Antimicrobial activities of some Thai traditional medical longevity formulations from plants and antibacterial compounds from Ficus foveolata

Abstract

Context: Medicinal plants involved in traditional Thai longevity formulations are potential sources of antimicrobial compounds.

Objective: To evaluate the antimicrobial activities of some extracts from medicinal plants used in traditional Thai longevity formulations against some oral pathogens, including Streptococcus pyogenes, Streptococcus mitis, Streptococcus mutans, and Candida albicans. An extract that possessed the strongest antimicrobial activity was fractionated to isolate and identify the active compounds.

Materials and methods: Methanol and ethyl acetate extracts of 25 medicinal plants used as Thai longevity formulations were evaluated for their antimicrobial activity using disc diffusion (5 mg/disc) and broth microdilution (1.2–2500 µg/mL) methods. The ethyl acetate extract of Ficus foveolata Wall. (Moraceae) stems that exhibited the strongest antibacterial activity was fractionated to isolate the active compounds by an antibacterial assay-guided isolation process.

Results and discussion: The ethyl acetate extract of F. foveolata showed the strongest antibacterial activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 19.5–39.0 and 39.0–156.2 µg/mL, respectively. On the basis of an antibacterial assay-guided isolation, seven antibacterial compounds, including 2,6-dimethoxy-1,4-benzoquinone (1), syringaldehyde (2), sinapaldehyde (3), coniferaldehyde (4), 3β-hydroxystigmast-5-en-7-one (5), umbelliferone (6), and scopoletin (7), were purified. Among these isolated compounds, 2,6-dimethoxy-1,4-benzoquinone (1) exhibited the strongest antibacterial activities against S. pyogenes, S. mitis, and S. mutans with MIC values of 7.8, 7.8, and 15.6 µg/mL, and MBC values of 7.8, 7.8, and 31.2 µg/mL, respectively. In addition, this is the first report of these antibacterial compounds in the stems of F. foveolata.

• Benzoquinone
• coniferaldehyde
• scopoletin
• sinapaldehyde
• syringaldehyde
• umbelliferone

Introduction

Nowadays, the incidence of multidrug-resistant microorganisms to commonly used antibiotics has become a major global healthcare problem that has been worsened by a steady decrease in the number of new antibiotics (Alanis, 2005; Shriram et al., 2008). This has necessitated and encouraged a search for new antimicrobial substances hopefully with new targets for their inhibitory activities from other sources that include plants.

For thousands of years, natural products have been a source of traditional herbal medicine for the treatment of various human diseases in many parts of the world (Devi et al., 2011). These natural products derived from medicinal plants have proven to be an abundant source of biologically active compounds. Some of these compounds have been found in vitro to have antimicrobial activities (Lewis & Ausubel, 2006; Palombo, 2011; Sibanda & Okoh, 2007). Thus, traditional medicine systems around the world that utilize medicinal plants are an important resource for the discovery of new antimicrobial agents (Okpekon et al., 2004).

In Thailand, traditional healers have used medicinal plants in Thai longevity formulations for the treatment of various ailments. Thai people believe that these formulations can exert health promotion activities and have tonic effects, i.e., make you feel better (Kraithep et al., 2008). Recently, there have been some reports on the antimicrobial activity of medicinal plants used in Thai longevity formulations. For examples, the extracts obtained from the aerial parts of Cyperus rotundus L. (Cyperaceae) exhibited antimicrobial activity against a number of bacterial strains including Staphylococcus aureus, Enterococcus faecalis, Salmonella enteritidis, and S. typhimurium (Kilani-Jaziri et al., 2011). In addition, crude extracts of Caesalpinia sappan L. (Leguminosae) showed antimicrobial activity against S. typhi, S. faecalis, Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, S. aureus, Aspergillus niger, and C. albicans (Srinivasan et al., 2012). However, only a few medicinal plants used in Thai longevity formulations have been studied for their antimicrobial activity.

