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Antimicrobial Activity of Herbal Extracts on Staphylococcus aureus and
Propionibacterium acnes

P. Leelapornpisid, S. Chansakao and T. Ittiwittayawat

Keywords: antimicrobial activity, Propionibacterium acnes, Staphylococcus aureus,
Excoecaria cochinchinensis, Salvia officinalis, gallic acid
Abstract
Excoecaria cochinchinensis Lour, Salvia officinalis Lour and Argyreia nervosa
(Burm.f) Bojer, leaves were extracted by various extracting procedures, using
different solvents for testing the antimicrobial activities against Staphylococcus
aureus (ATCC 25923) and Propionibacterium acnes isolated from patients. We
obtained 13 extracts. Using well diffusion method, ethanolic and water extracts of E.
cochinchinensis and S. officinalis were found to be effective against these organisms
whereas all extracts of A. nervosa were inactive. The chemical anti-acne agents such
as azelaic acid, benzoyl peroxide and commercial clindamycin phosphate were also
included in the experiment. The minimum inhibitory concentration (MIC) for the
active extracts was examined by broth dilution method. The results showed that the
MIC of water extracts from E. cochinchinensis and S. officinalis were 1.56 and 3.13
mg/ml for S. aureus (ATCC 25923), 1.56 and 6.25 mg/ml for P. acnes, respectively.
The minimal bactericidal concentration (MBC) were 3.13 and 6.25 mg/ml for S.
aureus (ATCC 25923), 6.25 and 12.5 mg/ml for P. acnes, respectively. These results
were comparable with azelaic acid, better than benzoyl peroxide but not as good as
clindamycin phosphate. Furthermore, the water extract from E. cochinchinensis
exhibited outstanding effective activities against clindamycin resistant S. aureus (P31
and Fl14) isolated from patients. The MIC and MBC were 0.78 and 3.13 mg/ml,
respectively. The phytochemical preliminary study on ethanolic and water extracts
of E. cochinchinensis was also performed for screening chemical constituents.
Tannin and phenolic compounds were found. From the thin layer chromatographic
pattern compared to standard tannic acid and gallic acid, the water extract revealed
the same Rf value (0.75) as gallic acid.
The purpose of this study was to find preliminary data for the development of
anti-acne products from natural plant extracts.
INTRODUCTION
Acne vulgaris is a common inflammatory disorder of the pilosebaceous unit in the skin. It is the most common of all skin problems. It occurs mostly on the face and, to a lesser extent, on the back, chest and shoulder. It is more common in males and typically begins at puberty. Main cause of acne is due to the effect of androgenic hormone that induced excessive sebum production together with the enlargement of sebaceous glands and form to comedones. The other major etiologic components of acne are abnormal desquamation of follicular epithelium and proliferation of Propionibacterium acnes (Bown, 1995; Sykes and Webster, 1994). Propionibacterium acnes (P. acnes) is an anaerobic gram-positive bacillus that lives as a normal commensal microorganism in the pilosebaceous unit. The abnormal desquamation of follicular epithelium causes an anaerobic condition in the follicle which promotes the proliferation of P. acnes. And P. acnes metabolizes the glycerol moiety of excessive sebaceous triglycerides into free fatty acids by excreting a bacterial lipase and chemotaxic factors. These excessive free fatty acids produced gather leukocytes around a Proc. WOCMAP III, Vol. 5: Quality, Efficacy, Safety, Processing & Trade in MAPs Eds. E. Brovelli, S. Chansakaow, D. Farias, T. Hongratanaworakit, M. Botero Omary, S. Vejabhikul, L.E. Craker and Z.E. Gardner follicle that cause inflammation. Therefore, inhibition of P. acnes will minimize the rupture of comedones into surrounding tissue and block the development of acne. Another organism that has also been implicated in the etiology of acne vulgaris is Staphylococcus aureus which promotes pustules and nodules. Many antibiotics have been used both orally and topically for acne treatment such as tetracycline, erythromycin as well as clindamycin. It was found that long term use of these broad-spectrum antibiotics often developed overgrowth of Candida albicans (Skyes and Webster, 1994) as well as bacterial resistance, and topical chemical anti-acne agents such as benzoyl peroxide, azelaic acid and retinoic acid caused skin irritation. This may decrease patient compliance and result in treatment failure. In recent years antimicrobial properties of herbs are increasingly reported from different parts of the world. These will be an alternative medicine for acne treatment and may decrease such problems. In this study, we investigated antimicrobial activities of three herbal extracts against S. aureus and P. acnes compared with some chemical agents for acne treatment.
