Professional Documents
Culture Documents
INTRODUCTION
In the recent years, the antimicrobial actions have received much attention. This is so because
of the increasing interest in human health and have been studied in vitro and in vivo by many
researchers. The natural antimicrobial agents protect living organisms from damages resulting in
antimicrobial properties that are supplied to human and animal organisms as specific
pharmaceutics. Although, much work has been done on the antimicrobial effects of different
plants species. It has been well known that essential oils and plant extracts have antimicrobial
effects.
This kind of Jasminum gathers more than 200 species originating from all the continents. The
species Jasminum sambac, probably originating in tropical India and Burma, is cultivated for its
perfume. Jasminum sambac is a persistent shrub, which often reaches 5 feet height in pots. The
sheets are persistent, green-beds, brilliant and opposite. The flowers are white. The flowers are
used in the manufacture of the perfumes and aromatizing. The plant is used in the form of liana
to decorate the hair and the neck in the form of collar. The traditional use of this plant suggests
tonic (uterine) effects. Essential oil of J. sambac is used as fragrance for skin care products.
Jasmine oil and absolute reduce skin inflammation, tones the skin and lifts up your mood.
1
Objectives of the Study
General Objective:
The study aims to evaluate the effectiveness of maceration method to draw out
Specific Objective:
• Compare the results of zones of inhibition between the Jasminum sambac extract
Null Hypothesis
2
Significance of the Study
researchers, medical students and patients. The result we gain from this study will provide
doctors new information regarding the emergence anti- microbial property that can be observe
from plant or plant materials Jasminum sambac extract thereby promoting the use and
prescription of organic herbal medications rather than of synthetic drugs. Researchers can
significantly benefit from this study since this can somehow provide idea and information
regarding the presence of antibacterial activity of Jasminum sambac extract. They can further
conduct, improve or modify a similar research. The result we gain from this study gives new
ideas to pharmaceutical companies to develop new drugs which is safer, more natural, and cost
efficient not only to consumers but also means financial gain to these companies. This study will
help the medical students provide knowledge with the use and properties of Jasminum sambac
extract. Students can now be able to apply what they learned and use them into practice in the
near future. This research would also promote the use of alternative medicine and become
actively involved in the field of research development. This would increase awareness regarding
the presence and effectiveness of the antibacterial activity of Jasminum sambac plant. Become
involve in promotion of the use of organic and alternative medicine rather than synthetically
prepared medicines which is more expensive, harder to prepare and may cause side effects.
3
Scope of Delimitation of the Study
This study focuses in determining the antibacterial activity of In vitro evaluation of crude
extract of Jasminum sambac on E. coli and S. Aureus based on the minimum inhibitory
concentration or (MIC). The effectiveness will be based on the ability of the extract to inhibit the
colonial growth reflected by the zone of inhibition of E. coli and S. areus after the administration
of the extract.
The study will be conducted in the Research and Microbiology laboratory of Remedios
Trinidad Romualdez Medical Foundation upon the preparation of bacteria, its cultivation and
preservation. Jasminum sambac will be obtained within the vicinity of Tacloban City.
4
Theoretical Framework
worldwide to discover new antimicrobial agents that are effective against the resistant microbial
pathogens. Introduction of plants with potential therapeutic values have been utilize for
preventing and treating various elements and food borne diseases. Flower extract and essential
oils are also considered to be potential natural antimicrobial agents. Available reports indicate
their efficacy and possess a broad spectrum of antimicrobial activity against various spoilage and
Introduction of antimicrobial
agents derived from plant
materials
From the information that was gathered from theoretical framework, we came up with a
conceptual framework of our study following the organization of theories. This represents the
predictive relationship among the different variables and how they correlate with each other.
Upon using S. aureus and E. coli this experimentation will provide us with the extract to inhibit
microbial growth. Upon reacting the extract to our prepared microbes it is appropriate in
reducing and inhibiting microorganism’s growth will determine how accurate and effective the
an(Nascimento and others 2000; Thaller and others 2010) foodborne illnesses can cause severe
health effects and can even lead to death among the residing population, especially in the
has prompted researchers’ world over to search for new antimicrobial agents that are more
effective against the resistant microbial pathogens Structural modification of the antimicrobials
(against which microbial resistance has been developed) is reported to improve the effectiveness
of antimicrobial agents against bacteria, fungi, and viruses. However, of late, research efforts
have been put forth to improve the effectiveness of antimicrobial drugs by developing novel and
a new class of antimicrobial drugs that can effectively work on multitargeted sites or organisms.
successfully utilized for preventing and treating various ailments and foodborne illnesses. Since
time immemorial, various plants and their products have been used in traditional medicine to
cure some of the common disorders and degenerative diseases in humans as well as in animals
(such as Ayurvedic and traditional Chinese medicinal practices). The effectiveness of these
procedures has been attributed mainly to the presence of active phytochemicals or bioactive
compounds in plants.