In this study, the antimicrobial activities of extracts from 25 medicinal plants used in Thai longevity formulations were investigated against some oral pathogens, including S. pyogenes, S. mitis, S. mutans, and C. albicans. In addition, in this report we have, for the first time, identified seven antibacterial compounds from the stems of F. foveolata.

Materials and methods

Plant materials

Twenty-five plants (Table 1) were collected from Nakhon Sri Thammarat Province, Thailand, in June 2011. The plants were identified by Associate Professor Pharkphoom Panichayupakaranant and deposited at the Herbarium of the Southern Center of Traditional Medicine, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. These plant materials were dried at 50 °C for 24 h in a hot air oven and reduced to powders using a grinder, and the powders were passed through sieve no. 45.

Table 1. Plant materials used in this study.

Family	Botanical name	Voucher number	Part used
Asclepiadaceae	Cryptolepis buchanani Roem. & Schult.	SKP 019 03 02 01	Vine
Betulaceae	Betula alnoides Buch.-Ham.	SKP 024 02 01 01	Bark
Compositae	Blumea balsamifera (L.) DC.	SKP 051 02 02 01	Leaves
Compositae	Spilanthes acmella L. Murr.	SKP 051 19 01 01	Leaves
Cyperaceae	Cyperus rotundus L.	SKP 060 03 18 01	Tubers
Ebenaceae	Diospyros rhodocalyx Kurz.	SKP 067 04 18 01	Bark
Gentianaceae	Fagraea fragrans Roxb.	SKP 077 06 20 01	Leaves
Leguminosae	Senna timoriensis (DC.) Irwin & Barneby	SKP 098 19 20 01	Heartwood
Leguminosae	Senna garrettiana (Craib) Irwin & Barneby	SKP 098 19 07 01	Heartwood
Leguminosae	Albizia procera (Roxb) Benth.	SKP 098 01 16 01	Bark
Leguminosae	Derris scandens (Roxb) Benth.	SKP 098 04 19 01	Vine
Leguminosae	Caesalpinia sappan L.	SKP 098 03 19 01	Heartwood
Leguminosae	Bauhinia strychnifolia Craib.	SKP 098 02 19 01	Vine
Menispermaceae	Stephania venosa (Blume) Spreng.	SKP 114 19 22 01	Tubers
Menispermaceae	Stephania suberosa Forman.	SKP 114 19 19 01	Tubers
Menispermaceae	Anamirta cocculus (L.) Wight & Arn.	SKP 114 01 03 01	Heartwood
Menispermaceae	Stephania pierrei Diels.	SKP 114 19 16 01	Tubers
Moraceae	Ficus foveolata Wall. ex Miq.	SKP 117 06 06 01	Stems
Moraceae	Artocarpus heterophyllus Lam.	SKP 117 01 08 01	Heartwood
Moraceae	Maclura cochinchinensis (Lour.) Corner.	SKP 117 13 03 01	Heartwood
Moraceae	Ficus chartacea Wall.	SKP 117 06 03 01	Heartwood
Opiliaceae	Lepionurus sylvestris Blume.	SKP 131 12 19 01	Stems
Piperaceae	Piper chaba Hunt.	SKP 146 16 03 01	Fruits
Piperaceae	Piper ribesoides Wall.	SKP 146 16 18 01	Vine
Simaroubaceae	Harrisonia perforata (Blanco) Merr.	SKP 178 08 16 01	Stems

Preparations of plant extracts

Dried plant powders were extracted twice with ethyl acetate under reflux conditions for 1 h. The extracts were combined and concentrated under reduced pressure to produce the ethyl acetate crude extract. The marcs were subsequently extracted with methanol and concentrated under the same conditions to produce the methanol extracts.

