Antibacterial Activity of Thai Medicinal Plant Extracts on the Skin ...

<strong>Antibacterial</strong> <strong>Activity</strong> <strong>of</strong> <strong>Thai</strong> <strong>Medicinal</strong> <strong>Plant</strong> <strong>Extracts</strong> on the Skin
Infectious Microorganisms
S. Chansakaow, P. Leelapornpisid and K. Yosprasit
Pharmaceutical Science
Faculty <strong>of</strong> Pharmacy
Chiang Mai University
Chiang Mai 50200
<strong>Thai</strong>land
P. Tharavichitkul
Department <strong>of</strong> Microbiology
Faculty <strong>of</strong> Medicine
Chiang Mai University
Chiang Mai 50200
<strong>Thai</strong>land
Keywords: antibacterial activity, medicinal plant, Punica granatum L. var. granatum
Abstract
Five <strong>Thai</strong> medicinal plants, Acanthus ilicifolius Linn. var. ilicifolius (leaves);
Argyreia nervosa (Burm. f.) Bojer (leaves); Punica granatum L. var. granatum (fruit
rind); Terminalia chebula Retz. var. chebula (fruits) and Zanthoxylum myriacanthum
Wall. ex Hook. F. (fruits) were extracted by several extracting procedures with
various solvents. We obtained 21 plant extracts that were tested for their
antibacterial activities against Staphylococcus aureus ATCC 25923 and Escherichia
coli ATCC 25922 by well diffusion method. The active extracts were found out for
minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) by broth dilution method. The results revealed that the ethanolic extract <strong>of</strong>
Punica granatum L. var. granatum by maceration (PG-1), possed the most
outstanding in vitro antibacterial activity. The MIC <strong>of</strong> PG-1 against S. aureus ATCC
25923 and E. coli ATCC 25922 were 0.39 and 12.5 mg/ml whereas the MBC were
1.56 and 12.5 mg/ml, respectively. The preliminary phytochemical study <strong>of</strong> PG-1
indicated that PG-1 contains both hydrolysable and condense tannin.
INTRODUCTION
<strong>Thai</strong> traditional medicines use a variety <strong>of</strong> herbal preparations to treat different
kinds <strong>of</strong> ailments, including microbial infections. Due to the indiscriminate use <strong>of</strong>
antimicrobial drugs the microorganisms have developed resistance. Additionally, using
antibiotics is sometime associated with adverse effects. Therefore, herbal medicine will
be an alternative for the treatment <strong>of</strong> bacterial infections and may decrease such
problems. This paper reports antibacterial properties <strong>of</strong> five <strong>Thai</strong> medicinal plants;
Acanthus ilicifolius Linn. var. ilicifolius (Mahidol, 1998d), Argyreia nervosa (Burm. f.)
Bojer (Mahidol, 1998b), Punica granatum L. var. granatum (Mahidol, 1998b),
Terminalia chebula Retz. var. chebula (Mahidol, 1998c; Malekzadeh et al., 2001; Sato et
al., 1997) and Zanthoxylum myriacanthum Wall. ex Hook. F. (Mahidol, 1998a) selected
on the basis <strong>of</strong> medicinal folklore reports and previous research reports (Mahidol, 1998ad;
Malekzadeh et al., 2001; Promjit et al., 1996; Sato et al., 1997).
MATERIALS AND METHODS
<strong>Plant</strong> Materials
A. ilicifolius, A. nervosa and P. granatum were collected in Chiang Mai. T.
chebula and Z. myriacanthum were purchased from Warorot market (Chiang Mai). The
voucher specimens were identified and kept at the Department <strong>of</strong> Pharmaceutical Science,
Faculty <strong>of</strong> Pharmacy, Chiang Mai University.
Preparation <strong>of</strong> <strong>Plant</strong> <strong>Extracts</strong>
The dried powder <strong>of</strong> plant materials except T. chebula was separately extracted by
maceration and continuous extraction. For maceration, portions <strong>of</strong> the powdered plant
materials were separately macerated three times with 95% ethanol for 2 days. The
combined macerate <strong>of</strong> each plant was passed through Whatman filter paper No. 1 and
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evarporated to dryness in vacuo using rotary evaporator at 45°C. For continuous
extraction, portions <strong>of</strong> the powdered plant materials were separately extracted until
exhausted by soxhlet apparatus using n-hexane, chlor<strong>of</strong>orm and 95% ethanol,
respectively. The extract solutions were filtered and then the solvents were removed by
evaporation at 45°C to yield the n-hexane extract, chlor<strong>of</strong>orm extract and ethanol extract
<strong>of</strong> four kinds <strong>of</strong> plant materials.
