Isolation and structure elucidation of highly antioxidative 3,8″-linked biflavanones and flavanone-C-glycosides from garcinia buchananii bark

Article abstract of DOI:10.1021/jf205175b

The aim of this study was to identify antioxidants from Garcinia buchananii bark extract using hydrogen peroxide scavenging and oxygen radical absorbance capacity (ORAC) assays. LC-MS/MS analysis, 1D- and 2D-NMR, and circular dichroism (CD) spectroscopy led to the unequivocal identification of the major antioxidative molecules as a series of three 3,8″-linked biflavanones and two flavanone-C-glycosides. Besides the previously reported (2R,3R,2″R, 3″R)-naringenin-C-3/C-8″ dihydroquercetin linked biflavanone (GB-2; 4) and (2R,3S,2″R,3″R)-manniflavanone (3), whose stereochemistry has been revised, the antioxidants identified for the first time in Garcinia buchananii were (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (1), (2R,3R)-aromadendrin-6-C-β-D-glucopyranoside (2), and the new compound (2R,3S,2″S)-buchananiflavanone (5). The H₂O₂ scavenging and the ORAC assays demonstrated that these natural products have an extraordinarily high antioxidative power, especially (2R,3S,2″R,3″R) -manniflavanone (3) and GB-2 (4), with EC₅₀ values of 2.8 and 2.2 μM, respectively, and 13.73 and 12.10 μmol TE/ μmol. These findings demonstrate that G. buchananii bark extract is a rich natural source of antioxidants.

Full text of DOI:10.1021/jf205175b

Article
pubs.acs.org/JAFC
Isolation and Structure Elucidation of Highly Antioxidative
3,8″-Linked Biflavanones and Flavanone-C-glycosides from
Garcinia buchananii Bark
Timo D. Stark,*,† Toshiaki Matsutomo,†,∥ Sofie Losch,† Paul A. Boakye,‡ Onesmo B. Balemba,‡
̈
Sofie P. Pasilis,§ and Thomas Hofmann†
†Lehrstuhl fur Lebensmittelchemie und Molekulare Sensorik, Technische Universitat Munchen, Lise-Meitner Str. 34, D-85354
̈
̈
̈
Freising, Germany
‡Department of Biological Sciences and Department of Chemistry, University of Idaho, Moscow, Idaho, United States
∥Central Research Institute, Wakunaga Pharmaceutical Co. Ltd., 1624 Shimokotachi, Kodacho, Akitakata, Hiroshima 739-1195, Japan
§
ABSTRACT: The aim of this study was to identify antioxidants from Garcinia buchananii bark extract using hydrogen peroxide
scavenging and oxygen radical absorbance capacity (ORAC) assays. LC-MS/MS analysis, 1D- and 2D-NMR, and circular
dichroism (CD) spectroscopy led to the unequivocal identification of the major antioxidative molecules as a series of three 3,8″-
linked biflavanones and two flavanone-C-glycosides. Besides the previously reported (2R,3R,2″R,3″R)-naringenin-C-3/C-8″
dihydroquercetin linked biflavanone (GB-2; 4) and (2R,3S,2″R,3″R)-manniflavanone (3), whose stereochemistry has been
revised, the antioxidants identified for the first time in Garcinia buchananii were (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (1),
(2R,3R)-aromadendrin-6-C-β-D-glucopyranoside (2), and the new compound (2R,3S,2″S)-buchananiflavanone (5). The H2O2
scavenging and the ORAC assays demonstrated that these natural products have an extraordinarily high antioxidative power,
especially (2R,3S,2″R,3″R)-manniflavanone (3) and GB-2 (4), with EC50 values of 2.8 and 2.2 μM, respectively, and 13.73 and
12.10 μmol TE/ μmol. These findings demonstrate that G. buchananii bark extract is a rich natural source of antioxidants.
