Medicinal plants with significant activity against important pathogenic bacteria

Abstract

Interest in the study of medicinal plants as a source of pharmacologically active compounds has increased worldwide. This study is a broad screening of 31 plant species against a wide range of pathogenic bacteria, including Acinetobacter baumannii, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella spp., Shigella flexneri, Staphylococcus aureus, Streptococcus mutans, and Streptococcus pyogenes. The agar disk diffusion method showed that most plants were more active against gram-positive than gram-negative bacteria. Streptococcus pyogenes was the most sensitive organism inhibited by nearly all of the extracts (97.6%), followed by Bacillus cereus (63.4%) and Staphylococcus aureus (61.0%). The ethanol extracts of Rhodomyrtus tomentosa (Aiton) Hassk. (Myrtaceae) (leaf) and Eleuterine americana Merr. (bulb) exhibited good antibacterial activity against gram-positive bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of Rhodomyrtus tomentosa ranged from 3.9 to 7.8 and 7.8 to 125 μg/mL, respectively, while those of Eleuterine americana were 120–125 and 250–1000 μg/mL, respectively. Quercus infectoria G. Olivier (Fagaceae) and Piper betle L. (Piperaceae) showed a broad spectrum of activity. The ethanol extract from the nut galls of Quercus infectoria demonstrated significant activity against all important pathogens. It inhibited the growth of all pathogens, with MIC values of 62.5–1000 μg/mL. The MBC ranged from 125 to 1000 μg/mL in most species, except Enterococcus faecalis. The ethanol extract from Piper betle (leaves) demonstrated antibacterial activity against almost all species with the same MIC and MBC values, ranging from 125 to 500 μg/mL.

Keywords::

• Antibacterial activity
• antibacterial assay
• pathogenic bacteria
• Piper betle Quercus infectoria
• Rhodomyrtus tomentosa
• Thai medicinal plants

Introduction

Interest in the study of medicinal plants as a source of pharmacologically active compounds is increasing worldwide. Medicinal plants have been used by the world population for their basic health care needs, including use against many infectious bacteria. It has been recognized that in most developing countries, plants are the main medicinal source used to treat infectious diseases. In Asia and Latin America, populations continue to use traditional medicine as a result of historical circumstances and cultural beliefs (World Health Organization (WHO), 2002a). Meanwhile, alternative medicine is becoming more and more popular in a number of developed countries. The percentage of the population that has used some form of alternative medicine is 48% in Australia, 70% in Canada, 42% in the USA, 38% in Belgium, and 75% in France (WHO, 2002a). In addition, a number of recent studies have documented the antibacterial activities of various species of medicinal plants against several pathogenic bacteria (Singh et al., 2005; Zaidi & Crow, 2005; Al-hebshi et al., 2006; Moon et al., 2006; Salazar et al., 2006).

The widespread use of antimicrobials has caused the emergence and spread of resistant bacteria to become a clinical problem (WHO, 2002b). Some pathogens rapidly become resistant to many of the first discovered effective drugs (Barbour et al., 2004). Many plants may have strong activity and good medical potential to be developed to effective drugs that may be substituted for antibiotic consumption and eventually reduce antibiotic resistant bacteria. Therefore, it is necessary to establish data to distinguish species that have weak antibiotic activities with no scientific significance and those with strong activities and good medical potential to be developed as effective drugs.

Forty-one extracts of 31 selected medicinal plant species used in Thai medicine against infections were studied against a wide range of important pathogens. Parts of plants were collected on the basis of traditional practices by Thai herbalists. They were tested against a wide range of important bacteria, including Acinetobacter baumannii, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella spp., Shigella flexneri, Staphylococcus aureus, Streptococcus mutans, and Streptococcus pyogenes.