7
Given the scope of searching new antimicrobial agents, antimicrobials derived from plant
materials are often regarded as natural and safe compared to industrial chemicals. Of late, plant-
based medicine has become more popular due to the increasing concern of consumers with
regard to the use of synthetic chemical preparations and use of artificial antimicrobial
Some of the hoped-for advantages of using natural antimicrobials include: reducing total
preservation technology, and strengthening immune system in humans Today, growing market
trends indicate a rapid increase in the number of natural plant-derived products (such as green
tea, herbal decoctions, or herbal medicines) that may include aerial parts, seeds, fruits, roots,
rhizomes, and flowers. Among these, flowers have attained high priority and found various
applications. Floral extracts and their isolated essential oils are traditionally believed to be rich in
phytochemicals exhibiting rich bioactivity. These compounds are of interest to the local industry
as well as to the general population and are actively being explored for various commercial
applications (such as tea, bakery products, and more). Floral extracts and essential oils are also
considered to be potential natural antimicrobial agents. Available reports indicate their efficacy
and to possess a broad spectrum of antimicrobial activity against various spoilage and pathogenic
microorganisms, which is attributed to their bioactive constituents. Based on these facts, the
present review focuses mainly on providing baseline information on exploring some of the
common and wild (edible and nonedible) flowers possessing potential antimicrobial activities.
The details on these aspects are hopefully expected to be useful for the commercial exploitation
8
of flowers to develop natural preservative preparations with applicability in the food and
pharmaceutical industries.
Although it has been reported that Sampaguita has possible anti-microbial activity against
Staphylococcus aureus and Escherichia coli, the active components have not been isolated and
several other plants have been reported. In the isolation, purification and characterization of the
major bioactive components of the rhizomes of Ethlingeria elatior, the sample was prepared by
air drying. The crude extracts of the air dried samples were extracted using DCM as solvent and
isolated using column chromatography (Budoy et al., 2003). The antioxidant compounds of
avocado, was extracted by solvent extraction by using organic and aqueous solvents. The crude
extract from avocado was partitioned between hexane and chloroform. Purification of the
chloroform fraction was done by isocratic and gradient elution column chromatography (De Asis
and Espeso, 2003). Adoption of the above mentioned extraction and isolation methods can be
done to sampaguita to successfully isolate, purify and partially characterize the components
responsible for its antibacterial activity against Staphylococcus aureus and Escherichia..
Maceration became a popular and inexpensive way to get essential oils and other plant
healing properties to the people. With the appearance of pharmaceutical drugs in the late 1880’s,
homemade preparations like macerations fell out of favor. Little was written down and much of
the process was lost. The 1960’s saw a questioning of “modern practices” and a turning to more
natural ways of living. The pharmaceutical industry was held in suspicion and making your own
herbal preparations were revisited. Many looked to the Native Americans and other ancient
9
cultures to relearn or regain information about making these preparations. The old ways of
producing essential oils inexpensively were lost, but are now being rediscovered.
Maceration oils, also called infused oils, are carrier oils that have been used as a solvent
to exctract the therapeutic properties of a certain plant or plants. The base oils commonly used
are Olive or Sunflower and the process is quite simple because of microbiological infection
caused by wet herbs infusing in oil, it is recommended to use only properly dried herbs and
Pathogens
Firmicutes, and is frequently found in the nose, respiratory tract and on the skin.
Escherichia coli and Staphylococcus aureus, are among the most prevalent species of
10
CHAPTER III
METHODOLOGY
Research Design
present in the macerated extract of Jasminum sambac (Sampaguita) against selected species of
The study focused on the antibacterial agent present in the macerated extract of
Jasminum sambac. After thorough extraction, antimicrobial susceptibility testing will follow;
utilizing the Kirby-Bauer method or Disc Diffusion method and then measurement of zones of
inhibition will proceed. The investigation used 1 specie of gram (-) and 1 gram (+) pathogens
namely: Escherichia coli and Staphylococcus aureus because both are common pathogens to
human.
