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In vitro antifungal activities of longan (Dimocarpus longan Lour.) seed extract
Fitoterapia 83 (2012) 545–553
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In vitro antifungal activities of longan (Dimocarpus longan Lour.) seed extract Nuchanart Rangkadilok a, b, 1, Songsak Tongchusak c, 1, Rachasak Boonhok c, Sansanee C. Chaiyaroj c, Varaporn B. Junyaprasert d, Waranun Buajeeb e, Jaratluck Akanimanee a, Thida Raksasuk a, Theeralaksna Suddhasthira e, Jutamaad Satayavivad a,b,⁎ a b c d e
Laboratory of Pharmacology, Chulabhorn Research Institute (CRI), Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand Environmental Toxicology Program, Chulabhorn Graduate Institute, Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Rajthevi, Bangkok 10400 Thailand Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajthevi, Bangkok 10400, Thailand Faculty of Dentistry, Mahidol University, Rajthevi, Bangkok 10400, Thailand
a r t i c l e
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Article history: Received 3 October 2011 Accepted in revised form 19 December 2011 Available online 8 January 2012 Keywords: Longan seed Ellagic acid Corilagin Dimocarpus longan Candida Cryptococcus neoformans
a b s t r a c t Longan, Dimocarpus longan Lour., contains polyphenolic compounds which exhibit several pharmacological properties. This study aims to evaluate antifungal activities of longan fruit extract in comparison to its active compounds. The results showed that longan seed exhibited antifungal activity against the opportunistic yeasts (Candida species and Cryptococcus neoformans). In contrast, longan pulp and whole fruit did not demonstrate any inhibitory effects. Ellagic acid showed the most potent antifungal activity followed by corilagin and gallic acid, respectively. Ellagic acid inhibited Candida parapsilosis and C. neoformans more effectively than Candida krusei and also some Candida albicans clinical strains. Baidam cultivar possessed higher antifungal activity (MIC= 500–4000 μg/ml) as it contained higher contents of ellagic acid and gallic acid than Edor (MIC= 1000–8000 μg/ml). For antibacterial activity, only corilagin and gallic acid possessed weak to moderate inhibitory effects against Staphylococcus aureus and Streptococcus mutans, respectively. Longan seed was then applied in the oral care products. Longan effervescent granule (5% extract) significantly reduced adhesion of C. albicans to acrylic strips. Mouthwash containing 0.5% extract exhibited good antifungal activity compared to a commercial product. These findings indicated that longan seed extract and its polyphenolic compounds can be used as an antifungal agent in oral care products for the treatment of opportunistic yeast infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Longan fruit (Dimocarpus longan Lour.) is a subtropical fruit, which belongs to the Sapindaceae family. Longan, known as ‘Lumyai’ in Thailand, is widely grown in China, Taiwan, and South East Asia including Thailand and Vietnam. Our previous study demonstrated that water extract of
⁎ Corresponding author at: Laboratory of Pharmacology, Chulabhorn Research Institute, Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand. Tel.: +66 2 5740622x3917; fax: +66 2 5742027. E-mail address: [email protected] (J. Satayavivad). 1 These authors contributed equally to this study. 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.12.023
longan fruit contained high levels of polyphenolic compounds such as corilagin, gallic acid, and ellagic acid [1] (Fig. 1). The contents of these polyphenolics depended on cultivars and plant tissues. Dried seed contained the highest levels of these three compounds followed by dried peel and dried pulp. Prasad et al. [2] used ultra-high-pressure-assisted extraction to extract phenolic compounds from longan fruit pericarp and found that it contained the highest content of corilagin compared to other phenolic compounds. In addition, Lin et al. [3] and Hsu et al. [4] also reported that longan seed contained corilagin and acetonylgeranin as active compounds. The methanolic extract of longan seed (0.1 and 0.2 mg/ml) exhibited ability to induce osteoclast differentiation in mouse
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HO
COOH
HO
OH O
O
OH
HO
Gallic acid HO
O
O
OH
Elagic acid HO
OH
HO
OH
HO
OH CO
CO
CH 2O O
O O C
OH OH
O OH OH
OH
Corilagin Fig. 1. The structures of gallic acid, ellagic acid, and corilagin.
bone marrow macrophages [5]. Jiang et al. [6] reported that longan fruit pericarp exhibited strong antioxidant activities in a concentration-dependent manner using DPPH and superoxide anion radical scavenging assays. From our previous study, dried longan seed extract of cultivar Edor exhibited the highest radical scavenging activities compared to fresh seed and dried pulp extracts [7]. In addition, longan seed extract also possessed anti-tyrosinase activity with IC50 = 2.9–3.2 mg/ml. Recent studies reported that polyphenol-rich longan seed extract inhibited the proliferation of human colorectal carcinoma cells [8] and longan seed polysaccharides (50–100 mg/kg) had antifatigue effects in mice by extending swimming time, increasing hepatic glucogen, reducing blood urea nitrogen and lactic acid [9]. These therapeutic effects of longan seed are attributed to various compounds present in this plant, including ellagic acid, gallic acid, and corilagin. The opportunistic fungi are common causes of serious morbidity and mortality in immunocompromised patients [10]. Candida is a genus of yeast, commonly part of the normal flora of the mouth, skin, intestinal tract and vagina, but can also cause several diseases. Candida albicans is one of the oral Candida species and it is responsible for most oral candidal infections [11]. Cryptococcus neoformans is an encapsulated yeast-like fungus living in both plants and animals and also an opportunistic fungal pathogen that causes central nervous system and pulmonary diseases among immunocompromised patients [12,13]. Medications associated with the emergence of clinical candidiasis are broad-spectrum antibiotics, corticosteroids, immunosuppressive agents (azathioprine), anticholinergic agents (e.g. tricyclic antidepressant) [14] whereas amphotericin B with/
without fluconazole is the treatment of choice for mild and localized C. neoformans infection [12]. The use of antifungal drugs is costly and long-term therapy may cause fungal resistance. Therefore, the development of antifungal agents from medicinal plants may be an alternative source for the inexpensive treatment of fungal infections. The antimicrobial activity of the three polyphenolic compounds (gallic acid, ellagic acid, and corilagin) was investigated on human pathogenic bacteria (Staphylococcus aureus and Corynebacterium accolans) and a plant pathogen (Erwinia carotovora) as well as human pathogenic yeast (C. albicans) [15]. The results showed that corilagin had the highest antimicrobial activity followed by ellagic acid and gallic acid. However, ellagic acid could not inhibit S. aureus in this study. From the previous study, there is limited scientific information for the use of these three compounds or longan seed extract in the treatment of microbial infections. The aim of this study was to investigate the antifungal activity of gallic acid, ellagic acid, and corilagin including different longan extracts against Candida species, C. neoformans and some dermatophytes. In addition, the antibacterial activity of these compounds and extracts and the potential use of longan seed extract in oral care products was also investigated. 2. Materials and methods 2.1. Chemicals Dimethyl sulfoxide (DMSO), absolute ethanol, and methanol (HPLC grade) were obtained from Merck (Darmstadt, FR Germany). 2,2′-Azobis(2-amidinopropane) dihydrochloride
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(AAPH) was purchased from Cayman (Ann Arbor, MI, USA). Chloramphenicol and ceftazidime were purchased from Oxoid (Hampshire, UK). Gallic acid, ellagic acid, amphotericin B, ketoconazole, and all other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA). Corilagin was extracted and purified from longan seeds by Laboratory of Pharmacology, Chulabhorn Research Institute, Thailand following the previous method reported by Latte and Kolodziej [16] with some modifications and used as a standard in this experiment (% purity = 98%). All chemicals for preparation of oral care products e.g. effervescent granule and mouthwash were purchased from Carlo Erba (Italy), S. Tong Chemicals Co, Ltd. (Bangkok, Thailand), Ajex Finechem (NSW, Australia), Fisher Scientific UK (Leicestershire, UK), and Thai-China Flavours and Fragrances Industry Co., Ltd. (Bangkok, Thailand). Milli-Q deionized water (Branstead, Newton, USA) was used throughout this experiment. 2.2. Plant material and extraction D. longan Lour. was identified by Dr. Somran Suddee (BKF Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Bangkok, Thailand). A voucher specimen (L. Worasuttayangkurn 01) was deposited at the BKF Forest Herbarium (Bangkok, Thailand). Longan cultivar Edor and Baidam were selected to use in this study. Edor is a commercial cultivar available in Thailand. Seed, pulp and whole fruit of Edor were used for extraction. Baidam contains high levels of gallic acid and ellagic acid in seed; therefore, it was used to compare the antifungal activity to Edor. Five hundred grams dried ground seed, pulp, or whole fruit (seed + pulp) of longan were extracted three times with 2 l of hot water (70–75 °C) for 1 h at each time. The extracts were then filtered and combined. The water extracts were then concentrated and dried using freeze-dryer (for pulp as it contained high sugar) and spray-dryer (1% lactose was added in seed extract and spray dried at 80 °C/120 °C). The percentage yield of extracts was between 25 and 35% depending on cultivars and plant samples. Plant extracts, 22–25 mg, were dissolved in 3.0 ml of hot water (70–75 °C) (2 replicates per sample) and vigorously shaken. The extracts were left at room temperature until they cooled down and then filtered through a 0.45 μm PVDF membrane (Chrom Tech, Apple Valley, MN) prior to HPLC analysis. These longan extracts were analyzed for the contents of three polyphenolic compounds (corilagin, gallic acid, and ellagic acid) by using our previous HPLC method modified by Rangsriwong et al. [17]. The HPLC analysis was performed using an HP1100 HPLC system with a thermostatically controlled column oven, a binary pump, and a diode-array detector (Hewlett Packard, USA). A 150 × 3.9 mm i.d., 5 μm reversed phase column, Symmetry® C18 (Waters Corporation, Milford, Massachusetts) was used for analysis of the active compounds in longan extract. The compounds were eluted with a gradient system of 0.1% formic acid (solvent A): methanol (solvent B) at a flow rate of 1.0 ml/min. The temperature of the column was 25 °C with the UV detection at 270 nm. The injection volume was 10 μl. The gradient system started with 4%B at 0 min to 80%B in 27 min. The total run time is 30 min. Retention times of gallic acid, corilagin, and ellagic acid are 6.69, 19.22, and 25.65 min, respectively (Fig. 2).