KEYWORDS: 3,8″-linked biflavanones, flavanone-6-C-glycosides, Garcinia buchananii, (2R,3S,2″R,3″R)-manniflavanone,
(2R,3S,2″S)-buchananiflavanone, antioxidants
anti-inflammatory,8 antimicrobial,9 and antioxidant proper-
INTRODUCTION
Oxidative stress is a serious condition that leads to chronic
■
ties.10−12
Stem and root bark extracts of G. buchananii are traditionally
used to treat diarrhea, dysentery, abdominal pain, and a range
of infectious diseases in Sub-Saharan Africa.13 It is thought that
the extract’s antidiarrhea effects reduce colon motility by
inhibiting neurotransmission and possibly 5-HT3 and 5-HT4
receptors.14,15 Furthermore, the extract has anti-inflammatory,
antinociceptive, and antidiarrheal effects.16,17 Initial compound
screening using standard chemical tests and preparative thin
layer chromatography methods suggest that the compounds
having antidiarrheal properties are flavonoids, or a combination
of flavonoids with alkaloids or steroids. Whether the flavonoids
found in G. buchananii bark extract have antioxidant activities
has never been determined.
Therefore, the purpose of the present study was to use
antioxidative activity-guided screening to determine if the stem
bark extract of G. buchananii has fractions that have
antioxidative activity and to isolate and elucidate the chemical
structures of compounds showing the most powerful
antioxidant activity.
metabolic and degenerative diseases. It is caused by
endogenous free radicals, generated in the human body as a
metabolic byproduct, or free radicals from exogenous sources
like ultraviolet light, ionizing radiation, chemotherapeutics,
environmental toxins, and inflammatory cytokines, which attack
various substrates in the body evoking irreparable injuries, cell
death, and necrosis. The targets of these free radicals are all
cellular components, e.g., proteins, carbohydrates, nucleic acids,
and polyunsaturated fatty acids. The destructive power of these
free radicals causing oxidative damage is associated with
coronary heart diseases, atherosclerosis, aging, cancer, and
inflammatory conditions.1,2
Natural phytochemicals having antioxidant activity, such as
vitamins (ascorbic acid and vitamin E), carotenoid terpenoids
(carotenoids), flavonoid polyphenolics, and alkyl sulfide reduce
risks of many degenerative and metabolic diseases. The
antioxidative power of phenolic compounds is mainly due to
their redox properties, which play an important role in
absorbing and neutralizing free radicals, quenching singlet and
triplet oxygen, or eliminating peroxides. In the past few years,
several investigations have shown that the antioxidant activity
of a plant extract is highly correlated with the extract’s phenolic
content.3−6
Received: December 19, 2011
Revised: January 16, 2012
Accepted: January 17, 2012
Published: January 17, 2012
About 400 species are known of the genus Garcinia, familiy
Guttiferae, in which extracts and pure isolates from Garcinia
species exhibited forms of biological activity such as anticancer,7
© 2012 American Chemical Society
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Table 1. Antioxidant Activities of Garcinia b. Extract and Fractions M1−M8
a
,
b
c,d
H2O2 assay
EC50 (dilution degree)
(10061.5−12721.7)
ORAC assay
each amount (mg)
100.0
(μmol TE/each amount)
EtOH extract
11320.5
8912.8
9879.8
609.4
1359.15
1102.95
1251.78
102.38
80.89
( 14.84)
( 46.09)
recombination of M1−M8
calculated sum of M1−M8
(7840.1−10040.1)
M1
M2
M3
M4
M5
M6
M7
M8
16.3
7.1
(534.1−702.0)
(600.6−748.9)
(5008.0−6473.8)
(639.9−812.1)
(894.4−1207.7)
(331.2−444.7)
(509.9−641.2)
(180.5−226.4)
( 3.05)
( 1.38)
( 27.87)
( 1.61)
( 3.44)
( 1.56)
( 2.97)
673.1
41.0
6.4
5679.3
722.3
744.83
86.89
10.3
5.2
1039.1
382.4
141.68
44.89
8.3
572.2
42.34
5.3
202.1
7.89
( 0.20)
a
b
c
Each sample was analyzed by means of the H2O2 assay by triplicate studies. The range in parentheses represents 95% confidence interval. Each
sample was analyzed by means of the ORAC assay by quadruplicate studies. The numerical value in parentheses represents SD.
d
Figure 1. Chemical structures of compounds 1−6.