Materials and methods

Medicinal plants

Thirty-one medicinal plants were used in this study: Aegle marmelos (L.) Corrêa (Rutaceae), Asclepias curassavica L. (Asclepiadaceae), Boesenbergia pandurata (Roxb.) Schltr. (Zingiberaceae), Cinnamomum bejolghota (Buch.-Ham.) Sweet (Lauraceae), Cinnamomum porrectum (Roxb.) Kosterm. (Lauraceae), Cleome gynandra L. (Cleomaceae), Eleutherine americana Merr. (Iridaceae), Gymnopetalum cochinchinensis (Lour.) Kurz (Cucurbitaceae), Manilkara achras (Mill.) Fosberg (Sapotaceae), Millingtonia hortensis L.f. (Bignoniaceae), Mimosa pudica L. (Mimosaceae), Mitragyna speciosa (Korth.) Havil (Rubiaceae), Morinda citrifolia L. (Rubiaceae), Murdannia loriformis (Hassk.) R.S. Rao & Kammathy (Commilinaceae), Oroxylum indicum (L.) Kurz (Bignoniaceae), Peltophorum pterocarpum (DC.) Backer ex K. Heyne (Fabaceae), Phyllanthus niruri L. (Euphorbiaceae), Piper betle L. (Piperaceae), Piper chaba Hunter (Piperaceae), Piper nigrum L. (Piperaceae), Piper sarmentosum Roxb. (Piperaceae), Quercus infectoria G. Olivier (Fagaceae), Quisqualis indica L. (Comcretaceae), Rhizophora mucronata Lam. (Rhizophoraceae), Rhodomyrtus tomentosa (Aiton) Hassk. (Myrtaceae), Terminalia bellirica (Gaertn.) Roxb. (Combretaceae), Terminalia chebula Retz. (Combretaceae), Terminalia sp. (Combretaceae), Theobroma cacao L. (Sterculiaceae), Walsura robusta Roxb. (Meliaceae), and Xylocarpus granatum J. König (Meliaceae). Botanical identification of the plant materials was authenticated by Professor Tanomjit Supavita, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University. Classified reference voucher specimens were deposited at the Herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla, Thailand.

All of the plant materials were cut into small pieces and dried at 60°C overnight. They were crushed in a mechanical mortar and soaked three times with ethanol for 7 days. The solvent was then distilled under reduced pressure in a rotary evaporator until it became completely dry.

Bacterial strains

Clinically bacterial isolates were obtained from the Regional Medical Sciences Center Songkhla and Songklanagarind Hospital, Thailand. Each was suspended in Mueller Hinton broth (MHB; Difco, France) and incubated at 37°C for 18 h. Mueller Hinton agar (MHA; Difco) was used for antibacterial assay. The antibiotic resistance profiles of 200 clinical isolates from each bacterial species are presented in Table 1.

Table 1.  Antibiotic resistance profile of pathogenic bacteria.

Pathogenic bacteria	Beta-lactam						Quinolone		Aminoglycoside		Glycopeptide	Miscellaneous
	Ampicillin	Amoxicillin	Ceftazidime	Cephalothin	Oxacillin	Penicillin	Ciprofloxacin	Norfloxacin	Amikacin	Gentamicin	Vancomycin	Clindamycin	Erythromycin	Chloramphenicol	Co-trimexazole	Tetracycline
Acinetobacter baumannii	38		65			100	65	77	61	67					69
Bacillus cereus	100				100	100	0			0	0	0	18	0		27
Enterococcus faecalis	14					28		66		36	3		92	31		83
Escherichia coli	81	35	18	70				43	5	30					62
Klebsiella pneumoniae	99	37	35	42				43	14	32					42
Listeria monocytogenes						30	10						30	0	0	10
Pseudomonas aeruginosa			27				24	45	20	30					97
Salmonella sp.	38							1						22	30	20
Salmonella Typhi	13							11						6	8
Salmonella Weltevreden	25							33						0	11
Shigella flexneri	44							0							76	88
Staphylococcus aureus (MRSA)	96	95			100	100					0	64	97		78
Staphylococcus aureus (MSSA)	0	1			0	94					0	5	11		3
Streptococcus mutans						0							0
Streptococcus pyogenes						0						100	0

% resistance from 200 clinical isolates.

Paper disk agar diffusion method

The paper disk agar diffusion method (Clinical and Laboratory Standards Institute (CLSI), 2006b) was used for preliminary screening of antibacterial activity of the extracts.

