium aqueum) leaf extract-based hand sanitizers as an effective
Received 19/08/2024 and safe natural alternative for maintaining hand hygiene. Given the
importance of hand hygiene in preventing the spread of disease,
Revised 26/09/2024
especially amid the COVID-19 pandemic, the use of hand sanitizers
Accepted 05/10/2024 has increased significantly. However, alcohol-based products are often
irritating to the skin. Therefore, this study aims to evaluate the
Available online 30/10/2024 potential of water guava leaf extract to improve the effectiveness and
convenience of hand sanitizers. Water guava leaves contain active
compounds such as flavonoids, tannins, and phenolics with
antibacterial and anti-inflammatory properties. The research method
includes extracting water guava leaves, hand sanitizer formulation,
and organoleptic, pH, homogeneity, and irritation testing. The tests
carried out were physical tests, including organoleptic tests (shape,
smell, and colour), pH, homogeneity, spreadability, adhesiveness, and
product irritation tests. The research was designed with a posttest-only
control group design with true experimental by analyzing the test
object after being given treatment. This research is expected to
produce products that are useful for the community. The results
showed that all hand sanitizer formulas tested had a pH within the safe
range of pH 5 for the skin without irritating the skin of the test animals.
Formulas with added guava extract showed improved aroma and anti-
irritation, with formulas F1 and F2 gaining the highest preference in
aroma and colour. This study concludes that water guava leaf extract
can be effectively used in the manufacture of hand sanitizers,
providing a gentler and skin-friendly alternative to alcohol-based
synthetic products.

Keywords: Syzygium aqueum, extract, hand sanitizer, flavonoids,

irritation

Citation: Sofya, S. W., & Muhsin, L. B. (2024) Physical test and irritation test of water guava leaf
extract (Syzygium aqueum) as a natural spray hand sanitizer. Acta Chimica Asiana, 7(2), 517–525.
[Link]

INTRODUCTION [4]. Bacteria are a group of single-celled

microorganisms with prokaryotic cell forms
World Health Organization (WHO) data states [5][6]. As living things, bacteria have genetic
that most diseases are transmitted through information in the form of DNA, but bacteria
hands [1]. Even in 2019, the WHO said that are not located in the nucleus or nuclear
the spread of COVID-19 was mostly membrane [7]. Bacterial DNA consists only of
transmitted through human-hand interaction exons and has no intron. Bacteria have long,
[2]. Diseases are generally caused by bacteria circular cells and are called nucleoids [8][9].
and viruses that attack the human body's Bacteria also have extrachromosomal DNA
immune system [3]. Based on research, incorporated in small circular plasmids [10].
bacteria are the main cause of human disease
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Commercial 4.0 International License
 Acta. Chim. Asiana., 2024, 7(2), 517–525 518

