Biosci. Biotechnol. Biochem., 74 (4), 878–880, 2010
Note
Inhibitory Activity against Urease of Quercetin Glycosides Isolated from Allium cepa and Psidium guajava Samah S HABANA, Azusa K AWAI, Kenji K AI, Kohki A KIYAMA, and Hideo H AYASHIy Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuenchou, Naka-ku, Sakai, Osaka 599-8531, Japan Received December 4, 2009; Accepted January 7, 2010; Online Publication, April 7, 2010 [doi:10.1271/bbb.90895]
Methanolic extracts of edible plants and seaweeds were tested for their inhibitory activity against Jack bean urease. Quercetin-40 -O- -D-glucopyranoside was isolated from Allium cepa as a urease inhibitor with an IC50 value of 190 M. Quercetin and two quercetin glycosides, avicularin and guaijaverin, were isolated from Psidium guajava as urease inhibitors with respective IC50 values of 80 M, 140 M, and 120 M. Key words:
urease inhibitor; quercetin; flavonoid; quercetin glycoside; edible plant
A variety of ureases are found in bacteria, fungi, higher plants, and in soil as soil enzymes.1) Bacterial ureases are medically important virulence factors implicated in the pathogenesis of many clinical conditions such as pyelonephritis, hepatic coma, peptic ulceration, and the formation of infection-induced urinary stones.2) High urease activity is responsible for significant environmental and economic problems in agriculture by causing the release of an abnormally large amount of ammonia into the atmosphere during urea fertilization; this induces plant damage, primarily by depriving plants of their essential nutrients and secondarily through ammonia toxicity, which increases the pH level of the soil.3) Urease inhibitors have recently attracted strong attention as potential new antiulcer drugs, and many urease inhibitors such as fluorofamide, hydroxyureas, and hydroxamic acids have been described in the last few decades; however, the in vivo use of some of these has been prohibited because of their toxicity or instability, for instance, acetohydroxamic acid has been demonstrated to be teratogenic in rats.4) Certain synthetic compounds have shown potential urease inhibition,3) while a diterpene ester with a myrsinol-type skeleton has been isolated from Euphorbia decipiens as the first naturally occurring urease inhibitor.5) More attention has been focused on exploring the novel biological properties of phytochemicals isolated from edible plants. Catechins in a green tea extract have been reported to strongly inhibit Helicobacter pylori urease.6) Another H. pylori urease inhibitor has been isolated from Rubus coreanus Miquel, and characterized as a proteinous substance.7) Edible plants may therefore be very useful resources for safe urease control. We screened in this study edible plants and plant-derived foodstuffs for their urease inhibitory activity, and isolated quercetin and quercetin y
glycosides from red onion peel and guava leaves as urease inhibitors. The plant-derived foodstuffs were soaked in methanol, and the methanol extract from each was adjusted to a concentration of 10 mg/ml and subjected to a Jack bean urease inhibition assay. Jack bean urease has been widely used as an enzyme for screening inhibitors. The edible parts of mung bean (Vigna radiata), pineapple (Ananas comosus), cinnamon (Cinnamomum zeylanicum) and guava (Psidium guajava), and the peel from avocado (Persea americana) showed high activity against Jack bean urease in the range of 70–100%. The edible parts of apple (Malus domestica), red onion (Allium cepa), pepper (Capsicum annuum), black gram (Vigna mungo), licorice (Glycyrrhiza glabra) and cummin (Cuminum cyminum), the peel from sudachi (Citrus sudachi), lemon (Citrus limon) and water melon (Citrullus lanatus), and coffee (Coffea arabica) beans showed moderate activity in the range of 30–70%. Such seaweeds as wakame (Undaria pinnatifida) and hitoegusa (Monostroma