Plant extracts were dissolved in dimethyl sulfoxide (DMSO; Merck, Germany) or ethanol before use. Ten microliters (250 mg/mL) of crude extracts were applied to sterile filter paper disks (Whatman No. 1; 6 mm in diameter) so that each disk was saturated with 2. 5 mg of the extract. Dry disks (dried at 37°C overnight) were applied onto the surface of MHA plates seeded with 3–5 h broth culture of the tested bacteria (5% blood was added to the MHA for Streptococcus mutans and Streptococcus pyogenes). The plates were then incubated for 18 h at 37°C and with 5% CO₂ in the case of Streptococcus mutans and Streptococcus pyogenes. Dimethyl sulfoxide and extractive solvents were used as controls. The antibacterial activity was evaluated by measuring the diameter of inhibition zones. The experiment was performed in duplicate and the means of the zones were calculated.

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

The broth microdilution method (CLSI, 2006a) was used to determine the MIC and MBC of the extracts that produced inhibition zones. The bacterial suspensions (10⁵ cfu/mL) were added to MHB supplemented with the plant extracts at concentrations ranging from 0.5 to 1000 μg/mL, and incubated at 37°C for 20 h (5% lysed blood was added to the MHB for Streptococcus mutans and Streptococcus pyogenes). The minimal inhibitory concentration was recorded as the lowest concentration that produced a complete suppression of visible growth. The minimal bactericidal concentration was performed with the extracts that gave significant MIC values by using a sterile loop streaking on fresh media.

Results and discussion

Many plants have been reported to possess antibacterial activity against pathogenic bacteria. In the present work, we aimed to screen for effective medicinal plant species on some clinically isolated pathogenic bacteria. The ethnobotanical data of 31 plant species commonly used in Thai traditional medicine for bacterial infections have been documented (Voravuthikunchai et al., 2007). The ethanol extract of Quercus infectoria gave the highest yield (57.15%), followed by Manilkara achras (26.77%) and Millingtonia hortensis (25.41%), respectively (data not shown).

Results for the antibacterial activities of extracts from the medicinal plants on selected pathogenic bacteria of medical importance were preliminarily tested according to the CLSI. The test strains used in the following experiment were selected according to their extremely multiple drug-resistant profiles as reported in Table 1. Among the plants tested, nearly all extracts produced good inhibition zones on Streptococcus pyogenes. The inhibition zones ranged from 7 to 26 mm. Piper betle, Quercus infectoria, and Eleutherine americana showed good activity with a zone diameter of 26, 23, and 23 mm, respectively (data not shown). In contrast, Murdannia loriformis did not have any antibacterial activity.

Preliminary data on zone inhibiton indicate that Streptococcus pyogenes is the most sensitive organism inhibited by nearly all of the plant extracts (97.6%), followed by Bacillus cereus (63.4%) and Staphylococcus aureus (61.0%) (Table 2). Quercus infectoria and Piper betle were the only two plant species that showed activity against most gram-negative bacteria. Many other workers have also reported better antibacterial activity of medicinal plants against gram-positive bacteria than gram-negative bacteria (McCutcheon et al., 1992; Bonjar, 2004). The study of 67 ethanol extracts from 50 plants used in North Cote-d’Ivoire as traditional remedies reported no antibacterial activity against Escherichia coli and Pseudomonas aeruginosa (Kone et al., 2004). The basis for their differences in susceptibility might be due to differences in their cell wall composition (Grosvenor et al., 1995).

Table 2.  Percentage of Thai medicinal plants which produced inhibition zones against pathogenic bacteria.