Microorganisms and media

Streptococcus pyogenes (DMST 17020), S. mutans (DMST 26095), S. mitis (ATCC 49456T), and C. albicans (TISTR 5779 and ATCC 90028) were from the Department of Medical Sciences, Thailand. Brain–heart infusion (BHI) and saboraud dextrose agar (SDA) were from the Becton, Dickinson Company (New York, NY).

Antimicrobial susceptibility testing

These experiments were performed by the disc diffusion method (NCCLS, 2008) with some modifications. Streptococcus pyogenes, S. mutans, and S. mitis were incubated on BHI agar at 37 °C under microaerophilic conditions for 24 h, C. albicans (TISTR 5779 and ATCC 90028) were incubated on SDA at 37 °C for 48 h. Inocula were prepared by mixing a few bacteria colonies with sterile ringer solution (0.85% NaCl) the volume was adjusted to yield approximately 10⁸ CFU/mL using the turbidity of the McFarland no. 0.5 standard as a reference. The prepared inoculum was streaked onto the surface of BHI or SDA with a cotton swab. A sterile paper disc (6 mm diameter) was impregnated with 10 µL of plant extract (500 mg/mL) and the disc was placed on the agar. The concentration of each plant extract was 5 mg/disc. DMSO was used as a negative control, whilst standard ampicillin and clotrimazole (10 µg/disc) were used as positive controls. Plates were then incubated under the most suitable conditions for each microbe. All disc diffusion tests were performed in triplicate and the antibacterial activity was expressed as the mean of their diameter of inhibition zones (mm).

Determination of the minimum inhibitory and bactericidal or fungicidal concentrations (MIC and MBC)

The MIC and MBC or MFC were determined by the broth microdilution assay (NCCLS, 2008). Cultures of microaerophilic bacteria and yeasts were 24 h and 48 h broth cultures, respectively. The MIC value was defined as the lowest concentration of the compound to inhibit the growth of microorganisms, and the MBC or MFC was defined as the lowest concentration of the compound required to kill the microorganisms.

Bioassay-guided isolation

The dried stem powder of F. foveolata (16 kg) was extracted with ethyl acetate (20 L × 5) under reflux conditions for 1 h, to obtain (after solvent evaporation) a dark-brown extract (82 g). The ethyl acetate extract (80 g) was subjected to silica gel vacuum chromatography. The extract was pre-adsorbed on silica gel, and applied to the top of the silica gel column (13 cm in diameter and 6 cm in height), and the column was subsequently eluted with 500 mL of solvent with the aid of a vacuum pump. The column was eluted using a step-gradient elution starting from the mixture of hexane and ethyl acetate (100, 50, and 20% v/v hexane) followed by the mixture of ethyl acetate and methanol (100, 90, and 50% v/v ethyl acetate). Based on the TLC chromatograms of each fraction (500 mL), seven pooled fractions (fractions 1–7) were obtained. The fractions were then subjected to an antibacterial assay. The antibacterial fraction (fraction 4; 16.8 g) was further fractionated by a silica gel column (7.5 × 60 cm) (1 g extract per 35 g silica gel). Mixtures of hexane and ethyl acetate (100, 90, 70, 50, 30, and 10% v/v hexane) and mixtures of ethyl acetate and methanol (100, 90, 80, 70, and 50% v/v ethyl acetate) were used for column elution, using a step-gradient elution to afford six pooled fractions (fractions 4.1–4.6). The fractions were then subjected to an antibacterial assay. The fractions 4.2 and 4.3 showed antibacterial activity.