Jasminum sambac (Sampaguita) is acquired from the sampaguita vendors at Sto. Nino
church of Tacloban City. The plant sample was presented to DENR, Ecosystems Research and
11
Development Service Region 8, Tacloban City for verification and certification by Ms. Emma
M. Germano as the Senior Science Research Specialist The collected plants were then washed
Extract Preparation
Maceration Method
were washed with tap water and was dried as possible. The plant material was chopped finely to
break the plant cell walls and encourage more of the plant's oil-soluble compound to infused into
the base oil. It is placed into a sterilized airtight container until the plant material was covered
with olive oil. The dried plant matter and oil in an airtight container such as glass jar was placed
in a warm sunny location for up to three weeks, the sunshine gently heats the oil and extract
many of plant's properties. Every week or so, the macerated flower material were replaced so
that it will continue to extract more therapeutic properties into base oil. This mixture is allowed
After some time has passed the macerated plant material is removed and the oil is filtered
and strained of any remaining plant particles. The resulting oil is now infused with and contains
Test Organism
The study utilized gram (-) and gram (+) pathogens as test organisms for the
antimicrobial susceptibility testing. The pure stock cultures of microorganisms were obtained in
12
the Laboratory of Doña Remedios Trinidad Romualdez Medical Foundation with permission.
The test organisms include gram (-), Escherichia coli and gram (+), Staphylococcus aureus.
Laboratory Assay
Standardization of Inoculums
The cultured microorganisms were standardized using of densitometer following the 0.5
Mcfarland standard prior to antimicrobial susceptibility testing. The medium to be used for
antimicrobial susceptibility testing was the standard agar based medium, Mueller-Hinton Agar
(MHA).
The antibiotic used as a positive control was Penicillin and Ampicillin. The chosen
controls was based on the recommended antibiotics for the selected microorganisms. For the
Antibacterial Assay
Antimicrobial susceptibility testing was done utilizing the Kirby-Bauer method following
the extraction. 6mm-filter paper discs were soaked in the base oil of Jasminum sambac
(Sampaguita) prior to the testing. A total of 3 MHA plates per bacteria were used to occupy 3 of
the soaked discs per MHA plate leaving a total of 9 tests/discs for each plant extract. After
placing of the discs accordingly, the plates were incubated at 37C for 24 hours. Positive and
13
negative control per test bacteria were incubated together with the test sample. Observation and
Statistical Analyses
After the antibacterial assay, the mean the zone of inhibition were calculated. To check
the variance of the data gathered, the standard deviation from the means of each sample trial was
calculated. And from this calculation, a two-tailed independent group t-test was followed to
analyze and check the differences of data gathered from the antibacterial assay which includes
the test, negative and positive control. The choice of test also involves the disparity between the
postive control and negative control when compared to test. Formula and calculations of data are
14
Chapter IV
Result:
Table 1: Average zone of ohobition of Macerated oil extract from Jasminum sambac
aureus
(Value of 6mm is the diameter of disc used which is equal to zero of inhibition)
Table 1 presents the result of the Jasminum sambac macerated extract against gram
negative & gram positive test organisms based on their average zone of inhibition together with
15
the positive control Ampicillin (gram +) & penicillin (gram -) and negative control (distilled
water). It was observed that both Escherichia coli & Staphylococcus aureus showed no
difference with mean zone of inhibition of 6mm on macerated oil extract. In the both positive
Table 2: Summary of computed t-value of test organisms between the macerated extract of
Average zone
of inhibition
Category Test (-)control SD DF Tabular Computed Interpretation
(distilled
water) (Test) Value t-value
A 6 6 0 58 2.0017 0 NS
B 6 6 0 58 2.0017 0 NS
Where:
A= Comparison between macerated extract and the negative control using S. aureus
B= Comparison between macerated extract and the negative control using E. coli
S = Significant
NS= Not Significant
16
Table 3: Summary of computed t-value of test organisms between the macerated extract of
Average zone of
inhibition
Category Test (+) control SD DF Tabular Computed Interpretation
(Ampicillin
& Penicillin) (Test) Value t-value
A 6 30 0 58 2.0017 0 NS
B 6 32 0 58 2.0017 0 NS
Where:
A= Comparison between macerated extract and the positive control (Ampicillin) using S. aureus
B= Comparison between macerated extract and the positive control (Penicillin) using E. coli
S = Significant
NS= Not Significant
Table 2 & 3 summarizes the comparison between the macerated extract of Jasminum
sambac against Negative & positive control using test organisms. Using two-tailed paired t-test,
it was determined that their was no significant difference between the computed t-values of
macerated extract against our positive controls (Ampicillin & Penicillin) as seen in the table.
Compared to the positive & negative control the mean zone of inhibition using macerated extract
17
on both E.coli & S.aureus yielded a computed t-value of zero. Since these computed t-values are
zero, therefore the results are considered not significant. Further, it implies that our macerated
Discussion:
The sampaguita flowers bloom either singly or as bundles of blossoms at the top of the
branches. Blooming all through the year, sampaguita are pure white, small, dainty, star-shaped
blossoms. The flowers open at night and wilt less than a day.
