MKG vol 39 no 1 Jan 2006 isi.pmd

Antimicrobial effects of Coleus amboinicus, Lour folium infusum
towards Candida albicans and Streptococcus mutans

Devi Rianti and Sri Yogyarti
Department of Dental Material and Technology
Faculty of Dentistry Airlangga University
Surabaya - Indonesia

ABSTRACT

A laboratory experimental study conducted on antimicrobial effects of Coleus amboinicus, Lour folium Infusum towards Candida
albicans and Streptococcus mutans (S. mutans). Effective concentration of Coleus amboinicus, Lour to decrease the quantities
Candida albicans and S. mutans colonies is expected to be found out in this study. This study was using Coleus Amboinicus, Lour
folium infusum with 12.5%, 15%, 17.5%, 20%, and 22.5% concentrations. Sterilized aquadest used as a control. Candida albicans
and S. mutans quantities was enumerated by counting the amount of Candida albicans and S. mutans growth in the Sabouraud ,s
dextrose agar and Tryptone and yeast Agar media, using Colony Forming Unit per milliliter (CFU/ ml) unit. Data analysis was using
a One-Way ANOVA and LSD with 5% degree of significance. The result showed 22.5% concentration of CAL folium infusum was the
most effective in decreasing the quantity Candida albicans and S. mutans colonies.

Key words: antimicrobial, coleus amboinicus, lour, candida albicans, streptococcus mutans

Correspondence: Devi Rianti, c/o: Bagian Ilmu Material dan Teknologi Kedokteran Gigi, Fakultas Kedokteran Gigi Universitas
Airlangga. Jln. Mayjend. Prof. Dr. Moestopo No. 47 Surabaya 60132, Indonesia.

INTRODUCTION

According to national health system guidance, the
traditional medication which can successfully, will be lead
and used for research and examination literally to
Indonesian herbs so it is safe to be used. Indonesian
government supports the use of traditional materials as one
of alternative medication, since Indonesia is enriched with
medical herbs.

One of the herbs usually plant in the garden and easily
growth is jinten leaf. According to Wijayakusuma et al.1

Jinten leaf in Latin called Coleus amboinicus, Lour and
simplified as Plectranthi amboinicus folium, is a plant
which peculiar for stomatitis and antifungal medication.
The chemical contents of Coleus amboinicus, Lour are
calium, essential oils which contains carvacrol, isoprophyl-
o-cresol, phenol and sineol. Weehuizen2 stated, that from
120 kg fresh leaf of Coleus amboinicus, Lour (CAL) will
be resulted essential oils such 25 ml, and 25 ml essential
oils equal to 0.2% essential oils which contains phenol
generations, i.e. isoprophyl-o-cresol, which has high
antiseptic effect.2 Preliminary examination of CAL showed,
that from Tawangmangu Central Java chemical content,
extracted with alcohol 96%, it contains alkaloids, saponin,
cardenolids, and bufadienolids together with polyphenol.3

Devi’s4 experiment stated that extract solution of CAL is
effective to decrease Candida albicans (C. albicans) in
acrylic resin after 2 hours soaking with 15% concentration.

Chemical component sineol has antifungal effects to
C. albicans, Trichiphyton metagrophytes and Cryptococcus
neoformans.5 Wisterich and Lechtman stated that Phenol

and Cresol can kill fungus vegetative cell and bacteria by
protein denaturation and surface tension reduction, so that
increases bacteria permeability.6

Streptococcus mutans (S. mutans) is a normal occupant
bacteria in oral, however if the atmosphere fits, and
population increases it becomes pathogen.7 S. mutans have
been admitted in the dentistry as the main cause of caries.7

Based on that, the growth of S. mutans should be obstructed
to become pathogen.

According to Bahalwan and Sjahbana8 the use of
traditional medication in Indonesia is as a prevention. Some
of herbal traditional medication can be used as a mouthwash
and they also work for antiseptic as if as a disinfectant are
semanggi leaf, jinten leaf (CAL), sirih leaf, gambir, saga
leaf and also kaca piring leaf.

