JMS Vol. 2 No. 2, hal. 57 - 63, Oktober 1997

Antimicrobial Activities of the Ethanol Extracts of Capsicum Fruits
with Different Levels of Pungency

S. Soetarno*, Sukrasno, E. Yulinah and Sylvia
Department of Pharmacy, Faculty of Mathematics and Natural Sciences,
Institut Teknologi Bandung
Jl. Ganesa 10, Bandung, 40132 - Indonesia

Diterima tanggal 7 Mei 1997, disetujui untuk dipublikasikan 27 September 1997

abstract
Ethanol extracts of the fruits of three kinds of Capsicum showed similar potencies in their
antimicrobial activities against Gram (+) and Gram (-) bacterias, and fungi, although they
contained different level of capsaicin. Bioautographic tests demonstrated that capsaicin was
the main antimicrobial component. At least two other non-polar components of ethanol
extract also contributed in the antimicrobial activity and very likely that these compounds
were responsible for the activity toward Pseudomonas aeruginosa.

KEYWORDS
Antimicrobial activity, ethanol extracts, fruits, Capsicum frutescens L., Capsicum annuum L,
Solanaceae.

1. Introduction

Pepper fruits are added at a substantial quantity to produce a characteristic taste of
cuisine of West Sumatran (Indonesia). It was observed that cuisine using a large amount of
chilli pepper could stand at long period without significant deterioration. Only a special
variety of chilli pepper was employed by this ethnic, i.e. Capsicum annuum L. var. longum
(Solanaceae) which has a curly shape. Chilli tinctur was previously reported to be able to
inhibit the growth of Staphyllococcus sp., Escherichia coli, Bacillus aureus and Bacillus
subtilis1). In Madura island (Indonesia), chilli pepper (Capsicum frutescens) is traditionally
used to treat ox cuts before race2). In Indonesia, chilli pepper is also traditionally used to treat
oral thrush which is usually caused by Candida albicans3).

This report presents the results of our study on the activity of the ethanol extracts of
Capsicum frutescens and two cultivars of Capsicum annuum, var. longum. The first pepper
fruit was very hot and one of the C. annuum was hot while the other one was moderately
hot.
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* To whom correspondence should be addressed

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2. Materials and Methods
2.1. Plant Materials

Three different pepper fruits were purchased from local market. Fruit-1 was from
Capsicum frutescens L. (chilli pepper), fruit-2 C. annuum L. var. longum (curly pepper), fruit-
3 C. annuum L. var. longum (sweet pepper). Taxonomic identification of fruits was conducted
at The Herbarium Bandungense, Department of Biology, Institut Teknologi Bandung and
Horticulture Research Center - Lembang, Indonesian Ministry of Agriculture. Voucher
specimens were deposited in the Herbarium of the Department of Pharmacy. The fruits were
dried at 60oC in an air oven and then ground to produce pepper powder.

2.2. Extraction

Chilli pepper fruit extracts were prepared by macerating pepper powder in ethanol for
24 h then filtered. The residue was washed twice with fresh ethanol and the filtrates combined
with the first filtrate. Combined filtrate was then evaporated to dryness to produce sticky
material of pepper extract.

2.3. Microorganisms

The microorganisms used in these tests include :
Gram (+) bacteria : Staphylococcus aureus (ATCC-14154)

Bacillus subtilis (ATCC-6633)

Sarcina lutea (Collection of Microbiology Lab. of Chemical
Engineering Department, Institut Teknologi Bandung)
Gram (-) bacteria
: Escherichia coli (ATCC-1698)

Pseudomonas aeruginosa (ATCC-23993)
Yeast :

Candida albicans (ATCC-14053)
Dermatophyte :
Microsporum gypseum (Biofarma-Bandung Collection)
Mold :
Aspergillus niger (ATCC-32611)
2.4. Media

Bacteria were grown on nutrient agar (Oxoid). Inoculum for the assay was prepared by
suspending bacterial cells 18-24 h old in nutrient broth (Oxoid) to yield 25% transmittance at

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580 nm. Fungi were grown on Sabouraud Dextrose Agar (Oxoid) and inoculum for the assay
prepared by suspending the 72 h old fungal cells in Sabouraud Dextrose Broth (SDB) to
produce 90% transmittance at 530 nm.

