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Table 2
References and notes
Contents of compounds 1, 2, 3, and 4 in kenaf leaf
extract
1. (a) Gupta, A. K.; Gover, M. D.; Nouri, K. N.; Taylor, S. J. Am. Acad. Dermatol. 2006,
55, 1048; (b) Dalton, S. R.; Gardner, T. L.; Libow, L. F.; Elston, D. M. J. Am. Acad.
Dermatol. 2005, 52, 859.
Compounds
Contents (mg/g)
2. Marmol, V. D.; Beermann, F. FEBS Lett. 1996, 381, 165.
3. Agbor, G. A.; Oben, J. E.; Nkegoum, B.; Takala, J. P.; Ngogang, J. Y. Pak. J. Biol. Sci.
2005, 8, 1397.
4. Agbor, G. A.; Oben, J. E.; Ngogang, J. Y.; Xinxing, C.; Vinson, J. A. J. Agric. Food
Chem. 2005, 53, 6819.
5. Agbor, G. A.; Oben, J. E.; Ngogang, J. Y. Afr. J. Biotechnol. 2005, 4, 833.
6. Lee, Y. G.; Byeon, S. E.; Kim, J. Y.; Lee, J. Y.; Rhee, M. H.; Hong, S.; Wu, J. C.; Lee, H.
S.; Kim, M. J.; Cho, D. H.; Cho, J. Y. J. Ethnopharmacol. 2007, 113, 62.
7. (a) Eom, S. H.; Park, H. J.; Seo, D. W.; Kim, W. W.; Cho, D. H. Food Sci. Biotechnol.
2009, 18, 362; (b) Lee, S.-C.; Jeong, S.-M.; Kim, S.-Y.; Nam, K. C.; Ahn, D. U. J.
Agric. Food Chem. 2005, 53, 1495; (c) Lee, S.-C.; Kim, S.-Y.; Jeong, S.-M.; Park, J.-
H. J. Agric. Food Chem. 2006, 54, 399.
Non-FIR
FIR
3.1
15.2
2.4
1 (kaempferitrin)
2 (kaempferol)
3 (afzelin)
29.3
—
—
4 (a-rhamnoisorobin)
—
5.7
—: not detected.
Table 3
Anti-tyrosinase activities of compounds 2, 3, and 4
8. (a) Preparation of kenaf leaf extract: kenaf (Hibiscus cannabinus L.) leaf
cultivated in the reclaimed land in Chuncheon, S. Korea, was harvested in the
second week of Sept. 2009. The leaves were sun dried and grinded to powder
by a grinder machine in the laboratory. The powdered leaf sample (50 g) was
suspended in 1 L of 70% ethanol (v/v) and kept overnight in a shaker at room
temperature. The extracts were filtered through Advantec 5B Tokyo Roshi
a
Compounds
IC50
1 (kaempferitrin)
2 (kaempferol)
3 (afzelin)
>400 lM
171.4 lM
>400
>400
l
l
M
Kaisha Ltd, Japan. The ethanol extract was dried using
a vacuum rotary
4 (a-rhamnoisorobin)
M (39.1%)b
evaporator (EYLA N-1000, Tokyo, Japan) in a 40 °C water bath. Dried samples
were weighed and kept at 4 °C for further analysis. (b) Preparation of FIR-
treated kenaf leaf extract: FIR was applied to powdered leaf sample (2 g
dissolve in 10 ml water and acetic acid, pH 3.3 each in a Glass Petri disc,
100 ꢀ 20 mm) in an FIR radiation chamber (Korea Energy Co., Seoul, Korea)
a
Values were determined from the logarithmic
concentration–inhibition curves and are given as the
mean values of the results of three experiments.
b
Inhibitory activity (% of control) at 400 lM.
emitting wavelengths of 3–1000
hour each emitting 8.4
was extracted with EtOH and the solvent was evaporated to obtain powder.
9. Measurement of mushroom tyrosinase: mushroom tyrosinase, -tyrosine were
purchased from Sigma Chemical. The reaction mixture for mushroom
tyrosinase activity consisted of 150 l of 0.1 M phosphate buffer (pH 6.5),
l of sample solution, 8 l of mushroom tyrosinase (2100 unit/ml, 0.05 M
phosphate buffer at pH 6.5), and 36 l of 1.5 mM -tyrosine. Tyrosinase activity
l
m. The sample was exposed at 60 °C for an
l
m wavelengths. After FIR treatment, kenaf powder
From HPLC results, we hypothesize that tyrosinase inhibitory
activity may have originated from kaempferol11b (major product)
and other minor products (afzelin 3 and a-rhamnoisorobin 4) after
the FIR treatment of kenaf leaf extract. In order to confirm our
hypothesis, anti-tyrosinase activities of deglycosylation products
(compounds 2, 3, and 4) were evaluated. The results are summa-
rized in Table 3. Specifically, compound 2 (kaempferol) exhibited
L
l
3
l
l
l
L
was determined by reading the optical density at 490 nm on a microplate
reader (Bio-Rad 3550, Richmond, CA, USA) after incubation for 20 min at 37 °C.
