Wasabi(Wasabia japonica), a member of the Brassicaceae, which is native to Japan is found growing in cool temperature climate. Its growth habitat requires shade, cold, running water year round. In Korea, wasabi plants are produced at cold mountain stream of Chuncheon, Cheorwon and Pyeongchang in Gan...
Wasabi(Wasabia japonica), a member of the Brassicaceae, which is native to Japan is found growing in cool temperature climate. Its growth habitat requires shade, cold, running water year round. In Korea, wasabi plants are produced at cold mountain stream of Chuncheon, Cheorwon and Pyeongchang in Gangwon province for the purpose of rhizome production, and at highland of Muju in Joellabukdo for the purpose of processed goods. However, the production area, production time and yield has decreased due to the sensitivity to growth environment. Currently, demand and market value for wasabi have risen. Wasabi contains a lot of biochemically active compounds, mainly isothiocyanate family. It is known to exert antibacterial and antifungal effect, and promoting effect on vitamin B1 biosynthesis, as well as biological potential like antioxidative. They could be applied to medicinal and pharmaceutical industry. In the present study, we have applied a plant tissue culture method to investigate suitable cultures in vitro producing useful chemical like sinigrin. We also developed a simultaneous determination of sinigrin and allyl isothiocyanate using ion-pair liquid chromatography. Finally antimicrobial activity from various tissues of Wasabi was examined with various microbes. 1. Tissue culture of Wasabi(Wasabia japonica) Explants of leaf blade, leaf petiole from greenhouse grown plants and from seedlings derived from seed in vitro were cultured on modified MS basal medium supplemented with auxins and cytokinins. Small pieces of leaf petiole from seedlings in vitro showed higher percentage of callus formation (95.9%) on MS liquid medium supplemented with 3% sucrose and 0.1 mg/L picloram plus 0.1 mg/L kinetin after 30 days of shake culture. Induction of adventitious roots was observed well on liquid shake culture for 5 weeks from roots at 1 cm in size of seedling. IBA is more effective than NAA for adventitious root culture. The optimal growth of adventitious roots was observed on MS medium supplemented with 3% sucrose and 4.0 mg/L NAA. Liquid medium for 2 weeks followed by semi-solid medium for 4 weeks containing 1.0 mg/L BA was the best for the induction of multi shoots to number of shoots (22.8) and shoot length (3.5 cm) as well as active growth of shoots. Shoot proliferated could be divided into ca 5 to 11 of cultures for the multiplication of plantlets. The growth of seedlings from shoots could be manipulated by the concentration ratio of NO₃^(-) and NH₄^(+) (10:50 mM) in the culture medium. 2. Simultaneous determination of sinigrin and allyl isothiocyanate in Wasabi(Wasabia japonica) using ion-pair liquid chromatography A reversed-phase ion-pair liquid chromatography was investigated for simultaneous determination of the glucosinolate sinigrin and its major breakdown product allylisothiocyanate (AITC) in wasabi. Both intact sinigrin and AITC were extracted by hot acetonitrile/water mixture while no sinigrin was hydrolyzed to AITC, and the extract was directly subjected to HPLC analysis. A reversed-phase octadecylsilyl column was used to separate sinigrin and AITC with the mobile phase of acetonitrile/20 mM phosphate buffer (40/60, v/v) containing 5 mM tetraheptylammonium as the counter ion at pH 6.2. The limit of determination based on 3% full-scale deflection was 30 and 45 mg/kg sample for sinigrin and AITC, respectively. Recoveries from fortified samples (n=9) ranged 89.3-97.8% and 92.5-98.6% for each analyte, and RSDs were all less than 5%. An alternative HPLC condition was also developed to positively confirm the sinigrin and AITC. 3. Sinigrin contents in different tissues of Wasabi and antimicrobial activity of water extracts Total sinigrin contents in rhizome, leaf petiole and leaf blade of wasabi (Wasabia japonica) were determined by simultaneous measurement of sinigrin and allyl isothiocyanate using HPLC. The antimicrobial activity of water extracts of the different tissues were also examined against Staphylococcus aureus, Streptococcus agalatiae, Listeria monocytogenes, Escherichia coli and Streptococcus mutans. The contents of total sinigrin including ally isothiocyanate were 15.22, 1.85, 1.95 mg·g^(-1) in rhizome, leaf petiole and leaf blade of wasabi, respectively. The contents were much higher than those found by other researchers, who determined sinigrin and isothiocyanates by measuring content of allyl isothiocyanate after conversion of sinigrin to allyl isothiocyanate. During the conversion, loss of the volatile compound iosthiocyanates and incomplete conversion of sinigrin to allyl isothiocynate might be possible, and these factors may be related to the differences in the total sinigrin measurements. Water extracts of wasabi rhizome, leaf petiole and leaf blade showed anti-microbial activity against Staphylococcus aureus, Streptococcus agalatiae, Listeria monocytogenes and Escherichia coli, and the highest activity was found in the rhizome extract, where the total sinigrin content was much higher than those found in leaf petiole and leaf blade. The main component related to antimicrobial activity in wasabi is known to be allyl isothiocyanate. Streptococcus mutans, whose growth was not affected by the treatment of wasabi extract, seems to be more resistant to isothiocyanates than other microbes. Although the contents of sinigrin and isothiocyanates in leaf petiole and leaf blade of wasabi are relatively lower than that in rhizome, the biomass of leaf petiole and leaf blade are greater than that of rhizome and also can be continuously harvested from the same root and rhizome system. Therefore, the aboveground parts of wasabi can be used in the extraction of sinigrin and isothiocyanates as commercial antimicrobial compounds.