2.5. Method of assay

The antimicrobial assay was performed by dispersing 100 µl of inoculum
homogeneously in 15 ml liquified nutrient agar (NA) or Sabouraud Dextrose Agar (SDA)
media and then left to solidify on Petri dishes. Six wells of 6.5 mm in diameter were prepared
on plate of each dish. Respectively 10 µl solution of pepper extract in DMSO at the desired
concentration was then added into each well. The assay dishes were then left for one hour and
subsequently incubated for 24 h at 37oC for bacteria and 22oC for fungi. The diameter of
inhibition was then observed and measured. As a comparison, tetracycline hydrochloride was
used as standard for antibacteria, nystatine for the activity against C. albicans and M.
gypseum, griseofulvin for A. niger.

2.6. Thin Layer Chromatography

Thin layer chromatography was performed on precoated silica gel GF-254 with 10
µm layer and benzene-acetic acid (9:2) as solvent system. Spot of capsaicin was visualized
with diazotised sulfanilamide which was prepared by freshly mixing 5 ml of 1% sulfanilamide
in 10% HCl, 5 ml of 5% sodium nitrite and 40 ml of metanol4)). Intensification of capsaicin
spot was performed by spraying with 5% sodium carbonate which yielded red color following
mild heating.

2.7. Determination of capsaicin content in the extract

Sample was prepared by dissolving extract in ethanol at concentration of 5 mg/ml and
15 µl solution was applied on TLC plate. Capsaicin isolated from fruits was used as a standard
for the measurement and applied in the range of 5 µg to 25 µg in each spot. After development
and visualization, the intensity and the area of the spots was measured using Spectrophoto
densitometer (Shimadzu CS-910) at 510 nm.

2.8. Bioautography

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To improve the separation and increase the loading capacity of the plate, the extract
was firstly fractionated into aqueous and dichlormethane fractions at pH 3 and the later
fractions collected and dried with sodium sulfate. Dichlormethane fraction was evaporated to
dryness at reduced pressure and redissolved in ethanol and applied on silica gel as a strip. TLC
chromatogram was dried using spray drier to eliminate solvent remaining on the plate and then
laid on the surface of NA/SDA media freshly inoculated with the assay microbes. The plate
was left for15-30 min to facilitate diffusion of substances in the plate to the gel and then
removed. Subsequently the assay gel was incubated at 37oC (bacteria) or 22oC (fungi) for 18-
24 h (bacteria) or 24-48 h (fungi).

3. Results and Discussion

In order to provide background information on the pepper fruits under our study,
following is a brief description of the three fruits. The first pepper was identified as one
cultivar of C. frutescens, since there are different type of fruits based on the color, size, form
and pungency. The other two fruits were all from C. annuum var longum, but they are very
different in shape and also pungency. Table 1 gives more detail description of the Capsicum
fruits used in our study.

All fruit extracts were active against most Gram (+) and Gram (-) bacteria tested.
Differ from fruit-1 and fruit-3 extracts, fruit-2 extract was inactive against P. aeruginosa.
Against fungi tested, i.e. C. albicans, M. gypseum and A. niger, the three fruit extracts were
only active to the first microorganism. These results suggest that all kinds of Capsicum fruits
tested are useful as antibacterial and anticandidal agents and not necessarily the most pungent
pepper as in the tradisional use2,3). Consistent with their pungency, the sequence of capsaicin
level in the three Capsicum fruit extracts was fruit-1 > fruit-2 > fruit-3 (see Table 1).

Based on their minimum inhibition concentrations (MIC), the activity of fruit-1 and
fruit-3 extracts to P. aeruginosa compared to the other bacteria tested and to C. albicans was
the lowest, while fruit-2 extract was inactive. Although fruit-3 contained the lowest level of
capsasicin, its extract exhibited the lowest MIC against S. lutea (see Table 1). Inactivity of
fruit-2 against P. aeruginosa, and the similarity in the activity of the three fruit extracts to the
other microrganisms suggest that capsaicin may not the only compound responsible for the
antimicrobial activity.

