Anti-Streptococcus pyogenes Activity of Selected Medicinal Plant Extracts Used in Thai Traditional Medicine

Purpose: To evaluate the anti-Streptococcus pyogenes activity of selected medicinal plants used in Thai traditional medicine. Methods: Sixty-nine extracts of 51 selected Thai medicinal plant species were tested for anti-S. pyogenes activity by paper disc agar diffusion and broth microdilution methods. Results: Ten plants including Boesenbergia pandurata (Roxb.) Schltr., Cinnamomum bejolghota (Buch.-Ham.) Sweet, Cinnamomum porrectum (Roxb) Kosterm, Eleutherine americana Merr., Gymnopetalum cochinchinensis (Lour.) Kurz, Piper betle L., Quercus infectoria G. Olivier, Quisqualis indica L, Rhodomyrtus tomentosa (Aiton) Hassk., and Walsura robusta Roxb. demonstrated good antibacterial activity against S. pyogenes NPRC 101. These plants were selected and further evaluated for their anti-S. pyogenes activity against 11 isolates of S. pyogenes from patients with upper respiratory tract infections. Three plants including Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa exhibited good antibacterial activity against all S. pyogenes isolates and produced similar activities against different tested isolates. Boesenbergia pandurata and Rhodomyrtus tomentosa demonstrated antibacterial activity with the same minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) range of 3.91 31.25 μg/ml whereas Eleutherine americana displayed MIC and MBC values of 250 and 250-500 μg/ml against all S. pyogenes isolates. Conclusion: Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa have great antibacterial potentials against S. pyogenes.


INTRODUCTION
Upper respiratory tract infections (URTIs) are the most common human infection, mostly caused by viruses and bacteria.Streptococcus pyogenes is a major upper respiratory tract bacterial pathogen that causes a wide variety of diseases.It is the most common cause of bacterial pharyngitis and is linked to many serious complications [1].Viral infections in upper respiratory tract are usually not an appropriate indication for the use of antibiotics.Bacteria that cause URTIs should be taken and cultured to determine the particular type of bacteria and antibiotics treatment are performed if necessary [2].However, unnecessary and irrational selfmedication with antibiotics seems to be common for URTIs [3,4] and these result in resistance to many bacteria including S. pyogenes [5,6].Antibiotic misuse for URTIs is a serious problem that not only results in selection of resistant strains of bacteria but also waste of resources.Correlation of antibiotic resistance in S. pyogenes with antibiotic consumption has been recorded.Increase in antibiotic use has resulted in increased prevalence of antibiotic-resistant S. pyogenes [7,8].Hence, the search for alternative treatment dealing with URTIs caused by S. pyogenes is necessary.Medicinal plants are a great source of alternative treatment for many infections.The use of medicinal plants may substitute antibiotic consumption for URTIs or decrease antibiotic-resistant bacteria.
In traditional Thai medicine, many medicinal plants have been in use since ancient times.Herbal medicines are relatively safer than synthetic drugs and offer profound therapeutic benefits [9].A number of Thai medicinal plants have been studied for their antibacterial activities.There are several reports on antibacterial activity of plants that inhibit various bacterial pathogens, but only limited number of studies on S. pyogenes, an important bacterial human pathogen, have been published.Therefore, this study was aimed to evaluate the antibacterial activity of selected medicinal plants commonly used in traditional Thai medicine for bacterial infections against S. pyogenes isolated from upper respiratory tract infections.

EXPERIMENTAL Preparation of plant extract
Fifty-one medicinal plant species used in traditional Thai medicine for bacterial infections were selected.The plant materials were collected from various areas of the southern region of Thailand from 2006 -2007.Quercus infectoria nut galls were purchased from an herb shop in Songkhla, Thailand.Botanical identification was performed by Dr. Oratai Neamsuvan, an ethnobotanist at the Faculty of Traditional Thai Medicine, Prince of Songkla University, where their voucher specimens are deposited.All plant materials were cut into small pieces and dried at 60 C overnight.The dried plant materials were crushed in a mechanical mortar and soaked in extracted solvent for 7 days (3 times).The solvent was then filtered and dried using a rotary evaporator.All extracts were stored at -4 C, and dissolved in dimethyl sulfoxide (DMSO, Merck, Germany) before use.The aliquots were checked for sterility by streaking with a sterile loop on brain heart infusion (BHI) agar and incubating at 37 °C overnight.

Bacterial strain and culture conditions
Eleven clinical isolates of S. pyogenes (NPRC 101-111) from patients with tonsillitis or pharyngitis were obtained from Department of Microbiology and Natural Products Research Center, Faculty of Science, Prince of Songkla University.All isolates were susceptible to erythromycin and penicillin G.These isolates were stored in BHI broth supplement with 20 % glycerol at -70 °C.All the isolates were routinely cultured in BHI broth or blood agar (BA) plates and incubated with 5 % CO 2 at 37 o C for 24 h.