The antibacterial fraction 4.2 (1 g) was further purified by a Sephadex LH-20 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (3 × 80 cm) using a mixture of chloroform and methanol (50% v/v chloroform) as an eluent to give four pooled fractions (fractions I–IV). The antibacterial fraction III (334 mg) was rechromatographed with the same Sephadex LH-20 column to produce three pooled fractions (fractions A–C). The antibacterial fraction B (285 mg) was further purified by a silica gel column (2.5 × 15 cm) using a mixture of hexane and chloroform (70% v/v hexane) as an eluent to produce three pooled fractions (fractions B1–B3). Compound 1 (17.2 mg) was obtained from the fraction B1 after recrystallization in a solution of hexane and methanol. The fraction B2 was rechromatographed several times on a silica gel column (2.5 × 12 cm) eluted with hexane and ethyl acetate (80% v/v hexane) to afford compounds 2 (26.2 mg), 3 (9.1 mg), and 4 (2.0 mg).

The antibacterial fraction 4.3 (1.2 g) was further fractionated by a silica gel column (7.5 × 50 cm) using a mixture of hexane and chloroform (50% v/v hexane) as an eluent to produce three pooled fractions (fractions I–III). The antibacterial fraction I (691 mg) was further purified by a Sephadex LH-20 column using a mixture of chloroform and methanol (50% v/v chloroform) as an eluent to give four pooled fractions (fractions A–D). The antibacterial fraction B (232 mg) was further purified by the silica gel column (2.5 × 12 cm) using a mixture of hexane and ethyl acetate (90%v/v hexane) as an eluent to afford three pooled fractions (fractions B1–B3). The antibacterial fraction B2 (108 mg) was further purified by the silica gel column (2.5 × 12 cm) eluted with hexane and ethyl acetate (80% v/v hexane) to produce compound 5 (91.1 mg). The antibacterial fraction D (130 mg) was purified by the Sephadex LH-20 column using a mixture of chloroform and methanol (50% v/v chloroform) as an eluent to produce two pooled fractions (fractions D1–D2). The antibacterial fraction D2 (103 mg) was purified by the silica gel column eluted with hexane and chloroform (80% v/v hexane) to afford compounds 6 (2.9 mg) and 7 (42.9 mg).

Identification of compounds 1–7

Compounds 1–7 were identified by ¹H NMR, ¹³C NMR, and EIMS by comparing with data in the literature (Kwon et al., 2001; Lee et al., 2010; Lim et al., 2005; Liu et al., 2008; Luoa et al., 2009; Wang et al., 2011; Zhao et al., 2005).

2,6-Dimethoxy-1,4-benzoquinone (1): yellow crystals; ¹H NMR (CD₃OD, 500 MHz) δ: 5.93 (2H, s, H-3, H-5), 3.82 (6H, s, OCH₃-2, OCH₃-6); ¹³C NMR (CD₃OD, 125 MHz) δ: 177.7 (C-1), 159.2 (C-2, C-6), 107.9 (C-3, C-5), 189.2 (C-4), 57.0 (OCH₃-2, OCH₃-6); EI-MS m/z (rel. int.%): 169 (8), 153 (5), 138 (25), 125 (17), 97 (14), 80 (32), 69 (100).

Syringaldehyde (2): yellow amorphous powders; ¹H NMR (CD₃OD, 500 MHz) δ: 7.22 (2H, s, H-2, H-6), 3.90 (6H, s, OCH₃-3, OCH₃-5), 9.75 (1H, s, CHO); ¹³C NMR (CD₃OD, 125 MHz) δ: 129.2 (C-1), 108.4 (C-2, C-6), 149.6 (C-3, C-5), 192.9 (CHO), 56.9 (OCH₃-3, OCH₃-5); EI-MS m/z (rel. int.%): 182 (94), 167 (11), 153 (5), 139 (13), 123 (8), 111 (38), 96 (46), 79 (61), 65 (100).