Staphylococcus aureus bacteria is one

of the most common bacteria found in the
human environment [11][12]. According to
Juliantina, Staphylococcus aureus bacteria are
arranged in groups, similar to grapes, and are
round or oval [13][14]. Staphylococcus aureus
bacteria are Gram-positive bacteria that do not
move and do not have spores. This group is
formed because daughter cells tend to be near
their parent cells when dividing [15]. One of
the diseases caused by Staphylococcus
aureus bacteria is diarrhoea and skin
diseases; this disease is experienced by many
people in general [16]. One way to minimize
the effects of bacteria is by using antibacterials Figure 1. Water guava plant
[17]. Antibacterials are substances that can kill
or suppress the growth and reproduction of
bacteria [18]. Antibacterials usually come from The classification of Syzygium aqueum is as
chemical synthesis, better known as follows: Kingdom: Plantae; Clade:
antibiotics. However, overuse of antibiotics Tracheophyta; Clade: Angiospermae; Clade:
causes resistance and side effects such as Eudicotyledons; Order: Myrtales; Family:
diarrhoea, allergies, and other toxic hazards Myrtaceae; Genus: Syzygium; Species: S.
[19]. Due to these side effects, treatments aqueum. Syzygium aqueum extract contains
using natural materials are becoming more tannins that have antibacterial activity against
popular because natural materials have S. aureus, E. coli, and P. aeruginosa as well
advantages that are not associated with side as antifungal activity against A. niger, C.
effects [20]. The problem in this study is albicans, and gram-positive S. aureus [11].
whether the plant extract is effective as a Water guava leaf, also known as Syzygium
stable antibacterial and anti-irritant material for aqueum, is one of the plants that has the
use on the skin. potential to be used in medicine, especially as
an antibacterial [23]. The secondary
One of the most common ways to metabolites in the ethanol extract of water
maintain hand hygiene is hand washing using guava leaves contain flavonoids, terpenoids,
antiseptic soap [20]. However, soap is and tannins [23]. Based on research on water
ineffective because it requires a lot of water to guava leaves, the leaf content has
wash hands, but this problem can be solved pharmacological effects, one of which is
with waterless handwashing products called antibacterial activity [24]. Water guava leaves
antiseptic hand sanitizers or hand sanitizers or Syzygium aqueum contain various active
[21]. Hand sanitizer is useful because it kills or compounds that are beneficial for health and
reduces the number of infection-causing make them an ideal choice for use in hand
bacteria quickly, conveniently, and practically sanitizer formulations. Guava leaves include
[21]. Preparations that can be used as an flavonoids, tannins, and phenolics which are
alternative to reduce bacteria are also known to have antibacterial and anti-
affordable and can be carried anywhere [16]. inflammatory activities. Flavonoids function by
However, using hand sanitizers from excess forming complexes with extracellular and
synthetic chemicals can cause skin disorders. soluble proteins, damaging bacterial cell
Therefore, it is necessary to make hand membranes, and inhibiting pathogenic
sanitizers from natural ingredients. Research microorganisms growth.
on making hand sanitizers from natural
ingredients is in line with Indonesia's biological Conversely, tannins work by
wealth [10]. In Indonesia, many plants contain constricting bacterial cell walls, disrupting cell
antimicrobial chemical compounds [16]. Water permeability, and inhibiting bacterial biological
guava leaves are one of them; water guava activities, resulting in bacterial death[18][20].
leaves have long been used as medicine to Phenolic compounds can inactivate bacterial
treat skin pain, bloating, loose stools, and enzymes and damage protein structures and
diarrhoea [11][12]. The classification of the cell membranes, contributing to antibacterial
water guava plant can be depicted in Figure 1. effectiveness[10]. In addition to antibacterial
properties, water guava leaf extract offers anti-
inflammatory and antioxidant benefits, which
can help reduce inflammation and maintain

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skin moisture balance. It is especially Methods