Screening for anti-S. pyogenes activity
The preliminary screening of all plant extracts for their anti-S.pyogenes activity was carried out by disc agar diffusion method [10].The extracts were dissolved in DMSO (250 mg/ml) and then 10 µl were applied to sterile filter paper discs (Whatman No. 1; 6 mm in diameter) so that each disc finally yielded 2.5 mg of the extract.Dry discs (dried at 37 °C overnight) were applied onto the surface of 5% blood Mueller Hinton agar (MHA) plates seeded with the culture of S. pyogenes.The plates were then incubated with 5% CO 2 at 37 °C for 20 h.Dimethyl sulphoxide, extraction solvents, and antibiotic discs including erythromycin (15 µg) and penicillin G (1 µg) were used as controls.The experiment was carried out in duplicate and the average diameter of inhibition zone was calculated.

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)
A broth microdilution method according to Clinical and Laboratory Standards Institute Guidelines (CLSI) was used to determine the MIC and MBC of the plant extracts against S. pyogenes [10].Erythromycin was used as reference antibiotic.MIC was recorded as the lowest concentration that produced a complete suppression of visible growth.MBC was taken as the concentration that gave significant MIC values using a sterile loop streaking on fresh media.All assays were carried out in triplicate.

Screening for anti-S. pyogenes activity
The antibacterial activities of 51 medicinal plant species used in Thai traditional medicine for bacterial infections against S. pyogenes NPRC 101 are presented in Table 1

DISCUSSION
A of plant extracts can be screened for their anti-S.pyogenes properties quickly using paper disc assay.However, this assay is not classically quantitative and using the size of inhibition zone to indicate relative antibacterial activity is not sufficient.The zone of inhibition may be affected by many factors such as the evaporation, solubility, and diffusion rate of the active components through test medium.Zone of inhibition testing is particularly appropriate for determining the ability of water-soluble antimicrobial compounds to inhibit the growth of microorganisms.Therefore, the MIC/MBC values and zone of inhibition of some of the plant extracts in this study did not correlate.For example, Boesenbergia pandurata extract generated a very small zone of inhibition (7 mm) but possessed very good MIC and MBC values.
In this study, we found that the extracts of Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa not only demonstrated good antibacterial activity against S. pyogenes isolated from upper respiratory tract infections but also produced similar activities against different S. pyogenes clinical isolates.
In Thai traditional medicine, the rhizomes of Boesenbergia pandurata are used to treat colic disorders, wound infections and inflammation [11].The antibacterial activities of this plant have been reported [12].Some antibacterial active components from this plant were isolated and studied for their activities.Pinostrobin isolated from this plant demonstrated anti-Helicobacter pylori activity [13].Panduratin A from this plant displayed significant antibacterial activity against a number of staphylococci and enterococci clinical isolates.Notably, the antibacterial activity of panduratin A was more potent than many reference antibiotics [14,15] Eleutherine americana is a herbal plant whose red bulb was used as a folk medicine and flavouring agent.In the screening test we found that the bulb extract of this plant demonstrated moderately strong activity against S. pyogenes when compared with other plant extracts.However, when we increased the number of bacterial isolates it could produce similar antibacterial activity against difference S. pyogenes isolates.The antibacterial activities of this plant have been previously reported against gram-negative [18] and gram-positive bacteria [19].Several compounds from this plant such as eleuthinone A, eleuthraquinone A and B, and eleucanarol have been isolated and studied for their antibacterial activities against Staphylococcus aureus [20].
Rhodomyrtus tomentosa is a Thai medicinal plant used to treat skin, oral, gastrointestinal, and urinary tract infections.In this study, we found that the extracts of flower, fruit, and leaf demonstrated good antibacterial activity against S. pyogenes.The leaf extract of this plant has been reported for its antibacterial activity against many bacterial pathogens [19].An isolated compound, named rhodomyrtone, from the leaf of this plant exhibited strong antibacterial activity against many pathogenic Gram-positive bacteria [21-23] including S. pyogenes [24].Moreover, rhodomyrtone possessed noteworthy activity against methicillin-resistant S. aureus, displaying a stronger activity than vancomycin, a reference antibiotic [24].

CONCLUSION
Our study demonstrated that the plants species including Boesenbergia pandurata, Eleutherine americana, and Rhodomyrtus tomentosa have great potentials as antibacterial agents against S. pyogenes.Thus, these plants may yield biologically active compounds that might be valuable in the treatment of the diseases caused by S. pyogenes.Their active components, however, need to be isolated, and their toxicity and therapeutic activity in vivo evaluated.