The selection of herbs is based on their traditional use in Thailand and reported
antimicrobial activity. However, these herbs were not previously screened against acne
related organisms. Preliminary phytochemical screening and thin layer chromatographic
pattern were also studied in order to determine some of extract constituents that will be a
basis standard for next extractions.
MATERIALS AND METHODS

Plant Materials
Leaves of Excoecaria cochinchinensis and leaves of Agyreia nervosa were collected from Chiang Mai Province, Leaves of Salvia officinalis were purchased from
Royal Project Foundation, Chiang Mai.
Preparation of Plant Extracts
For maceration, each herb was dried, reduced to powder then macerated with 95% ethanol for 2 days, filtered the extract and repeated for 2 times. Concentrated all the collected filtrated by Rotary evaporator (Eyela N-N series) and stored at 2-8°C until further use. For continuous extraction, hexane, chloroform, 95% ethanol and distilled water were prepared for extraction solvents. Each herb was reduced to powder. Each solvent was added to each herb with 10-fold weight, and extracted until exhausted. Each filtrate was concentrated under vacuum except water filtrate was concentrated by freeze dryer. For reflux, each herb was minced then reflux for 4 hrs by using water as a solvent. The filtrate was collected and concentrated by freeze dryer.
Chemicals and Test Substances
Solution of each extract, 5% w/v, was prepared in the solvent of each extract as test substance. Except chloroform extract and ethanol extract from maceration method
were prepared in DMSO and 95% ethanol:DMSO (1:1), respectively.
Test Organisms
Propionibacterium acnes (isolated from patients), Staphylococcus aureus (ATCC 25923), S. aureus Fl14 and S. aureus P31 (isolated from patients) were cultivated on
tryptone soya agar (Oxoid Ltd, Basingstoke) plates. For working cultures, 3-4 colonies
were suspended in tryptone soya broth, incubated for 1 hr for S. aureus and 24 hrs for P.
acnes at 37°C (over night culture, ONC) (Raman et al., 1995) Then diluted to provide a
final inoculum of approximately 106 CFU/ml.
Tests for Antimicrobial Activities, Well Diffusion Method
Antimicrobial activities of all test substances against S. aureus and P. acnes were screened by well diffusion method (Bown, 1995; Sharma et al., 1998). A 0.1 ml volume
of broth organism cultured was seeded into 10 ml of molten and cool (45°C) agar then
poured in plate that has been poured with 10 ml agar and placed four sterile 12-mm
diameter cups prior. Cups were taken off and 0.05 ml of test substances were added into
each well with duplicate experiments. The plates were allowed to stand for 30 minutes at
room temperature, and then incubated aerobically for S. aureus, anaerobically for P.
acnes at 37°C. The diameter of inhibition zone was measured, including well size, after
24 hrs incubation for S. aureus and 72 hrs incubation for P. acnes
Minimal Inhibitory Concentration (MIC) Test, Broth Dilution Method
Antimicrobial activities against S. aureus and P. acnes were evaluated by broth dilution method. (Raman et al., 1995; Wiriyachitra et al., 1984) Cultures of S. aureus and
P. acnes were diluted to density of 105 CFU/ml in MHB and TSB, respectively. The test
tubes containing 2-folds dilution of each test substance were inoculated by the diluents. S.
aureus was incubated aerobically at 37°C for 24 hrs and P. acnes was incubated
anaerobically at 37°C for 72 hrs. The MICs were determined by detecting the turbidity.
Minimal Bactericidal Concentration (MBC) Test
All clear tubes from MIC test were steaked on TSA plate using 10 µl loop. S. aureus was incubated aerobically at 37°C for 24 hrs and P. acnes was incubated
anaerobically at 37°C for 72 hrs. The MBC were determined by colony count of more
than 99.9% killing.