Usually, extract materials are hard to find, the alternative
ways to process the herbs are needed. In one of research,
infusum technique for herbs is better than boiled.9 The
advantage of infusum, is easier to use and it has been
socialized, cheaper because there is no special treatment
and complicated instrument is needed as well.

In some of literatures said, that CAL has an antiseptic
character because of it chemical contents. Some of
researches stated that it could decrease the growth of
C. albicans, Trichiphyton metagrophytes and Cryptococcus
neoformans, either ways there are some possibilities of CAL
to have antibacterial character towards S. mutans. The best
technique to use and to, reduce the cost and to be more
socialized, the infusum technique is chosen. The minimum
concentration of CAL folium infusum to decrease S. mutans
and C. albicans colonies is unknown yet. The aims of this

13Rianti and Yogyarti: Antimicrobial effects of Coleus amboinicus

article is to investigate if CAL folium infusum concentration
variations effect towards C. albicans and S. mutans colonies
and to find the concentration of CAL folium infusum that
has an effective antimicrobial character towards C. albicans
and S. mutans.

MATERIALS AND METHODS

The research is an experimental laboratories and the
research design is post test only control group. Independent
variable is CAL folium infusum with 12.5%, 15%, 17.5%,
20%, 22.5% concentration. Dependent variable is the
amount of C. albicans and S. mutans colonies. The control
variables are the similarity of planting area, crops time,
drying time, temperature and time of C. albicans and
S. mutans proliferation, growth media, counting C. albicans
and S. mutans, and CAL folium infusum process, in
accordance with Farmakope Indonesia.10 This research has
been done in phytochemical Laboratory Faculty of
Pharmacy Airlangga University, Oral Microbiology
Laboratory Faculty of Dentistry Airlangga University,
Surabaya.

Materials those being used in these research are
C. albicans suspension, S. mutans, Sabouraud’s dextrose
media and Sabouraud’s broth, Tryptone and Yeast Agar
(TYC) media and Brain Hearth Infusion (BHI) and CAL
leaf from central traditional medication research garden.
Instruments to count C. albicans and S. mutans are:
incubator (Precision, Japan), 0,2 μm milipore filter unit
(Millex-HA, Bedford), reaction tube, stopwatch, petridish
(Anumbra), autoclave (Foundry), ose, spreader, pinset,
vibrator (Vortex), 5 cc injection syringe (Terumo), 1 cc
tuberculine syringe (Terumo), Ependorff micropipette
(Titertek, England), anaerobic jar, mechanic counter
instrument (Hand Tally model H-102, Japan).

The research methods are making of CAL folium
infusum in accordance with Farmakope Indonesia, and
counting of S. mutans and C. albicans colonies. Counting
S. mutans colonies is by preparing 7 reaction tubes and
giving numbers by order 1 until 7. Reaction tubes 1 until 5
are filled with test materials i.e. 1 ml CAL folium infusum
in 2 ml Brain Hearth Infusion Broth (BHIB) media and
S. mutans with a density of 3 × 108 that already liquefied
by 10–7 as much as 0.1 ml. CAL folium infusum
concentrations in reaction tubes number 1 until 5 are 22.5%,
20%, 17.5%, 15%, 12.
5%. Reaction tube number 6 is not filled with test material,
but it is added with S. mutans with 3 × 108 densities that
already liquefied as positive control. Reaction tube number
7 filled with only BHIB media without S. mutans as
negative control. Ten times of replication are done. All of
reaction tubes are placed in an anaerobic jar, in anaerobic
environment and incubated as long as 24 hours on 37 °C.
The result is read by observing whether there is a deposit
or turbid. To count the S. mutans colonies, all test materials
are planted in TYC media in petridish. Each of petridish

is filled with the liquid in the tube (CAL folium infusum +
BHIB media + S. mutans) using pipette which have the
same number as the reaction tube, then spread using
spreader at the surface of solid TYC media. All the
petridish to put in anaerobic jar incubated in 37 °C degree,
as long as 48 hours. Result reading is by observing whether
there is a colony proliferation at the surface of TYC, and
counting the colonies (CFU/ml).