Sampaguita’s distinct sweet, heady fragnance is its unique feature. The essential oil from
the flowers is similar jasmine (Jasminum grandiflores). Sampaguita flowers do not bear seeds,
therefore the plant is cultivated by cuttings. Sampaguita was imported into the Philippines in 7th
century from Himalayan areas. The Sampaguita is a native part of the Philippine landscape for
centuries. The plant is originally from India and is grown throughout India today. About eight
cultivars are generally listed for Sampaguita. Some varieties Sampaguita can grow as large as
parturient, uterine etc. The Jasminum sambac is used for removing intestinal worms and is also
used for jaundice and venereal diseases. The flower buds are useful a treating ulcers, vesicle,
boils, skin disease and eye disorders. The leaves extracts against breast tumours. The leaves are
antiseptic and are useful for wounds and acne when used as a poultice. Drinking Jasmine tea
18
The dried flowers of Jasminum sambac are used by the Chinese to flavour Jasmine tea.
Jasmine tea is most commonly consumed with or after meals as a digestive aid. It aslo used for
making perfumes, creams, shampoos, soaps and incense. Its flowers are used to flavour Jasmine
flavonoid, terpines, tannin, resin and salicylic acid. Study showed all extracts with antimicrobial
activity against pathogen, scoring highest with S.typhi and lowest with S.aureus. the study
The test organisms utilized in this study, Escherichia coli and Staphylococcus aureus,are
among the most prevalent species of gram-positive and gram-negative bacteria, respectively that
induce mastititis.
Thus, this present study aims to evaluate if there is antimicrobial activity exhibited by
the macerated extract of Jasminum sambac. The extraction of said compounds can only be done
by using the right method as the components of phytochemicals can only be extracted upon the
After that thorough extraction, testing antimicrobial property followed which utilized the
modified Kirby-Bauer method or disc diffusion method. A standard 6-mm whatmann filter paper
disc were soaked in the extract and the other for the negative control were soaked in distilled
water. Penicillin and Ampicillin disc was the choice of the antibiotic as the positive control. The
test were inoculated on to the surface of the Mueller-Hinton Agar followed by the addition of
negative control, positive control and the sterile paper disc containing the plant extract. It was
incubated for 24 hours at 37C. The inhibition of microbial growth was indicated by a clear area
19
(zone of inhibition) around the disc. The zone of inhibition was measured to its diameter which
is expressed in millimeter.
The utilization of standard analytical quantization technique was employed to obtain their
statistical values and somehow to determine their significant difference between the mean of
macerated extract, positive and negative control based on the zone of inhibition. The one-sample
independent t-test was utilized, a summarized in Tables 2, 3, and 4, respectively. It showed that
all result from using macerated Jasminum sambac extract as antibacterial have statistical
similarity when compared to negative control. This means that macerated Jasminum sambac
extract do not possess potent antibacterial properties against the two test organisms used in the
study. However, upon comparing the test result against that of the positive control (Ampicillin
and Penicillin), a high statistical difference was conveyed, therefore the macerated Jasminum
sambac extract is not potent to be used as antibiotic to Escherichia coli and Staphylococcus
aureus.
20
CHAPTER V
Conclusions
Based on the zone of inhibition obtained using maceration method for the extraction of
Jasminum sambac (Sampaguita) against the gram negative and gram positive test organisms, it
was found out that Escherichia coli and Staphylococcus aureus exhibited no average zone of
inhibition of 6mm.
Among the test organisms that were used, it was found out that Jasminum sambac extract
showed no bactericidal effect on both Staphyloccocus aureus and Escherichia coli in terms on the
Recommendations
The researchers recommend that further test on antimicrobial property from Jasminum
It is also highly recommended exploring other means of extraction that would enable the
extract to express its beneficial property at its best and to properly isolate the active components
21
Bibliography
Betty A. Forbes, Daniel F. Sahm, Alice S. Weissfel. Bailey and Scott’s Diagnostic Microbiology,
Twelfth Edition, Morsby Publishing. P157
Hasina Yasmi1, Md. Abdul Kaisar2, Md. MoklesurRahman Sarker3, Mohammed Shakifur
Rahmman4 and Mohammad A. Rashid (2009). Preliminary anti-bacterial activity of some
indigenous plants of Bangladesh, dhak. Univ. J. Pharm. Sci. 8(1):61-65.