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2.3. Fungi and bacteria strains C. albicans (ATCC90028), Candida parapsilosis (ATCC20019), and Candida krusei (ATCC6258) were purchased from the American Type Culture Collection and C. albicans (ATCC10231) was from the Department of Medical Science, Ministry of Public Health, Thailand (DMST). Total 7 clinical isolates of C. albicans, 4 clinical isolates of C. neoformans and filamentous fungi including Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum gypseum, Microsporum canis, Epidermophyton floccosum, Pseudallescheria boydii, Penicillium siamensis, Penicillium pinophilum, Penicillium marneffei, Aspergillus nidulans, Aspergillus niger and Aspergillus fumigatus were received from Mycology Section, Division of Clinical Pathology, Department of Medical Sciences, National Institute of Health, Thailand and 2 clinical strains of C. albicans (for effervescent granule testing) were isolated from oral lesions of patients of the Out Patient Clinic at the Faculty of Dentistry, Mahidol University, Thailand. Filamentous fungi were grown on Sabouraud Dextrose Agar slant (SDA; 4% dextrose, 2% neopeptone and 1.7% agar) at room temperature for one or four weeks (depending on fungal species). Conidia were harvested by flooding with 0.01% Tween-20 on fungal colony. Conidia were counted on a hemacytometer chamber and adjusted to a final concentration of 106 cells/ml or McFarland standard solution no. 0.5. Yeasts were cultured on Sabouraud Dextrose Agar plate (SDA; 4% dextrose, 2% neopeptone and 1.7% agar) and incubated at 37 °C for 24 h or 48 h. A single colony was then subcultured onto Sabouraud Dextrose Broth (SDB) and incubated at 37 °C in shaking incubator for 24 h or 48 h. Before antifungal activity testing, the concentration of yeasts was adjusted so they were equal to the turbidity of McFarland standard solution no. 0.5. The bacterial reference strains were obtained from the Thailand Institute of Scientific and Technological Research (TISTR) and the Department of Medical Science, Ministry of Public Health, Thailand (DMST). The following microorganisms were used for determining antibacterial activity: Escherichia coli TISTR 887 (ATCC25922), S. aureus TISTR 517 (ATCC25923), Pseudomonas aeruginosa TISTR 1467 (ATCC27853), Salmonella typhimurium TISTR 292 (ATCC13311), Streptococcus mutans DMST 18777 (ATCC25175T). Bacterial inoculum suspension was prepared as described in Performance Standards for Antimicrobial Disk Susceptibility Tests; Ninth Edition [18]. Each tested culture should be streaked onto trypticase soy agar to obtain isolated colonies and incubated at 35 °C for 18–24 h. For S. mutans, bacteria were streaked on trypticase soy agar with 5% sheep blood and incubated at 35 °C in a candle jar for 18–24 h. Four or five well-isolated colonies were then selected and transferred to a tube of sterile saline or Mueller–Hinton broth. Turbidity was measured and adjusted to match a 0.5 McFarland density standard resulting in inoculums containing 1.5×108 cells/ml. This suspension was used to directly inoculate agar plates for disc diffusion method. 2.4. Disc–agar diffusion method For antifungal activity, all longan extracts and the reference compounds were tested on standard antimicrobial assay (AA) disc at a final concentration of 1 mg/disc (Whatman, Brentford, UK). Ketoconazole was used at a concentration of 200 μg/disc. Aqueous solvent, ethanol, and 10% aqueous dimethyl sulfoxide
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Dried seed Gallic acid
Corilagin
Ellagic acid
Dried pulp
Whole fruit
Fig. 2. HPLC chromatograms of different parts of longan fruit: dried seed, dried pulp and whole fruit. Retention time (RT) of gallic acid = 6.69 min, corilagin = 19.22 min, and ellagic acid = 25.65 min.
(DMSO) control discs were prepared using the same volume of extracts and reference compounds. All discs were prepared aseptically and dried at 37 °C before testing. SDA plates were prepared in 20 cm diameter plastic Petri dishes by fixing the level of media at 4 mm height. In order to culture the yeasts, a wound cotton swab was used for streaking fungal preparation on the surface of media with 60° difference for 3 plans of streak. Testing discs were placed on the surface of media and incubated at 37 °C for 24 h (C. albicans) or 48 h (C. neoformans). Experiment was carried out in triplicate. The antifungal activity was evaluated by measuring the inhibition zone diameter. For antibacterial activity, the entire surface of Mueller–Hinton agar plates was inoculated simultaneously in three directions by using a cotton swab. All plant extracts were reconstituted in distilled water at the stock concentration at 10 mg/ml and filtersterilized through a 0.45 μm membrane filter. The three reference compounds (corilagin, gallic acid, ellagic acid) were dissolved in 10% DMSO to make the stock concentrations of 10 and 50 mg/ml. A sterile paper disc (6 mm in diameter) was
impregnated with 20 μl of suspension containing plant extracts (longan solutions at 100, 200, 500 and 1000 μg/disc) in water and three standard compounds (at concentrations of 100 and 200 μg/disc for corilagin; and 100–1000 μg/disc for ellagic acid and gallic acid). The discs were then placed on the agar surface. Plates were incubated at 35 °C for 18–24 h. For S. mutans, plate was incubated at 35 °C for 18–24 h in candle jar. The antibacterial activity was evaluated by measuring the inhibition zone diameter. The solvents used for each plant extract and standard compound preparation were used as the negative control. Chloramphenicol (30 μg/disc) and ceftazidime (30 μg/disc) were used as the positive control. 2.5. Determination of Minimum Inhibitory Concentration (MIC) by agar dilution method The Minimum Inhibition Concentration (MIC) of the longan extracts and reference compounds (gallic acid, ellagic acid, and corilagin) were determined by the agar dilution method. Plant
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extracts were dissolved in distilled water and serial two fold dilution was performed. For antifungal testing, the range of concentration of the tested compounds was 15.63–16,000 μg/ml and the ranges of concentrations for reference compounds were 0.98–1000, 1.95–2000, and 31.25–16,000 μg/ml for ellagic acid, corilagin, and gallic acid, respectively. The MIC was determined as the lowest concentration of the sample that resulted in complete inhibition of microbial growth. Testing media were left at room temperature allowing media surface to dry. Fungi were inoculated on medium surface for 3 μl (~4.5×105 cells) and incubated at 37 °C for 24 h (C. albicans) or 48 h (C. neoformans). For control testing, SDA alone or SDA+EtOH and SDA+DMSO were used. Ketoconazole was used as a positive control (at 0.0075–125 μg/ml). 2.6. Preparation of effervescent granules for denture cleanser For effervescent granule base, citric acid, tartaric acid and sodium bicarbonate were homogeneously mixed at 1:2:3.44 (by weight). Subsequently, 8% sorbitol and 0.01% sodium lauryl sulfate (SLS) were added to the mixture. An appropriate amount of 75% ethanol was then used as a binder by slowly adding to the mixture to produce a damp mass which was then passed through a 14-mesh sieve to produce granules. The obtained granules were dried in an oven at 45 °C for 2 h. Afterwards, the dried granules were sprayed with solution of 0.1% thymol and 0.8% peppermint oil and dried again at 45 °C for 15 min. Finally, the effervescent granules were packed in aluminum foil and kept in a desiccator until use. In the case of longan seed extract effervescent granules, 5% spray-dried longan seed extract was mixed with sodium bicarbonate and the granules were prepared as previously described. These longan effervescent granules were then tested for appearance, disintegration time, moisture content, and flow ability. 2.7. Adherence assay Acrylic strips were placed vertically in the plastic well (15 mm diameter) (Cellstar, Grenier labortechnik, Denmark). Approximately 200 μl of yeast suspension was added to each well. Thereafter, the whole assembly was incubated at 37 °C for 1 h with gentle agitation at 120 rev/min. The strips were then recovered from the wells and washed 3 times by dipping gently in sterile PBS, which helped to dislodge the nonadherent cells. Then the strips were placed in new wells containing 200 μl of longan extract formulation and incubated at 37 °C for 30 or 60 min. Sterile distilled water and granule base were used as negative controls, whereas 0.2% chlorhexidine was used as a positive control. The strips were then recovered from the wells and washed 3 times in sterile PBS again. They were dried and stained using gram stain without counterstain. After air-drying at room temperature, they were mounted on glass slides and the adherent yeasts were quantified. 2.8. Microscopic quantification of adherent yeasts A light microscope (Nikon E100, Japan) was used for the quantitative estimation of the adherent yeasts. Twenty fields of view were randomly counted in each strip at ×400 magnification. The mean number of yeasts was finally expressed as