To count C. albicans colonies is by preparing 7 reaction
tubes and giving numbers 1 until 7. Reaction tubes number
1 until 5 are filled with 1 ml CAL folium infusum in 2 ml
Sabouraud’s broth media and with C. albicans density of
3 × 108 that already liquefied by 10–7 as much as 0,1 ml.
CAL folium infusum concentrations in reaction tubes 1
until 5 are 22.5%, 20%, 17.5%, 15%, 12.5%. Reaction tube
number 6 is not filled with test material, but it is added
with C. albicans which has 3 × 108 densities that already
liquefied as positive control. Reaction tube number 7 filled
with Sabouraud’s broth media without C. albicans as
negative control. Replications are done in ten times. The
result is read by observing whether there is a deposit or
turbid. To count the C. albicans colonies, all test materials
are planted in Sabouraud’s dextrose media in petridish.
Each of petridish is filled with the liquid in the tube (CAL
folium infusum + Sabouraud’s broth media + C. albicans)
using pipette which have the same number as the reaction
tube, then spread using spreader at the surface of
Sabouraud’s dextrose media media. All the petridish to put
in incubator, incubated in 37 °C degree, as long as 48 hours.
Result reading is by observing whether there is a colony
proliferation at the surface of Sabouraud’s dextrose media,
and counting the colonies (CFU/ml).

The result is being tabulated according to each group
then statistic test is held using One-Way ANOVA, with
deviation degrees 5%. However, if there is a significant
result, it will be continued by LSD test.

RESULTS

The average result and standard deviation C. albicans
and S. mutans colonies after contacted with CAL folium
infusum in concentration 12.5%, 15%, 17.5%, 20%, 22.5%
can be seen at table 1 and 2.

Table 1. Average and Standard deviation from total C. albicans
colony (CFU/ml) after contact with CAL folium
infusum

Concentration N X SD

Control
12,5%
15%
17,5%
20%
22,5%

10
10
10
10
10
10

479.50
244.60
183.30
114.30
55.20
18.60

18.91
36.04
10.79
10.66
10.43
9.43

14 Maj. Ked. Gigi. (Dent. J.), Vol. 39. No. 1 January–March 2006: 12–15

Table 2. Average and Standard deviation from total S. mutans
colony (CFU/ml) after contacted with CAL folium
infusum

Concentration N X SD

Control
12,5%
15%
17,5%
20%
22,5%

10
10
10
10
10
10

495
286.80
233.40

165
120.30
55.70

42.52
30.36
26.66
11.24

13
12.09

Notes: N: Samples amount X: Average SD: Standard deviation

Before parametric test is done, to know the significant
difference result, a normality test using Kolmogorov-
Smirnov test should be done. The result is, 5 treatment
group and control are normally distributed (p > 0.05),
moreover, comparison test to 5 treatment group and control
is done by One-Way ANOVA test. From One-Way
ANOVA test, it appears that there is a significant result
among treatments with p = 0.001 (p < 0.05), to find out
further the differences among treatments, the analysis is
continued with Least Significant Difference (LSD) test.
LSD test shows that there is significant result in each
concentration groups; 12.5%, 15%, 17.5%, 20%, 22.5%
and control. In other words the increase of CAL folium
infusum concentration will affect C. albicans and S. mutans
colonies.

DISCUSSION

The use of natural herbs medication has already been
socialized and barely a new thing, but it has been exercised
long time ago to fulfill the need of medication in solving
health problem, and the experience has been inherited. The
benefit is admitted by a lot of people, but still there is not
lot of literatures.

