key: cord-0005515-yy6ig6q7
authors: Lee, Hyang Burm; Kim, Jin Cheol; Lee, Sang Myung
title: Antibacterial activity of two phloroglucinols, flavaspidic acids AB and PB, from Dryopteris crassirhizoma
date: 2009-05-27
journal: Arch Pharm Res
DOI: 10.1007/s12272-009-1502-9
sha: a3eeaa271941b86bb7865a44add8d480ec3b2c9f
doc_id: 5515
cord_uid: yy6ig6q7

The antimicrobial effect of solvent extracts from the rhizome of a thick-stemmed wood fern (Dryopteris crassirhizoma) was evaluated and its phloroglucinol components, flavaspidic acids PB and AB. Flavaspidic acids PB and AB were isolated from the D. crassirhizoma rhizomes by methanol extraction, followed by silica gel and Sephadex LH-20 column chromatography. The chemical structures were characterized by spectral techniques, including ESI-MS, UV, (1)H- and (13)C-NMR spectrum analysis. When the antimicrobial activity of the extracts and compounds was tested by the paper disc method, the extracts and compounds were highly active against Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus KCTC 1928 (a MRSA bacterium), Streptococcus mutans and Bacillus subtilis. The extracts and compounds were not active against fungi and chlorella. Our study revealed that the antibacterial activity of samples from D. crassirhizoma was mainly related to the flavaspidic acids.

The thick-stemmed wood fern (Dryopteris crassirhizoma Nakai, Dryopteridaceae) is a semi-evergreen plant that grows on the deciduous forest floor as a pteridophyte. Two ferns, Dryopteris crassirhizoma and Osmunda japonica, are commonly used as antiinfection agents, especially for the common cold and flu, and are frequently collectively referred to as the fern (Dharmananda, 2003) . Recently, the fern has also been used as the major plant with six Chinese herbs (Astragalus, Atractylodes, Red Atractylodes, Pogostemon, Adenophora, Lonicera), a combination that was recommended as a prescription formula to prevent SARS. Its rhizomes have also been used in a vermicide (Namba, 1993) . In the search for natural products with antimicrobial activity, methanol extracts of the D. crassirhizoma rhizome exhibited antimicrobial activity against some bacteria. Phloroglucinols (albaspidin, aspidin, flavaspidic acids and dryocrassin) and kaempferol acetyl rhamnosides (crassirhizomosides AC and sutchuenoside A) have been isolated from D. crassirhizoma (Noro et al., 1973; Widen et al., 1996; Min et al., 2001) . Recently, acylphloroglucinols isolated from D. crassirhizoma were reported to inhibit fatty acid synthase (Na et al., 2006) . In addition, the compound showed a cytoprotective effect against oxidative stress-induced cell damage via catalase activation (Kang et al., 2006) . The phloroglucinol composition of 18 species (including subspecies) that belong to Dryopteris Adanson sect. Fibrillosae Ching has been investigated on a world-wide basis (Widen et al.,, 1996) . Phloroglucinols were observed to have anti-tumorpromoting activity (Govind et al., 1996) , nitric oxide inhibitory effect (Rie et al., 2001) , anti-reverse transcriptase activity (Hideo et al., 1991) and antioxidant activity (Lee et al., 2003) . Since Namba (1982) (Do, 1993) . Antimicrobial activity of phloroglucinols was once reported by Abbey et al. (2000) . However, these phloroglucinols were acylated phloroglucinols from Helichrysum caespititium. The objectives of this study were to evaluate the antimicrobial potential of rhizome extracts and flavaspidic acids PB and AB from D. crassirhizoma against Gram-positive and -negative bacteria, fungi and chlorella.

Rhizome of Dryopteris crassirhizoma was collected in Mt. Sulak, Korea in July 2002 and identified by Prof. Bae, College of Pharmacy, Chungnam National University. A voucher specimen (CNU 1011) was deposited in the herbarium of the College of Pharmacy, Chungnam National University.

As shown in Fig. 1 , the dried rhizomes (1 kg) of Dryopteris crassirhizoma were extracted with methanol (3 L, 48 h ×2) at room temperature, and the extract was concentrated to dryness in vacuo to yield a dark brown syrupy residue (150 g). The methanol extract (150 g) was suspended in H 2 O (1 L) and then partitioned successively with hexane (1 L × 2), ethyl acetate (1 L × 2), and BuOH (1 L × 2). The dry ethyl acet-ate extract (80 g) was subjected to column chromatography on silica gel (70-230 mesh, Merck, Germany). A step gradient was used for elution; each step constituted a 10% increase in acetone (in 1 L volumes) with hexane (10% acetone in 1 L volume), up to 80% acetone. Eleven 1-L fractions were collected. These fractions were tested by in vitro antimicrobial assays, and the active fractions (fr. 5, 6) were combined. Using methanol as a solvent, the active fraction (8 g) was subjected to column chromatography on Sephadex LH-20 to afford two active compounds: flavaspidic acid PB (1, 300 mg) and flavaspidic acid AB (2, 150 mg), which were characterized by spectral methods (Noro et al., 1973; Do, 1993) .

