IC-GWBT2012, Ahmad Dahlan University, March 23-24, 2012

ANTIBACTERIAL ACTIVITY TEST OF 70% ETHANOL

EXTRACT OF ROSELLE CALYX FLOWER PURPLE (Hibiscus
sabdariffa L.) AGAINTS Salmonella typhi In vitro AND ITS
CHROMATOGRAPHY PROFILE
Eriska Nofriani, Dwi Suhartanti
Ahmad Dahlan University Faculty of Pharmacy
Yogyakarta

Abstract
Rosella (Hibiscus sabdariffa. L) is one of the plants that have antibacterial activity. Antibacterial
activity is caused by the presence of compounds such as anthocyanins, flavonoids, saponins, and
tannins. The pupose of this study to determine the antibacterial activity of 70% ethanol extract of
roselle calyx purple flowers against Salmonella typhi by liquid dilution method and sterilization stages
(Tyndalization). The compounds in the roselle flower extracted using maceration method with 70%
ethanol solvent. Screening phytochemical compounds conducted on anthocyanins, polyphenols,
flavonoids, saponins, and tannins. Antibacterial activity levels seen from the value of the Minimum
Inhibitory Concentrate (MIC) and Minimum Bacterisid Concentrate (MBC). The results indicate
screening phytochemcal compounds anthocyanins, flavonoids, saponins, and tannins are thought to
have antibacterial activity. Microbiological test results obtained from MBC of the sterilization process
with Tyndalization method of 1,75% w/v. While the MIC can not be determined because the extract of
purple-black.
Key words : Purple Roselle (Hibiscus sabdariffa, Linn), Salmonella typhi, liquid dilution method,
Tyndalization.

1 Introduction
Typhoid fever is an infectious disease that is widely found in tropical and sub tropical
areas, especially in the quality of water resources with inadequate hygiene and
sanitation standards are low (Simanjuntak, 1990).
Typhoid fever caused by Salmonella typhi. These organisms can survive longer in dry
and frozen environments, sensitive to chlorination and pasteurization at a temperature
of 63oC, survive several weeks in water, ice, dust, dry waste, raw garbage, clothing,
and can survive and multiply in milk, meat , eggs or products without changing the
color or shape (Baralli, 2000).
Until now the management of typhoid fever are giving antibiotics to stop the spread
of germs and destroy. Antibiotics can be used include Chloramphenicol, Ampicillin /
Amoxicillin, Cotrimoxazole, and Sefalosforin generation II and III (Mansjoer, 2000).
However, synthetic drugs usually can cause side effects, so people start looking for
another alternative treatment that is the traditional medicines that are safer and give
effect to a level with synthetic drugs (Fudholi, 2001).
One of the plants are efficacious for the treatment of roselle (Hibiscus sabdariffa L.
Traditionally, rosella flowers efficacious as a cure various diseases such as
antispasmodics,diuretics,antihypertensives, antelmentik, antiseptics, sedatives and
antibacterial (Morton, 1987).
According Chumsri, et al (2007) the content contained on rosella flowers will be
stable at a temperature not exceeding 70oC. Temperatures above the fear can lead to

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IC-GWBT2012, Ahmad Dahlan University, March 23-24, 2012

damage to their chemical structure to allow decreasing the activity of these

compounds.
Microbiological testing did not escape from the sterile state, be it of tools, materials,
places and processes. All must be in sterile condition to avoid any influence from
outside the study of microorganisms. The problem here is in the process of
sterilization material. It is known that temperature affects the stability of the ethanol
extract of roselle flower, while in general many microbiological testing of
sterilization using high heat. Sterilization methods should be considered in
accordance with the stability of the materials to be used and is expected to give better
results.
Therefore, pursuant to the above description it is necessary to test research on the
antibacterial activity of 70% ethanol extract of roselle purple flowers and its content
detection in order to know the chemical class of antibacterial activity of 70% ethanol
extract of roselle purple flowers against Salmonella typhi and its kromatogramnya
profile. These results can be used as a basis for new fitofarmaka search so that it can
be used for antibacterial treatment.
2 Metode Research
2.1 Material
The materials used include rosella flower petals purple, 70% ethanol as a solution
penyari, the bacteria Salmonella typhi, BHI media, BHI medium DS, Mc Conkey
media, NaCl 0.9%, Chloramphenicol, sterile distilled water, and standards of Mc
Farland .
2.2 Tool
The tools used in the test include glass beaker, Buchner funnel, analytical scales, rod,
measuring cups, stirer, porcelain bowls, tubes, oven, pots, micropipette, rack tubes,
petri dishes, incubators, blue tip, yellow tip, autoclave, the light spirits, and loop
round, laver chromatography, capillary tube, measuring pipette, UV light, and spray
reagents.
3 Research procedures
3. 3.1 Collection Simplicia
Rosella dried flower petals purple used was taken in the hamlet Nglangu Rt. 3, Rw. 5,
Village Pohsarang, Cement District, District Karachi in March 2011. Rosella flowers
that have been dried and then in powder.
3.3.2
Determination of Moisture
Simplicia rosella flower petal powder placed in plate purple aluminum, and then
inserted into the tool Halogen Moisture Analyzer, so that the water content of the
powder can be known simplicia. When the number of eligible tertetapkan moisture
content of less than 10%.
3.3.3
Determination of Ash Content
Weighted crucible set in kromatorium silicate. Weigh 2-3 grams of sample in the
crucible thoroughly. Incandescent at 900oC in the kromatorium to ashes. Put in
eksikator, wait until cool, then weigh up to a constant weight. Difference of two
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weighings last no more than 0.25% of the weight-weighted.

