Investigation of Antimicrobial Activity of 13 Thai Medicinal Plants against Bacteria and Fungi

Thirteen medicinal plants used in Thai traditional medicine for the treatment of inflammatory diseases were screened for in vitro antibacterial and antifungal activities. The agar disc diffusion method was employed against five strains of bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) and four strains of fungi (Candida albicans, Candida tropicalis, Candida glabrata and Candida guilliermondii). The 13 plants were extracted with methanol and the antimicrobial results were evaluated as the diameter of the inhibition zone of microbial growth. The results showed that the extract with the greatest activity against the gram-positive bacteria was Garcinia mangostana (peri-carb). The extract with the greatest activity against the gram-negative bacteria was Psidium guajava (leaves). The extracts with the greatest activity against Candida was Psidium guajava (leaves) and Allium sativum (bulbs). The results support the ethnopharmacological uses of the tested plants, especially G. mangostana, P. guajava and A. sativum.

Since ancient times, plants have been used by Thai communities to treat a large number of diseases, including inflammation and infections.The 13 plants used in this study exist in many Thai traditional books.Plants have been known to be an important source of new structures and starting materials for the discovery of new therapeutic agents since bacteria resistant to conventional medicines started to increase.Medicinal plants may enhance the effects of conventional antimicrobials that will probably decrease costs and shorten the duration of sickness in some aspects.However, several plants may interfere with antibiotics when they are used together 1 .
Natural products and/or natural product structures continue to play a highly significant role in the drug discovery and development process 2 .About 25% of drugs in the modern pharmacopoeia are derived from plants, including several anticancer drugs currently in clinical use 3 .Besides, about 12.5% of the 422,000 plant species documented worldwide are reported to have medicinal value 4 .Many prescription drugs originate from chemicals found in plants and today are simple synthetic modifications or copies of the naturally obtained substances.
In developing countries, high throughput screening for new drugs or new compounds from plants is rather difficult as the budget is limited.Therefore, a multidisciplinary approach is a tool that can help in discovering new compounds.Ethnobotanical data can help to find a link between the ancient usage and the activities of such plant products.In addition, the long history of use may prove that the plant is safe to consume.The 13 plant species in this study were selected based on their anti-inflammatory use or anti-infectious use from some Thai ancient textbooks.It is expected that the long history of their usage might offer opportunities for the discovery of novel antimicrobial agents.

Collection of plant materials
All 13 plant materials were collected in Thailand, 100-200 g per sample.

Preparation of extracts
All plant materials were collected, washed with water, dried at about 35-40 ºC for several days and then ground to a powder using a laboratory scale mill.Twenty grams of dried powder of each plant was extracted with 90% methanol and dried under pressure using a rotary evaporator.All dried extracts were then kept in tightly fitting stopper bottles in a freezer (-4 ºC) until used.Thereafter, the extracts were re-dissolved in 10 % dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ ml and then cold sterilized by filtration through a mini-disk filter (0.45 µm) and stored in amber glass bottles for antimicrobial testing.

Microorganisms and media
The five bacterial strains used (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonalla typhi) were cultured on nutrient agar at 35ºC for 18 hours.
The bacterial colonies were placed into test tubes containing sterile normal saline solution (0.85% NaCl) to make a suspension equal to the turbidity of McFarland standard No. 0.5 (approximately 1-5 x 10 8 CFU/ml of bacteria) and spread on Mueller Hinton agar using a sterile cotton swab.
The four yeast strains used (Candida albicans, Candida tropicalis, Candida glabrata and Candida guilliermondii) were cultured on Sabouraud dextrose agar at 35ºC for 24-48 hours.The yeast colonies were placed into test tubes containing a sterile normal saline solution (0.85% NaCl) to make a suspension equal to the turbidity of McFarland standard No. 0.5 (approximately 1-5 x 10 6 CFU/ml of yeast) and spread on Sabouraud dextrose agar using a sterile cotton swab.

Antimicrobial screening
The agar disc diffusion method 10,11 was used to screen the antimicrobial activity of the herb extracts.Sterile 6-mm diameter paper discs (Schleicher and Schuell, Germany) were impregnated with 10 µl of herb extracts (100 mg/ ml) and placed on the inoculated Mueller Hinton agar (MHA) and Sabouraud dextrose agar (SDA) plates.Lincomycin (5 mg/mL) antibiotic discs were used as antibacterial agents.Amphotericin B (5 mg/mL) and garlic extract were used as antifungal agents.Discs impregnated with 10 µl of DMSO were used as negative controls.Plates were incubated at 35ºC for 18 h for bacteria and 48 h for yeasts.The inhibition zone diameter was measured.The tests were performed in triplicate and the results were averaged.