For T. chebula, extraction was done by continuous extraction and the solvent
extraction method adopted from Sato et al. (1997). Continuous extraction was done by
using methanol as solvent. For solvent extraction method (Sato et al., 1997), the powder
<strong>of</strong> T. chebula was extracted three times with 50% ethanol under reflux for 2 hours. The
extract solutions were combined, concentrated in vacuo to evaporate all ethanol and then
extracted three times with diethyl ether and n-butanol, respectively. The combined <strong>of</strong>
each extract solutions was evaporated or freeze dried to dryness (Table 1).
Test Organisms
The test organisms used, S. aureus (ATCC 25923) and E. coli (ATCC 25922),
were obtained from the Department <strong>of</strong> Microbiology, Faculty <strong>of</strong> Medicine, Chiang Mai
University.
The inoculum size <strong>of</strong> each test strain was standardized according to the Committee
for Clinical Laboratory Standard (NCCLS, 1993). The test bacterial strain was inoculated
into Mueller Hinton broth, MHB (Oxoid) medium and incubated for 3-6 hrs at 35°C until
the culture attained a turbidity <strong>of</strong> 0.5 McFarland unit. The final inoculum was adjusted to
5 x 10 5 cfu/ml.
Screening for <strong>Antibacterial</strong> <strong>Activity</strong>
The extracts were evaluated for antibacterial activity by agar well diffusion
method. A molten 6 ml MHA was poured in a sterile petri dish. After agar hardening, four
cups <strong>of</strong> 12 mm were placed. 0.1 ml <strong>of</strong> the standard suspension (5 x 10 5 cfu/ml) <strong>of</strong> each
test bacterial strain was inoculated to molten 14 ml MHA (about 50°C) and then the
mixture was poured in the prepared petri dish. After agar hardening and cup removing,
each well was applied with 0.1 ml <strong>of</strong> each 0.1% extract. The solvents were used as
control. The applied plates were incubated at 35°C for 18-24 hrs and then inhibition zone
diameters (IZD) were measured.
The minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) were determined for the extracts that have inhibition zone diameter
more than 20 mm. The MIC was determined by macrobroth dilution method and
compared with commercial antibiotic Grammicin (gentamicin sulfate) as positive control.
Each extract was reconstitued with its solvent, then two-fold serial dilutions <strong>of</strong> the
extracts and control drug were prepared in tubes with MHB as diluent. Each dilution was
seeded with test organism to the standard concentration (5 x 10 5 cfu/ml). The MIC was
taken as the last dilution showing no noticeable growth (turbidity).
The MBC determination was determined by subculturing 10 µl <strong>of</strong> the culture
medium from each tube (in macrobroth MIC assay) showing no apparent growth on MHA
and incubated at 35°C for 18-24 hrs. The MBC was read as the least concentration
showing the growth <strong>of</strong> not more than five colonies <strong>of</strong> each organism (99.9% were dead).
Phytochemical Screening
The high potential fraction, PG-1, was preliminary studied for phytochemical
constituents using standard method.
RESULTS AND DISCUSSION
In this present study, five <strong>Thai</strong> medicinal plants were separately extracted with
various methods to yield 21 kinds <strong>of</strong> extracts. All extracts were evaluated for their
antibacterial activity against S. aureus and E. coli, representing gram positive and gram
negative bacteria. The results are shown in Table 1 and 2. Three out <strong>of</strong> 21 kinds <strong>of</strong>
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extracts (PG-1, PG-4 and AN-3) showed definite antibacterial activity. Four kinds <strong>of</strong>
extracts (PG-3, TC-2, ZM-2 and ZM-3) exhibited moderate inhibition <strong>of</strong> the growth <strong>of</strong>
representative gram positive and gram negative bacteria. The remaining 14 kinds <strong>of</strong>
extracts showed no antibacterial activity.
The MIC <strong>of</strong> ethanol extracts <strong>of</strong> P. granatum from maceration (PG-1) and
continuous extraction (PG-4) to S. aureus were equal but MBC <strong>of</strong> PG-1 is lower than PG-
4. The MIC and MBC value to E. coli <strong>of</strong> ethanol extracts <strong>of</strong> P. granatum by two different
methods were equal. It is to say that ethanolic extract <strong>of</strong> P. granatum acts as broad
spectrum antibacterial agent. Therefore the PG-1 extract was the most interesting for
further topical formulation for skin infectious disease.
Hydrolysable and condense tannin were found in the preliminary phytochemical
studies <strong>of</strong> the ethanolic extracts from P. granatum. The results are shown in Table 3.
CONCLUSION
From 21 kinds <strong>of</strong> extracts <strong>of</strong> <strong>Thai</strong> medicinal plants, PG-1 showed a broad
spectrum <strong>of</strong> antibacterial activities. Therefore PG-1 will be a valuable alternative
medicine for the treatment <strong>of</strong> bacterial infections. Further investigation on PG-1 extract
for phytochemical control such as chromatographic fingerprint should be performed for
developing to topical preparation as well as product stability testing.
Literature Cited
Mahidol University Faculty <strong>of</strong> Pharmacy. 1998a. Samunprai Maipeunban V1.