3.3.4
Preparation of Extracts
Preparation of the extract using the method maceration with 70% ethanol. Remaserasi
performed 3 times. The filtrate is then combined and evaporated on a waterbath with
the temperature maintained no more than 60 C to obtain a concentrated extract.
3.3.5
Phytochemical screening
Phytochemical screening carried out using a test tube and Thin Layer
Chromatography using mobile phase n-Butanol: glacial acetic acid: water (4:1:5)
upper phase and stationary phase silica gel 60 F254. The test is performed to identify
the presence of polyphenols, anthocyanins, flavonoids, saponins and tannins.
3.3.6. Microbiology testing
3.3.6.1 Making Concentration and Control
Ethanol extract of roselle purple flowers sterile and dissolved in distilled water after it
was made in a variety of concentrations, namely 50.0% w / v; 25.0% w / v; 12.5% w /
v; 6.25% w / v; 3.125% w / v and 1.563% w / v. The next step is to control the
manufacture, which consists of media control, solvent, extracts, bacteria and drug
control.
Each - each tube is placed in a pot of boiling water berdandang with the temperature
measured on the sample (T 60 C). Heat for 30 minutes, after that in an incubator
at 37 C for 18-24 hours. Perform repetitions for 3 consecutive days (Tyndalisasi).
3.3.6.2.Determination of Minimum inhibitory Levels
Salmonella typhi bacteria suspension included in the test tube containing the test
solution in Various concentrations. Then incubated at 37 C for 18-24 hours.
Salmonella typhi bacteria growth observations made on each tube with a view to
determining the turbidity levels of the Minimum Inhibitory (MIC).
3.3.6.3.Determination of Minimum Levels of Suicide
Minimum levels of suicide is determined by the scratch test solution from each tube
on solid media Mc Conkey and then incubated at 37 C for 18-24 hours. By looking
at the presence or absence of bacterial growth in the scratches on the media compared
with controls, it can be determined the lowest concentration of 70% ethanol extract of
roselle petals purple flower that can kill bacteria (KBM).
3.4. Data Analysis
The active compound was identified by phytochemical screening methods that
test tubes and thin-layer chromatography.
For sterilization and control samples using the gradual sterilization (Tyndalisasi) and
microbiological test method used is the dilution liquid in which the MIC is known by
observing the clarity of the solution, while KBM can be known only after etched on
Mc Conkey media with presence or absence of bacterial growth of Salmonella typhi.
4. Result and Discussion

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4.1. Identification Simplicia

Identification of plants made to ensure the correctness simplicia to be used in
this study so as to avoid mistakes in the selection of the next simplicia can disrupt the
course of the study. Plants performed at the Laboratory Determination of Natural
Science Faculty of Mathematics and Science University Ahmad Dahlan in
Yogyakarta. Based on the results of determination acquired the certainty that the plant
used in this study is really Hibisscus sabdariffa L.
4.2. Results Determination of Moisture
Water levels are set to determine the water content is in simplicia after the
drying process. Water content remained high on simplicia can be mold and mildew
growth media, but it also can occur enzymatic reactions that can describe the active
substance in simplicia. Standard moisture content in the dried simplicia ie less than
10% (anonymous 2009). Water content contained in simplicia rosella flower petals
purple is 4.63%.
4.3. Results Determination of Ash Content
Test with mineral ash content of a material. The minerals contained in a material
is usually in the form of salt, the salts of organic and inorganic salts. In addition to
these two salts, sometimes - sometimes found as a mineral complex organic
compounds. To determine the amount of mineral in its original form is very difficult,
it is done by setting the burning remnants of mineral salts, namely, the process
Pengabuan (Sudarmadji, S, et al, 1989). Total ash content is high, can be caused by
unclean simplicia, there are dust or parts that have not been washed. By Anonymous
(2008), ash content requirements of each simplicia is different. Based on the plant
part used and the location of growth. Test results of total ash content of rosella flower
petals purple is 5.89%.
4.4. Phytochemical screening
Aimed to determine phytochemical screening of chemical substances contained
in extracts of roselle petals purple flowers that are likely related to the biological
activity of these extracts. Phytochemical screening results may give a compound
contained in extracts of rosella flower petals purple efficacious as antibacterial. The
results of phytochemical screening test tube and TLC 70% ethanol extract of roselle
petals purple flowers can be seen in Table I.
Table I. The results of phytochemical screening of 70% ethanol extract of roselle
flower petals purple
Contents
Result
Antosianin
+
Polifenol
+
Flavonoid
+
Saponin
+
Tanin
+
Alkaloid
-