nitidum) exhibited only slight inhibition. The active constituents of A. cepa and P. guajava were investigated in this study. Freshly collected peel from red onion (A. cepa) was soaked in methanol, and the aqueous concentrate obtained was partitioned with ethyl acetate. The active ethyl acetate extract was purified in a silica gel C-200 column with various solvents to afford active compound 1. Compound 1 was identified as quercetin-40 -O--Dglucopyranoside by comparing its MS, 1 H-NMR, and 13 C-NMR data with those previously reported.8,9) Dried guava (P. guajava) leaves were soaked in methanol, and the aqueous concentrate obtained was partitioned with ethyl acetate. Repeated chromatography of the active ethyl acetate extract in silica gel and Sephadex LH-20 columns led to the isolation of compounds 2, 3, and 4. Compounds 2, 3, and 4 were respectively identified as quercetin-3-O--L-arabinofuranoside (avicularin),10,11) quercetin-3-O--L-arabinopyranoside (guaijaverin),10,11) and quercetin12) by comparing their MS, 1 H-NMR, and 13 C-NMR data with those previously reported. The inhibitory activities of quercetin and its glycosides, together with isoquercitrin (quercetin-3-O--Dglucoside, 5) and quercitrin (quercetin-3-O--L-rhamnoside, 6), against Jack bean and Lactobacillus fermentum ureases are summarized in Table 1. L. fermentum urease was used as a comparison because this enzyme exhibits activity in the same pH range as H. pylori urease.
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Urease Inhibitors from Allium cepa and Psidium guajava
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OH O HO
O
OH
OH
OH HO
O
HO
OH
O O HO
OH OH
OH
OH
O
O
OH O
OH HO
OH OH HO
OH
O
O
OH
O
OH OH
OH
O
O
3
4 OH
HO
O
OH O HO
OH
OH
2
1
HO
OH
O
OH HO
O
OH
OH OH
O
OH
O
O HO OH
OH
O
O
5
6
OH
Fig. 1. Chemical Structures of Compounds 1–6.
Table 1. Inhibitory Activity of Quercetin and Its Glycosides against Jack Bean and Lactobusillus fermentum Ureases
Compounds
Quercetin-40 -O--D-glucoside Avicularin Guaijaverin Quercetin Isoquercitrin Quercitrin Acetohydroxamic acid
(1) (2) (3) (4) (5) (6)
Jack bean urease IC50
L. fermentum urease Inhibition (%)a
190 mM 140 mM 120 mM 80 mM 160 mM 200 mM 20 mM
53 48 17 12 —b — 70
a The inhibitory activity was evaluated using inhibitors at a concentration of 100 mM. b — indicates that the inhibitory activity is less than 10%.
Quercetin (4) showed the strongest inhibitory activity against Jack bean urease with an IC50 value of 80 mM, while it showed slight activity against L. fermentum urease. Glycosidation of the 3-hydroxy and 40 -hydroxy groups decreased the inhibitory activity against Jack bean urease. Avicularin (2) and guaijaverin (3) were slightly more active than the other glycosides. In the case of L. fermentum urease, quercetin-40 -O--D-glucoside (1) and avicularin (2) showed inhibitory activity at a concentration of 100 mM, while the other compounds showed little or no activity, suggesting that the inhibitory activity of quercetin glycosides depended on the site of glycosidation and location of sugar molecules. Compounds 1, 2, 3, and 4 (quercetin) were respectively isolated from A. cepa,8) Picea abies9) species, the apple pomace extract11) and P. guajava,10,13) the activity of quercetin (4) having already been reported.14) In addition to quercetin, kampferol, apigenin, luteolin, and baicalein have also been reported to exhibit inhibitory activity against urease,14) indicating that the 5- and 7-hydroxy groups in the A-ring of flavone played an important role