Sinapaldehyde (3): a yellow oil; ¹H NMR (CD₃OD, 500 MHz) δ: 6.98 (2H, s, H-2, H-6), 7.58 (1H, d, J = 15.5 Hz, H-1′), 6.66 (1H, dd, J = 15.5, 8.0, H-2′), 9.58 (1H, d, J = 8.0 Hz, H-3′), 3.88 (6H, s, OCH₃-3, OCH₃-5); ¹³C NMR (CD₃OD, 125 MHz) δ: 126.5 (C-1), 107.7 (C-2), 149.6 (C-3), 140.3 (C-4), 149.6 (C-5), 107.7 (C-6), 156.3 (C-1′), 127.1 (C-2′), 196.0 (C-3′), 56.9 (OCH₃); EI-MS m/z (rel. int. %): 208 (100), 180 (28), 165 (35), 137 (20).

Coniferaldehyde (4): a yellow oil; ¹H NMR (CD₃OD, 500 MHz) δ: 7.25 (1H, d, J = 2.0 Hz, H-2), 6.86 (1H, d, J = 8.5 Hz, H-5), 7.16 (1H, dd, J = 8.5, 2.0 Hz, H-6), 7.59 (1H, d, J = 16.0 Hz, H-1′), 7.65 (1H, dd, J = 16.0, 8.0 Hz, H-2′), 9.58 (1H, d, J = 8.0 Hz, H-3′), 3.90 (3H, s, OCH₃-3); ¹³C NMR (CD₃OD, 125 MHz) δ: 126.7 (C-1), 112.3 (C-2), 151.7 (C-3), 149.5 (C-4), 116.7 (C-5), 125.1 (C-6), 156.2 (C-1′), 127.6 (C-2′), 196.1 (C-3′), 56.5 (OCH₃); EI-MS m/z (rel. int.%): 178 (100), 161 (18), 147 (34), 135 (44), 107 (35).

3β-Hydroxystigmast-5-en-7-one (5): white needles; ¹H NMR (CD₃OD, 500 MHz) δ: 5.64 (1H, d, J = 1.5 Hz, H-6), 3.55 (1H, m, H-3), 0.71 (3H, s, H-18), 1.22 (3H, s, H-19), 1.04 (3H, d, J = 7.0 Hz, H-21), 0.87 (3H, d, J = 7.5 Hz, H-26), 0.81 (3H, d, J = 7.0 Hz, H-27), 0.94 (3H, t, J = 6 Hz, H-29). ¹³C NMR (CD₃OD, 125 MHz) δ: 37.59 (C-1), 31.92 (C-2), 71.20 (C-3), 42.76 (C-4), 169.03 (C-5), 126.29 (C-6), 204.61 (C-7), 46.61 (C-8), 52.76 (C-9), 39.67 (C-10), 22.27 (C-11), 39.96 (C-12), 44.27 (C-13), 51.43 (C-14), 26.51 (C-15), 29.52 (C-16), 56.15 (C-17), 12.37 (C-18), 17.69 (C-19), 37.34 (C-20), 19.45 (C-21), 35.17 (C-22), 27.43 (C-23), 47.29 (C-24), 30.75 (C-25), 21.52 (C-26), 20.18 (C-27), 24.18 (C-28), 12.64 (C-29). EI-MS m/z (rel. int.%): 428 [M]⁺ (100), 414 (58), 395 (19), 383 (20), 365 (8).

Umbelliferone (6): white crystals; (CD₃OD, 500 MHz) δ: 6.18 (1H, d, J = 9.5 Hz, H-3), 7.85 (1H, d, J = 9.5 Hz, H-4), 7.45 (1H, d, J = 8.5 Hz, H-5), 6.79 (1H, dd, J = 8.5, 2.0 Hz, H-6), 6.70 (1H, d, J = 2.0 Hz, H-8); ¹³C NMR (CD₃OD, 125 MHz) δ: 163.6 (C-2), 103.4 (C-3), 146.0 (C-4), 130.6 (C-5), 114.5 (C-6), 157.2 (C-7), 112.4 (C-8), 163.2 (C-9), 114.5 (C-10); EI-MS m/z (rel. int.%): 162 (100), 134 (81), 105 (17), 78 (35).

