Antioxidative effects of 6-methoxysorigenin and its derivatives from Rhamnus nakaharai

Article abstract of DOI:10.1248/cpb.55.382

In the search for potential antioxidants, the naphthalenic compounds, 6-methoxysorigenin (2) and its glycosides [i.e. 6-methoxysorigenin-8-O-glucoside (3), α-sorinin (4), and 6-methoxysorigenin-8-rutinoside (5)] isolated from Rhamnus nakaharai together with two acylates (peracetate and perpropionate) of 2 were evaluated for antioxidant activities using 2,2-diphenyl-1- picrylhydrazyl (DPPH), metal chelating, and electron spin resonance (ESR) assays as well as anti-lipid peroxidation assay. The results showed that 2 possesses the most potent DPPH radical scavenging, metal chelating, and anti-lipid peroxidation activities with IC₅₀ values of 3.48, 615.90, and 5.95 μg/ml, respectively. The glycosides 3, 4, and 5 showed decreasing antioxidant activity that was related to an increased substitution at 1,8-dihydroxyl with sugar molecules. This suggests the importance of 1,8-dihydroxyl of 2 in the antioxidative effect. The iron chelation result could further explain the main cause of increasing antioxidant activity in 2. The acylates of 2 (2a peracetate and 2b perpropionate), although lacking a free hydroxyl, also exhibited significant anti-lipid peroxidation effect. ESR results further demonstrated that 2 possesses strong antioxidant activities. Taken together, this study shows that 2 is a potent antioxidant and may also be used for designing new iron chelators for clinical applications.

Full text of DOI:10.1248/cpb.55.382

382
Chem. Pharm. Bull. 55(3) 382—384 (2007)
Vol. 55, No. 3
Antioxidative Effects of 6-Methoxysorigenin and Its Derivatives from
Rhamnus nakaharai
Lean-Teik NG,a Chun-Ching LIN,b and Chai-Ming LU*,c
a Department of Biotechnology, Tajen University; No. 20 Weishin Road, Yanpu Shiang, Pingtung 907, Taiwan: b College of
c
Pharmacy, Kaohsiung Medical University; No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan: and Department of
Pharmacy, Tajen University; No. 20 Weishin Road, Yanpu Shiang, Pingtung 907, Taiwan.
Received August 25, 2006; accepted November 6, 2006
In the search for potential antioxidants, the naphthalenic compounds, 6-methoxysorigenin (2) and its glyco-
sides [i.e. 6-methoxysorigenin-8-O-glucoside (3), a-sorinin (4), and 6-methoxysorigenin-8-rutinoside (5)] isolated
from Rhamnus nakaharai together with two acylates (peracetate and perpropionate) of 2 were evaluated for an-
tioxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), metal chelating, and electron spin resonance
(ESR) assays as well as anti-lipid peroxidation assay. The results showed that 2 possesses the most potent DPPH
radical scavenging, metal chelating, and anti-lipid peroxidation activities with IC50 values of 3.48, 615.90, and
5.95 mg/ml, respectively. The glycosides 3, 4, and 5 showed decreasing antioxidant activity that was related to an
increased substitution at 1,8-dihydroxyl with sugar molecules. This suggests the importance of 1,8-dihydroxyl of
2 in the antioxidative effect. The iron chelation result could further explain the main cause of increasing antioxi-
dant activity in 2. The acylates of 2 (2a peracetate and 2b perpropionate), although lacking a free hydroxyl, also
exhibited significant anti-lipid peroxidation effect. ESR results further demonstrated that 2 possesses strong an-
tioxidant activities. Taken together, this study shows that 2 is a potent antioxidant and may also be used for de-
signing new iron chelators for clinical applications.
