Antimicrobial Activities of Diterpene Dialdehydes, Constituents from Myoga (Zingiber Mioga Roscoe), and Their Quantitative Analysis

Biosci. Biotechnol. Biochem., 68 (7), 1601–1604, 2004 Note Antimicrobial Activities of Diterpene Dialdehydes, Constituents from Myoga (Zingiber mioga Roscoe), and Their Quantitative Analysis

y Masako ABE,1 Yoshio OZAWA,1; Yasushi UDA,2 Fuminori YAMADA,3 Yasujiro MORIMITSU,4 Yoshimasa NAKAMURA,5 and Toshihiko OSAWA5

1Department of Health and Nutrition, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan 2Deparment of Bioproductive Sciences, Utsunomiya University, Utsunomiya 321-8505, Japan 3Department of Horticultural Breeding, Gunma Agricultural Technology Center, 493 Azuma-mura, Sawagun 379-2224, Japan 4Laboratory of Food Chemistry, Ochanomizu University, 211 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan 5Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan

Received February 25, 2004; Accepted April 2, 2004

The antimicrobial activities of the three diterpene wrapped with the leaves of the plant are called dialdehydes, miogadial, galanal A and galanal B, myogabochi, a traditional food in Japan. It is believed isolated from flower buds of the myoga (Zingiber mioga that provisions can be preserved for a long time by Roscoe) plant were investigated with some strains of wrapping them with the leaves of the plant. It is, bacteria, yeasts and molds. Among the three com- therefore, worthwhile to confirm whether the flower pounds, miogadial exhibited relatively greater antimi- buds and leaves of the myoga plant have antimicrobial crobial activity than the others against Gram-positive activities. We have demonstrated that galanals A and B bacteria and yeasts. Galanals A and B also behaved as isolated from flower buds of the myoga plant induced antimicrobial agents against Gram-positive bacteria caspase-3-dependent apoptosis in p53-mutated Jurkat and yeasts. The content of miogadial in the flower buds cells.3) With respect to the antimicrobial activity of (E)- was much higher than that in the leaves, whereas 8--(17)-epoxylabd-12-ene-15, 16-dial (miogadial), on- galanals A and B were contained at high levels in the ly a few investigations have been reported.4–6) However, leaves and rhizomes. no report on the antibacterial activities of galanals A and B have been presented. This paper describes the Key words: Zingiber mioga; antimicrobial activity; mio- antimicrobial activities of miogadial, galanal A and gadial (aframodial); galanal A; galanal B galanal B, and the contents of these compounds in different parts of the plant of different varieties. Myoga (Zingiber mioga Roscoe, Zingiberaceae fam- Fresh flower buds of myoga for the antimicrobial test ily) is a perennial herb native to Eastern Asia, and were purchased from a market in Takasaki City. To widely cultivated in Japan. It has a stalk which extends compare the antimicrobial activity of a methanol extract up to 1 m long, with slender leaves reaching 30 cm. The with that of an ethyl acetate extract, sliced flower buds inflorescence (flower bud) of the plant arises from buds of myoga (1 kg) were pulped in 3 liters of methanol or on the rhizome, and flower buds grow 7–10 cm in length ethyl acetate with a blender. After filtration, the filtrate with a shape like a pinecone at the edible stage. The was concentrated under a reduced pressure, and then the flower buds are used as spices or pickles in Japan, concentrate was freeze-dried to use for the antimicrobial because of their pungent and pleasant flavor. It has been assay. Both extracts were assayed for antimicrobial reported that the characteristic pleasant and pungent activity against Bacillus cereus by an agar diffusion flavor of the flower buds was due to such constituents as method.7) The bacterial cells were grown in 1 ml of 2-alkyl-3-methoxypyrazine (alkyl: isobutyl, isopropyl) nutrient broth (Nissui Co.) at 37 C for 7 h while and (E)-8--(17)-epoxylabd-12-ene-15, 16-dial (mioga- shaking. An aliquot (0.2 ml) of the culture was then dial, aframodial), respectively.1,2) We have found that mixed with 2 ml of 0.6% top agar, and the mixture was miogadial played the major role in the pungent taste of layered on soybean-casein-digest agar (Daigo Co.; pH the flower buds.2) In addition, the leaves of the myoga 5.0) plate at 37 C. Each extract (1 mg) dissolved in plant are used as a wrapping material for ‘‘manjyu,’’ a 20 l of methanol or ethyl acetate was dropped on a bun filled with sweetened bean paste. Such manjyu paper disk (6 mm in diameter). After removing the

y To whom correspondence should be addressed. Fax: +81-27-353-2055; E-mail: [email protected] 1602 M. ABE et al. Table 1. MICs of Miogadial, Galanal A and Galanal B