important in hand sanitizer products, as it can
The research methods are scientific ways to
reduce the risk of irritation and dryness often
obtain data for specific purposes and benefits
caused by excessive alcohol use. By
[19]. Researchers used quantitative research
harnessing this wealth of compounds, water
methods to provide a researcher's perspective
guava leaf extract not only improves the
on the problem under study. Extraction
effectiveness of hand sanitizers in killing
research, physical tests, and irritation tests of
germs but also ensures that the product is
Jambu air (Syzygium aqueum) leave as a
gentle and does not damage skin health,
natural spray hand sanitizer designed with
making it suitable for regular use.
Posttest Only Control Group Design is a true
With the increasing awareness of the experiment by analyzing the test object after
importance of hand hygiene in preventing the being given treatment [20].
spread of diseases, hand sanitizer has
The research begins with identifying
become one of the most widely used practical
water guava leaf plants; water guava leaves
solutions[14]. However, with so many products
that have been obtained are then dried in the
in the market, consumers are increasingly
sun or dried to dry; drying is done to reduce
concerned about the effectiveness, safety, and
the water content contained in water guava
impact of the ingredients used. One of the
leaves. The next step is making simplisia,
natural ingredients increasingly attracting
which results from grinding water guava leaves
attention in hand sanitizer formulations is
into powder. The water content test was
water guava leaf extract. Water guava leaf,
carried out on the simplisia to determine the
known in the herbal world for its diverse
water content in the simplisia. Furthermore,
properties, offers tremendous potential as an
the powdered simplisia was extracted using
additive in hand sanitizer products. With its
the maceration method with 70% ethanol
antioxidant content and anti-inflammatory
solvent and obtained water guava leaf extract.
properties, this extract can provide additional
benefits such as reducing irritation and
Physical Test
maintaining healthy skin. This article will
discuss the process of making hand sanitizer The physical test of water guava leaf extract
from water guava leaf extract, reveal the as a natural hand sanitizer begins with
advantages and benefits of using this natural organoleptic tests (shape, smell, and colour),
ingredient, and how proper formulation can pH, homogeneity, spreadability, adhesiveness,
improve the effectiveness and convenience of and protective power carried out on the
the product. preparation. pH testing using pH paper is a
The current research focuses on simple and effective method to determine the
making natural hand sanitizers using water acidity or alkalinity of a solution. The pH paper
guava leaves by looking at their effectiveness is treated with indicators that change colour
and safety, including phytochemical screening, when immersed in a solution, allowing for a
physical tests, and product irritation tests. For visual comparison against a standard pH
many years, metals have been crucial to scale. To conduct the test, a strip of pH paper
medicine. In varied amounts, many are is dipped into the solution for a few seconds,
necessary in and the resulting colour is matched to the
scale to determine the pH value. This method
MATERIALS AND METHODS is widely used in laboratories, educational
settings, and household applications, providing
Materials quick and reliable results for various solutions.
This test aims to determine the effectiveness
Syzgium auqeum leaves were collected in of the hand sanitizer preparation produced
Penujak village, Central Lombok Regency, [13][14].
West Nusa Tenggara. The plant was identified
at Bumigora University. The materials required Irritation Test
are Alcohol Denat, Glycerine, Fragrance,
Triethanolamine, Acrylates/C10-30 Alkyl The irritation test was carried out by the Draize
Acrylate Crosspolymer, Sygium Aqueum test, originally developed to assess the
Extract, Aqua Demineralisata and three irritancy of substances, involves testing on
Rabbits as an animal test. The practical tools rabbits to evaluate the severity of irritation
that are used are a Rotary Evaporator, Water caused by chemicals applied to the skin or
bath, Ph meter, and extraction tools. eyes. This method measures the extent of
damage and irritation, providing valuable data