Pathogenic bacteria	Resistance pattern to antibiotics^a	% Active
		Plant species (31 species)	Plant extracts (41extracts)
Acinetobacter baumannii NPRCM0192	Amp, Cef, Pen, Cip, Nor, Ami, Co-tri	16.1	12.2
Bacillus cereus NPRCM 0380	Amp, Oxa, Pen, Ery, Chl	58.1	63.4
Enterococcus faecalis NPRCM 0573	Amp, Pen, Nor, Gen, Van, Ery, Chl, Tet	29.0	26.8
Escherichia coli ATCC 25922		6.5	4.9
Klebsiella pneumoniae NPRCM 0777	Amp, Amo, Cef, Cep, Nor, Ami, Gen	6.5	4.9
Listeria monocytogenes NPRCM 0982	Pen, Cip, Ery, Tet	22.6	22.0
Pseudomonas aeruginosa NPRCM 1201	Cip, Nor, Ami, Gen, Co-tri	12.2	9.8
Salmonella sp. NPRCM1333	Amp, Nor, Chl, Co-tri, Tet	6.5	4.9
Salmonella Typhi NPRCM 1565	Amp, Nor, Chl, Co-tri	6.5	4.9
Salmonella Typhimurium ATCC 13311		6.5	4.9
Salmonella Weltevreden NPRCM 1780	Amp, Nor, Chl, Co-tri	6.5	4.9
Shigella flexneri NPRCM 1872	Amp, Co-tri, Tet	16.1	14.6
Staphylococcus aureus ATCC 25923		64.5	61.0
Streptococcus mutans NPRCM 2010		29.0	29.3
Streptococcus pyogenes NPRCM 2105	Clin	96.8	97.6

^aAmp = ampicillin, Amo = amoxicillin, Cef = ceftazidime, Cep = cephalothin, Oxa = oxacillin, Pen = penicillin, Cip = ciprofloxacin, Nor = norfloxacin, Ami = amikacin, Gen = Gentamicin, Van = vancomycin, Ery = erythromycin, Chl = chloramphenicol, Co-tri = co-trimexazole, Tet = tetracycline, Clin = clindamycin.

The MIC and MBC values for six effective plants against representative multiple drug-resistant strains are shown in Table 3. The ethanol extract of Rhodomyrtus tomentosa leaf showed very good antibacterial activity against gram-positive bacteria. The extract exhibited very low MIC values of 3.9–7.8 μg/mL against Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Streptococcus mutans, and Streptococcus pyogenes; and the MBC ranged from 7.8 to 125 μg/mL. The ethanol extract of Eleuterine americana had MIC and MBC against gram-positive bacteria in the range 120–125 and 250–1000 μg/mL, respectively. Quercus infectoria inhibited the growth of all bacterial strains, with MIC values of 62.5–1000 μg/mL and MBCs ranging from 125 to 1000 μg/mL. Piper betle demonstrated antibacterial activity against almost all pathogens with the same MIC and MBC values (125–500 μg/mL).

Table 3.  Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of ethanol extracts of effective plant species on pathogenic bacteria.

Pathogenic bacteria	MIC and MBC (μg/mL)
	Eleutherine americana (bulb) SV0627^a	Piper betle (leaf) SV0645	Quercus infectoria (nut gall) SV0652	Rhizophora mucronata (bark/fruit) SV0654	Rhodomyrtus tomentosa (Leaf/stem) SV0655	Terminalia sp. (fruit) SV0659
Acinetobacter	NA	125^b	125	1000/>1000^d	NA	250
baumannii	NA	125^c	125	1000/>1000	NA	250
Bacillus cereus	250	250	250	>1000/500	7.8/1000	500
	1000	250	250	>1000/>1000	7.8/1000	1000
Enterococcus	>1000	NA	1000	>1000/500	62.5/>1000	>1000
faecalis	>1000	NA	1000	>1000/>1000	1000/>1000	>1000
Escherichia coli	NA	250	1000	NA	NA	NA
ATCC 25922	NA	250	1000	NA	NA	NA
Klebsiella	NA	250	500	NA	NA	NA
pneumoniae	NA	250	500	NA	NA	NA
Listeria	500	125	62.5	62.5/1000	7.8/>1000	NA
monocytogenes	1000	125	1000	1000/>1000	62.5/>1000	NA
Pseudomonas	NA	125	65.5	NA	NA	125
aeruginosa	NA	250	250	NA	NA	250
Salmonella sp.	NA	500	250	NA	NA	NA
	NA	500	500	NA	NA	NA
Salmonella Typhi	NA	250	250	NA	NA	NA
	NA	250	250	NA	NA	NA
Salmonella	NA	125	250	NA	NA	NA
Typhimurium ATCC 13311	NA	250	250	NA	NA	NA
Salmonella	NA	250	500	NA	NA	NA
Weltevreden	NA	250	1000	NA	NA	NA
Shigella flexneri	NA	125	125	NA	1000/500	250
	NA	125	250	NA	1000/1000	500
Staphylococcus	250	125	125	1000/250	7.8/1000	250
aureus ATCC 25923	250	125	500	>1000/1000	15.6/>1000	>100
Streptococcus	125	250	125	>1000/500	3.9/500	500
mutans	500	250	500	>1000/>1000	125/>1000	>1000
Streptococcus	250	500	500	>1000/1000	7.8/1000	>1000
pyogenes	500	500	1000	>1000/>1000	62.5/>1000	>1000