Scopoletin (7): white needles; (CD₃OD, 500 MHz) δ: 6.20 (1H, d, J = 9.5 Hz, H-3), 7.85 (1H, d, J = 9.5 Hz, H-4), 7.10 (1H, s, H-5), 6.76 (1H, s, H-8), 3.9 (3H, s, OCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ: 164.0 (C-2), 112.7 (C-3), 146.0 (C-4), 110.0 (C-5), 147.0 (C-6), 151.4 (C-7), 104.0 (C-8), 152.9 (C-9), 112.5 (C-10), 56.9 (OCH₃); EI-MS m/z (rel. int.%): 192 [M]⁺ (100), 177 (86), 164 (31), 149 (46), 121 (45).

Results and discussions

Among the 50 extracts of the 25 medicinal plants involved in traditional longevity formulations that were investigated for antimicrobial activity against the oral pathogens, 33 plant extracts including the ethyl acetate extracts of Cryptolepis buchanani Roem. & Schult. (Asclepiadaceae), Betula alnoides Buch.-Ham. (Betulaceae), Cyperus rotundus L. (Cyperaceae), Blumea balsamifera (L.) DC. (Compositae), Fagraea fragrans Roxb (Gentianaceae), Senna garrettiana (Craib) Irwin & Barneby (Leguminosae), Derris scandens (Roxb) Benth. (Leguminosae), Caesalpinia sappan L. (Leguminosae), Bauhinia strychnifolia Craib. (Leguminosae), Stephania venosa (Blume) Spreng. (Menispermaceae), Stephania suberosa Forman. (Menispermaceae), Anamirta cocculus (L.) Wight & Arn. (Menispermaceae), Stephania pierrei Diels. (Menispermaceae), Ficus foveolata Wall. ex Miq. (Moraceae), Artocarpus heterophyllus Lam. (Moraceae), Maclura cochinchinensis (Lour.) Corner. (Moraceae), Lepionurus sylvestris Blume. (Opiliaceae), Piper ribesoides Wall. (Piperaceae), Harrisonia perforata (Blanco) Merr. (Simaroubaceae), and the methanol extracts of B. alnoides, C. rotundus, B. balsamifera, S. garrettiana, D. scandens, C. sappan, S. venosa, S. suberosa, S. pierrei, F. foveolata, A. heterophyllus, M. cochinchinensis, L. sylvestris, and P. ribesoides inhibited the growth of one or more tested pathogens at the concentration of 5 mg/disc. Only the ethyl acetate and methanol extracts of S. pierrei exhibited inhibitory effect against all tested pathogens. Six medicinal plants involved in traditional longevity formulations, including Spilanthes acmella L. Murr. (Compositae), Diospyros rhodocalyx Kurz. (Ebenaceae), Senna timoriensis (DC.) Irwin & Barneby (Leguminosae), Albizia procera (Roxb) Benth. (Leguminosae), Ficus chartacea Wall. (Moraceae), and Piper chaba Hunt. (Piperaceae) showed no inhibitory activity against the tested pathogenic microbes.

The extracts that possessed inhibitory effects (inhibition zones > 10 mm) were tested for their MIC and MBC by the broth microdilution method. The MICs and MBCs or MFCs of the plant extracts are shown in Tables 2 and 3. Only the ethyl acetate extracts of S. pierrei exhibited satisfactory antimicrobial activity against all the tested microbes. However, the ethyl acetate extract of F. foveolata exhibited the highest antibacterial activity against S. pyogenes, S. mitis, and S. mutans with MICs of 19.5, 19.5, and 39.0 µg/mL, and MBCs of 39.0, 78.1, and 156.2 µg/mL, respectively, followed by the methanol extract of C. sappan that had MICs of 39.0, 39.0, and 78.1 µg/mL, and MBCs of 78.1, 78.1, and 156.2 µg/mL. Recently, there have also been two reports of potent and selective butyrylcholinesterase inhibitors (Sermboonpaisarn & Sawasdee, 2012), and cytotoxic compounds against cancer cell lines (Somwong et al., 2013) obtained from F. foveolata stems. This is the first report of the antibacterial activity of a F. foveolata stem extract. The ethyl acetate extract of F. foveolata was, therefore, subjected to isolation of antibacterial compounds using the antibacterial assay-guided isolation as described.

