Nisarat Siriwatanametanon1*, Wanwisa Dodgson2
and Jolyon L.A. Dodgson2

1Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.
2Microbiology and Applied Microbiology Research Unit, Faculty of Science, Mahasarakham University, Mahasarakham, Thailand.

Abstract

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 employedagainstfivestrains of bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosaandSalmonellatyphi) and fourstrains of fungi(Candida albicans, Candida tropicalis,Candida glabrata and Candida guilliermondii). The13plants wereextracted 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 Garciniamangostana (peri-carb). The extract with the greatest activity against the gram-negative bacteria was Psidiumguajava (leaves). The extracts with the greatest activity against Candida was Psidiumguajava(leaves) and Allium sativum (bulbs). The results support the ethnopharmacological uses of the tested plants, especiallyG.mangostana, P.guajavaand A.sativum.

Keywords: Antibacterial, Antifungal, Garciniamangostana,Psidiumguajava, Allium sativum.

INTRODUCTION

Since ancient times, plants have been used by Thai communities to treat a large number of diseases, including inflammation and infections. The13 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 agentssince 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 mayinterfere with antibiotics when they are used together1.

Natural products and/or natural product structures continue to play a highly significant role in the drug discovery and development process2.About 25% of drugs in the modern pharmacopoeia are derived from plants, including several anticancer drugs currently in clinical use3. Besides, about 12.5% of the 422,000 plant species documented worldwide are reported to have medicinal value4.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 atool 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.

EXPERIMENTAL

Collection of plant materials
All 13 plant materials were collected in Thailand, 100–200 g per sample.Basellaalba, Basellarubra, Gynurapseudochina and Rhinacanthusnasutus were collected from farmland in Buriram Province.Oroxylumindicum andCayratiatrifolia were collected in suburban areas. Pouzolziaindica and Muehlenbeckiaplatyclada were collected from the Sirirukhachart Botanical Garden, Mahidol University. Morusalba and Psidiumguajava were collected from Roi-Et Province. Garciniamangostana was collected from ChantaburiProvince and Vernonianeinerea was collected from MahasarakhamProvince.Bulbs of Allium sativum were purchase from a local market inMahasarakhamProvince.

Their scientific names, local names and medicinally used parts are detailed in Table 1. Their macroscopic characteristics were identified according to Thai herbal pharmacopeia guidelines. The plants were gathered during August 2015. The fresh and dried plants were identified by comparisonwith the plant specimens at the Forest Herbarium of the ThaiRoyal Forest Department, Bangkok, Thailand.

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

Plant species
FAMILY
Local names
Parts used
Traditional uses
Allium sativumL.

AMARYLLIDACEAE

Kra-tiem
Bulbs
Regulating blood pressure, lowering blood sugar and cholesterol levels, effective against bacterial, viral, fungal and parasitic infections5.
Basella albaL.

BASELLACEAE

Pak-pang -kaow
Leaves
Treatment of infectious disease, e.g., appendicitis, small pox, skin infections, eye infections and mouth ulcers6.
BasellarubraL.

BASELLACEAE

Pak-pang-daeng
Leaves
Similar to B. alba but more for urticarial, skin burns and tumors6.
Cayratia trifolia (L.) Domin.

VITACEAE

Thow-kan-kaow
Leaves
Used externally for nose ulcers, muscle pain, abscess, fever and asthma and used as expectorant, carminative and blood purifier6.
GarciniamangostanaL.

GUTTIFERAE

Mang-kood
Peri-carb
Treatment of anti-inflammatory diseases and skin infections, wounds, dysentery, different urinary disorders, cystitis and gonorrhoea7.
Gynurapseudochina (L.) DC.

ASTERACEAE

Wan-hua-nuam
Underground root/stem
Treatment of herpes, fevers, pimples, sore throat, bruises and used as homeostatic and against breast tumors6.
Morus alba L.

MORACEAE

Mon
Leaves
Cancer preventative, relieves fever, indigestion and flatulence8.
Muehlenbeckia platyclada  (F.Muell.) Meisn.

POLYGONACEAE

Tra-kab-bin
Leaves
Applied externally for skin swelling, sores and insect bites, poisonous snake bites and fracture injuries, alleviating fever and detoxification6.
Oroxylumindicum(L.) Kurz.

BIGNONIACEAE

Pae-ka
Stem bark
Used against abscesses, skin inflammation, purifying blood and expectorant, fevers, tongue inflammation, bruises and swellings, vomiting, diabetes,diarrhea, rheumatism, stomachache, arthritis, antimalarial, antibacterial and antiviral6.
Pouzolziaindica(L.)Gaudich.

URTICACEAE

Kob-cha-nang-daeng
Leaves
Applied as emmenagogue, galactogogue, diuretic, insecticide, used against stomachache, sores,ulcers, gangrene, gonorrhoea, syphilis and wounds6.
PsidiumguajavaL.

MYRTACEAE

Fa-rang
Leaves
Used for diarrhea, skin infections, head lice, parasites and as mouthwash to relieve bad smell.
Rhinacanthusnasutus (L.) Kuntze.