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on the safety of various products and essential oils with antimicrobial properties,
chemicals[15]. Despite its historical further contributing to the fruit's protective
significance, the test has faced criticism for benefits. Moreover, saponins are compounds
ethical concerns regarding animal welfare, that can disrupt bacterial membranes, leading
leading to the exploration of alternative to cell death. Quercetin, another flavonoid
methods for irritation assessment. The first found in guava, has demonstrated antibacterial
step taken was to divide the rabbit group in a activity against several pathogens. Research
ratio of 1: 1, namely as control animals and suggests that these compounds collectively
animal tests [15]. Treatment performed on the make guava effective against various bacteria,
animal test is to shave the rabbit's back hair including those responsible for foodborne
until clean. After that, the animal's back is illnesses and infections.
divided into four square sections, each The weight of syzgium aquem extract
measuring 1 by 1 inch [14][24]. Before produced in this study is 45 grams, calculated
applying the test material, the rabbit's skin was with the weight of raw materials using the yield
cleaned with a cotton swab soaked in water. formula, it produces a 6% yield with 750 grams
The results of the hand sanitizer product were of raw materials. Every plant that is grown in a
applied to gauze and then attached to the different location and with a different
rabbit's back for 3 x 24 hours. Then, the environment may contain different chemical
animal tests were returned to their cages and composition. Uniformity in the production of
examined the results again the next day. After herbal medicines is required to guarantee the
that, symptoms were observed as initial quality, effectiveness, and formulation of
irritation in the form of neuritis and swelling at herbal ingredients in compliance with
hours 24, 48, and 72 or for 3 days [14][16]. established standards [20]. A key element in
Based on the initial irritation test, irritation is establishing the calibre and purity of the
calculated as swelling and erythema. The finished product is standardization, a
irritation test formula can be depicted in Figure procedure for gauging the potency of herbal
2. remedies [21]. Standardization aims to
produce herbal plant extracts that are widely
utilized for formal health services and self-
medication and whose quality, efficacy, and
safety are ensured through scientific testing.
The antibacterial effect of ethanol
extract from water guava leaves (Syzygium
aqueum) against clinical isolate bacteria is
Figure 2. Eritema and Edema Scoring Formula. caused by active compounds that are soluble
in ethanol. These compounds are thought to
RESULTS AND DISCUSSION include flavonoids, phenolics, and tannins.
This research is green economy research Flavonoids work as antibacterials by forming
based on the use of natural materials in the complexes with extracellular and soluble
form of plants, which are very easy to obtain proteins, thereby damaging the bacterial cell
and can be used to make very useful items. membrane and causing the release of
Based on the description of the problem and intracellular compounds [16]. Phenolic
urgency obtained, research into making compounds, according to Singh and Bharate
natural hand sanitizer from water guava leaves (2005), inhibit bacterial growth by inactivating
needs to be done to answer today's proteins (enzymes) in the cell membrane.
challenges. It is hoped that this research will Phenol binds to proteins through hydrogen
become the basis for further research to bonds, damaging the protein structure
produce natural hand sanitizer products that because most bacteria's cell walls and
are effective and easy for the general public to cytoplasmic membranes consist of proteins
obtain. Syzgium auqeum is known for its rich and fats. Damage to the cell wall and
composition of active compounds that exhibit cytoplasmic membrane disrupts the function of
notable antibacterial properties. Among these, selective permeability, active transport, and
phenolic compounds such as flavonoids and control of protein composition, which ultimately
tannins have been shown to effectively inhibit causes leakage of macromolecules and ions
the growth of various bacteria. Additionally, from the cell. As a result, bacteria lose their
vitamin C, recognized for its antioxidant shape and undergo lysis. Tannins work as
effects, may also enhance antibacterial antibacterials by shrinking the bacterial cell
activity. Extracts from guava leaves contain wall, disrupting cell permeability, and inhibiting
its life activities, resulting in bacterial
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death[18]. Sari and Sari (2011) stated that contain 8% Alcohol Denat, 2% Glycerine, 1%
tannins target cell wall polypeptides, forming Fragrance, 2% Triethanolamine, and 1%
imperfect cell walls and ultimately causing Acrylates/C10-30 Alkyl Acrylate Crosspolymer,
bacterial cell lysis due to osmotic or physical while Aqua Demineralisata is used as much as
pressure so that the bacteria die. 60 ml in each formula. The difference lies in
All hand sanitizer formulas (F0, F1, the Sygium Aqueum extract, which varied from
F2, F3) have almost the same ingredient 0% in F0 to 1% in F3. The hand sanitizer
composition, except for Sygium Aqueum formula can be described in Table 1.
extract and Aqua Demineralisata. All formulas

Table 1. Formula Handsanitizer

Ingredients F0 F1 F2 F3
Alcohol Denat 8 8 8 8
Glycerine 2 2 2 2
Fragrance 1 1 1 1
Triethanolamine 1 1 1 1
Acrylates/C10-30 Alkyl acrylate Crosspolymer 1 1 1 1

Sygium Auqeum Extract 0 0.1 0.5 1

Aqua Demineralisata Add up to Add up to Add up to Add up to
60 ml 60 ml 60 ml 60 ml

Hand sanitizers that are made with natural showed increasing turbidity along with the
ingredients tend to be gentler on the skin and addition of Sygium Aqueum extract. The
have an aromatherapy effect. NaturaI aroma in F0 was dominated by alcohol with a
ingredients like the extract of sygium aqeum, hint of fragrance, while F1 to F3 showed an
and another herbal extract can help keep hand increase in leaf extract aroma, with F3 having
skin moisturized and healthy, reducing the risk the most dominant aroma. All formulas had
of dryness and irritation that often occurs with cool and non-sticky properties on the skin,
alcohol-based products that can reduce the consistent in user experience. From the
risk of allergies and irritation. Natural organoleptic test results by respondents, F1
ingredients often contain fewer additives and received the highest preference from 3
preservatives that can cause allergies or respondents in terms of colour, while 2
irritation. It makes them a better choice for respondents preferred F2. F0 and F3 had no
individuals with sensitive skin or allergic clear preference. For aroma, F1 and F2 were
reactions to synthetic chemicals. Sygium each favoured by 2 respondents, while F0 was
aqeum leaf contains antimicrobial only favoured by 1 respondent, and F3 did not
effectiveness that can kill bacteria and viruses. get a preference. Regarding properties, F1
While they may not be as powerful as alcohol and F2 were also favored by 2 respondents,
in killing germs, combining several natural indicating a better user experience in terms of
ingredients can provide effective protection. non-stickiness and cooling effect. The
Organoleptic test results revealed that organoleptic test can be described in the table
all formulas were clear in colour, but F1 to F3 2.