Table 2. Antimicrobial activity of the plant extracts and standard antibiotics.

Plants	Minimum inhibitory concentrations (µg/mL)
	Ethyl acetate extracts					Methanol extracts
	Smu	Smi	Spy	Ca5	Ca9	Smu	Smi	Spy	Ca5	Ca9
A. heterophyllus	312.5	19.5	19.5	–	–	312.5	312.5	312.5	–	–
F. foveolata	39.0	19.5	19.5	–	–	1250	–	1250	–	–
C. sappan	156.2	39.0	78.1	–	–	78.1	39.0	39.0	–	–
S. pierrei	1250	19.5	78.1	2500	2500	>2500	2500	>2500	>2500	>2500
F. fragrans	–	39.5	156.2	–	–	–	–	–	–	–
M. cochinchinensis	312.5	78.1	156.2	–	–	312.5	78.1	156.2	–	–
S. garrettiana	312.5	78.1	156.2	–	–	312.5	156.2	312.5	–	–
B. balsamifera	–	156.2	1250	–	–	78.1	625.0	156.2	–	–
B. strychnifolia	–	–	1250	–	–	–	–	–	–	–
L. silvestris	–	19.5	–	–	–	–	78.1	625.0	–	–
S. suberosa	–	2500	–	–	–	1250	625	625.0	–	–
D. scandens	–	–	–	–	–	>2500	2500	>2500	–	–
H. perforata	>2500	2500	–	–	–	–	–	–	–	–
S. venosa	–	156.2	–	–	–	–	156.2	–	–	–
P. ribesoides	–	–	–	2500	2500	–	–	–	>2500	>2500
Ampicillin	0.1	0.1	0.1	–	–	0.1	0.1	0.1	–	–
Clotrimazole	–	–	–	2.0	2.0	–	–	–	2.0	2.0

Smu, S. mutans (DMST 26095); Smi, S. mitis (ATCC 49456T); Spy, S. pyogenes (DMST 17020); Ca5, C. albicans (TISTR5779); Ca9, C. albicans (ATCC 90028); –, not performed. Data are mean values (n = 3).

Table 3. Minimum bactericidal or fungicidal concentrations of the plant extracts and standard antibiotics.

Plants	Minimum bactericidal or fungicidal concentrations (µg/mL)
	Ethyl acetate extracts					Methanol extracts
	Smu	Smi	Spy	Ca5	Ca9	Smu	Smi	Spy	Ca5	Ca9
A. heterophyllus	312.5	78.1	39.0	–	–	312.5	312.5	312.5	–	–
F. foveolata	156.2	39.0	78.1	–	–	>2500	–	>2500	–	–
C. sappan	156.2	312.5	78.1	–	–	156.2	78.1	78.1	–	–
S. pierrei	>2500	19.5	78.1	2500	2500	>2500	2500	>2500	>2500	>2500
F. fragrans	–	78.1	156.2	–	–	–	–	–	–	–
M. cochinchinensis	1250	312.5	156.2	–	–	1250	156.2	312.5	–	–
S. garrettiana	625.0	312.5	312.5	–	–	625.0	625.0	312.5	–	–
B. balsamifera	–	156.2	1250	–	–	78.1	625.0	156.2	–	–
B. strychnifolia	–	–	2500	–	–	–	–	–	–	–
L. silvestris	–	19.5	–	–	–	–	156.2	625.0	–	–
S. suberosa	–	2500	–	–	–	1250	625.0	1250	–	–
D. scandens	–	–	–	–	–	>2500	2500	>2500	–	–
H. perforata	>2500	2500	–	–	–	–	–	–	–	–
S. venosa	–	625.0	–	–	–	–	>2500	–	–	–
P. ribesoides	–	–	–	>2500	2500	–	–	–	>2500	>2500
Ampicillin	0.1	0.1	0.1	–	–	0.1	0.1	0.1	–	–
Clotrimazole	–	–	–	4.0	2.0	–	–	–	4.0	2.0

–, not performed. Data are mean values (n = 3).