MIC (g/ml) Microorganism Miogadial Galanal A Galanal B Bacteria Bacillus cereus ATCC 10702 25 200 200 Bacillus subtilis ATCC 6633 25 500 300 Staphlococcus aureus ATCC 12600 60 500 500 Staphlococcus aureus ATCC 361 25 500 300 Staphlococcus epidermidis ATCC 14990 25 500 300 Staphlococcus faecalis RIMD 3116001 125 >1000 >1000 Escherichia coli ATCC 27166 >1000 >1000 >1000 Escherichia coli ATCC 25922 >1000 >1000 >1000 Escherichia coli ATCC 11775 >1000 >1000 >1000 Enterobacter cloacae ATCC 13047 >1000 >1000 >1000 Pseudomonas fluorescenes Type 1 >1000 >1000 >1000 Pseudomonas marginalis No. 804 >1000 >1000 >1000 Pseudomonas aeruginosa ATCC 9271 >1000 >1000 >1000 Salmonella typhimurium ATCC 13311 >1000 >1000 >1000 Yeasts Candida albicans NBRC 2091 50 200 200 Candida tropicalis NBRC 66029 50 200 200 Candida glabata NBRC 2001 100 500 500 Zygosaccharomyces rouxii NBRC 28253 50 200 200 Saccharomyces cerevisiae NBRC 2601 50 200 200 Molds Aspergillus fumigatus NBRC 7080 500 >1000 >1000 Penicillium frequentans NBRC 7919 500 >1000 >1000 Penicillium martensii NBRC 8142 >1000 >1000 >1000 Mucor racemosus NBRC 5403 >1000 >1000 >1000 Cladosporium colocasiae NBRC 6698 >1000 >1000 >1000 Eurotium chevalieri NBRC 4090 >1000 >1000 >1000 solvent, the paper disk was placed at the center of the dextrose agar slants (Difco Co.) at 28 C for ten days. agar plate seeded with the cells. After 24 h of incubation Each culture was diluted to 106 CFU/ml with sterile at 37 C, the diameter of the inhibited growth zone was saline. Each of the test compounds was dissolved at measured. The results show that the ethyl acetate extract 1 mg/ml in DMSO, and used after twice diluting with strongly inhibited bacterial growth (growth-inhibition DMSO. One hundred l of the solution was mixed with zone of 8 mm), while the methanol extract showed weak 100 l of each microbial suspension in 3.8 ml of antimicrobial action (growth-inhibition zone of 2 mm). top agar, and the mixtures were layered on the surface It was thus assumed that miogadial played an important of agar plates (90 mm 20 mm). The bacteria were role in the antimicrobial activity, because the ethyl cultured on soybean-casein-digest agar (Daigo CO.; acetate extract of the flower buds contained a consid- pH 5.0) plates at 37 C for 48 h, the yeasts were erable amount of miogadial.2) On the other hand, cultivated on Y-1 agar plates (pH 6.0) at 28 C for methanol can extract miogadial together with such 120 h, and the molds were grown on potato dextrose miogadial-reactive substances as amines and sulfhydryl agar plates (pH 5.0) at 28 C for 120 h. After cultivation, compounds8) in the flower buds, and miogadial should MICs were evaluated by the detection of no colonies be converted into an inactive form during the concen- with the naked eye. tration process. It was noted that the freeze-dried powder MICs of the tested compounds are shown in Table 1. of the methanol extract had no pungent taste. The values for miogadial against the Gram-positive Next, the minimum inhibitory concentrations (MICs) bacteria were 25–125 g/ml. Galanals A and B ex- of miogadial and its structurally related compounds, hibited MICs ranging from 200 to 500 g/ml against the galanals A and B, were measured by using the micro- Gram-positive bacteria, B. cereus, B. subtilis, St. aureus organisms listed in Table 1 by the twofold dilution and St. epidermidis. The antibacterial properties of method with triplicate plates.9) Miogadial (225 mg), galanals A and B were found for the first time in the galanal A (25.6 mg) and galanal B (52.2 mg) were present study. However, MICs of miogadial, galanal A isolated from the ethyl acetate extract of the flower buds and galanal B against the Gram-negative bacteria were by HPLC as reported in our previous paper.2) Their over 1000 g/ml, suggesting that these compounds had structures are shown in Fig. 1. The bacterial cells were little or no activity against the Gram-negative bacteria. cultured in nutrient broth (Nissui Co.) at 37 C for 6 h Similarly, the growth of molds, except for Pen. while shaking. The