in exhibiting this activity. The inhibitory activity of compounds 1–3 against urease is reported for the first time in this study. Many synthetic polyphenols based on isoflavones have shown inhibitory activity against H. pylori.4) Datisdirin, a flavone isolated from Datisca cannabina, has been reported to show inhibitory activity against Jack bean urease with an IC50 value of 83.9 mM.15) Apple peel extracts containing rutin (quercetin-3-Orutinoside), hyperoside (quercetin-3-O-galactoside), isoquercitrin (quercetin-3-O--D-glucoside), two quercetin3-O-pentosides, and quercitrin have recently been shown to exhibit inhibitory activity against H. pylori urease, although the active constituents were not identified.16) It has recently been reported that quercetin inhibited melanogenesis by melanoma cells, and that this inhibition was due to the inhibition of both tyrosinase activity and protein expression.17) Our findings in this study seem to reveal further diversity of the physiological functions exhibited by quercetin and its glycosides. Experimental Jack bean urease was purchased from Toyobo (Japan) and Lactobacillus fermentum urease was kindly provided by Nagase ChemteX Corporation (Japan). Quercetin-3--D-glucoside, quercitrin hydrate, and acetohydroxamic acid were respectively obtained from Sigma Aldrich (Germany), Sigma Aldrich (India), and ACROS Organics (USA). Plant material. P. guajava leaves were collected at a farm in Qalyubia (North Cairo, Egypt) in April 2008. The fresh leaves were washed and carefully dried in the sun. Fresh A. cepa bulbs were purchased at a vegetable shop in Sakai (Osaka, Japan) in August 2007. Extraction and isolation of the active compounds. The fresh bulbs of red onion (A. cepa; 2 kg) were soaked in methanol (7 liters). The methanol extract was evaporated in vacuo, and the aqueous concentrate obtained was partitioned with ethyl acetate to afford an active ethyl acetate extract (700 mg). This extract was subjected to successive chromatography on silica gel C-200 (eluted by 10% stepwise ethyl acetate-methanol), silica gel G-60 (toluene-ethyl acetate), and Sephadex LH-20 (90% aqueous methanol) to yield active compound 1 (18 mg).
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The dried guava leaves (P. guajava; 2 kg) were soaked in methanol (10 liters). The methanol extract was evaporated in vacuo, and the aqueous concentrate obtained was partitioned with ethyl acetate to afford an active ethyl acetate extract (7 g). This extract was subjected to chromatography on silica gel C-200 (eluted by 10% stepwise n-hexane-ethyl acetate-methanol) to obtain 20–40% ethyl acetate eluates and an 80% methanol eluate. The 80% methanol eluate (250 mg) was subjected to successive chromatography on silica gel G-60 (n-hexane-ethyl acetate-methanol (2.5:2:1, v/v)), ODS (watermethanol), and silica gel G-60 (ethyl acetate-methanol) to give active compounds 2 (8 mg) and 3 (3 mg). The 20–40% EtOAc eluates (430 mg) were successively purified by using silica gel G-60 (n-hexane-acetone), silica gel G-60 (n-hexane-ethyl acetate), and ODS (water-methanol) to give active compound 4 (10 mg). Assay for urease inhibition. Reaction mixtures comprising 0.2 ml (0.47 U) of a Jack bean urease solution and 1.2 ml of buffer of a 60 mM MES buffer at pH 6.0 were incubated for 5 min with 0.1 ml of a sample solution at 37 C in test tubes. After a preincubation, 0.5 ml (0.05 mM) of urea was added to the reaction mixture, and the whole incubated for 20 min. Urease activity was determined by measuring the ammonia produced with the indophenol method described by Weatherburn.18) Briefly, 1 ml each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and an alkaline reagent (1% w/v NaOH and 0.075% active chloride NaOCl) were added to each test tube. The increase in absorbance at 640 nm was measured after 30 min, all reactions being performed in triplicate in a final volume of 4 ml. Percentage inhibition was calculated by using the formula 100 ðODsample =ODcontrol Þ 100. Acetoxyhydroxamic acid was used as the standard inhibitor of urease. In the case of L. fermentum urease, the enzyme dissolved in a 100 mM acetate buffer at pH 4.0 was used.
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