Key words Rhamnus nakaharai; Rhamnaceae; 6-methoxysorigenin; antioxidant activity
Oxidative stress is well known as a major cause of various isolated from R. nakaharai, showed antioxidant7) and platelet
chronic diseases, namely cardiovascular diseases, rheuma- aggregation inhibitory8) activities. 6-Methoxysorigenin (2),
tism, diabetes mellitus, and cancer.1,2) Reactive oxygen isolated from the wood of R. nakaharai, possesses a structure
species (ROS) are generated in vivo during shock, inflamma- closely analogous to 1 (Fig. 1) and is rich in glycosides.9)
tion, and ischemia/reperfusion injury.3) Antioxidant therapy
To evaluate the antioxidant effect of 2 and its derivatives,
has been clinically demonstrated to be valuable in the pre- glycosides from the wood of R. nakaharai were isolated and
vention of certain chronic diseases.3,4) Antioxidants also play characterized. They were 6-methoxysorigenin-8-O-glucoside
an important role in the food industry, because excessive for- (3), a-sorinin (4), 6-methoxysorigenin-8-O-rutinoside (5). In
mation of free radicals can accelerate oxidation of lipids in addition, peracylates 2a and 2b, and 2 were prepared and
foods and thereby decreases food quality.
evaluated for antioxidant activities using 2,2-diphenyl-1-
Rhamnus nakaharai HAYATA (Rhamnaceae) is traditionally picrylhydrazyl (DPPH), metal chelating, and electron spin
used as a folk medicine in Taiwan for treating constipation, resonance (ESR) assays as well as FeCl2-ascorbic acid-in-
inflammation, malignant tumors, and asthma.5) 3-O-Methy- duced lipid peroxidation assay in rat liver homogenates in
quercetin, the main constituent of the plant, has been re- vitro. Their antioxidant activities were compared with those
ported to inhibit cAMP- and cGMP-phosphodiesterase activ- of trolox and with EDTA for results of metal chelating assay.
ity of guinea pig trachealis.6) It was also demonstrated a se-
lective and competitive inhibitor of phosphosdiesterase sub- Results and Discussion
type 3/subtype 4.5) Isotorachrysone (1), an acetonaphthone
DPPH· is a stable free radical that accepts an electron or
hydrogen radical to become a stable diamagnetic molecule.10)
In this assay, all test compounds effectively reduced the sta-
ble radical DPPH to the yellow-colored diphenylpicrylhy-
drazine and their scavenging effect was dose-dependent. It
was found that compound 2 possesses the most potent DPPH
radical-scavenging activity, with an IC50 value of 3.48 mg/ml,
which is close to that of trolox (IC50: 3.18 mg/ml) (Table 1).
Chelating agents, which form s-bonds with a metal, are
effective as secondary antioxidants because they reduce the
redox potential thereby stabilizing the oxidized form of the
metal ion.11) Chelation may inhibit lipid oxidation by stabiliz-
ing transition metals. In this study, all test compounds in-
cluding trolox and EDTA interfered with the formation of
ferrous and ferrozine complexes, suggesting that they have
chelating activity and are able to capture ferrous ion before
ferrozine. Compound 2 (IC50: 615.90 mg/ml) showed a
stronger iron-binding capacity than trolox (IC50: 1848.83 mg/
Fig. 1. Chemical Structures of Isotorachrysone (1), 6-Methoxysorigenin
(2) and Its Derivatives
∗ To whom correspondence should be addressed. e-mail: cmlu@mail.tajen.edu.tw
© 2007 Pharmaceutical Society of Japan

March 2007
383
Table 1. IC50 Values of 6-Methoxysorigenin (2) and Its Derivatives (2a, 2b, 3—5) by Several Antioxidant Assays
IC50 (mg/ml)a)
2
2a
2b
3
4
5
Trolox
EDTA
ND
DPPH radical
scavenging
3.48ꢂ0.02
224.32ꢂ4.76
210.43ꢂ3.34
170.76ꢂ1.49
63.99ꢂ1.48 107.14ꢂ2.73
3.18ꢂ0.05
Metal chelating 615.90ꢂ5.79
Anti-lipid 5.95ꢂ0.37
peroxidation
846.87ꢂ29.79 1626.94ꢂ13.25 1914.90ꢂ27.09 2071.33ꢂ5.18 1451.51ꢂ25.04 1848.83ꢂ49.93 127.92ꢂ2.67
21.81ꢂ1.09 13.59ꢂ0.41 43.94ꢂ1.98 127.22ꢂ2.25 105.00ꢂ1.91 9.66ꢂ0.13 ND
a) Values are meanꢂS.E. ND: not determined.