The inhibitory activity of the sample was expressed as the concentration that
inhibits 50% of the enzyme activity (IC50).
10. Compound 1: 1H NMR (500 MHz, CD3OD) d 7.80 (d, 2H, J = 9.5 Hz), 6.95 (d, 2H,
J = 9.5 Hz), 6.72 (s, 1H), 6.46 (s, 1H), 5.56 (s, 1H), 5.40 (s, 1H), 4.20 (s, 1H), 4.00
(s, 1H), 3.83–3.81 (m, 1H), 3.71–3.70 (m, 1H), 3.60–3.57 (m, 1H), 3.48 (t, 1H,
J = 9.2 Hz), 3.34–3.32 (m, 2H), 1.27 (d, 3H, J = 6.5 Hz), 0.93 (d, 3H, d, J = 6.5 Hz).
13C NMR (125 MHz, CD3OD) d 179.9, 163.5, 163.0, 161.8, 159.8, 158.1, 136.5,
131.9, 122.4, 116.6, 107.6, 103.5, 100.6, 99.9, 95.6, 73.6, 73.2, 72.2, 72.1, 72.0,
71.9, 71.7, 71.3, 18.0, 17.6. FABMS: (m/e) 577 [MꢁH]+. (a) Urgaonkar, S.; Shaw,
J. T. J. Org. Chem. 2007, 72, 4582. (b) Tzeng, Y.-M.; Chen, K.; Rao, Y. K.; Lee, M.-J.
Eur. J. Pharmacol. 2009, 607, 27.
inhibitory activities with IC50 values of 171.4
pound 3 showed no inhibitory activity. The weak inhibitory activ-
ity was shown in compound 4 ( -rhamnoisorobin). Compound 4
showed 39.1% inhibition of tyrosinase at 400 M concentration.
lM. However, com-
a
l
All these data suggested that treatment with FIR irradiation
might serve as not only an efficient deglycosylation technique
but also a potent tool for enhancing the biological activities of nat-
ural extracts.
In conclusion, the present investigation reported for the first
time deglycosylation of kaempferol glycoside by FIR irradiation.
FIR-treated kenaf leaf extract was more active than kenaf leaf ex-
tract against tyrosinase because derhamnosylation of kaempferi-
trin occurred to give kaempferol and other minor products
11. (a) Park, J. S.; Rho, H. S.; Kim, D. H.; Chang, I. S. J. Agric. Food Chem. 2006, 54,
2951; (b) Kubo, I.; Kinst-Hori, I.; Chaudhuri, S. K.; Kubo, Y.; Sanchez, Y.; Ogura,
T. Bioorg. Med. Chem. 2000, 8, 1749.
12. Compound 3: 1H NMR (500 MHz, CD3OD) d 7.77 (d, 2H, J = 8.4 Hz), 6.94 (d, 2H,
J = 8.4 Hz), 6.38 (s, 1H), 6.20 (s, 1H), 5.38 (s, 1H), 4.22 (s, 1H), 3.75 (m, 1H),
3.39–3.31 (m, 2H), 0.94 (d, 3H, d, J = 6.5 Hz). 13C NMR (125 MHz, CD3OD) d
179.6, 165.9, 163.2, 161.6, 159.3, 158.7, 136.2, 131.9, 122.7, 116.5, 106.0, 103.5,
99.8, 94.8, 73.2, 72.2, 72.0, 71.9, 17.6. FABMS: (m/e) 431 [MꢁH]+. (a) Xu, G.-H.;
Ryoo, I.-J.; Kim, Y.-H.; Choo, S.-J.; Yoo, I.-D. Arch. Pharm. Res. 2009, 32, 275. (b)
Lee, S.-Y.; Min, B.-S.; Kim, J.-H.; Lee, J.; Kim, T.-J.; Kim, C.-S.; Kim, Y.-H.; Lee, H.-
K. Phytother. Res. 2005, 19, 273.
(afzelin and a-rhamnoisorobin). Further studies on deglycosylation
of other natural glycosides are underway in our laboratory.
13. Compound 4: 1H NMR (500 MHz, CD3OD) d 8.02 (d, 2H, J = 8.4 Hz), 6.82 (d, 2H,
J = 8.4 Hz), 6.64 (s, 1H), 6.32 (s, 1H), 5.45 (s, 1H), 3.92 (s, 1H), 3.72 (m, 1H),
3.40–3.50 (m, 1H), 3.35–3.42 (m, 1H), 1.17 (d, 3H, d, J = 6.5 Hz). 13C NMR
(125 MHz, CD3OD) d 177.5, 163.3.9, 162.3, 160.7, 157.7, 148.7, 137.5, 130.8,
123.5, 116.3, 106.2, 99.98, 99.88, 95.3, 73.6, 72.1, 71.7, 71.2, 18.0. FABMS: (m/e)
431 [MꢁH]+.
Supplementary data
Supplementary data associated with this article can be found, in