Seven known compounds: 2,6-dimethoxy-1,4-benzoquinone (1), syringaldehyde (2), sinapaldehyde (3), coniferaldehyde (4), 3β-hydroxystigmast-5-en-7-one (5), umbelliferone (6), and scopoletin (7) were identified (Figure 1). All these isolated compounds possessed antibacterial activities. This is the first report of these compounds in F. foveolata. The antibacterial activities of these compounds against the oral pathogenic bacteria are shown in Table 4. The 2,6-dimethoxy-1,4-benzoquinone exhibited the strongest antibacterial activities against S. pyogenes, S. mitis, and S. mutans with MIC values of 7.8, 7.8, and 15.6 µg/mL, and MBC values of 7.8, 7.8, and 31.2 µg/mL, respectively, while the others showed medium to weak antibacterial activity. It was previously reported that 2,6-dimethoxy-1,4-benzoquinone isolated from the wheat germ extract exhibited strong antibacterial activity against Gram-positive bacteria, including S. aureus, Bacillus cereus, and Gram-negative bacteria including S. typhimurium and E. coli (Kim et al., 2010). In addition, this compound also exhibited cytotoxic effect in Ehrlich ascites tumor cells and thereby inhibited tumor propagation (Pethig et al., 1983, 1985).

Figure 1. Chemical structures of the compounds isolated from F. foveolata.

Table 4. Antibacterial activity of the isolated compounds and a standard antibiotic.

	S. pyogenes		S. mitis		S. mutans
Compounds	MICs µg/mL (µM)	MBCs µg/mL (µM)	MICs µg/mL (µM)	MBCs µg/mL (µM)	MICs µg/mL (µM)	MBCs µg/mL (µM)
1	7.8 (46)	7.8 (46)	7.8 (46)	7.8 (46)	15.6 (93)	31.2 (186)
2	62.5 (343)	62.5 (343)	125 (687)	250 (1374)	250 (1374)	250 (1374)
3	31.2 (150)	31.2 (150)	125 (601)	125 (601)	125 (601)	125 (601)
4	62.5 (351)	62.5 (351)	62.5 (351)	125 (702)	250 (1405)	250 (1405)
5	125 (292)	125 (292)	n.a.	–	125 (292)	125 (292)
6	15.6 (96)	15.6 (96)	250 (1543)	250 (1543)	250 (1543)	250 (1543)
7	125 (651)	125 (651)	n.a.	–	n.a.	–
Ampicillin	0.1 (0.3)	0.1 (0.3)	0.1 (0.3)	0.1 (0.3)	0.1 (0.3)	0.1 (0.3)

2,6-Dimethoxy-1,4-benzoquinone (1), syringaldehyde (2), sinapaldehyde (3), coniferaldehyde (4), 3β-hydroxystigmast-5-en-7-one (5), umbelliferone (6), scopoletin (7). n.a., no activity; –, not perform.

Our findings indicated that the stems of F. foveolata, a medicinal plant used in Thai longevity formulations, may be involved in health promotion by protecting against some GI infections. In addition, 2,6-dimethoxy-1,4-benzoquinone may be used as an indicative marker for the quality control of F. foveolata stem extracts as well as a lead compound for the development of anti-infectious drugs.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the article.

Acknowledgements

The authors wish to thank the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission for support in the form of a research grant. Also thanks to Dr. Brian Hodgson for assistance with the English.