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Bioautography of chromatogram of the three extracts which was conducted by
contacting the chromatogram with the assay gel for 15-30 min followed by incubation and
observation the inhibion area showed that capsaicin was the main compound for antimicrobial
activity. Bioautographic tests against S. aureus and B. subtilis gave the same pattern of results,
while against C. albicans, good result was not obtained although several repetition had been
made. Apart from capsaicin, in extract of fruit-1 another component having hRx 123.3
(relative to capsaicin) also showed antimicrobial activity. In fruit-2 capsaicin was the only
antimicrobial compound, while in fruit-3, two other active components i.e. spots with hRx
153.8 and 165.4 were also active. The higher Rf value of the other active components than
capsaicin in the TLC system used suggest that these compounds are less polar than capsaicin.
The bioautographic data also suggest that these compounds may be responsible for the activity
toward P. aeruginosa since fruit-2 extract which contained capsaicin as the main active
component was inactive. Clearly further tests are needed to confirm this possibility. The
similarity in the antimicrobial activity of the hottest and the least hot pepper fruit extracts
tested suggests the presence of synergism between capsaicin and the other components in the
pepper fruit extracts.

Compared to the activity of the standard antibiotics, the activity of pepper fruit extracts
was still much lower. Relative activity fruit-1 extract compared to tetracycline HCl for
example only approximately 10-5 times and variation existed depended on the bacterial test
used. The relative activity of Capsicum fruit extracts to nystatine was in the order of 10-3 time
magnitude which was higher than to tetracycline. These results support the traditional use of
Capsicum fruits to treat oral thrush caused by C. albicans3) .

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JMS Vol. 2 No. 2, Oktober 1997
Table 1. Botanical description of the three Capsicum fruits, antimicrobial activity and
bioautographic data of their ethanol extracts

Criteria evaluated
Fruit-1
Fruit-2
Fruit-3
Species
C. frutescens
C. annuum
C.annuum
Local name
cabe rawit
cabe keriting
cabe besar
Length/diameter (cm)
3.5-5.0/1.0-1.2
6.5-10.0/0.5-1.0 13.0-15.0/2.0-2.5
Color red
(mature)
red (mature)
red (mature)
green (raw)
green (raw)
green (raw)
Shape stright
curly
stright
Pungency and capsaisin content
very hot
hot
moderately hot
In extract
8.49
4.28
2.18
MIC {% (µg/well)}



S. aureus
0.40 (80)
0.40 (80)
0.40 (80)
E. coli
0.06 (12)
0.08 (16)
0.06 (12)
P. aeruginosa
5.00 (1.000)
-
5.00 (1.000)
B. subtilis
0.40 (80)
0.20 (40)
0.60 (120)
S. lutea
0.10 (20)
0.10 (20)
0.08 (16)
C. albicans
0.06 (12)
0.04 (8)
0.06 (12)
HRx of active bioautographic spots
100 (capsaicin)
100 (capsaicin)
100 (capsaicin)
to B. subtilis and S. aureus
123.3
153.8
165.4

Acknowledgement

The authors are gratefully thank The Herbarium Bandungense, Department of Biology,
Institut Teknologi Bandung, and Horticulture Research Center - Lembang, Indonesian
Ministry of Agriculture for the assistance in taxonomic identification.

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References
1. Siehta, A.S., Magdoub, M.W.I., Fayed, E.O., and Hoft, A..A. "Effect of added salt and
Capsicum tincture on non lactic acid bacteria in pickled Domiati Cheese", Annual of
Agricurtural Science, 29: 793-810 (1984).
2. Djarwaningsih and Uji. T. "The utilization of Indonesian traditional medicines for
treatment of cattle diseases in three villages of East Java, in: National Seminar and
Workshop Proceeding on Ethnobotany", Ministry of Education and Culture, Ministry of
Agriculture, Indonesian Research Institute, Indonesian National Library, Cisarua-Bogor:
112-115 (1992)
3. Syamsuhidayat, S.S., and Hutapea, J.R. "Inventarization of Indonesian Medicinal Plants",
Vol. 1, Indonesian Ministry of Health, Jakarta: 112-115 (1991).
4. Medziak, I. and Waksmundski, A. "Selection of composition and concentration of the
mobile phase for separation of phenol derivatives by TLC", Chem. Anal., 28: 251-257
(1983).

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