Table 2. Superoxide Anion Radical Scavenging (SOD-Like) Activity of Compound 2 as Analyzed by Electron Spin Resonance Spectrometry
Signal peak height
Concentration
(g/ml)
SOD-like activity
(unit/g)
IC50
(mM)
Compound
SOD activity
Mn2ꢃ
Radical
95.60
2
0.0493
132.00
9.374
1.9ꢄ104
1.04 or 0.256 mg/ml
Calibration curve: Yꢅ0.235197ꢁ0.061957X; rꢅ0.997799; Yꢅ[I0/Iꢁ1]; Xꢅ[SOD (unit)]. I0: blank; I: the average peak height of the treated compound.
ml) but a weaker activity than EDTA (IC50: 127.92 mg/ml).
Thiobarbituric acid reactive substances (TBARS) method
has been widely used in studies of anti-lipid peroxidation ac-
tivity of natural products/phytochemicals in rat liver ho-
mogenate.12,13) Our results showed that inhibition of malondi-
aldehyde (MDA) formation increases with increasing con-
centrations of test compounds and trolox. The peracylates 2a
and 2b also showed notable inhibitory effects with IC50 val-
ues 21.81 and 13.59 mg/ml, respectively. However, glycosides
Fig. 2. ESR Spectra of 6-Methoxysorigenin (2) and Blank
3, 4, and 5 showed only moderate activity. The potency ported by the decreased inhibitory effect on lipid peroxida-
of anti-lipid peroxidation activity was in the order of tion of the 8-O-glycosides of 2 (3, 4, 5). These glycosides,
2ꢀtroloxꢀ2bꢀ2aꢀ3ꢀ5ꢀ4. Based on the IC50 values, com- especially the huge di-glycosides 4 and 5, can become hin-
pound 2 (IC50: 5.95 mg/ml) demonstrated the best inhibitory drances of chelation and result in a dramatic decrease of anti-
effect against lipid peroxidation, which was about two-fold lipid peroxidative activity. On the other hand, the sugar moi-
more potent than trolox (IC50: 9.66 mg/ml). The potent anti- eties of the glycosides might also act as lipophobic sub-
lipid peroxidation activity of 2 could be related to its effec- stituents that can prevent contact of the antioxidant with the
tive iron-binding capacity.
lipid membrane as well as breaking the chain reaction.
ESR spectral method is considered one of the most effec-
Acylates 2a and 2b, which lack a donable hydrogen atom,
tive methods in the study of free radical-scavenging activity retained apparent inhibitory effect on lipid peroxidation. This
of natural products.14,15) The superoxide anion (·Oꢁ2 ) is gen- activity seemed correlated with the length of the fatty acid
erated by the hypoxanthine–xanthine oxidase system and chain. The manner of lipid peroxidation inhibition of acylates
trapped by DMPO (5,5-dimethyl-1-pyrroline-1-oxide). The 2a and 2b could be different from 2 but similar to the perac-
DMPO–OOH adduct exhibits an electron spin resonant etate of isotorachrysone (1), which has been shown to pos-
effect that can be detected by ESR spectrometry. If the sess anti-lipid peroxidative effect.7) Its activities could be de-
DMPO–OOH adduct is reduced, the magnitude of ESR rived from the membrane-stabilizing mechanism that is simi-
would be decreased. The superoxide anion free radical-scav- lar to those of cholesterol and tamoxifen.16) The antioxidant
enging effect of the tested compound can be calculated by a property of 2 could be similar to isotorachrysone (1) since
calibration curve that has been established with various con- these molecules are structural analogues.
centrations of SOD in the same manner. The ESR spectrum
Rhamnaceous-derived crude drugs have long been used as
of 2 showed an obvious signal reduction (Fig. 2) and SOD- laxatives,17) which were found to contain 6-methoxysorigenin
like scavenging activity (Table 2). This observation further (2) and its derivatives.8,9,18,19) To date, bioactivities of 6-
supports the contention that 2 is an excellent antioxidant.
methoxysorigenin and its derivatives remain largely un-
Isotorachrysone (1) has been described as a potent antioxi- known. This is the first report on their antioxidant activities.