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yeast cell per unit square mm. All experiments were repeated on 3 separate occasions with duplicate determinations on each occasion. 2.9. Assessment of the antimicrobial activity of mouthwash The tested microorganisms S. mutans DMST 18777 (ATCC25175T), C. albicans DMST 8684 (ATCC90028) and C. albicans DMST 5815 (ATCC10231) were used in this study. S. mutans was cultured overnight at 35 °C (in candle jar) on blood agar and Candida spp. were cultured overnight at 35 °C on Sabouraud dextrose agar (Becton, Dickinson and company, USA). A sensitivity test was carried out according to the agar dilution method described by National Committee for Clinical Laboratory Standards (NCCLS document M7-A5) (at present known as Clinical and Laboratory Standards Institute; CLSI) [19] and some modifications of Yoshida et al. [20]. The mouthwash was added aseptically to sterile molted Mueller–Hinton agar (Oxoid, Basingstoke, England) supplemented with 5% sheep blood (for S. mutans) and Sabouraud dextrose agar (for C. albicans) in appropriate volumes to produce the concentrations of 1:4, 1:6, 1:8, 1:12, 1:16, 1:24 of each mouthwash. The resulting agar solutions were immediately poured into Petri-plates after vortexing. Inocula were prepared by suspending growth from overnight cultures of S. mutans in Mueller–Hinton broth (Oxoid, Basingstoke, England) and C. albicans in normal saline to a turbidity of a 0.5 McFarland standard. The prepared suspensions were further diluted 1:100 for S. mutans and 1:10 for C. albicans. The 10 μl of cell suspension was applied to each agar plate, with a final inoculum of approximately 10 4 cells per spot for bacteria and 10 3 cells per spot for yeast. For C. albicans, plates were incubated overnight at 35 °C in air but for S. mutans, the plates were incubated overnight at 35 °C in a candle jar. The lowest concentration of mouthwash solution showing no growth was read as the Minimum Inhibitory Concentration (MIC). Control cultures containing only the Mueller–Hinton agar supplemented with 5% sheep blood/Sabouraud dextrose agar, were also prepared. A commercial mouthwash containing zinc chloride, benzoic acid, eucalyptol oil, thymol, and menthol was used as a positive control. 2.10. Statistical analysis Data were expressed as mean ± SEM. The statistical analyses were performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, Illinois, USA). The Kruskal–Wallis and Wilcoxon Mann–Whitney non-parametric test were used to compare the means of adherence of C. albicans on acrylic resin (cells/ mm 2) between the treatments of control and product samples. A p value b 0.05 was regarded as statistically significant. 3. Results and discussion The active compounds present in longan seed were different from those in longan pulp (Fig. 2). Gallic acid, ellagic acid and corilagin are the major polyphenolic compounds present in dried longan seed extracts while longan pulp extract contained a small amount of ellagic acid but no gallic acid and corilagin (Table 1). Dried seed of Baidam contained the highest contents of gallic acid and ellagic acid with the same
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Table 1 The contents of gallic acid, corilagin, and ellagic acid in different longan fruit extracts (mean ± SEM; n = 3). Extracts
Gallic acid (mg/g DW)
Corilagin (mg/g DW)
Ellagic acid (mg/g DW)
Dried seed Edora Dried pulp Edorb Dried fruitb (seed + pulp Edor) Dried seed Baidamb
9.82 ± 0.87 ND 1.64 ± 0.01
14.84 ± 0.31 ND 0.43 ± 0.01
3.15 ± 0.03 0.13 ± 0.03 0.10 ± 0.00
21.89 ± 0.76
14.81 ± 0.32
9.76 ± 0.28
ND = not detectable. a Spray dried water extract of longan seed cultivar Edor. b Freeze dried water extracts of longan seed, pulp, and whole fruit cultivar Edor and Baidam.
amount of corilagin as cultivar Edor. The contents of these three compounds were present in small amounts in whole fruit of longan (Fig. 2). 3.1. Antifungal and antibacterial activities of polyphenolic compounds Ellagic acid and longan seed extracts showed antifungal activity against C. albicans and C. neoformans at 1 mg/disc (Table 2). It was found that longan seed extract was more potent than pure ellagic acid. However, both longan extract and ellagic acid exhibited lower antifungal activity than ketoconazole (a positive antifungal drug). Using agar dilution method, all polyphenolic compounds (gallic acid, ellagic acid, and corilagin) possessed antifungal activity against Candida species and C. neoformans (Table 3). The antifungal activities of these compounds in decreasing order were ellagic acid > corilagin > gallic acid. Ellagic acid inhibited Candida parapsilosis (MIC = 7.81 μg/ml) and C. neoformans (MIC = 15.63 μg/ml except for C. neoformans 1112) more effectively than C. krusei (MIC = 125 μg/ml). For C. albicans, ellagic acid showed greater activity to two clinical strains (C. albicans 1555 and 2923) with MIC = 7.81 μg/ml than other C. albicans strains (MIC = 15.63– 125 μg/ml). However, the antifungal activities of ellagic acid against Candida species and C. neoformans were less potent than an antifungal drug, ketoconazole (MIC = 0.06–31.25 μg/ ml). Gallic acid exhibited antifungal activity against C. parapsilosis (MIC = 4000 μg/ml) and most of C. albicans strains (ATCC 90028, 1435, 1434; MIC= 4000 μg/ml) higher
Table 2 Antifungal activities of longan extracts and ellagic acid against the tested microorganisms (mean ± SEM; n = 3). Compounds
10% DMSO Ethanol Distilled water Longan seed extract (cultivar Edor) Longan seed extract (cultivar Baidam) Ellagic acid Ketoconazole
Inhibition zone diameter (mm) Candida albicans (90028)
Cryptococcus neoformans (CI1435)
0 0 0 13.33 ± 0.57
0 0 0 26.33 ± 0.57
11.33 ± 1.15
28.33 ± 0.57
8.75 ± 0.50 21.5 ± 0.57
15.50 ± 1.52 70.00 ± 0.00
than C. krusei (MIC= 8000 μg/ml) and C. neoformans (MIC = 8000–> 16,000 μg/ml). For corilagin, the MIC against both Candida species and C. neoformans was about 1000 μg/ ml. It should be noted that ellagic acid, a dimer form of gallic acid, showed better antifungal activity than gallic acid (lower MIC values). In contrast, Latte and Kolodziej [16] indicated that the antifungal effect of gallic acid against C. neoformans (MIC=31 μg/ml), C. krusei (MIC=125 μg/ml), and C. albicans (MIC=500–>2000 μg/ml) was similar to that of corilagin. They reported that corilagin showed prominent antifungal activity against Candida glabrata strains with the MIC similar to the antifungal drugs, amphotericin B and sertaconazole. However, the MIC values of both gallic acid and corilagin in their study were lower than the MIC values in the present study. This may be due to the differences in the isolates of fungi, yeasts, and testing methods. Most of our isolates are clinical isolates but their study used mostly reference strains. The report from Fogliani et al. [15] which was different from our results, also found that corilagin had the highest inhibitory activity against C. albicans followed by ellagic acid and gallic acid. The present study showed that at the highest tested concentration (200 μg/ml), ellagic acid exhibited weak antifungal activity against dermatophytes species (Trichophyton rubrum, M. gypseum and E. floccosum) while gallic acid and corilagin could not inhibit the growth of these dermatophytes (data not shown). These results agree with the previous results from Latte and Kolodziej [16] who reported that neither gallic acid nor corilagin possessed any activity against filament fungi e.g. E. floccosum, M. canis, T. mentagrophytes, T. rubrum, A. fumigatus, Mucor racemosus or Rhizopus nigricans as reflected by MIC >2000 μg/ ml. For bacteria (E. coli, S. typhimurium, P. aeruginosa, and S. aureus), the results showed that only corilagin possessed moderate inhibitory effect against S. aureus (inhibition zone at 200 μg/disc =13 mm) compared to chloramphenicol (inhibition zone at 30 μg/disc = 20 mm). In addition, gallic acid showed weak antibacterial activity against S. mutans (inhibition zone at 1000 μg/disc = 7 mm) compared to chloramphenicol (inhibition zone at 30 μg/disc =30 mm) while ellagic acid did not show any inhibitory effect to all tested bacteria (maximum tested concentration =1000 μg/disc). 3.2. Antifungal and antibacterial activities of longan extracts Longan seed extract exhibited the highest antifungal activity while pulp and whole fruit extracts did not show any effects at the concentration tested (16,000 μg/ml) (Table 3). The results showed that freeze-dried extract of longan seed cultivar Baidam possessed a higher antifungal activity than the extracts of seed, pulp, and whole fruit of Edor. It should be noted that longan seed extract (Baidam) was found to be more active against C. parapsilosis (MIC= 250 μg/ml) than C. albicans (MIC = 500–1000 μg/ml except C. albicans 1555), C. neoformans (500–1000 μg/ml), and C. krusei (MIC = 1000 μg/ml). These results were related to the results of gallic acid. Longan seed extract (Edor) exhibited antifungal activity against C. parapsilosis (MIC=500 μg/ml), C. krusei (MIC=4000 μg/ml), and C. neoformans (MIC=4000 μg/ml) less effectively than the results of Baidam (MIC=250–1000 μg/ml). For C. albicans, Edor seed extract possessed the same antifungal activity or less than (MIC=500–1000 μg/ml) those of Baidam. Higher contents
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Table 3 In vitro antifungal activities of gallic acid, corilagin, ellagic acid and various longan fruit extracts compared to ketoconazole (a positive antifungal drug) using agar dilution method. The values represent Minimum Inhibition Concentration; MIC in μg/mL for duplicates. Microorganismsa
C. C. C. C. C. C. C. C. C. C. C. C. a b c
krusei ATCC 6258 parapsilosis ATCC 20019 albicans ATCC 90028 albicans 1435 albicans 1555 albicans 1434 albicans 1818 albicans 2923 neoformans 1795 neoformans 1640 neoformans 1221 neoformans 1112
Tested samples Ketoconazole
Ellagic acid
Gallic acid
Corilagin
Longan seed (Edor)b
Longan pulp (Edor)c
Longan seed + pulp (Edor)c
Longan seed (Baidam)c
0.24 0.12 15.63 31.25 0.06 31.25 3.91 3.91 0.06 0.12 0.24 7.81
125 7.81 62.50 125 7.81 125 15.63 7.81 15.63 15.63 15.63 62.50
8000 4000 4000 4000 8000 4000 16,000 16,000 16,000 >16,000 8000 8000
1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
4000 500 1000 1000 500 500 1000 1000 4000 4000 4000 4000
>16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000
>16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000
1000 250 500 1000 125 1000 500 1000 500 500 500 1000
Microorganisms: C. krusei: Candida krusei; C. parapsilosis: Candida parapsilosis; C. albicans: Candida albicans; C. neoformans: Cryptococcus neoformans. Spray-dried water extract of longan seed (cultivar Edor). Freeze-dried water extracts of longan pulp, pulp and seed, and seed (cultivar Baidam).