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4.5. Microbiology Test Results

Microbiological testing is inseparable from the process of sterilization either
sample or media to be used. Therefore appropriate to consider sterilization for
samples that are not resistant to the compounds contained heating is not damaged by
the heat so as to provide optimal results.
One of the efforts to control microorganisms without damaging compounds
contained in the sample is to use the gradual method of sterilization (Tyndalisasi).
Tyndalisasi using hot steam from boiling water with a measured 60 C heat for 30
minutes and be repeated for 3 consecutive days.
On the first day Tyndalisasi process aims to kill the vegetative cells of bacteria,
incubated at 37 C for 24 hours is intended to provide the opportunity for bacteria to
form vegetative cells to be easily killed, but it is thought there are still bacteria
present in the form of spores. A second day of the same treatment intended to kill the
spores had become vegetative cells. The third day is an effort to optimize the results
of sterilization. Microbiological test results can be seen in Tabel II.
Table II. The results of microbiological tests rosella flower petals purple
Result
Consentration
b
(% /v)
KHM KBM`
I
3.25
X
II
2.75
X
III
2.25
X
IV
1.75
X
V
1.25
X
++
VI
0.75
X
++++
VII
K1
J
VIII
K2
J
IX
K3
J
X
K4
K
++++
XI
K5
J
NO

Information:
X: not observed clarity and turbidity
J: Clear
K: cloudy
+: Growth of bacteria
-: No growth of bacteria
Based on the above table in the levels of 1.75% w / v have been able to kill the
bacteria Salmonella typhi. Control results are as expected where there is only contained
in the control of bacterial colonies. On the antibacterial activity assay uses five control
the media control, solvent control, control extracts, bacterial control, and drug control.
Control medium was 2.0 ml BHI digunkan DS without a suspension of bacteria. This
control used for know whether the media that we use in a sterile condition. Solvent
control consisting of 1.0 ml of sterile distilled water + 1.0 ml of BHI DS is used as a

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comparison to determine whether the use of sterile distilled water. Control extracts
used to determine that the ethanol extract of roselle petals purple flower that is used to
test the antibacterial activity was sterile, containing 1.0 ml of extract + 1.0 ml of BHI
DS. Control of bacterial suspension containing 2.0 ml of bacteria in BHI, used to see
whether the media used to support the growth of bacteria or not. Drug control
containing 1.0 ml of the drug chloramphenicol 0.25% w / v + 1.0 ml bacterial
suspension, is intended as a comparison between the extract and medicinal potential of
the bacterium Salmonella typhi.
5. Conclusion and Recommendation
Tube test results indicate a class of polyphenolic compounds, flavonoids, tannins
and saponins. Test thin-layer chromatography showed the compound anthocyanin
flavonoids, tannins and saponins. 70% ethanol extract of roselle petals purple flowers
can inhibit and kill the bacteria Salmonella typhi.
Tyndalisasi as an alternative method of sterilization of the extract to give good
results. KHM rates of 70% ethanol extract of roselle petals purple flowers can not be
determined, because the purple-black, and KBM rates of 70% ethanol extract of
roselle petals purple flowers against Salmonella typhi was 1.75% w / v.
Suggestions for further research, more research needs to be done about the
amount of content contained in 70% ethanol extract of roselle petals purple flowers
that have antibacterial activity.
Further research needs to be done any compound of 70% ethanol extract of roselle
petals purple flowers that were damaged by high heat sterilization.
Further research is necessary to use methods other than that of liquid dilution
MIC of 70% ethanol extract of roselle petals purple flowers can be determined.
6.

References

Baralli, 2000, Salmonella infection, http://www.emedicine.com/emerg/topicsis.

Diakses pada tanggal 20 Februari 2011
Chumsri, P., Anchalee, S., dan Arunporn, I.,2007, Studies on the optimum condition
for the extraction and concentration of Roselle (Hibiscus sabdariffa Linn.)
extract. Terdapat pada http://www.rdoapp.psu.ac.th. Diakses pada tanggal 15
Juni 2011.
Fudholi, A., 2001, Teknologi dan Formulasi Sediaan Obat Alam dan
Permasalahannya, Majalah Pharmacon, volume 2, No. 1, 25-29, Yogyakarta.
Mansjoer, Arif dkk, 2000, Kapita Selekta Kedokteran, Jilid I, Edisi III, 422-424,
Penerbit Media Aesculapius, Jakarta.
Morton, J., 1987, Roselle ( Fruits of warm climates ), p 281-286
htt:/www.hort.purdue.edu/newcorp/morton/Roselle.html,
Diakses
pada
tanggal 11 Februari 2011.
Pelczar, J. M., E. C. S. Chan., 1988, Dasar-Dasar Mikrobiologi, Jilid II,
Diterjemahkan oleh Hadioetomo, R.S., Penerbit UI, Jakarta
Simanjuntak. C., 1990, Masalah Demam Tifoid di Indonesia, Majalah Cermin Dunia
Kedokteran No. 60, PT. Kalbe Farma, Jakarta.

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