NA, not applicable (no inhibition zone).

^aSpecimen voucher number; ^bMIC; ^cMBC; ^dtwo different parts of plants tested.

This high activity of Rhodomyrtus tomentosa against Gram-positive bacteria is very interesting. Active principles from the ethanol extract of Rhodomyrtus tomentosa were identified as rhodomyrtone and acylphloroglucinol derivatives (data not presented). This plant species should be further investigated; it may substitute the use of currently prescribed antibiotics and could reduce problems due to antibiotic-resistant gram-positive bacteria. Eleuterine americana is a herbal plant whose red bulbs are commonly employed in Asian cuisine. Bioactive compounds such as anthraquinones, bi-eleutherol, and elecanacin have been previously documented (Nakatani, 1994). This study supports the use of Eleuterine americana as a food additive to prevent a number of foodborne infections.

The antibacterial activity of Quercus infectoria has been previously reported against both Gram-positive (Nimri et al., 1999; Hwang et al., 2004; Voravuthikunchai et al., 2004b; Basri & Fan, 2005; Voravuthikunchai & Kitpipit, 2005) and Gram-negative bacteria (Nimri et al., 1999; Voravuthikunchai et al., 2004a; Singh et al., 2005; Voravuthikunchai & Limsuwan 2006). In the present communication, a wide range of important pathogenic bacteria were further studied. Results from the determination of MICs and MBCs clearly demonstrated that the ethanol extract of Quercus infectoria exhibited a broad spectrum against all tested pathogenic bacteria. The main constituents found in Quercus infectoria are 50–70% tannins (Ikram & Nowshad, 1977). Several tannins have been reported to have antibacterial activities (Machado et al., 2003; Fogliani et al., 2005; Hatano et al., 2005; Kaur et al., 2008). We have isolated ellagic tannins from this plant species (unpublished data). The ethanol extract of Piper betle also demonstrated significant activity against almost all species, except Enterococcus faecalis. The antibacterial and antifungal activities of Piper betle have been reported (Shitut et al., 1999; Nalina & Rahim, 2006). The main component of the leaves of this plant is a volatile oil known as ‘betel oil’. Essential oil from this plant showed an effect against both gram-positive and gram-negative bacteria (Wannissorn et al., 2005). In addition, phenolic compounds from Piper betle leaves have been reported to have antibacterial activity (Ramji et al., 2002). Though tannins from Quercus infectoria or phenolic compounds (in oils) from Piper betle do not have good potential to be used as antibiotics, this finding provides an insight into usage of the crude extract in the treatment of wounds or burns associated with bacterial infections, especially for multiresistant bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

Conclusions

Rhodomyrtus tomentosa showed remarkably good antibacterial activity against Gram-positive bacteria, with MICs and MBCs in the range of 3.9–7.8 and 7.8– 125 μg/mL, respectively. It shows promise as a good candidate in the search for new principles. The extract from Eleuterine americana may be applied as a food additive to prevent a number of foodborne infections. The broad spectra of activities of the extracts from Quercus infectoria and Piper betle support their usage in the treatment of multiple drug-resistant bacterial infections.

Declaration of interest: This work was supported by the Thailand Research Fund: Royal Golden Jubilee, PhD grant (PHD/0029/2548), Fiscal year 2005–2010 and Basic Research Grant (BRG 4880021), Fiscal year 2005–2008.