John Bernard Henr, M.D. Clinical Diagnosis and Management by Laboratory Methods,
Nineteenth Edition. W.B SAUNDERS COMPANY. Part 6, Medical Microbiology,
p. 1152-1153
Nimri, LF; Meqdam, MM and Alkofahi, A (1999). Antibacterial activity of Jordanian medicinal
plants. Pharmacologycal Biology. Vol. 37(3), 196-201
Websources:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2792499/
https://en.m.wikipedia.org/wiki/Minimum_inhibitory_concentration-Minimum-inhibitory
concentration
https://cvi.asm.org/content/11/3/463.full
https://www.stuartxchange.com/Sampaguita
https://everything-lavender.com/methods-of-producing-essential-oils.h
https://www.airase.com/extraction-methods-for-essential-oils/
https://www.ethnoleaflets.com/leaflets/naveen.html
https://www.naha.org/explore-aromatherapy/about-aromatherapy/how-are-essential-oils-
extracted
22
APPENDICES A
Escherichia coli
EXTRACT
(Penicillin)
23
Staphylococcus aureus
24
APPENDICES B
Step 1: Hypothesis
𝑯𝒐 : 𝑿𝟏 = 𝑿𝟐 (There is no significant difference in the average zones of inhibition
between the test organisms)
𝑯𝑨 : 𝑿𝟏 ≠ 𝑿𝟐 (There is a significant difference in the average zones of inhibition
between the test organisms)
Step 4: Critical Value: These is derived from table of critical values. The critical value of t-
distribution 3+3-2 degrees of freedom is 2.000
Step 5: Formulas:
𝟐
∑(𝑿 − 𝑿𝟐 )𝟐
𝑺 =
𝒏−𝟏
𝑿𝟏 − 𝑿𝟐
𝒕=
(𝒏𝟏− 𝟏)𝒔𝟏 𝟐 𝒏𝟏+𝒏𝟐
√[ 𝒏𝟏+𝒏𝟐 −𝟐
][
𝒏𝟏𝒏𝟐
]
25
Legend:
𝑿𝟏 = mean of sample 1
𝑿𝟐= mean of sample 2
𝒏𝟏= number of subjects in sample one
𝒏𝟐= number of subjects of sample two
𝒔𝟏= standard deviation of sample one
𝒔𝟐= standard deviation of sample two
26
APPENDICES C
CALCULATION
I. Variance
Jasminum sambac Macerated extract (mm)
(𝑿𝟏 − 𝑿𝟐 ) (𝑿𝟏 − 𝑿𝟐 )
E.coli S.aureus
X X
1 6 0 6 0 6 32 30
2 6 0 6 0 6 32 30
3 6 0 6 0 6 32 30
𝑋1 6 0 6 0 6 32 30
∑ 0 0 0 0 0
𝑆2 0 0 0 0 0
27
I. Variance/ Standard Deviation
A. Macerated extract using Escherichia coli
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2
B.Maceration extract using Staphylococcus aureus
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2
C.Positive Control (Penicillin & Ampicillin)
∑(𝑋1− 𝑋2 )2 0 0
𝑆2 = = = =0
𝑁−1 3−1 2
2
∑(𝑋1− 𝑋2 )2 0 0
𝑆 = = = =0
𝑁−1 3−1 2
II. T-test Calculations
Escherichia coli
Sample x (Zone of inhibition) ∑(𝑋1− 𝑋2 )2 𝑆2
Macerated extract 6 0 0
Positive Control 32 0 0
Negative Control 6 0 0
28
A. Macerated extract (𝑿𝟏 )vs Negative Control (𝑿𝟐 )
𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2
6−6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)
0
𝑡= 0 6
=0
√[ ][ ]
4 9
𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2
32 − 6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)
26
𝑡= 0 6
=0
√[ ][ ]
4 9
29
Staphylococcus aureus
Sample x (Zone of inhibition) ∑(𝑋1− 𝑋2 )2 𝑆2
Macerated extract 6 0 0
Positive Control 10 0 0
Negative Control 6 0 0
𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2
6−6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)
0
𝑡= 0 6
=0
√[ ][ ]
4 9
30
B. Positive Control (𝑿𝟏 ) vs Macerated extract (𝑿𝟐 )
𝑋1 − 𝑋2
𝑡=
(𝑛1 −1)𝑠1 2 (𝑛1− 1)𝑠2 2 𝑛1+𝑛2
√[ ][ ]
𝑛1+𝑛2 −2 𝑛1𝑛2
10 − 6
𝑡=
(3−1)0(3−1)0 3+3
√[ ][ ]
3+3−2 (3)(3)
4
𝑡= 0 6
=0
√[ ][ ]
4 9
APPENDIX D
COMMUNICATION LETTERS
31
APPENDIX E
DOCUMENTATION
32