of gallic acid and ellagic acid present in Baidam seed extract may result in higher inhibitory effect on C. krusei, C. parapsilosis and C. neoformans than Edor seed extract (Table 1). Longan pulp and whole fruit contained low amounts of these polyphenolic compounds, thus none of these extracts had antifungal activity (MIC >16,000 μg/ml). Interestingly, it was also found that longan seed extract showed weak antifungal effect on a few species of dermatophytes including T. rubrum, M. gypseum and E. floccosum (data not shown). The fungi showed retarded growth and did not grow as a fluffy colony in medium containing seed extract (at 200 μg/ml) compared to the control. However, microscopic examination did not show any morphological changes of these fungi. These results suggested that the active compounds especially ellagic acid and gallic acid in longan seed extract may affect the physiological growth of these filamentous fungi. None of the tested longan extracts possessed antifungal activity against A. niger, A. nidulans, A. fumigatus, P. marneffei, P. siamensis, P. pinophilum, T. mentagrophytes, T. rubrum, M. gypseum, M. canis, and P. boydii at the tested concentration in this study (200 μg/ ml). For antibacterial activity, none of the longan extracts possessed an inhibitory effect on the tested bacteria strains at the concentration of 1000 μg/ml. 3.3. Antifungal activity of oral care products containing longan seed extract The color of effervescent granules became darker depending on the concentrations of longan seed extract added into preparation (5%). The effervescent granules possessed good characteristics as well as acceptable color and odor. The moisture content of granule base was 4.7% while that of granule containing 5% longan extract was 4.8%. The disintegration time for all granules, by dissolving granules in water until the clear solution obtained, was less than 3 min which was acceptable according to the British Pharmacopoeia (2004). The flow ability of all effervescent granules by angle of repose (θ) (determined by the funnel method) was found to be in a range of 27.8–28.4 indicating excellent flow. The physical
properties of these effervescent granules were acceptable since they had rapid disintegration, low moisture content, and good flow ability. After 30 and 60 min immersion of antifungal testing, chlorhexidine strongly reduced adherence of C. albicans on acrylic resin compared to non-treated control and water (Table 4). For effervescent granules, granule base and 5% extract granules also significantly reduced (p b 0.05) adherence of C. albicans with the most sensitive strains of ATCC13803 and clinical isolate I compared to nontreated control at both 30 and 60 min immersion. The microscopic pictures of adherence of C. albicans on acrylic resin were shown in Fig. 3. The results showed that both chlorhexidine (Fig. 3B) and longan effervescent granules (Fig. 3C) reduced the adherence of C. albicans on acrylic resin compared to granule base (Fig. 3A). The adherent reduction property of 5% longan extract granule may depend on the duration of immersion. The longer the immersion time, the less adherence of C. albicans on the acrylic resin was shown. If the immersion time is longer (>60 min), the adherence of these three strains of C. albicans on acrylic resin may be reduced at the same level. These were also supported by the antimicrobial effect of mouthwash (0.5% longan extract) which was determined after incubation of the longan mouthwash with fungal for 24 h (Table 5). In addition, each strain of C. albicans may have its own adherent property. For antifungal and antibacterial activities of mouthwash containing 0.5% longan extract (pH of mouthwash was 7.5– 7.8), C. albicans ATCC90028 and S. mutans were inhibited by 1:8 dilution and C. albicans ATCC10231 at 1:12 dilution (Table 5). Mouthwash with 0.5% longan extract showed the same potency or a little higher than a commercial mouthwash product for both C. albicans and S. mutans. A mouth rinse of 10% concentrated Terminalia chebula containing high polyphenols also showed a decrease in the microbial count for S. mutans (65%) and lactobacilli (71%) [21]. In addition, Nayak et al. [22] reported a significant reduction in S. mutans count at 5 and 60 min after rinsing with the T. chebula extract and salivary pH remained alkaline for a period of 1 h. The active compounds present in T. chebula Retz.
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Table 4 The adherence of Candida albicans on acrylic resin (cells/mm2) at 30 and 60 min (mean ± SEM; n = 3). C. albicans
Non-treated control
Chlorhexidine
5% longan granule
Granule base
Water
After 30 min immersion Clinical isolate I Clinical isolate II ATCC13803
499.76 ± 55.04 392.19 ± 27.59 131.59 ± 59.25
261.63 ± 35.46 a,b 345.91 ± 72.87 35.34 ± 25.36 a,b
389.81 ± 65.25 a,b 469.43 ± 50.45 a,b 51.57 ± 32.49a
372.19 ± 76.68 a,b 325.40 ± 74.45 33.95 ± 16.99 a,b
559.87 ± 50.97 386.91 ± 49.78 119.37 ± 57.14
After 60 min immersion Clinical isolate I Clinical isolate II ATCC13803
499.76 ± 55.04 392.19 ± 27.59 131.59 ± 59.25
23.89 ± 12.09 a,b 169.30 ± 66.94 a,b 31.44 ± 25.15 a,b
109.43 ± 44.38a 279.62 ± 76.99a 45.28 ± 27.19a
56.60 ± 32.98a 377.48 ± 72.18 55.70 ± 20.19a
88.04 ± 39.02a 330.43 ± 47.12a 77.98 ± 34.04
a b
Significantly different from the control (p b 0.05). Significantly different from distilled water (p b 0.05).
were gallic acid, chebulic acid, punicalagin, corilagin, ellagic acid, chebulagic acid, chebulinic acid, and chebulanin [23]. Some of these active compounds were similar to those of longan seed extract. Although, the antifungal activity of these three polyphenolic compounds was already well known, the present study reported the antifungal activity of the longan seed extracts for the first time. However, the finding of the susceptibility of these opportunistic yeasts to longan seed extract and its polyphenolic compounds needs further attention. We are also currently investigating anticancer activity and oral toxicity of longan seed extract. In conclusion, the present study therefore strongly suggested the potential use of longan seed extract (waste of longan fruit can production) or its polyphenolic compounds as an antimicrobial agent for oral care products such as mouthwash and effervescent granule for denture cleanser to treat the infection caused by opportunistic fungi and yeasts such as candidosis and cryptococcosis.
Acknowledgments The authors would like to thank Dr. Nopporn Thasana (Laboratory of Medicinal Chemistry) and Mr. Supachai Ritruechai (Laboratory of Pharmacology), Chulabhorn Research Institute, for purification and identification of polyphenolic compounds from longan seed. We also gratefully acknowledge the Faculty of Science, Mahidol University for their financial support for this project. Some results from this work were also the basis for a petty patent in Thailand (no. 5164 granted on December, 14, 2009).
Table 5 Minimum Inhibition Concentration (MIC) of mouthwash containing 0.5% longan seed extract against the tested microorganisms.
Fig. 3. Microscopic pictures of the adherence between Candida albicans (ATCC13803) and acrylic resin after rinsing with (A) granule base; (B) 0.2% chlorhexidine; (C) effervescent longan granules for 60 min. Twenty fields of view were randomly counted in each strip at ×400 magnification.