RESULTS
The results, evaluated as the diameter of the inhibition zone of microbial growth, showed that most extracts tested were active against grampositive bacteria (both B. cereus and S. aureus).The extracts with the greatest activity against both gram-positive bacteria tested were G. mangstana and P. guajava with similar inhibition zone diameters that ranged from 11.7-14.8mm.
For gram negative bacteria, four plant species (C.trifolia, G. mangostana, P. indica and P. guajava) were active against E. coli.However, only C. trifolia was active against S. typhi.The extracts that showed the strongest inhibition against gram negative P. aeruginasa were O. indicum followed   Note: Lincomycin was a control for bacteria, Amphotericin B was a control for fungi and Allium sativum Linn.(garlic) was a control for microbes.ND = not determined by G. pseudochina and P. guajava.The extract with the greatest activity against the gram-negative bacteria overall was P. guajava (diameter of the inhibition zone was 10-13.3mm).
For fungi, the extract with the greatest activity against C. albicans was G. mangostana, while P. guajava had the greatest activity against C. tropicalis and C. guilliermondii.A. sativum was also active against C. tropicalis but was not good for other fungal strains.For C. grabrata, only C. trifolia and G. mangostana were active, while the other plants showed mild activity.

DISCUSSION
A. sativum, or garlic, has been widely used in Thai traditional medicine as a potent antifungal remedy.Thus, it was selected to be a positive control for the natural products in this study with the hope that it would show the greatest activity against all the Candida strains tested.It was the best against C. tropicalis but not the other strains tested, including C. albicans, while previous studies found that allicin from garlic has antifungal activity particularly against C. albicans 12 .However, the concentration of allicin can vary depending on the location that the garlic grows.The allicin compound is also instable and decomposes 13 .
C. trifolia, which is commonly known as fox grape in English, in a previous study, was found to possess the most potent DPPH free radical scavenging activity and strongly inhibited lipid peroxidation as well as containing the highest amount of phenolic compounds 6 .The bark extract showed antiviral, antibacterial, antiprotozoal, hypoglycemic, anticancer and diuretic activities 14 .In this study, its methanolic extract showed good antibacterial activity against both gram positive and gram negative bacteria.In addition, it showed potency against all Candida spp., especially C. tropicalis.C. trifolia has been reported to contain yellow waxy oil, steroids, terpenoids, flavonoids and tannins 15 .
G. mangostana, or mangosteen, is the queen of fruit in Thailand, which has a wide range of medicinal properties including antibacterial and antifungal 7 .In this study, its extract showed the greatest activity against B. cereus, S. aureus and C. albicans, which is in agreement with many previous studies.Recently, it was found that the crude chloroform extract of mangosteen pericarp showed an effective zone of inhibition against Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis and Lactobacillus acidophilus 16 .Thus, the mangosteen pericarp extract showed promising activity against dental pathogens and would aid in designing a novel drug for the treatment of dental infections.
P. guajava, or guava leaves, tea is commonly used as a medicine against gastroenteritis and diarrhea in children by those who cannot afford or do not have access to antibiotics 17 .Of the bacteria tested in this study, the S. aureus strain was inhibited the most by the extracts followed by the E. coli.This result is in agreement with many previous studies in which the guava extracts were very active antibacterially and that the ethanol and methanol were better than n-hexane and water for the extraction of the antibacterial properties of guava 18 .One study found that the essential oil extracted from guava showed inhibitory activity against S. aureus, Salmonella spp.and E. coli isolated from seabob shrimp, which caused diarrhea 17 .These data supported the use of guava leaf-made medicines in diarrhea cases where access to commercial antibiotics is restricted.

CONCLUSION
The findings of this study generally support the popular use of some plant species that show great activity against bacteria and fungi as antibiotic folk medicines, especially A. sativum, C. trifolia, G. mangostana and P. guajava.In addition, this paper also provides preliminary information about thirteen plant species.Such information may serve as basic knowledge for further insight for future pharmacological, phytochemical and clinical investigations.

Table 1 .
Plants species, their names, family, local names, parts used and traditional uses.

Table 2 .
Antimicrobial activities of Thai herb extracts against bacteria and fungi.