Guided by national health system, it is stated that a
useful traditional medication will be lead and used for some
scientific research and test to Indonesian plants. One of
traditional medication herbs that usually planted in the
garden and easily to grow is jinten leaf, in latin it is called
Coleus amboinicus, Lour (CAL), which have chemical
content, phenol which is as an antiseptic.1,2 Preliminary
examination on chemical content of CAL material from
Tawangmangu Central Java filtered with alcohol 96%, it
is appears containing alkaloids, saponin, kardenolids,
bufadienolids also polyphenol.3 Base on that, there are some
possibilities those materials can be used for oral
antimicrobial as antiseptic in dentistry.

Result from the research shows the average C. albicans
and S. mutans colonies after contact with CAL folium
infusum in increasing infusum concentration, i.e. 12.5%,
15%, 17.5%, 20%, 22.5% will decrease. On the contrary,
in lower concentration the total C. albicans and S. mutans
colonies increased constantly.

This is caused by Coleus amboinicus, Lour contains
phenol and phenol generations which are useful
antiseptic.1,2 Supported from Devi research’s, stated that,
in dry CAL leaf extract contains 5.15% phenol and 3.65%
sineol.4 Increasing CAL folium infusum concentration will
cause decreasing C. albicans and S. mutans colonies, since
the increased concentration will increase the phenol and
sineol in infusum. The increased phenol and sineol, will
increase the antimicrobial effect of infusum towards
C. albicans and S. mutans. It is further explained by Regezi
and Sciubba11 that C. albicans is a very sensitive species
towards phenol. Besides phenol, Hammerschimdt et al.5

stated that the other chemical materials, sineol, also have
antifungus activity towards C. albicans, Trichiphyton
metagrophytes and Cryptococcus neoformans. Hugo and
Rusell,12 stated that phenol generally can be used as
disinfectant which have antimicrobial activity and fast
bactericide, the activity usually decreased by watering. It
is also supported by Siswandono and Soekarjo,13 who stated
that materials effectiveness affected by concentration, time
and temperature.

In these research, infusum pH is approximately between
4.78–4.80, which is possibly has good effect in
antimicrobial activities of CAL folium infusum, since
phenol appears more effective in acid pH and moreover, it
is explained that disinfection process influenced by several
factors such as, concentration, temperature, and pH.12

Other factor which possibly the cause is the presence
of alkaloid contents. Alkaloid is a chemical compound
contains nitrogen, and this group can be diverged from other
plant component based on its alkali characteristic. This
compound is found in plant as salt of various organic acids,
which is functioned to protect plant from parasites and
predator, and also effective as antimicrobial.14

Until now, there still no references for antimicrobial
mechanism of this alkaloid. Based on antimicrobial
mechanism, phenol can eliminate fungus vegetative and
bacteria by protein denaturation and surface tension
reduction which caused the bacteria and fungus
permeability are increased.6 It is explained by Melville and
Russel15 that reaction with cell protein, is an obstruction or
elimination process by ruining the colloid system using
protein coagulation and precipitation. Microbial cell protein
coagulation will cause metabolism distraction and
membrane cell permeability change is by decreasing the
surface tension which increase the membrane cell
permeability causing liquid enters and eliminates the
microbe.

Renner et al.16 proved that people using denture without
pathologic change of its supporting mukosa orally, as if
the C. albicans colonies are not more than 100 CFU/ml.
Candida species experiment will be decided as positive, if
the amount is more than 100 colonies of subject samples
with Sabouraud’s dextrose breeding, or if the amount is
more than 500 CFU/ml saliva. According to Brightman &
Greenberg17 C. albicans is assumed as a normal flora part
in oral, as it can be found in some healthy individuals and

15Rianti and Yogyarti: Antimicrobial effects of Coleus amboinicus

carriers if it not more than 200 CFU/ml, detected by oral
swapping inoculation of Sabouraud’s dextrose agar
breeding. Average value of C. albicans colonies in this
research are 244.60 CFU/ml, 183.30 CFU/ml, 114.30 CFU/
ml, 55.20 CFU/ml, 18.60 CFU/ml (Tabel 1), from contact
with CAL infusion in 12.5%, 15%, 17.5%, 20%, 22.5%
concentrations. C. albicans colonies which are contacted
with CAL folium infusum with a 15% concentrate are less
than total colonies of a healthy individual. From those
scholars’ research and opinion comparison, the use of 15%
concentrate of CAL folium infusum is found has been
effective to decrease the amount of C. albicans colonies.