Table 2. Organoleptic Test

F0 F1 F2 F3
Colour Clear Slightly cloudy Slightly cloudy Dark green
clear clear
Aroma The smell of The smell of The smell of The smell of extract
fragrance mixed fragrance mixed fragrance mixed
with alcohol with extract with extract
Nature Cold not sticky Cold not sticky Cold not sticky Cold not sticky

The organoleptic test results for the natural showed a slightly cloudy but still clear colour,
hand sanitizer showed marked variations in while F3 had a darker dark green colour. The
some important aspects of the product. aroma of each sample was also different: F0
Sample F0 had a clear colour, while F1 and F2 had a mixed odour between fragrance and

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alcohol, F1 and F2 created a mixed odour of Maintaining these qualities helps ensure that
fragrance with extract, and F3 had a more the hand sanitizer is effective in killing germs
dominant extract odour. All samples, although and provides a pleasant and discomfort-free
different in colour and scent, had the same user experience, ensuring that the benefits of
properties in terms of texture, i.e. they all felt guava leaf extract, such as anti-irritant and
cool and non-sticky when used. These data antioxidant properties, are optimized.
show that despite differences in colour and The next test is the homogeneity test.
scent, all samples have consistent texture Homogeneity testing in natural hand sanitizers
characteristics, which is important for user is an important step in ensuring that the
experience. product has an even consistency and quality
Maintaining the scent, shape, and throughout the production batch. In natural
spray properties of hand sanitizer made from hand sanitizers, which often contain
water guava leaf extract is essential to ensure ingredients such as alcohol and essential oils,
a pleasant and effective user experience. The a homogeneity test helps to ensure that all
scent of the hand sanitizer, which is influenced components are well mixed and that there is
by water guava leaf extract, not only affects no separation or unevenness. By conducting
the pleasure of use but can also provide a this test, the researcher identify if any issues in
calming effect thanks to the natural properties the mixing or formulation process could affect
of the extract. In addition, the hand sanitizer's the effectiveness and safety of the product.
spray form affects the product's distribution The results of the homogeneity test ensure
and application; with guava leaf extract having that each application of hand sanitizer will
certain characteristics, it is important to ensure have a consistent concentration of active
that the spray form remains consistent to ingredients, which is important for effectively
spread evenly and effectively during use. killing germs and ensuring that the product
Spray properties also affect practicality and meets established quality and safety
convenience, ensuring the product is easy to standards. The homogeneity test can be
use without leaving unwanted residue. described in Table 3.