Products
MIC (S. mutans)
MIC (C. albicans ATCC90028)
MIC (C. albicans ATCC10230)
Base mouthwash Mouthwash 0.5% longan extract Commercial mouthwash
1:8 1:8
1:6 1:8
1:8 1:12
1:6
1:8
1:6
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[13] Lindenberg AS, Chang MR, Paniago AM, Lazéra MS, Moncada PM, Bonfim GF, et al. Clinical and epidemiological features of 123 cases of cryptococcosis in Mato Grosso do Sul, Brazil. Rev Inst Med Trop Sao Paulo 2008;50: 75–8. [14] Muzyka BC, Glick M. A review of oral fungal infections and appropriate therapy. J Am Dent Assoc 1995;126:63–72. [15] Fogliani B, Raharivelomanana P, Bianchini JP, Bouraïma-Madjèbi S, Hnawia E. Bioactive ellagitannins from Cunonia macrophylla, an endemic Cunoniaceae from New Caledonia. Phytochemistry 2005;66:241–7. [16] Latté KP, Kolodziej H. Antifungal effects of hydrolysable tannins and related compounds on dermatophytes, mould fungi and yeasts. Z Naturforsch C 2000;55:467–72. [17] Rangsriwong P, Rangkadilok N, Satayavivad J, Goto M, Shotipruk A. Subcritical water extraction of polyphenolic compounds from Terminalia chebula Retz. fruits. Sep Purif Technol 2009;66:51–6. [18] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests; Approved standard-Ninth Edition. CLSI document M2-A9. Wayne, PA: Clinical and Laboratory Standards Institute, 2006. [19] National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard-Fifth Edition. NCCLS document M7-A5. Wayne, PA: NCCLS, 2000. [20] Yoshida T, Jono K, Okonogi K. Modified agar dilution susceptibility testing method for determining in vitro activities of antifungal agents, including azole compounds. Antimicrob Agents Chemother 1997;41: 1349–51. [21] Carounanidy U, Satyanarayanan R, Velmurugan A. Use of an aqueous extract of Terminalia chebula as an anticaries agent: a clinical study. Indian J Dent Res 2007;18:152–6. [22] Nayak SS, Kumar BR, Ankola AV, Hebbal M. The efficacy of Terminalia chebula rinse on Streptococcus mutans count in saliva and its effect on salivary pH. Oral Health Prev Dent 2010;8:55–8. [23] Juang LJ, Sheu SJ. Chemical identification of the sources of commercial fructus chebulae. Phytochem Anal 2005;16:246–51.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
In vitro antifungal activities of longan (Dimocarpus longan Lour.) seed extract Nuchanart Rangkadilok a, b, 1, Songsak Tongchusak c, 1, Rachasak Boonhok c, Sansanee C. Chaiyaroj c, Varaporn B. Junyaprasert d, Waranun Buajeeb e, Jaratluck Akanimanee a, Thida Raksasuk a, Theeralaksna Suddhasthira e, Jutamaad Satayavivad a,b,⁎ a b c d e
Laboratory of Pharmacology, Chulabhorn Research Institute (CRI), Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand Environmental Toxicology Program, Chulabhorn Graduate Institute, Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Rajthevi, Bangkok 10400 Thailand Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajthevi, Bangkok 10400, Thailand Faculty of Dentistry, Mahidol University, Rajthevi, Bangkok 10400, Thailand
a r t i c l e
i n f o
Article history: Received 3 October 2011 Accepted in revised form 19 December 2011 Available online 8 January 2012 Keywords: Longan seed Ellagic acid Corilagin Dimocarpus longan Candida Cryptococcus neoformans
a b s t r a c t Longan, Dimocarpus longan Lour., contains polyphenolic compounds which exhibit several pharmacological properties. This study aims to evaluate antifungal activities of longan fruit extract in comparison to its active compounds. The results showed that longan seed exhibited antifungal activity against the opportunistic yeasts (Candida species and Cryptococcus neoformans). In contrast, longan pulp and whole fruit did not demonstrate any inhibitory effects. Ellagic acid showed the most potent antifungal activity followed by corilagin and gallic acid, respectively. Ellagic acid inhibited Candida parapsilosis and C. neoformans more effectively than Candida krusei and also some Candida albicans clinical strains. Baidam cultivar possessed higher antifungal activity (MIC= 500–4000 μg/ml) as it contained higher contents of ellagic acid and gallic acid than Edor (MIC= 1000–8000 μg/ml). For antibacterial activity, only corilagin and gallic acid possessed weak to moderate inhibitory effects against Staphylococcus aureus and Streptococcus mutans, respectively. Longan seed was then applied in the oral care products. Longan effervescent granule (5% extract) significantly reduced adhesion of C. albicans to acrylic strips. Mouthwash containing 0.5% extract exhibited good antifungal activity compared to a commercial product. These findings indicated that longan seed extract and its polyphenolic compounds can be used as an antifungal agent in oral care products for the treatment of opportunistic yeast infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Longan fruit (Dimocarpus longan Lour.) is a subtropical fruit, which belongs to the Sapindaceae family. Longan, known as ‘Lumyai’ in Thailand, is widely grown in China, Taiwan, and South East Asia including Thailand and Vietnam. Our previous study demonstrated that water extract of
⁎ Corresponding author at: Laboratory of Pharmacology, Chulabhorn Research Institute, Kamphaengphet 6 Road, Laksi, Bangkok 10210, Thailand. Tel.: +66 2 5740622x3917; fax: +66 2 5742027. E-mail address: [email protected] (J. Satayavivad). 1 These authors contributed equally to this study. 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.12.023
longan fruit contained high levels of polyphenolic compounds such as corilagin, gallic acid, and ellagic acid [1] (Fig. 1). The contents of these polyphenolics depended on cultivars and plant tissues. Dried seed contained the highest levels of these three compounds followed by dried peel and dried pulp. Prasad et al. [2] used ultra-high-pressure-assisted extraction to extract phenolic compounds from longan fruit pericarp and found that it contained the highest content of corilagin compared to other phenolic compounds. In addition, Lin et al. [3] and Hsu et al. [4] also reported that longan seed contained corilagin and acetonylgeranin as active compounds. The methanolic extract of longan seed (0.1 and 0.2 mg/ml) exhibited ability to induce osteoclast differentiation in mouse
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HO
COOH
HO
OH O
O
OH
HO
Gallic acid HO
O
O
OH
Elagic acid HO
OH
HO
OH
HO
OH CO
CO
CH 2O O
O O C
OH OH
O OH OH
OH
Corilagin Fig. 1. The structures of gallic acid, ellagic acid, and corilagin.
bone marrow macrophages [5]. Jiang et al. [6] reported that longan fruit pericarp exhibited strong antioxidant activities in a concentration-dependent manner using DPPH and superoxide anion radical scavenging assays. From our previous study, dried longan seed extract of cultivar Edor exhibited the highest radical scavenging activities compared to fresh seed and dried pulp extracts [7]. In addition, longan seed extract also possessed anti-tyrosinase activity with IC50 = 2.9–3.2 mg/ml. Recent studies reported that polyphenol-rich longan seed extract inhibited the proliferation of human colorectal carcinoma cells [8] and longan seed polysaccharides (50–100 mg/kg) had antifatigue effects in mice by extending swimming time, increasing hepatic glucogen, reducing blood urea nitrogen and lactic acid [9]. These therapeutic effects of longan seed are attributed to various compounds present in this plant, including ellagic acid, gallic acid, and corilagin. The opportunistic fungi are common causes of serious morbidity and mortality in immunocompromised patients [10]. Candida is a genus of yeast, commonly part of the normal flora of the mouth, skin, intestinal tract and vagina, but can also cause several diseases. Candida albicans is one of the oral Candida species and it is responsible for most oral candidal infections [11]. Cryptococcus neoformans is an encapsulated yeast-like fungus living in both plants and animals and also an opportunistic fungal pathogen that causes central nervous system and pulmonary diseases among immunocompromised patients [12,13]. Medications associated with the emergence of clinical candidiasis are broad-spectrum antibiotics, corticosteroids, immunosuppressive agents (azathioprine), anticholinergic agents (e.g. tricyclic antidepressant) [14] whereas amphotericin B with/
without fluconazole is the treatment of choice for mild and localized C. neoformans infection [12]. The use of antifungal drugs is costly and long-term therapy may cause fungal resistance. Therefore, the development of antifungal agents from medicinal plants may be an alternative source for the inexpensive treatment of fungal infections. The antimicrobial activity of the three polyphenolic compounds (gallic acid, ellagic acid, and corilagin) was investigated on human pathogenic bacteria (Staphylococcus aureus and Corynebacterium accolans) and a plant pathogen (Erwinia carotovora) as well as human pathogenic yeast (C. albicans) [15]. The results showed that corilagin had the highest antimicrobial activity followed by ellagic acid and gallic acid. However, ellagic acid could not inhibit S. aureus in this study. From the previous study, there is limited scientific information for the use of these three compounds or longan seed extract in the treatment of microbial infections. The aim of this study was to investigate the antifungal activity of gallic acid, ellagic acid, and corilagin including different longan extracts against Candida species, C. neoformans and some dermatophytes. In addition, the antibacterial activity of these compounds and extracts and the potential use of longan seed extract in oral care products was also investigated. 2. Materials and methods 2.1. Chemicals Dimethyl sulfoxide (DMSO), absolute ethanol, and methanol (HPLC grade) were obtained from Merck (Darmstadt, FR Germany). 2,2′-Azobis(2-amidinopropane) dihydrochloride
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(AAPH) was purchased from Cayman (Ann Arbor, MI, USA). Chloramphenicol and ceftazidime were purchased from Oxoid (Hampshire, UK). Gallic acid, ellagic acid, amphotericin B, ketoconazole, and all other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA). Corilagin was extracted and purified from longan seeds by Laboratory of Pharmacology, Chulabhorn Research Institute, Thailand following the previous method reported by Latte and Kolodziej [16] with some modifications and used as a standard in this experiment (% purity = 98%). All chemicals for preparation of oral care products e.g. effervescent granule and mouthwash were purchased from Carlo Erba (Italy), S. Tong Chemicals Co, Ltd. (Bangkok, Thailand), Ajex Finechem (NSW, Australia), Fisher Scientific UK (Leicestershire, UK), and Thai-China Flavours and Fragrances Industry Co., Ltd. (Bangkok, Thailand). Milli-Q deionized water (Branstead, Newton, USA) was used throughout this experiment. 2.2. Plant material and extraction D. longan Lour. was identified by Dr. Somran Suddee (BKF Forest Herbarium, Department of National Parks, Wildlife and Plant Conservation, Bangkok, Thailand). A voucher specimen (L. Worasuttayangkurn 01) was deposited at the BKF Forest Herbarium (Bangkok, Thailand). Longan cultivar Edor and Baidam were selected to use in this study. Edor is a commercial cultivar available in Thailand. Seed, pulp and whole fruit of Edor were used for extraction. Baidam contains high levels of gallic acid and ellagic acid in seed; therefore, it was used to compare the antifungal activity to Edor. Five hundred grams dried ground seed, pulp, or whole fruit (seed + pulp) of longan were extracted three times with 2 l of hot water (70–75 °C) for 1 h at each time. The extracts were then filtered and combined. The water extracts were then concentrated and dried using freeze-dryer (for pulp as it contained high sugar) and spray-dryer (1% lactose was added in seed extract and spray dried at 80 °C/120 °C). The percentage yield of extracts was between 25 and 35% depending on cultivars and plant samples. Plant extracts, 22–25 mg, were dissolved in 3.0 ml of hot water (70–75 °C) (2 replicates per sample) and vigorously shaken. The extracts were left at room temperature until they cooled down and then filtered through a 0.45 μm PVDF membrane (Chrom Tech, Apple Valley, MN) prior to HPLC analysis. These longan extracts were analyzed for the contents of three polyphenolic compounds (corilagin, gallic acid, and ellagic acid) by using our previous HPLC method modified by Rangsriwong et al. [17]. The HPLC analysis was performed using an HP1100 HPLC system with a thermostatically controlled column oven, a binary pump, and a diode-array detector (Hewlett Packard, USA). A 150 × 3.9 mm i.d., 5 μm reversed phase column, Symmetry® C18 (Waters Corporation, Milford, Massachusetts) was used for analysis of the active compounds in longan extract. The compounds were eluted with a gradient system of 0.1% formic acid (solvent A): methanol (solvent B) at a flow rate of 1.0 ml/min. The temperature of the column was 25 °C with the UV detection at 270 nm. The injection volume was 10 μl. The gradient system started with 4%B at 0 min to 80%B in 27 min. The total run time is 30 min. Retention times of gallic acid, corilagin, and ellagic acid are 6.69, 19.22, and 25.65 min, respectively (Fig. 2).