Preliminary Phytochemical Test
Preliminary phytochemical screening for some constituents of extracts were under taken using standard method as described by various authors (Xie et al., 1989; Sharma et
al., 1998; Boonton et al., 1984; Nisit et al., 1999; Wiriyachitra et al., 1984). The plant
extracts were screened for the presence of biologically active compounds like glycoside,
alkaloids, tannins and phenolic compounds.
Thin-layer Chromatographic Pattern
The extracts with outstanding antimicrobial activity extracts were carried out for TLC. Difference solvent systems were developed for finding the best manifest
chromatogram. TLC was performed on silica gel 60 F254 (0.25 mm) using iso-butyl
alcohol:glacial acetic acid:water (3:1:1) as mobile phase. Gallic acid and tannic acid were
used as reference standard. TLC spot were visualized under ultraviolet light at 254 and
365 nm and were sprayed with 5% ferric chloride in ethanolic reagent.
RESULTS AND DISCUSSION
Thirteen extracts were obtained from three different extracting procedures of three herbs. All the extracts as well as chemical antiacne agents were tested for screening the antimicrobial activities against S. aureus and P. acnes. Six extracts from Excoecaria cochinchinensis and Salvia officinalis exhibited antimicrobial activity which were comparable to chemical agents. All of Argyreia nervosa extracts showed no activity against the tested bacteria. The results as shown in Table 1. The extracts with more than 20 mm of inhibition zone (EC-1, EC-5, EC-6, SO-1, SO-2) were selected for minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) testing compared with chemical agents by broth dilution method. Besides EC-1 and SO-1 which were tested by well diffusion method due to the precipitation in broth medium. The results were given in Table 2. Furthermore, the EC-5 extracts exhibited outstanding activity against S. aureus (P31 and Fl14) that resist to clindamycin, erythromycin, oxacillin and co-trimoxazole, The MIC and MBC were given in Table 3. It was observed that ethanolic and water extracts of the leaves of E. cochinchinensis showed markedly activity against S. aureus and P. acnes. Furthermore, its water extracts also revealed activity against S. aureus that resisted to clindamycin, erythromycin, oxacillin and co-trimoxazole. Whereas hexane and chloroform extracts showed no antimicrobial activity. This might explain the active constituents of this plant were soluble in polar solvents. The MIC of water extracts from continuous extractions (EC-5) and reflux method (EC-6) were equal but MBC of EC-6 was higher than EC-5. Thus, may due to the difference in method of extraction. For S. officinalis, only ethanolic (SO-1) and water (SO-2) extracts were tested for antimicrobial activity, followed folklore use. The results showed no differences in antimicrobial activity of both extracts. When antimicrobial activity of the plant extracts were compared to chemical agents, it has been found that extracts of E. cochinchinensis and S. officinalis revealed good activity equal to azelaic acid and benzoyl peroxide but lower than clindamycin. Especially, the water extract of E. cochinchinensis by continuous extraction (EC-5) was equal to azelaic acid, better than benzoyl peroxide and effective against S. aureus that resisted to clindamycin, erythromycin, oxacillin and co-trimoxazole. So, the extract of E. cochinchinensis was then carried out for phytochemical study. The preliminary phytochemical studies on E. cochinchinensis extracts with outstanding antimicrobial activity revealed the present of cyanogenic glycoside, leucoanthocyanin, saponin in ethanolic extract; antracene in water extract and tannins and phenolic compounds in both ethanolic and water extracts. All the extracts contained no alkaloids as shown in Table 4. As for TLC pattern (Fig. 1) the water extract revealed more than one constituent that can absorb ultraviolet light at 254 nm and found only one constituent that can
fluorescent in yellow to green at 365 nm. When sprayed with 5% ferric chloride in
ethanolic reagent, were appeared black spot and tails. Black spot of water extract has the
Rf value of about 0.75 as same as gallic acid that correlated with the observation of
Wiriyachitra et al. (1984).
CONCLUSION
From the herbs investigated for antimicrobial activity against acne related organisms; S. aureus and P. acnes. We found that the water extracts from E.
cochinchinensis and S. officinalis leaves revealed outstanding activity that equal to some
chemical agents which caused skin irritation. Furthermore, the water extract from E.
cochinchinensis showed good activity against S. aureus that resisted to clindamycin,
which was now the most effective antibiotic in acne treatment. So these extracts would be
valuable as the alternative in the treatment of acne in the future.