yeasts were cultivated in Y-1 broth frequentans and Asp. Fumigatus, was little influenced at 28 C for 48 h, while the molds were grown on potato by the three diterpene dialdehydes at a dose level over Antimicrobial Activities of Constituents from Myoga 1603 A and B, however, having the @,-unsaturated dialde- hyde group similar to miogadial, exhibited less anti- microbial activity than miogadial against all of the microorganisms tested. These data suggest that the relative position of the two aldehyde groups was important for strong antimicrobial activity. In addition, it has been found that the introduction of a hydroxy group in sesquiterpene dialdehyde compounds such as warburganal and mukaadial possessing an @,-unsat- urated-1,4-dialdehyde moiety decreased the antimicro- bial activity in comparison with that of polygodial.6) Considering these facts together with the present results, it is suggested that presence of an @,-unsaturated-1,4- dialdehyde moiety and a hydroxy group in the molecule affected the antimicrobial activity of the diterpene dialdehydes. Thus, we assume that the relatively high activity of miogadial in comparison with that of galanals A and B was mainly due to the @,-unsaturated-1,4- dialdehyde moiety. To investigate the quantitative distribution of the antimicrobial diterpene dialdehydes in myoga plants, four varieties (jindawase 1, jindawase 2, jindawase 3 and akimyoga) were analyzed for their contents of mioga- dial, galanal A and galanal B in the leaves, stems, flower buds and rhizomes. Among them, jindawase 1, jinda- wase 2 and jindawase 3 were harvested from an experimental field at Gunma Agricultural Technology Center at the beginning of August in 2002, while the akimyoga variety was harvested from the same field in the middle of September in 2002. The leaves, stems, flower buds and rhizomes were separated, and then Fig. 1. Chemical Structures of Miogadial, and Galanals A and B. 100 g of each was homogenized with a blender and extracted three times with 100 ml each of ethyl acetate. The combined extract was concentrated under reduced 1000 g/ml. In respect of the yeast strains, miogadial, pressure to dryness and then dissolved in 10 ml of ethyl galanal A and galanal B suppressed their growth, acetate. A portion (0.5 ml) of the solution was applied to miogadial inhibiting growth in a dose range of 50– a silica gel cartridge (Waters) that had been precondi- 100 g/ml, and galanals A and B exhibiting MICs in the tioned with n-hexane. After the cartridge had been range of 200–500 g/ml. Miogadial had 4–20 times washed with 2 ml of n-hexane, it was twice eluted with higher activity against the Gram-positive bacteria and 1 ml each of ethyl acetate/n-hexane (1:1, v/v). The yeasts than galanals A and B. These results demonstrate eluate (20 l) was submitted to HPLC by a Shimadzu that the three diterpene dialdehydes behaved as growth LC-10AT instrument equipped with an autosampler and inhibitors for some Gram-positive bacteria and yeasts. a Tosoh silica 60 column (4:5 250 mm). Miogadial The MIC levels obtained in the present study were (tR ¼ 9:7 min), galanal B (tR ¼ 19:1 min), and galanal A 4,5) relatively higher than those from previous works; it is (tR ¼ 26:0 min) were eluted with 7.5% acetone in known that the antimicrobial activities varied with hexane at a flow rate of 2 ml/min, and the peaks were medium components, culture time, and inoculum size monitored at 232 nm. Miogadial, and galanals A and B of the microorganisms.6) were quantified on the basis of their peak areas by From the viewpoint of the structure-activity relation- comparison with calibration curves obtained from the ship, it is interesting to compare the antimicrobial corresponding standards. The analysis of each sample activities between miogadial and galanals A and B. was conducted three times in the same manner. Table 2 Miogadial has a characteristic @,-unsaturated-1,4-di- shows the contents of miogadial, and galanals A and B aldehyde structure like polygodial. On the other hand, in the flower buds, rhizomes, stems