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2.3. Fungi and bacteria strains C. albicans (ATCC90028), Candida parapsilosis (ATCC20019), and Candida krusei (ATCC6258) were purchased from the American Type Culture Collection and C. albicans (ATCC10231) was from the Department of Medical Science, Ministry of Public Health, Thailand (DMST). Total 7 clinical isolates of C. albicans, 4 clinical isolates of C. neoformans and filamentous fungi including Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum gypseum, Microsporum canis, Epidermophyton floccosum, Pseudallescheria boydii, Penicillium siamensis, Penicillium pinophilum, Penicillium marneffei, Aspergillus nidulans, Aspergillus niger and Aspergillus fumigatus were received from Mycology Section, Division of Clinical Pathology, Department of Medical Sciences, National Institute of Health, Thailand and 2 clinical strains of C. albicans (for effervescent granule testing) were isolated from oral lesions of patients of the Out Patient Clinic at the Faculty of Dentistry, Mahidol University, Thailand. Filamentous fungi were grown on Sabouraud Dextrose Agar slant (SDA; 4% dextrose, 2% neopeptone and 1.7% agar) at room temperature for one or four weeks (depending on fungal species). Conidia were harvested by flooding with 0.01% Tween-20 on fungal colony. Conidia were counted on a hemacytometer chamber and adjusted to a final concentration of 106 cells/ml or McFarland standard solution no. 0.5. Yeasts were cultured on Sabouraud Dextrose Agar plate (SDA; 4% dextrose, 2% neopeptone and 1.7% agar) and incubated at 37 °C for 24 h or 48 h. A single colony was then subcultured onto Sabouraud Dextrose Broth (SDB) and incubated at 37 °C in shaking incubator for 24 h or 48 h. Before antifungal activity testing, the concentration of yeasts was adjusted so they were equal to the turbidity of McFarland standard solution no. 0.5. The bacterial reference strains were obtained from the Thailand Institute of Scientific and Technological Research (TISTR) and the Department of Medical Science, Ministry of Public Health, Thailand (DMST). The following microorganisms were used for determining antibacterial activity: Escherichia coli TISTR 887 (ATCC25922), S. aureus TISTR 517 (ATCC25923), Pseudomonas aeruginosa TISTR 1467 (ATCC27853), Salmonella typhimurium TISTR 292 (ATCC13311), Streptococcus mutans DMST 18777 (ATCC25175T). Bacterial inoculum suspension was prepared as described in Performance Standards for Antimicrobial Disk Susceptibility Tests; Ninth Edition [18]. Each tested culture should be streaked onto trypticase soy agar to obtain isolated colonies and incubated at 35 °C for 18–24 h. For S. mutans, bacteria were streaked on trypticase soy agar with 5% sheep blood and incubated at 35 °C in a candle jar for 18–24 h. Four or five well-isolated colonies were then selected and transferred to a tube of sterile saline or Mueller–Hinton broth. Turbidity was measured and adjusted to match a 0.5 McFarland density standard resulting in inoculums containing 1.5×108 cells/ml. This suspension was used to directly inoculate agar plates for disc diffusion method. 2.4. Disc–agar diffusion method For antifungal activity, all longan extracts and the reference compounds were tested on standard antimicrobial assay (AA) disc at a final concentration of 1 mg/disc (Whatman, Brentford, UK). Ketoconazole was used at a concentration of 200 μg/disc. Aqueous solvent, ethanol, and 10% aqueous dimethyl sulfoxide
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Dried seed Gallic acid
Corilagin
Ellagic acid
Dried pulp
Whole fruit
Fig. 2. HPLC chromatograms of different parts of longan fruit: dried seed, dried pulp and whole fruit. Retention time (RT) of gallic acid = 6.69 min, corilagin = 19.22 min, and ellagic acid = 25.65 min.
(DMSO) control discs were prepared using the same volume of extracts and reference compounds. All discs were prepared aseptically and dried at 37 °C before testing. SDA plates were prepared in 20 cm diameter plastic Petri dishes by fixing the level of media at 4 mm height. In order to culture the yeasts, a wound cotton swab was used for streaking fungal preparation on the surface of media with 60° difference for 3 plans of streak. Testing discs were placed on the surface of media and incubated at 37 °C for 24 h (C. albicans) or 48 h (C. neoformans). Experiment was carried out in triplicate. The antifungal activity was evaluated by measuring the inhibition zone diameter. For antibacterial activity, the entire surface of Mueller–Hinton agar plates was inoculated simultaneously in three directions by using a cotton swab. All plant extracts were reconstituted in distilled water at the stock concentration at 10 mg/ml and filtersterilized through a 0.45 μm membrane filter. The three reference compounds (corilagin, gallic acid, ellagic acid) were dissolved in 10% DMSO to make the stock concentrations of 10 and 50 mg/ml. A sterile paper disc (6 mm in diameter) was
impregnated with 20 μl of suspension containing plant extracts (longan solutions at 100, 200, 500 and 1000 μg/disc) in water and three standard compounds (at concentrations of 100 and 200 μg/disc for corilagin; and 100–1000 μg/disc for ellagic acid and gallic acid). The discs were then placed on the agar surface. Plates were incubated at 35 °C for 18–24 h. For S. mutans, plate was incubated at 35 °C for 18–24 h in candle jar. The antibacterial activity was evaluated by measuring the inhibition zone diameter. The solvents used for each plant extract and standard compound preparation were used as the negative control. Chloramphenicol (30 μg/disc) and ceftazidime (30 μg/disc) were used as the positive control. 2.5. Determination of Minimum Inhibitory Concentration (MIC) by agar dilution method The Minimum Inhibition Concentration (MIC) of the longan extracts and reference compounds (gallic acid, ellagic acid, and corilagin) were determined by the agar dilution method. Plant
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extracts were dissolved in distilled water and serial two fold dilution was performed. For antifungal testing, the range of concentration of the tested compounds was 15.63–16,000 μg/ml and the ranges of concentrations for reference compounds were 0.98–1000, 1.95–2000, and 31.25–16,000 μg/ml for ellagic acid, corilagin, and gallic acid, respectively. The MIC was determined as the lowest concentration of the sample that resulted in complete inhibition of microbial growth. Testing media were left at room temperature allowing media surface to dry. Fungi were inoculated on medium surface for 3 μl (~4.5×105 cells) and incubated at 37 °C for 24 h (C. albicans) or 48 h (C. neoformans). For control testing, SDA alone or SDA+EtOH and SDA+DMSO were used. Ketoconazole was used as a positive control (at 0.0075–125 μg/ml). 2.6. Preparation of effervescent granules for denture cleanser For effervescent granule base, citric acid, tartaric acid and sodium bicarbonate were homogeneously mixed at 1:2:3.44 (by weight). Subsequently, 8% sorbitol and 0.01% sodium lauryl sulfate (SLS) were added to the mixture. An appropriate amount of 75% ethanol was then used as a binder by slowly adding to the mixture to produce a damp mass which was then passed through a 14-mesh sieve to produce granules. The obtained granules were dried in an oven at 45 °C for 2 h. Afterwards, the dried granules were sprayed with solution of 0.1% thymol and 0.8% peppermint oil and dried again at 45 °C for 15 min. Finally, the effervescent granules were packed in aluminum foil and kept in a desiccator until use. In the case of longan seed extract effervescent granules, 5% spray-dried longan seed extract was mixed with sodium bicarbonate and the granules were prepared as previously described. These longan effervescent granules were then tested for appearance, disintegration time, moisture content, and flow ability. 2.7. Adherence assay Acrylic strips were placed vertically in the plastic well (15 mm diameter) (Cellstar, Grenier labortechnik, Denmark). Approximately 200 μl of yeast suspension was added to each well. Thereafter, the whole assembly was incubated at 37 °C for 1 h with gentle agitation at 120 rev/min. The strips were then recovered from the wells and washed 3 times by dipping gently in sterile PBS, which helped to dislodge the nonadherent cells. Then the strips were placed in new wells containing 200 μl of longan extract formulation and incubated at 37 °C for 30 or 60 min. Sterile distilled water and granule base were used as negative controls, whereas 0.2% chlorhexidine was used as a positive control. The strips were then recovered from the wells and washed 3 times in sterile PBS again. They were dried and stained using gram stain without counterstain. After air-drying at room temperature, they were mounted on glass slides and the adherent yeasts were quantified. 2.8. Microscopic quantification of adherent yeasts A light microscope (Nikon E100, Japan) was used for the quantitative estimation of the adherent yeasts. Twenty fields of view were randomly counted in each strip at ×400 magnification. The mean number of yeasts was finally expressed as
549