Literature Cited
Boonton, P., Hajiwangoh, H., Wiriyachitra, P., Adolf, W., Oferkuck, H. and Hecker, E.
1984. Isolation and structural studies of chemical constituent of latex of Excoecaria bicolor Zoll.ex Hassk. Abstr. 10th Conf. on Science and Technology of Thailand. p.205-208. Bown, D. 1995. Encyclopedia of Herbs and Their Used. Lee, D., Yoon, E., Park, S., Lee, K., Pyo, H. and Lee, B. 2001. The anti-acne activity of natural plant extracts and applications in cosmetic product. Discover the Secret of Asian Natural Beauty, 5thASCS Conf. in Bangkok, Thailand. Holt, J.G., Krieg, N.R., Sneath, P.H.A., Statey, J.T. and Williams, S.T. 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Keville, K. 1991. The Illustrated Herbs Encyclopedia Guide to herbs. Marino, M., Bersani, C. and Comi, G. 2001. Impedance measurements to study the antimicrobial activity of essential oils from Lamiaceae and Compositae. Intl. J. Food Microbiol. 67:187-195. Murray, P.R., Baron, E.J., Pfaller, M.A. et al. 1994. Manual of Clinical Microbiology. 6th Nisit, P., Sirinthorn, P. and Pimporn, L. 1999. Development of Litsea glutinasa Leaf Extract for Medical Preparation and Cosmetics. Part I: Pharmacognostic and Preliminary Chemical Constituents Study. Naresuan Univ. J. 7(July-Dec):64-78. Raman, A., Weir, U. and Bloomfield, S.F. 1995. Antimicrobial effects of tea-tree oil and its major components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes. Letter in Appl. Microbiol. 21:242-245. Sykes, N.L. and Webster, G.F. 1994. Acne a review of optimum treatment. Drugs Sharma, O.P., Bhat, T.K. and Singh, B. 1998. Thin-layer chromatography of gallic acid, methyl gallate, pyrogallol, phloroglucinol, catechol, resorcinol, hydroquinone, catechin, epicatechin, cinnamic acid, p-coumaric acid, ferulic acid and tannic acid. J. Chromatography A. 822:167-171. Wiriyachitra, P., Boonton, P., Hajiwangoh, H. et al. 1984. Uterotonic substance from Excoecaria agallocha, E. bicolor and E. oppositifolia. Abstr. 10th Conf. on Science and Technology of Thailand. p.214-215. Xie, J.M., Chen, Y.S., Zhao, S.N. and Zhou, X.D. 1989. Studies on the chemical constituents of Excoecaria cochinchinensis Lour. var. viridis Merr. China J. Chinese Materia Medica 14(May):292-294. Tables

Table 1. Antimicrobial activity to S. aureus and P. acnes of herbal extracts and chemical
5% Ethanolic extract of E. cochinchinensis 5% Hexane extract of E. cochinchinensis 5% Chloroform extract of E. cochinchinensis 5% Ethanolic extract of E. cochinchinensis 5% Water extract of E. cochinchinensis 5% Water extract of E. cochinchinensis 5% Ethanolic extract of A. nervosa 5% Chloroform extract of A. nervosa 5% Ethanolic extract of A. nervosa 5% Ethanolic extract of S. officinalis 5% Water extract of S. officinalis *The inhibition zone of all solvents were zero Table 2. The MIC and MBC value of herbal extracts and chemical agents to S. aureus Table 3. The MIC and MBC value of EC-5 extracts to S. aureus by broth dilution method. P31 and Fl14 were isolated from pus and fluid of patients resistant to clindamycin, erythromycin, oxacillin and co-trimoxazole but susceptible to fosfomycin and vancomycin Table 4 Preliminary phytochemical test of E. cochinchinensis extracts. 1. Glycoside
2. Alkaloids
3. Tannins & phenolic cpd
+ = positive, − = negative, / = not interpret

Fig. 1. TLC pattern of E. cochinchinensis extracts and reference standard: (A) chloroform
extract; (B) ethanolic extract; (C) water extract; (D) gallic acid; (E) tannic acid.

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