ACANTHACEAE

Thong-pan-chang
Leaves
Treatment of ringworm, eczema, tinea, herpes infections and many skin disorders, antipyretic, anti-inflammatory, detoxicant, cancers, hepatitis, diabetes, hypertension, mental disorders, rheumatism, circulatory problems, asthma and bronchitis, epilepsy and immune system deficiencies6.
Vernonianeinerea L.

ASTERACEAE

Mo-noi
Aerial parts
Used as aid in smoking cessation for relieving cigarette craving. Treatment of asthma, cough, fever, malaria, arthritis and urinary calculi9.

 

Preparation of extracts
All plant materials were collected, washed with water, driedat about 35–40 ºC for several days and then ground toa powder using a laboratory scale mill. 20 g of dried powderof 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 bottlesin a freezer (−4ºC) until used.Thereafter, the extracts were re-dissolved in 10 % dimethyl sulfoxide(DMSO) at aconcentration of 100 mg/ml and then cold sterilized by filtration througha mini-disk filter (0.45µm) 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 Salmonallatyphi) 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 108 CFU/ml of bacteria) and spread on Mueller Hintonagar using a sterile cotton swab.

The four yeast strains used (Candida albicans, Candida tropicalis, Candida glabrataand 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 106 CFU/ml of yeast) and spread on Sabouraud dextrose agar using a sterile cotton swab.

Antimicrobial screening
The agar disc diffusion method 10,11was 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 gram-positive bacteria(both B. cereus and S. aureus). The extracts with the greatest activity against bothgram-positive bacteria tested wereG.mangstanaand P. guajavawith similar inhibition zone diameters thatranged from 11.7-14.8 mm.

For gram negative bacteria, four plant species (C.trifolia, G.mangostana, P. indica and P. guajava) were active against E.coli. However, only C.trifoliawas active against S. typhi. The extracts that showed the strongest inhibition against gram negativeP. aeruginasa wereO. indicumfollowed byG. pseudochinaand P. guajava.The extract with the greatest activity against the gram-negative bacteria overall was P.guajava (diameter of the inhibition zone was 10-13.3 mm).

For fungi, the extract with the greatest activity against C. albicans wasG. mangostana, whileP.guajavahad the greatest activity against C. tropicalisandC. guilliermondii.A.sativumwas also active against C.tropicalis but was not good for other fungal strains.For C. grabrata, only C.trifoliaand G. mangostana were active, while the other plants showed mild activity.

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

Extract Inhibition zone (mm)
B. cereus S. aureus E. coli S. typhi P. aeruginosa C. albicans C. tropicalis C. glabrata C.guilliermondii
Allium sativum ND ND ND ND ND 10.5 17.4 7.7 9.0
Basella alba 8.8 9.3
Basellarubra 7.5
Cayratiatrifolia 12.0 11.3 12.3 9.0 12.6 14.1 11.3 12.0
Garciniamangstana 14.5 11.8 10.5 14.4 11.4 11.0 12.3
Gynurapseudochina 10.0 9.5 7.7 9.0
Morus alba 9.7 8.8 9.8 10.6 9.0
Muehlenbeckiaplatyclada 10.2 9.0 11.9 10.9 7.7 10.5
Oroxylumindicum 8.0 12.0 10.4 15.8 7.0 9.5
Pouzolziaindica 10.5 9.0 10.5 11.9 11.8
Psidiumguajava 11.7 14.0 13.3 10.0 13.2 17.4 9.0 14.3
Rhinacanthusnasutus
Vernonianeinerea 9.5 8.5 9.9 8.0 11.0
Lincomycin 11.0 12.0 12.0 11.0 12.0
Amphotericin B ND ND ND ND ND 11.0 10.0 10.0 9.3

Note:Lincomycinwas a control for bacteria, Amphotericin Bwas a control for fungi and Allium sativumLinn. (garlic)was a control for microbes. ND = not determined.

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 Candidastrains tested. It was thebest against C.tropicalis but not the other strains tested, including C.albicans,whileprevious studies found that allicin from garlic has antifungal activity particularly against C. albicans12. However, the concentration of allicin can vary depending on the location that the garlic grows. The allicin compound is also instable and decomposes13.

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 lipidperoxidation as well as containing the highest amount of phenolic compounds6. The bark extract showed antiviral, antibacterial, antiprotozoal, hypoglycemic, anticancer and diuretic activities14. 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., especiallyC.tropicalis. C.trifolia has been reported to contain yellow waxy oil, steroids, terpenoids, flavonoids and tannins15.

G.mangostana, or mangosteen, is the queen of fruit in Thailand, which has a wide range of medicinal properties including antibacterial and antifungal7. 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 oralisand Lactobacillus acidophilus16. 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 antibiotics17.Of the bacteria tested in this study, the S.aureusstrain was inhibited the mostby the extracts followed by theE.coli. This result is in agreement with many previous studies in which the guava extracts were very activeantibacteriallyand that the ethanol and methanol were better than n-hexane and water for the extraction of the antibacterial properties of guava18. 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 diarrhea17. 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 supportthe popular use of some plant species that show great activity against bacteria and fungi as antibiotic folk medicines, especially A.sativum,C.trifolia,G.mangostanaand P.guajava.In addition, this paper also provides preliminary information about thirteen plant species. Such information may serve as basic knowledge for furtherinsight for futurepharmacological,phytochemical and clinical investigations.

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