yeast cell per unit square mm. All experiments were repeated on 3 separate occasions with duplicate determinations on each occasion. 2.9. Assessment of the antimicrobial activity of mouthwash The tested microorganisms S. mutans DMST 18777 (ATCC25175T), C. albicans DMST 8684 (ATCC90028) and C. albicans DMST 5815 (ATCC10231) were used in this study. S. mutans was cultured overnight at 35 °C (in candle jar) on blood agar and Candida spp. were cultured overnight at 35 °C on Sabouraud dextrose agar (Becton, Dickinson and company, USA). A sensitivity test was carried out according to the agar dilution method described by National Committee for Clinical Laboratory Standards (NCCLS document M7-A5) (at present known as Clinical and Laboratory Standards Institute; CLSI) [19] and some modifications of Yoshida et al. [20]. The mouthwash was added aseptically to sterile molted Mueller–Hinton agar (Oxoid, Basingstoke, England) supplemented with 5% sheep blood (for S. mutans) and Sabouraud dextrose agar (for C. albicans) in appropriate volumes to produce the concentrations of 1:4, 1:6, 1:8, 1:12, 1:16, 1:24 of each mouthwash. The resulting agar solutions were immediately poured into Petri-plates after vortexing. Inocula were prepared by suspending growth from overnight cultures of S. mutans in Mueller–Hinton broth (Oxoid, Basingstoke, England) and C. albicans in normal saline to a turbidity of a 0.5 McFarland standard. The prepared suspensions were further diluted 1:100 for S. mutans and 1:10 for C. albicans. The 10 μl of cell suspension was applied to each agar plate, with a final inoculum of approximately 10 4 cells per spot for bacteria and 10 3 cells per spot for yeast. For C. albicans, plates were incubated overnight at 35 °C in air but for S. mutans, the plates were incubated overnight at 35 °C in a candle jar. The lowest concentration of mouthwash solution showing no growth was read as the Minimum Inhibitory Concentration (MIC). Control cultures containing only the Mueller–Hinton agar supplemented with 5% sheep blood/Sabouraud dextrose agar, were also prepared. A commercial mouthwash containing zinc chloride, benzoic acid, eucalyptol oil, thymol, and menthol was used as a positive control. 2.10. Statistical analysis Data were expressed as mean ± SEM. The statistical analyses were performed using SPSS 11.5 for Windows (SPSS Inc., Chicago, Illinois, USA). The Kruskal–Wallis and Wilcoxon Mann–Whitney non-parametric test were used to compare the means of adherence of C. albicans on acrylic resin (cells/ mm 2) between the treatments of control and product samples. A p value b 0.05 was regarded as statistically significant. 3. Results and discussion The active compounds present in longan seed were different from those in longan pulp (Fig. 2). Gallic acid, ellagic acid and corilagin are the major polyphenolic compounds present in dried longan seed extracts while longan pulp extract contained a small amount of ellagic acid but no gallic acid and corilagin (Table 1). Dried seed of Baidam contained the highest contents of gallic acid and ellagic acid with the same
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Table 1 The contents of gallic acid, corilagin, and ellagic acid in different longan fruit extracts (mean ± SEM; n = 3). Extracts
Gallic acid (mg/g DW)
Corilagin (mg/g DW)
Ellagic acid (mg/g DW)
Dried seed Edora Dried pulp Edorb Dried fruitb (seed + pulp Edor) Dried seed Baidamb
9.82 ± 0.87 ND 1.64 ± 0.01
14.84 ± 0.31 ND 0.43 ± 0.01
3.15 ± 0.03 0.13 ± 0.03 0.10 ± 0.00
21.89 ± 0.76
14.81 ± 0.32
9.76 ± 0.28
ND = not detectable. a Spray dried water extract of longan seed cultivar Edor. b Freeze dried water extracts of longan seed, pulp, and whole fruit cultivar Edor and Baidam.
amount of corilagin as cultivar Edor. The contents of these three compounds were present in small amounts in whole fruit of longan (Fig. 2). 3.1. Antifungal and antibacterial activities of polyphenolic compounds Ellagic acid and longan seed extracts showed antifungal activity against C. albicans and C. neoformans at 1 mg/disc (Table 2). It was found that longan seed extract was more potent than pure ellagic acid. However, both longan extract and ellagic acid exhibited lower antifungal activity than ketoconazole (a positive antifungal drug). Using agar dilution method, all polyphenolic compounds (gallic acid, ellagic acid, and corilagin) possessed antifungal activity against Candida species and C. neoformans (Table 3). The antifungal activities of these compounds in decreasing order were ellagic acid > corilagin > gallic acid. Ellagic acid inhibited Candida parapsilosis (MIC = 7.81 μg/ml) and C. neoformans (MIC = 15.63 μg/ml except for C. neoformans 1112) more effectively than C. krusei (MIC = 125 μg/ml). For C. albicans, ellagic acid showed greater activity to two clinical strains (C. albicans 1555 and 2923) with MIC = 7.81 μg/ml than other C. albicans strains (MIC = 15.63– 125 μg/ml). However, the antifungal activities of ellagic acid against Candida species and C. neoformans were less potent than an antifungal drug, ketoconazole (MIC = 0.06–31.25 μg/ ml). Gallic acid exhibited antifungal activity against C. parapsilosis (MIC = 4000 μg/ml) and most of C. albicans strains (ATCC 90028, 1435, 1434; MIC= 4000 μg/ml) higher
Table 2 Antifungal activities of longan extracts and ellagic acid against the tested microorganisms (mean ± SEM; n = 3). Compounds
10% DMSO Ethanol Distilled water Longan seed extract (cultivar Edor) Longan seed extract (cultivar Baidam) Ellagic acid Ketoconazole
Inhibition zone diameter (mm) Candida albicans (90028)
Cryptococcus neoformans (CI1435)
0 0 0 13.33 ± 0.57
0 0 0 26.33 ± 0.57
11.33 ± 1.15
28.33 ± 0.57
8.75 ± 0.50 21.5 ± 0.57
15.50 ± 1.52 70.00 ± 0.00
than C. krusei (MIC= 8000 μg/ml) and C. neoformans (MIC = 8000–> 16,000 μg/ml). For corilagin, the MIC against both Candida species and C. neoformans was about 1000 μg/ ml. It should be noted that ellagic acid, a dimer form of gallic acid, showed better antifungal activity than gallic acid (lower MIC values). In contrast, Latte and Kolodziej [16] indicated that the antifungal effect of gallic acid against C. neoformans (MIC=31 μg/ml), C. krusei (MIC=125 μg/ml), and C. albicans (MIC=500–>2000 μg/ml) was similar to that of corilagin. They reported that corilagin showed prominent antifungal activity against Candida glabrata strains with the MIC similar to the antifungal drugs, amphotericin B and sertaconazole. However, the MIC values of both gallic acid and corilagin in their study were lower than the MIC values in the present study. This may be due to the differences in the isolates of fungi, yeasts, and testing methods. Most of our isolates are clinical isolates but their study used mostly reference strains. The report from Fogliani et al. [15] which was different from our results, also found that corilagin had the highest inhibitory activity against C. albicans followed by ellagic acid and gallic acid. The present study showed that at the highest tested concentration (200 μg/ml), ellagic acid exhibited weak antifungal activity against dermatophytes species (Trichophyton rubrum, M. gypseum and E. floccosum) while gallic acid and corilagin could not inhibit the growth of these dermatophytes (data not shown). These results agree with the previous results from Latte and Kolodziej [16] who reported that neither gallic acid nor corilagin possessed any activity against filament fungi e.g. E. floccosum, M. canis, T. mentagrophytes, T. rubrum, A. fumigatus, Mucor racemosus or Rhizopus nigricans as reflected by MIC >2000 μg/ ml. For bacteria (E. coli, S. typhimurium, P. aeruginosa, and S. aureus), the results showed that only corilagin possessed moderate inhibitory effect against S. aureus (inhibition zone at 200 μg/disc =13 mm) compared to chloramphenicol (inhibition zone at 30 μg/disc = 20 mm). In addition, gallic acid showed weak antibacterial activity against S. mutans (inhibition zone at 1000 μg/disc = 7 mm) compared to chloramphenicol (inhibition zone at 30 μg/disc =30 mm) while ellagic acid did not show any inhibitory effect to all tested bacteria (maximum tested concentration =1000 μg/disc). 3.2. Antifungal and antibacterial activities of longan extracts Longan seed extract exhibited the highest antifungal activity while pulp and whole fruit extracts did not show any effects at the concentration tested (16,000 μg/ml) (Table 3). The results showed that freeze-dried extract of longan seed cultivar Baidam possessed a higher antifungal activity than the extracts of seed, pulp, and whole fruit of Edor. It should be noted that longan seed extract (Baidam) was found to be more active against C. parapsilosis (MIC= 250 μg/ml) than C. albicans (MIC = 500–1000 μg/ml except C. albicans 1555), C. neoformans (500–1000 μg/ml), and C. krusei (MIC = 1000 μg/ml). These results were related to the results of gallic acid. Longan seed extract (Edor) exhibited antifungal activity against C. parapsilosis (MIC=500 μg/ml), C. krusei (MIC=4000 μg/ml), and C. neoformans (MIC=4000 μg/ml) less effectively than the results of Baidam (MIC=250–1000 μg/ml). For C. albicans, Edor seed extract possessed the same antifungal activity or less than (MIC=500–1000 μg/ml) those of Baidam. Higher contents
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Table 3 In vitro antifungal activities of gallic acid, corilagin, ellagic acid and various longan fruit extracts compared to ketoconazole (a positive antifungal drug) using agar dilution method. The values represent Minimum Inhibition Concentration; MIC in μg/mL for duplicates. Microorganismsa
C. C. C. C. C. C. C. C. C. C. C. C. a b c
krusei ATCC 6258 parapsilosis ATCC 20019 albicans ATCC 90028 albicans 1435 albicans 1555 albicans 1434 albicans 1818 albicans 2923 neoformans 1795 neoformans 1640 neoformans 1221 neoformans 1112
Tested samples Ketoconazole
Ellagic acid
Gallic acid
Corilagin
Longan seed (Edor)b
Longan pulp (Edor)c
Longan seed + pulp (Edor)c
Longan seed (Baidam)c
0.24 0.12 15.63 31.25 0.06 31.25 3.91 3.91 0.06 0.12 0.24 7.81
125 7.81 62.50 125 7.81 125 15.63 7.81 15.63 15.63 15.63 62.50
8000 4000 4000 4000 8000 4000 16,000 16,000 16,000 >16,000 8000 8000
1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
4000 500 1000 1000 500 500 1000 1000 4000 4000 4000 4000
>16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000
>16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000 >16,000
1000 250 500 1000 125 1000 500 1000 500 500 500 1000
Microorganisms: C. krusei: Candida krusei; C. parapsilosis: Candida parapsilosis; C. albicans: Candida albicans; C. neoformans: Cryptococcus neoformans. Spray-dried water extract of longan seed (cultivar Edor). Freeze-dried water extracts of longan pulp, pulp and seed, and seed (cultivar Baidam).