Wasabi(Wasabia japonica), a member of the Brassicaceae, which is native to Japan is found growing in cool temperature climate. Its growth habitat requires shade, cold, running water year round. In Korea, wasabi plants are produced at cold mountain stream of Chuncheon, Cheorwon and Pyeongchang in Gangwon province for the purpose of rhizome production, and at highland of Muju in Joellabukdo for the purpose of processed goods. However, the production area, production time and yield has decreased due to the sensitivity to growth environment. Currently, demand and market value for wasabi have risen. Wasabi contains a lot of biochemically active compounds, mainly isothiocyanate family. It is known to exert antibacterial and antifungal effect, and promoting effect on vitamin B1 biosynthesis, as well as biological potential like antioxidative. They could be applied to medicinal and pharmaceutical industry. In the present study, we have applied a plant tissue culture method to investigate suitable cultures in vitro producing useful chemical like sinigrin. We also developed a simultaneous determination of sinigrin and allyl isothiocyanate using ion-pair liquid chromatography. Finally antimicrobial activity from various tissues of Wasabi was examined with various microbes. 1. Tissue culture of Wasabi(Wasabia japonica) Explants of leaf blade, leaf petiole from greenhouse grown plants and from seedlings derived from seed in vitro were cultured on modified MS basal medium supplemented with auxins and cytokinins. Small pieces of leaf petiole from seedlings in vitro showed higher percentage of callus formation (95.9%) on MS liquid medium supplemented with 3% sucrose and 0.1 mg/L picloram plus 0.1 mg/L kinetin after 30 days of shake culture. Induction of adventitious roots was observed well on liquid shake culture for 5 weeks from roots at 1 cm in size of seedling. IBA is more effective than NAA for adventitious root culture. The optimal growth of adventitious roots was observed on MS medium supplemented with 3% sucrose and 4.0 mg/L NAA. Liquid medium for 2 weeks followed by semi-solid medium for 4 weeks containing 1.0 mg/L BA was the best for the induction of multi shoots to number of shoots (22.8) and shoot length (3.5 cm) as well as active growth of shoots. Shoot proliferated could be divided into ca 5 to 11 of cultures for the multiplication of plantlets. The growth of seedlings from shoots could be manipulated by the concentration ratio of NO₃^(-) and NH₄^(+) (10:50 mM) in the culture medium. 2. Simultaneous determination of sinigrin and allyl isothiocyanate in Wasabi(Wasabia japonica) using ion-pair liquid chromatography A reversed-phase ion-pair liquid chromatography was investigated for simultaneous determination of the glucosinolate sinigrin and its major breakdown product allylisothiocyanate (AITC) in wasabi. Both intact sinigrin and AITC were extracted by hot acetonitrile/water mixture while no sinigrin was hydrolyzed to AITC, and the extract was directly subjected to HPLC analysis. A reversed-phase octadecylsilyl column was used to separate sinigrin and AITC with the mobile phase of acetonitrile/20 mM phosphate buffer (40/60, v/v) containing 5 mM tetraheptylammonium as the counter ion at pH 6.2. The limit of determination based on 3% full-scale deflection was 30 and 45 mg/kg sample for sinigrin and AITC, respectively. Recoveries from fortified samples (n=9) ranged 89.3-97.8% and 92.5-98.6% for each analyte, and RSDs were all less than 5%. An alternative HPLC condition was also developed to positively confirm the sinigrin and AITC. 3. Sinigrin contents in different tissues of Wasabi and antimicrobial activity of water extracts Total sinigrin contents in rhizome, leaf petiole and leaf blade of wasabi (Wasabia japonica) were determined by simultaneous measurement of sinigrin and allyl isothiocyanate using HPLC. The antimicrobial activity of water extracts of the different tissues were also examined against Staphylococcus aureus, Streptococcus agalatiae, Listeria monocytogenes, Escherichia coli and Streptococcus mutans. The contents of total sinigrin including ally isothiocyanate were 15.22, 1.85, 1.95 mg·g^(-1) in rhizome, leaf petiole and leaf blade of wasabi, respectively. The contents were much higher than those found by other researchers, who determined sinigrin and isothiocyanates by measuring content of allyl isothiocyanate after conversion of sinigrin to allyl isothiocyanate. During the conversion, loss of the volatile compound iosthiocyanates and incomplete conversion of sinigrin to allyl isothiocynate might be possible, and these factors may be related to the differences in the total sinigrin measurements. Water extracts of wasabi rhizome, leaf petiole and leaf blade showed anti-microbial activity against Staphylococcus aureus, Streptococcus agalatiae, Listeria monocytogenes and Escherichia coli, and the highest activity was found in the rhizome extract, where the total sinigrin content was much higher than those found in leaf petiole and leaf blade. The main component related to antimicrobial activity in wasabi is known to be allyl isothiocyanate. Streptococcus mutans, whose growth was not affected by the treatment of wasabi extract, seems to be more resistant to isothiocyanates than other microbes. Although the contents of sinigrin and isothiocyanates in leaf petiole and leaf blade of wasabi are relatively lower than that in rhizome, the biomass of leaf petiole and leaf blade are greater than that of rhizome and also can be continuously harvested from the same root and rhizome system. Therefore, the aboveground parts of wasabi can be used in the extraction of sinigrin and isothiocyanates as commercial antimicrobial compounds.
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