In conclusion, increasing of CAL folium infusum
concentration, i.e. 12.5%, 15%, 17.5%, 20%, 22.5% will
decrease the total C. albicans and S. mutans colonies. The
most effective CAL folium infusum to decrease C. albicans
and S. mutans colonies is 22.5%.

More experiments are needed to find out the
biocompatibility of Coleus amboinicus, Lour folium
infusum to be used as an alternative mouth wash.

REFERENCES

1. Wijayakusuma H, Dalimartha S, Wirian AG, Tanaman obat
berkhasiat Indonesia IV. Edisi 1. Jakarta: Pustaka Kartini; 1996.
p. 38–41.

2. Heyne K. Tumbuhan berguna Indonesia. Jilid III. Jakarta: Badan
Litbang Kehutanan. Yayasan Sarana Wana Jaya; 1987. p. 1698.

3. Hutapea JR. Pemeriksaan pendahuluan golongan kandungan kimia
tanaman Coleus Amboinicus, Lour (Labiatae) asal Tawangmangu.
Balai Penelitian Tanaman Obat Tawangmangu, Pusat Penelitian
Farmasi, Badan Penelitian dan Pengembangan Kesehatan,
Departemen Kesehatan RI; 1982. p. 389–94.

4. Devi R. Daya antimikroba ekstrak Coleus Amboinicus, Lour terhadap
Candida Albicans pada resin akrilik. J Kedokteran Gigi Indonesia
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5. Hammerschmidt FJ, Clark AM, Soliman FM, El-Kashoury ES, Kawy
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6. Rahardjo MB. Perbedaan daya antibakteri Allium Sativum Linn dan
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7. Kidd EAM, Bechal SJ. Dasar-dasar karies penyakit dan
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8. Bahalwan R, Sjabana D. Mengkudu seri referensi herbal. Jakarta:
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9. Eha D. Khasiat obat kumur infusa daun Kacapiring terhadap
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Majalah Ilmiah Kedokteran Gigi Fakultas Kedokteran Gigi Usakti
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10. Dep Kes RI. Farmakope Indonesia. Edisi 3. Jakarta: Dep Kes RI;
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11. Regezi JA, Sciubba JJ. Oral pathology: Clinical pathologic
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12. Hugo WB, Russel AD. Pharmaceutical microbiology. 4th ed. Oxford,
London, Edinburgh, Boston, Melbourne: Blackwell Scientific
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13. Siswandono, Soekarjo B. Kimia medisinal. Cetakan I. Surabaya:
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14. Robinson T. Kandungan organik tumbuhan tinggi. Padmawinata K.
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15. Melville PH, Russel C. Microbiology for dental student. 3rd ed.
London: Williem Heinewmann Medical Book Ltd; 1981. p. 155–76.

16. Boedi S. Aspek klinis dan penetapan diagnosis kandidiasis mulut.
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17. Brightman VJ, Greenberg MS. Candidiasis. In: Lynch MA, editor.
Burket’s oral medicine diagnosis and treatmen. 8th ed. Philadelphia:
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/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
/NOR <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>
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/ENU (Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
/ErrorControl /WarnAndContinue
/FlattenerIgnoreSpreadOverrides false
/IncludeGuidesGrids false
/IncludeNonPrinting false
/IncludeSlug false
/Namespace [
(Adobe)
(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /NoConversion
/DestinationProfileName ()
/DestinationProfileSelector /NA
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure true
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles true
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /NA
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /LeaveUntagged
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice