Isolation, Identification and Tyrosinase Inhibitory Activities of the Extractives from Allamanda cathartica 168
O O
OH OH
3 5
6 8
9 10
1 2
3 5
6 1
4 5
4 7
2
3 2
4
O O
1 2
3 4
6 7
8 9
1 2
3 4
5 6
7 8
9
O O
HO OH
O
O OH
OH HO
OH
2 3
4 5
6 7
8 9
10 1
2 3
4 5
6
O OH
O OH
HO
2 3
4 5
6 7
9 10
1 2
3 4
5 6
8
O O
O O
O O
OH
1 3
4 5
6 7
8 9
10 11
12 13
14
H H
A: glabridin B: new lignan
1-3-4-allyl-2,6-dimethoxyphenoxy -4-methoxyphenylpropane-1,2,diol
C: kaempferol
D: naringeni E: allamandicin
Figure 1. Structure of compound A-E.
1002002.
2.2. Extraction and Fractionation of A. cathartica
Stem Powder
A. cathartica stem powder 385.4 g was extracted with methanol. The methanol extract was fractionated with
ethyl acetate. The ethyl acetate soluble fraction was separated with silica gel column chromatography 69 mm
φ × 510 mm L. Eluted with n-hexane, EtOAc, MeOH to obtain Fr.1-Fr.8. The Fr.3 was separated with preparative
HPLC[ODS-3 20 mm φ × 250 mm L MeOH0.05
TFA aq.soln. = 1090 0 min, 1000 60 min, 1000 80 min] to obtain Fr.3-1-Fr.3-4. Finally, compound A, B, C,
D, and E were isolated from Fr.3-3, Fr.3-1, Fr.3-4, Fr.3-4, and Fr.3-4 respectively by preparative HPLC [ODS-3 10
mm
φ × 250 mm L MeOH0.05 TFA aq.soln. = 1090
0 min, 1000 60 min, 1000 80 min] Figure 2. 2.3. Tyrosinase Activity Assay
The tyrosinase activity method performed based on Ba- tubara et al. 2010 [10]. Briefly, sample 70
μl was put in 96-well plate. Tyrosinase 30
μl 333 unitml in phosphate buffer 50 mM pH 6.5 and 110
μl of substrates L-tyro- sine 2mM or L-DOPA 12mM were added. After incuba-
tion at 37 ˚C for 30 min, the absorbance at 510 nm was
determined using a micro plate reader. Moreover IC
50
value concentration of inhibitor showing 50 inhibition was calculated.
2.4. Identification of Compounds
Compound A-E were identified by
1
H-NMR,
13
C-NMR,
1
H-
1
H-COSY, HMQC, HMBC, and LC-MS. Aceton-d6 was used as the solvent for all compounds. These NMR
measurements were performed by using JEOL EC600- NMR.LC-MS measurements Waters Waters
®
Xevo
TM
QTof MS was performed using column C
18
2.1 × 100 mm with MeOHwater = 6040 0 min, 1000 10 min,
1000 13 min as eluent. The NMR data of compounds isolated from A. cathar-
tica stem is shown in Table 1. LC-MS: ES
－
data of Compoun A, B, C, D, and E were mz: 323 M-1, 373,
285, 271, 307 respectively.
3. Results and Discussion
3.1. Compounds Identification
Allamanda cathartica contains hydrocarbonslong chain esters, e.g. 1-triacontanol, 1-dotriacontanol, docosanoic-,
tetracosanoic- and hexacosanoic acid in the root; β-si-
tosterol and triterpenes e.g. ursolic acid and β-amyrin in
the leaves or stem, and lupeol in the roots [1-3]. Other components isolated from the roots include series of iri-
doid lactones: allamadin, allamandicin, plumericin, iso- plumericin, plumeieride and fluvoplumierin [11,12].
Compound A concluded as glabridin, while compound C, D and E was kaempferol, naringenin, and alla-
mandicin respectively. Interestingly, Kaempferol have
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Isolation, Identification and Tyrosinase Inhibitory Activities of the Extractives from Allamanda cathartica 169
P-HPLC Preparative HPLC P-HPLC
P-HPLC P-HPLC
Stem powder of A .catharitica 385.4 g
Insoluble fraction Extracting with EtOAc 12 h ×3
Residue MeOH extract ASM
29.9 g
Soluble fraction Silicagel
column chromatography 69 mmφ×510 mm L
Fr.3-1 Fr.3-2 Fr.3-3 Fr.3-4 Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6 Fr.7 Fr. 8
Extracted with MeOH 12 h ×3
Compound B Compound A Compound C,D,E
Figure 2. Isolation scheme of the compounds from A. catharitica stem powder.
been found in petals of this plant, and allamandicin have been found in roots[4]. However, it was revealed that the
two compounds are also contained in stem. Moreover, glabridin and naringenin are found the first time in this
plant.
NMR spectrum of glabridin was also searched, and tried to compare to data of compound A and glabridin.
The NMR spectrum data from glabridin was similar to that of compound A. Equally, compound C, D, E were
identified as keampferol, naringenin, allmandicin respec- tively [13-15].
Compound B was found to be a novel compound. Ac- cording to NMR data for compound B, 5.11 and 5.07ppm
protons were geminal and alkene protons because of chemical shift and HMQC data. The two protons of 6.55
ppm peeks were equivalen in aromatic ring protons, be- cause it appeared as singlet proton. The three protons of
6.72, 6.94, 6.68 ppm were also the aromatic protons in- dicating ortho-metha, ortho, and metha coupling. Ac-
cording to the HMBC spectrum of compound B Figure 3
, long-range correlations were observed between H-1 and C-2, H-2 and C-1, C-3, C-4, H-3 and C-1, C-2, C-4,
H-5 and C-3, C-4, C-6, 6-OMe and C-6, 8-OMe and C-8, H-9 and C-8, C-4, C-3, H
3
-1’ and C-2’, C-3’, H-2’ and C-3’, H-3’ and C-4’, C-9’, H-5’ and C-3’, C-4’, C-6’,
C-7, 7’-OMe and C-7’, H-8’ and C-7’, C-9’ and between H-9’ and C-8’, C-3’, C-4’. These NMR and MS data
showed us the compound B is lignan 1-[3-4-allyl-2, 6-dimetoxyphenoxy-4-methoxyphenyl]propane-1,2,diol
shown Figure 1.
3.2. Tyrosinase Inhibitory Activity