Table 3. Homogeneity Test

Homogeneity F0 F1 F2 F3
textures soft soft soft soft
grain No coarse grains No coarse grains No coarse grains No coarse grains
absorbing speed Quick Quick Quick Quick
color Clear Clear Clear Clear
and colour, supporting product quality and
The results of the Ph test are described in consistency. The pH test showed that all
Table 4. formulas had a pH between 5 to 7. F0 had a
higher pH of 7, while F1, F2, and F3 had a
lower pH, around 5, indicating that the addition
Table 4. Ph Test of Sygium Aqueum extract slightly lowered the
F0 F1 F2 F3 pH, making it more acidic than F0. pH test can
Ph test
7 5 5 5
be captured in Figure 3.
The composition of natural guava leaf
The homogeneity test results for natural hand extract in hand sanitizer plays an important
sanitizers showed that all samples, namely F0, role in keeping the pH of the product balanced
F1, F2, and F3, met the same criteria in and skin-friendly. Water guava leaf extract is
several important aspects. The texture of all known to have acidic properties that can help
samples was identified as soft, with no coarse balance the pH of the hand sanitizer, bringing
grains that could affect the comfort of use. The it closer to the natural pH of human skin, which
absorption speed of each sample was also ranges. Keeping the pH of hand sanitizer at
consistent, with all showing the ability to the right level is essential for skin health, as an
absorb quickly on the skin. In addition, the unbalanced pH can disrupt the skin's natural
colour of all samples remained clear and protective layer and cause irritation or
showed no differences, ensuring a uniform damage. Human skin has a naturally slightly
appearance across production batches. Thus, acidic pH, usually ranging from 4.5 to 5.5,
these data indicate that the natural hand which acts as a barrier to protect against
sanitisers tested have good homogeneity, with infection and maintain moisture. If the pH of
no significant variations in texture, absorption,
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the hand sanitizer is too high or too low, it can as irritation or dermatitis. Therefore, keeping
disrupt this balance, causing the skin to the pH of hand sanitisers within a safe range
become dry, itchy, or even inflamed. Hand and in line with the skin's natural pH is a
sanitizers with the right pH help ensure that crucial step to ensure the product effectively
the skin remains healthy and protected after kills germs and gently and harmless to the
use and minimize the risk of side effects such skin.
F0 F1 F2 F3

Figure 3. Ph Test

Table 5. Irritation Test 3x24 Hours

Irritation Test F0 F1 F2 F3
Erythema No No No No
Edema No No No No
Result No irritation No irritation No irritation No irritation

The irritation test showed no signs of erythema or by active ingredients such as alcohol. When
or irritation on the skin of the animal test skin is exposed to harsh cleansing ingredients,
(rabbits), indicating that all formulas were safe water guava extract works to neutralize those
to use and did not cause irritating reactions. negative effects by helping to maintain
The result of the irritation test is described in moisture balance and reduce redness or
Table 5. dryness. By adding water guava extract to the
In conclusion, F1 and F2 appeared to hand sanitizer formula, manufacturers ensure
be preferred over F0 and F3, especially in that the product is effective in killing germs and
aroma and colour, with F1 showing the most maintaining the health of the user's skin,
positive results overall. The addition of Sygium making it more convenient to use regularly
Aqueum extract increased the turbidity and without compromising skin condition.
changed the scent of the hand sanitizer, with
formulas containing higher extracts (F2 and CONCLUSION
F3) providing a more pleasant scent to some The study concludes that hand-borne
respondents. The addition of extracts affected diseases, such as those caused by bacteria,
pH and lower pH formulas may be more are a significant health concern. Hand
suitable for maintaining skin pH balance. All sanitizers are a practical solution to eradicate
formulas were found to be safe and non- these bacteria, but synthetic chemical-based
irritating, so they can be used without risk of products often cause side effects. To address
skin irritation. The irritation test can be shown this issue, this study aimed to develop a
in Figure 4. natural hand sanitiser from water guava leaf
The use of water guava extract as an extract with antimicrobial compounds such as
anti-irritant ingredient in hand sanitizers is very tannins and saponins. The study involved
important as it can help reduce the risk of physical and irritation testing of the product
irritation and increase the comfort of using the and statistical analysis to determine the
product. Water guava extract has anti- effectiveness and safety of the hand sanitizer.
inflammatory and antioxidant properties that Based on the results obtained, all tests
can soothe the skin and reduce inflammation, responded well to both the physical test and
making it an ideal choice to address potential the irritation test, and it can be concluded that
irritation caused by frequent hand sanitizer use
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the hand sanitizer from water guava extract is suitable for use.
F0 F1 F2 F3
Time

1x24
hour
s

2x24
hour
s

3x24
hour
s

Figure 4. Irritation test with 3 animal tests (3x24 hours)

[5] Magani AK, Tallei TE, Kolondam BJ. Uji

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Special thanks to the Ministry of Education and terhadap Pertumbuhan Bakteri
Culture through the PDP program and thanks Staphylococcus aureus dan Escherichia
to all those who have helped in the success of coli. Jurnal Bios Logos. 2020 Jan
this research. 25;10(1):7-12.
[6] Mulqie L, Suwendar S, Choesrina R,
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