dant with iron chelating and potential free radical-scavenging Recently, iron chelators have been suggested clinically bene-
effects on superoxide anion, hydroxyl (·OH), hydroperoxyl ficial in the treatment of iron overload disease and cancer.20)
(·OOH), and DPPH radicals, etc. It was suggested that the Structurally, many of these synthetic iron chelators possess a
unsubstituted 1,6-dihydroxyl and the 2-acetyl substitution of naphthalene nucleus. The present study concludes that 6-
1 contribute to these effects.7) The structure of 2 could even methoxysorigenin (2) is a potent antioxidant and may also be
more feasibly behave as a chelator of iron ions because of the used for designing new iron chelators for clinical applica-
1,8-dihydroxyl and g-lactonic carbonyl that constitute two tions.
chelation sites (Fig. 1). This mechanism could also be sup-

384
Vol. 55, No. 3
ules (MeOH); [a]D22 ꢁ58° (cꢅ0.1, MeOH); mp 186—187 °C.
Experimental
Chemicals L-(ꢃ)-Ascorbic acid, thiobarbituric acid (TBA), and ethyl-
enediamine tetraacetic acid (EDTA) were purchased from Sigma Chemical
Co. (St. Louis, MO, U.S.A.). Trolox and ferrous chloride were obtained from
Wako Pure Chemical Industries (Osaka, Japan). 2,2-Diphenyl-1-picrylhy-
drazyl (DPPH) was obtained from MP Biomedicals Inc. (Eschwege, Ger-
many). All other chemicals used were of analytical grade.
Plant Material The heartwood of R. nakaharai was collected from
Yang-Ming Shan, Taipei, Taiwan. After confirming the authenticity of the
species, a voucher specimen was deposited in the herbarium (T89-Rh-001)
of Tajen University, Pingtung, Taiwan.
Analytical Apparatus Melting points were determined by an Elec-
trothermal IA9100 melting point apparatus (ESSLAB, Essex, U.K.). 1H-
NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Var-
ian Mercury-400 (400 MHz; Varian Inc., CA, U.S.A.) spectrometer whereas
the EI-MS was taken on a TurboMass mass spectrometer and FAB-MS on a
JMS-HX 110 mass spectrometer (Perkin Elmer Inc., MA, U.S.A.). Optical
specific rotation was measured by Jasco model DIP-370 Digital Polarimeter
(JASCO Inc, MD, U.S.A.).
6-Methoxysorigenin-8-O-rutinoside 5: Brownish needles (pyridine–
MeOH); mp 257 °C.
DPPH Radical-Scavenging Activity DPPH radical-scavenging activity
was determined according to the methods of Blois21) and Chang et al.22)
Metal Chelating Activity The chelation of ferrous ions by various
compounds was estimated by the method of Dinis et al.23) with slight modifi-
cations.
Anti-lipid Peroxidation Assay The effect of 6-methoxysorigenin and
its derivatives on FeCl2-ascorbic acid-induced lipid peroxidation in rat liver
homogenate was determined by the methods of Janero12) and Lu et al.13)
Electron Spin Resonance (ESR) Spectrometry ESR assay was used
further to confirm the antioxidant activity of compound 2. The experimental
procedure was conducted according to the method as described by Kohno et
al.24)
Acknowledgements The authors would like to thank the National Sci-
ence Council for providing financial support for this study (NSC89-2314-B-
127-001), and Mr. B. L. Hua and Mr. C. H. Chen for collecting the plant ma-
terials.
Extraction and Isolation The wood was dried and chipped into small
pieces followed by weighing about 5 kg for extraction. This was extracted
with 10 l of methanol at room temperature for 1 week. The methanolic ex-
tract was condensed to obtain a residue of 186 g. This residue was chro-
matographed on a silica gel (900 g, 70—230#, Merck, Germany) column
and eluted with a solvent system of CH2Cl2–MeOH. The elution began with
CH2Cl2 (1500 ml) then with CH2Cl2 : MeOH (9 : 1; 2000 ml), followed by
CH2Cl2 : MeOH (8 : 1; 2000 ml) to obtain 6-methoxysorigenin-8-O-gluco-
side (3, 2.1 g). After discarding the solution obtained with CH2Cl2 : MeOH
(8 : 1—6 : 1; each 1500 ml), elution with CH2Cl2 : MeOH (4 : 1; 2500 ml) was
performed to obtain 6-methoxysorinin (4, 4.2 g). The same solution of 4 was
further analyzed with RP-18 TLC to obtain another blue-fluorescing spot
with Rf 0.56, which was close to that of 4. Hence it was further chro-
matographed on a 25ꢄ600-mm RP-18 column (60 mm, Merck, Germany).