Prachachon, Bangkok. p.259-260.
Mahidol University Faculty <strong>of</strong> Pharmacy. 1998b. Samunprai Maipeunban V2.
Prachachon, Bangkok. p.259-260.
Mahidol University Faculty <strong>of</strong> Pharmacy. 1998c. Samunprai Maipeunban V4.
Prachachon, Bangkok. p.469-477.
Mahidol University Faculty <strong>of</strong> Pharmacy. 1998d. Samunprai Maipeunban V5.
Prachachon, Bangkok. p.263-265.
Malekzadeh, F., Ehsanifar, H., Shahamat, M., Levin, M. and Colwell, R.R. 2001.
<strong>Antibacterial</strong> activity <strong>of</strong> black myrobalan (Terminalia chebula Retz) against
Helicobacter pylori. Int. J. Antimicrob. Agents. 18:85-88.
Promjit, S., Wongsatit, C., Rungravi, T. and Terry, C. 1996. <strong>Medicinal</strong> <strong>Plant</strong>s in <strong>Thai</strong>land
V2. Amarin Printing and Public, Bangkok. p.32.
Sato, Y., Oketani, H., Singyouchi, K., Ohtesuro, T., Kihara, M., Shibata, H. and Higuti, T.
1997. Extraction and Purification <strong>of</strong> Effective Antimicrobial Constituents <strong>of</strong>
Terminalia chebula Retz. against Methicillin-resistant Staphylococcus aureus. Biol.
Pharm. Bull. 20(4):401-404.
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Tables
Table 1. Percent yield and inhibition zone diameter (IZD) <strong>of</strong> 1% <strong>of</strong> plant extracts against test bacterial strains.
Botanical name <strong>Thai</strong> name Part used
Acanthus ilicifolius Linn.
var. ilicifolius
Argyreia nervosa (Burm. f.)
Bojer
Punica granatum L. var.
granatum
Terminalia chebula Retz.
var. chebula
Zanthoxylum myriacanthum
Wall. ex Hook. F.
Ngueak plamo
Bai rabat
Thap thim
Samo thai
Ma khwaen
leaves
leaves
pericarps
fruit
fruit
* M: maceration; C: continuous extraction; S: solvent extraction
** IZD <strong>of</strong> all solvents were zero
● : black sticky extract, ❍ : oily extract
Extract Extracting IZD ± SD (mm) **
code method * Solvent % yield
S. aureus E. coli
AI – 1 M 95% ethanol 13.98 0 0
AI – 2 C hexane 6.83 0 0
AI – 3 C chlor<strong>of</strong>orm 2.03 0 0
AI – 4 C 95% ethanol 5.85 0 0
AN – 1 M 95% ethanol 8.77 0 0
AN – 2 C hexane 3.52 0 0
AN – 3 C chlor<strong>of</strong>orm 2.56 0 25
AN – 4 C 95% ethanol 5.08 0 0
PG – 1 M 95% ethanol 34.09 21.33 ± 0.58 25.66 ± 0.58
PG – 2 C hexane 1.15 0 0
PG – 3 C chlor<strong>of</strong>orm 0.65 0 15.66 ± 0.58
PG – 4 C 95% ethanol 42.80 20 24
TC – 1 C methanol 48.66 0 0
TC –2 S diethyl ether 3.22 15.33 ± 0.58 0
TC – 3 S n- butanol 18.34 0 0
TC – 4 S water 25.02 0 0
ZM – 1● M 95% ethanol 3.54 0 0
ZM – 2 ❍ M 95% ethanol 3.52 19.33 ± 0.58 18
ZM – 3 C hexane 14.14 16 17.66 ± 0.58
ZM – 4 C chlor<strong>of</strong>orm 6.14 0 0
ZM – 5 C 95% ethanol 9.74 0 0
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Table 2. MIC and MBC (mg/ml) <strong>of</strong> PG-1, PG-4, AN-3 and gentamicin against test
bacterial strains.
Antimicrobial
S. aureus E. coli
MIC MBC MIC MBC
PG – 1 0.39 1.56 12.5 12.5
PG – 4 0.39 3.13 12.5 12.5
Gentamicin 0.001 0.004 0.004 0.016
Table 3. Phytochemical screening <strong>of</strong> PG-1.
Chemical constituent Method Result
Alkaloid
Mayer’s reagent
Dragendorff’s reagent
- ve
- ve
Flavonoid Shinoda’s test - ve
Coumarin NaOH-paper - ve
Saponin Froth test - ve
Cardiac glycoside
Liebermann-Burchard test
Keller-kiliani test
- ve
- ve
Cyanogenic glycoside Picrate paper - ve
Anthraquinone glycoside Borntrager’s test - ve
Tannin
Gelatin solution
FeCl 3 TS
Formaldehyde-HCl test
Vanillin-HCl test
Lime water
Lead acetate solution
+ ve
+ ve
+ ve
+ ve
+ ve
+ ve
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