of gallic acid and ellagic acid present in Baidam seed extract may result in higher inhibitory effect on C. krusei, C. parapsilosis and C. neoformans than Edor seed extract (Table 1). Longan pulp and whole fruit contained low amounts of these polyphenolic compounds, thus none of these extracts had antifungal activity (MIC >16,000 μg/ml). Interestingly, it was also found that longan seed extract showed weak antifungal effect on a few species of dermatophytes including T. rubrum, M. gypseum and E. floccosum (data not shown). The fungi showed retarded growth and did not grow as a fluffy colony in medium containing seed extract (at 200 μg/ml) compared to the control. However, microscopic examination did not show any morphological changes of these fungi. These results suggested that the active compounds especially ellagic acid and gallic acid in longan seed extract may affect the physiological growth of these filamentous fungi. None of the tested longan extracts possessed antifungal activity against A. niger, A. nidulans, A. fumigatus, P. marneffei, P. siamensis, P. pinophilum, T. mentagrophytes, T. rubrum, M. gypseum, M. canis, and P. boydii at the tested concentration in this study (200 μg/ ml). For antibacterial activity, none of the longan extracts possessed an inhibitory effect on the tested bacteria strains at the concentration of 1000 μg/ml. 3.3. Antifungal activity of oral care products containing longan seed extract The color of effervescent granules became darker depending on the concentrations of longan seed extract added into preparation (5%). The effervescent granules possessed good characteristics as well as acceptable color and odor. The moisture content of granule base was 4.7% while that of granule containing 5% longan extract was 4.8%. The disintegration time for all granules, by dissolving granules in water until the clear solution obtained, was less than 3 min which was acceptable according to the British Pharmacopoeia (2004). The flow ability of all effervescent granules by angle of repose (θ) (determined by the funnel method) was found to be in a range of 27.8–28.4 indicating excellent flow. The physical
properties of these effervescent granules were acceptable since they had rapid disintegration, low moisture content, and good flow ability. After 30 and 60 min immersion of antifungal testing, chlorhexidine strongly reduced adherence of C. albicans on acrylic resin compared to non-treated control and water (Table 4). For effervescent granules, granule base and 5% extract granules also significantly reduced (p b 0.05) adherence of C. albicans with the most sensitive strains of ATCC13803 and clinical isolate I compared to nontreated control at both 30 and 60 min immersion. The microscopic pictures of adherence of C. albicans on acrylic resin were shown in Fig. 3. The results showed that both chlorhexidine (Fig. 3B) and longan effervescent granules (Fig. 3C) reduced the adherence of C. albicans on acrylic resin compared to granule base (Fig. 3A). The adherent reduction property of 5% longan extract granule may depend on the duration of immersion. The longer the immersion time, the less adherence of C. albicans on the acrylic resin was shown. If the immersion time is longer (>60 min), the adherence of these three strains of C. albicans on acrylic resin may be reduced at the same level. These were also supported by the antimicrobial effect of mouthwash (0.5% longan extract) which was determined after incubation of the longan mouthwash with fungal for 24 h (Table 5). In addition, each strain of C. albicans may have its own adherent property. For antifungal and antibacterial activities of mouthwash containing 0.5% longan extract (pH of mouthwash was 7.5– 7.8), C. albicans ATCC90028 and S. mutans were inhibited by 1:8 dilution and C. albicans ATCC10231 at 1:12 dilution (Table 5). Mouthwash with 0.5% longan extract showed the same potency or a little higher than a commercial mouthwash product for both C. albicans and S. mutans. A mouth rinse of 10% concentrated Terminalia chebula containing high polyphenols also showed a decrease in the microbial count for S. mutans (65%) and lactobacilli (71%) [21]. In addition, Nayak et al. [22] reported a significant reduction in S. mutans count at 5 and 60 min after rinsing with the T. chebula extract and salivary pH remained alkaline for a period of 1 h. The active compounds present in T. chebula Retz.
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Table 4 The adherence of Candida albicans on acrylic resin (cells/mm2) at 30 and 60 min (mean ± SEM; n = 3). C. albicans
Non-treated control
Chlorhexidine
5% longan granule
Granule base
Water
After 30 min immersion Clinical isolate I Clinical isolate II ATCC13803
499.76 ± 55.04 392.19 ± 27.59 131.59 ± 59.25
261.63 ± 35.46 a,b 345.91 ± 72.87 35.34 ± 25.36 a,b
389.81 ± 65.25 a,b 469.43 ± 50.45 a,b 51.57 ± 32.49a
372.19 ± 76.68 a,b 325.40 ± 74.45 33.95 ± 16.99 a,b
559.87 ± 50.97 386.91 ± 49.78 119.37 ± 57.14
After 60 min immersion Clinical isolate I Clinical isolate II ATCC13803
499.76 ± 55.04 392.19 ± 27.59 131.59 ± 59.25
23.89 ± 12.09 a,b 169.30 ± 66.94 a,b 31.44 ± 25.15 a,b
109.43 ± 44.38a 279.62 ± 76.99a 45.28 ± 27.19a
56.60 ± 32.98a 377.48 ± 72.18 55.70 ± 20.19a
88.04 ± 39.02a 330.43 ± 47.12a 77.98 ± 34.04
a b
Significantly different from the control (p b 0.05). Significantly different from distilled water (p b 0.05).
were gallic acid, chebulic acid, punicalagin, corilagin, ellagic acid, chebulagic acid, chebulinic acid, and chebulanin [23]. Some of these active compounds were similar to those of longan seed extract. Although, the antifungal activity of these three polyphenolic compounds was already well known, the present study reported the antifungal activity of the longan seed extracts for the first time. However, the finding of the susceptibility of these opportunistic yeasts to longan seed extract and its polyphenolic compounds needs further attention. We are also currently investigating anticancer activity and oral toxicity of longan seed extract. In conclusion, the present study therefore strongly suggested the potential use of longan seed extract (waste of longan fruit can production) or its polyphenolic compounds as an antimicrobial agent for oral care products such as mouthwash and effervescent granule for denture cleanser to treat the infection caused by opportunistic fungi and yeasts such as candidosis and cryptococcosis.
Acknowledgments The authors would like to thank Dr. Nopporn Thasana (Laboratory of Medicinal Chemistry) and Mr. Supachai Ritruechai (Laboratory of Pharmacology), Chulabhorn Research Institute, for purification and identification of polyphenolic compounds from longan seed. We also gratefully acknowledge the Faculty of Science, Mahidol University for their financial support for this project. Some results from this work were also the basis for a petty patent in Thailand (no. 5164 granted on December, 14, 2009).
Table 5 Minimum Inhibition Concentration (MIC) of mouthwash containing 0.5% longan seed extract against the tested microorganisms.
Fig. 3. Microscopic pictures of the adherence between Candida albicans (ATCC13803) and acrylic resin after rinsing with (A) granule base; (B) 0.2% chlorhexidine; (C) effervescent longan granules for 60 min. Twenty fields of view were randomly counted in each strip at ×400 magnification.
Products
MIC (S. mutans)
MIC (C. albicans ATCC90028)
MIC (C. albicans ATCC10230)
Base mouthwash Mouthwash 0.5% longan extract Commercial mouthwash
1:8 1:8
1:6 1:8
1:8 1:12
1:6
1:8
1:6
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