Elution with 60% MeOH–H2O solvent system 250 ml led to the isolation of
6-methoxysorigenin 8-O-rutinoside (5, 87 mg). Structure determination of 3,
4, and 5 was characterized by spectrometric analyses and further confirmed
with the reported data.18,19) Compound 2 was obtained from acid hydrolysis
of the glycoside 3. The preparation of acylates (2a, 2b) was achieved by
treating 2 with pyridine and related acid anhydrides.
Characterization of Compounds 2, 2a, 2b 6-Methoxysorigenin 2:
Brownish powders (CHCl3–MeOH); mp ꢀ300 °C; 1H-NMR (400 MHz,
pyridine-d5): d 3.71 (3H, s, OCH3), 5.07 (2H, s, g-lactonic CH2), 6.58 (1H,
s, H-4), 6.63 (1H, d, Jꢅ2.0 Hz, H-7), 6.68 (1H, d, Jꢅ2.0 Hz, H-5), 14.50
(1H, br, OH), 15.10 (1H, br, OH); 13C-NMR (100 MHz, pyridine-d5): d
172.2 (C-1), 113.7 (C-2), 144.0 (C-3), 103.1 (C-4), 98.1 (C-5), 164.5 (C-6),
98.3 (C-7), 162.5 (C-8), 102.8 (C-9), 143.4 (C-10), 176.6 (g-lactonic CꢅO),
70.2 (g-lactonic CH2), 55.0 (OCH3); EI-MS m/z: 246 [M]ꢃ, 217 (base peak).
6-Methoxysorigenin Peracetate 2a: Colorless needles (CHCl3–MeOH);
mp 244 °C; 1H-NMR (400 MHz, CDCl3): d 2.41, 2.45 (each 3H, s, COCH3),
3.95 (3H, s, OCH3), 5.52 (2H, s, g-lactonic CH2), 7.13 (1H, d, Jꢅ2.4 Hz, H-
7), 7.50 (1H, d, Jꢅ2.4 Hz, H-5), 7.97 (1H, s, H-4); EI-MS m/z: 330 [M]ꢃ,
288 [Mꢁ42]ꢃ, 246 [Mꢁ84]ꢃ (base peak).
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6-Methoxysorigenin Perpropionate 2b: Colorless masses (CHCl3–
1
MeOH); mp 189 °C; H-NMR (400 MHz, CDCl3): d 1.33, 1.35 (each 3H, t,
Jꢅ7.6 Hz, propionate CH3), 2.69 (2H, m, propionate CH2), 2.82 (2H, br,
propionate CH2), 3.89 (3H, s, OMe), 5.18 (2H, s, g-lactonic CH2), 6.86 (1H,
d, Jꢅ2.4 Hz, H-7), 6.97 (1H, d, Jꢅ2.4 Hz, H-5), 7.46 (1H, s, H-4); 13C-NMR
(100 MHz, CDCl3): d 9.0, 9.2 (propionate CH3), 27.7, 27.8 (propionate
CH2), 56.0 (OMe), 68.7 (g-lactonic CH2), 104.9 (C-7), 113.3 (C-9), 114.8
(C-5), 117.1 (C-2), 117.7 (C-4), 141.6 (C-3), 142.1 (C-10), 146.1 (C-8),
148.4 (C-1), 159.9 (C-6), 168.3 (g-lactonic CꢅO), 172.7, 173.1 (propionate
CꢅO); EI-MS m/z: 358 [M]ꢃ, 302 [MꢁC2H5COꢃ1]ꢃ, 246 [aglycone]ꢃ.
6-Methoxysorigenin-8-O-glucopyranoside 3: Brownish granules (CH2Cl2–
MeOH); mp 223 °C.
24) Kohno M., Yamada M., Mitsura K., Mizuta Y., Yoshikawa T., Bull.
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6-Methoxysorigenin-8-O-premeveroside (a-Sorinin) 4: Brownish gran-