and leaves of the galanals A and B have an @,-unsaturated-1,6-dialde- four varieties of myoga plant. Miogadial was mainly hyde structure with a hydroxy group in their molecules. distributed in the flower buds and rhizomes, its content The @,-unsaturated aldehyde moiety in the sesquiter- in the flower buds being 2.3–2.9 times higher than that pene dialdehyde molecules has been found to be in the rhizomes in the three varieties harvested at the responsible for their antimicrobial activity.8) Galanals beginning of August. On the contrary, akimyoga, which 1604 M. ABE et al. Table 2. Contents of Miogadial, Galanal A and Galanal B in Myoga References Plants 1) Sakakibara, H., Yanai, T., Yajima, I., and Hayashi, K., Content (mg/100 g of fresh sample) Cultivar Part Volatile flavor components of myoga (Zingiber mioga). Miogadial Galanal A Galanal B Agric. Biol. Chem., 55, 1655–1657 (1991). jindawase 1 2) Abe, M., Ozawa, Y., Uda, Y., Yamada, Y., Morimitsu, flower bud 24.7 3.4 1.7 Y., Nakamura, Y., and Osawa, T., Labdane-type diter- rhizome 9.8 9.9 5.1 pene dialdehydes, pungent principle of myoga, Zingiber stem 0.2 0.5 0.1 mioga Roscoe. Biosci. Biotechnol. Biochem., 66, 2698– leaf 0.1 10.7 4.4 2700 (2002). jindawase 2 3) Miyoshi, N., Nakamura, Y., Ueda, Y., Abe, M., Ozawa, flower bud 21.5 4.0 2.4 Y., Uchida, K., and Osawa, T., Dietary ginger constit- rhizome 9.5 8.2 3.4 uents, galanals A and B, are potent apoptosis inducers in stem 0.7 2.0 0.5 human T lymphoma jurkat cells. Cancer Lett., 199, 113– leaf 3.0 7.1 3.2 jindawase 3 119 (2003). flower bud 27.3 4.9 2.7 4) Morita, H., and Itokawa, H., Cytotoxic and antifungal rhizome 9.3 10.0 4.1 diterpene from the seeds of Alpinia galanga. Planta stem 1.3 4.2 1.2 Med., 54, 117–120 (1988). leaf 1.8 6.8 2.8 5) Haraguchi, H., Kuwata, Y., Inada, K., Shingu, K., akimyoga Miyahara, K., Nagao, M., and Yagi, A., Antifungal flower bud 8.5 8.6 4.2 activity from Alpinia galanga and the competition for rhizome 17.3 10.9 6.6 incorporation of unsaturated fatty acids in cell growth. stem 0.2 0.1 0.1 Planta Med., 62, 308–313 (1996). leaf 1.9 15.4 6.2 6) Kubo, I., Fujita, K., and Lee, S. H., Antifungal mechanism of polygodial. J. Agric. Food Chem., 49, 1607–1611 (2001). had been harvested in the middle of September, 7) Uda, Y., Matsuoka, H., Shima, H., Kumagami, H., and contained twice the amount of miogadial in the rhizomes Maeda, Y., Antimicrobial activity of water-soluble than that in the flower buds. The leaves and stems all products derived from radish mustard oil and identifica- had small amounts of miogadial. On the other hand, tion of an active component therein. Nippon Shokuhin large amounts of galanal A and B were found in the Kogyo Gakkaishi, 40, 801–806 (1993). rhizomes and leaves. The contents of galanal A and B 8) Taniguchi, M., Adachi, T., Oi, S., Kimura, A., and Kubo, detected in the leaves of akimyoga were higher than I., Structure-activity relationship of the Warburgia those in the leaves of the other varieties. In addition, the sesquiterpene dialdehydes. Agric. Biol. Chem., 48, 73– leaves of the myoga plant contained 2.0–10.0 times 78 (1984). more galanal A than miogadial. These data suggest that 9) Matsuoka, H., Takita, J., Kawakami, S., Takahashi, A., the leaves of the myoga plant acted as an antimicrobial Ozawa, Y., and Uda, Y., Antimicrobial actions of !- alkenyl and !-methylthioalkyl isothiocyanates. J. Anti- material for food wrapping. It was confirmed that the bact. Antifung. Agents (in Japanese), 27, 81–87 (1999). contents of miogadial, galanal A and galanal B varied 10) Ficker, C. E., Smith, M. L., Susiarti, S., Leaman, D. J., with variety and part of the myoga plant. In conclusion, Irawati, C¸ ., and Arnason, J. T., Inhibition of human the varieties belonging to the Zingiberaceae family are pathogenic fungi by members of Zingiberaceae used by generally regarded as safe for human consumption;10) it the Kenyan (Indonesia Borneo). J. Ethnopharmacol., 85, is thus considered that the myoga plant is a suitable herb, 289–293 (2003). spice and wrapping material for food.