Flavaspidic acid PB (1) 

Melting point was measured on an Electrothermal instrument (Dubuque, IA, USA). UV spectra were obtained on a Milton Roy 3000 spectrometer (Ivyland, PA, USA). 1 H-and 13 C-NMR spectra were recorded on a DRX 300 MHz (Bruker, Karlsruhe, Germany) with CDCl 3 as a solvent. ESI-MS spectra were measured on JMS 700 mass spectrometer (JEOL, Tokyo, Japan).

The antimicrobial activity of the crude extracts and purified materials (flavaspidic acids AB and PB) was tested by the paper disc method. All samples were dissolved in trace ethanol. Sterile filter paper discs (Whatman No. 1, 8 mm diameter) were impregnated with 200 µg of each sample (50 µL, 4 mg mL -1 ) per paper disc and dried under the laminar flow cabinet KCTC 1940 , and Chlorella regularis EML-CR02. The medium for Bacillus subtilis and Staphylococcus aureus was Nutrient agar (pH 7.0) with 5 g peptone, 3 g meat extract and 15 g agar per liter. The medium for Candida albicans was YMPG agar with 3 g yeast extract, 3 g malt extract, 5 g soybean peptone, 10 g glucose and 15 g agar per liter. The medium for Aspergillus flavus was YpSs agar consisting of 4 g yeast extract, 15 g soluble starch, 1 g K 2 HPO 4 , 0.5 g MgSO 4 × 7 H 2 O and 15 g agar per liter. The medium for Chlorella regularis was Arnon's A5 medium (pH 6.5) consisting of 1 mL Arnon's A5 solution, 1 g KH 2 PO 4 , 1 g MgSO 4 ·7H 2 O, 0.005 g FeSO 4 ·7H 2 O, 5 g yeast extract, 20 g glucose and 20 g agar per liter. All assays were performed in duplicate, so that four inhibition zone measurements were obtained for each test combination. These values were averaged to obtain the final inhibitory activity results. For each assay, two control plates were inoculated with ethanol, but without actual extracts, and were treated in the same manner as the test plates. , and δ C 8.1 were indicative of C-11' and C-11, respectively. δ H 1.40 [6H, s] and δ C 24.7 indicated gem-dimethyl at C-7, 8. In addition, δ H 2.05 [3H, s], δ C 7.4 and δ H 3.55 [2H, s] were indicative of C-11' and C-12. Thus, the structure of 1 was determined to be flavaspidic acid PB (Fig. 2) . This was confirmed by comparison of the physiochemical and spectral data with published data (Noro et al., 1973) .

Flavaspidic acid AB (2) corresponded to gem-dimethyl at C-7, 8. δ H 1.87 [3H, s] and δ C 7.9 indicated an aromatic methyl (C-7'). The spectral data mentioned above were similar to flavaspidic acid PB (1), but the secondary methyl (C-10) signals in 1 did not appear in 2. Thus, the structure of 2 was determined to be flavaspidic acid AB (Fig. 2) . This was confirmed by comparison of the physiochemical and spectral data comparison with published data (Noro et al., 1973; Do, 1993) .

The methanol and ethyl acetate extracts from the Dryopteris crassirhizoma rhizome were highly active against bacteria. As shown in Table I on kinds of microorganisms tested (data not shown). Interestingly, the flavaspidic acids were considerably more active against the MRSA bacterium, Staphylococcus aureus KCTC 1228, than against Staphylococcus aureus KCTC 1916. Also, flavaspidic acid PB was somewhat more active against Bacillus subtilis than flavaspidic acid AB. However, both compounds were moderately to slightly active against a Gramnegative bacterium, E. coli, and were not active against a fungus, Aspergillus flavus or an alga, Chlorella regularis.

This study reports the antibacterial activity of plant-derived phloroglucinols. We found that the ethylacetate fraction of the Dryopteris crassirhizoma rhizome exhibited antimicrobial activity and yielded two phloroglucinols, flavaspidic acid PB and flavaspidic acid AB. The identity of the compounds was first confirmed through interpretation of their spectral characters in comparison with reported data (Noro et al., 1973) . Do (1993) once reported that flavaspidic acids PB and AB had an MIC value of 12.5 µg per ml toward Streptococcus mutans OMZ 176. However, detailed antimicrobial activities against other bacteria were not investigated.

Recently, MRSA bacteria have become more resistant to vancomycin antibiotics. Interestingly, the flavaspidic acids were highly active against Grampositive and MRSA bacteria including Staphylococcus aureus, but not against fungi. Our study revealed that thick-stemmed wood fern extracts may be applied to development of natural functional products with antibacterial activity. More studies on the antibacterial spectrum, the susceptibility of various bacteria to the compounds, and the mode of action are now under way.

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This work was supported in part by a grant (20070301034004) from BioGreen21 program, Rural Development Administration, Republic of Korea.