G2 HDMS mass spectrometer (Waters, Manchester, UK) coupled to
an Acquity UPLC core system (Waters, Milford, MA, USA). For CD
spectroscopy, methanolic solutions of the samples were analyzed by
MATERIALS AND METHODS
■
Chemicals. The following reagents were obtained commercially:
hydrogen peroxide (Merck, Hohenbrunn, Germany); 2,2′-azino-bis(3-
means of a Jasco J810 Spectro polarimeter (Hachioji, Japan). HPLC
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
separations were performed using a preparative HPLC system
peroxidase from horseradish (HRP), ( )-6-hydroxy-2,5,7,8-tetrame-
thylchromane-2-carboxylic acid (Trolox), fluorescein sodium salt (FL),
(PrepStar, Varian, Darmstadt, Germany). MPLC separations were
performed on a Buchi Sepacore (Flawil, Switzerland) system using PP
̈
2,2′-azobis(2-methylpropinamidine) (AAPH), quercetin, (−)-epicate-
chin, ( )-naringenin, methyl iodide, ascorbic acid (Sigma-Aldrich,
Steinheim, Germany), rutin, and (+)-(2R,3R)-taxifolin (AppliChem,
Darmstadt, Germany). Water for chromatographic separations was
purified with a Milli-Q Gradient A10 system (Millipore, Schwalbach,
Germany), and solvents used were of HPLC-grade (Merck,
Darmstadt, Germany). Deuterated solvents were obtained from
Euriso-Top (Gif-sur-Yvette, France).
cartridges (id. 40 mm, l. 150 mm) and LiChroprep RP18, 25−40 μm
mesh material (Merck, Darmstadt, Germany).
Plant Material. Garcinia buchananii stem bark was collected from
plants in their natural habitats in Karagwe, Tanzania, and processed as
described previously.14 A sample of bark powder was deposited at the
University of Idaho Stillinger herbarium (voucher # 159918).
Extraction and Isolation. G. buchananii bark powder (10 g) was
suspended in a mixture of ethanol/water (50 mL, 70/30, v/v),
sonicated (10 min), stirred at RT (20 min), and filtered. The filtrate
was extracted with hexane (50 mL), the ethanol/water extract was
evaporated, and then the sample was freeze-dried. Aliquots (1 g) of the
freeze-dried ethanol/water extract were dissolved in a water/methanol
mixture (10 mL, 50/50, v/v) and fractionated using MPLC.
Chromatography was performed starting with a mixture (65/35, v/v)
of aqueous formic acid (0.1% in water, pH 2.5) and MeOH, increasing
General Experimental Procedure. 1D and 2D NMR spectros-
1
1
1
copy H, H-1H-gCOSY, gHSQC, gHMBC, and 13C, H-1H rotating
frame nuclear Overhauser enhancement spectroscopy (phase-sensitive
ROESY) NMR measurements were performed on an Avance III 500
MHz spectrometer with a CTCI probe or an Avance III 400 MHz
spectrometer with a BBO probe (Bruker, Rheinstetten, Germany).
Mass spectra of the compounds were measured on a Waters Synapt
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the MeOH content up to 55% within 25 min, and in 5 min to 100%.
Eight fractions (M1−M8, 160, 70, 401, 63, 101, 51, 51, and 52 mg,
Table 1) were collected, concentrated under reduced pressure, and
freeze-dried.
glucopyranoside (1, Figure 1) and (2R,3R)-aromadendrin-6-
C-β-D-glucopyranoside (2, Figure 1). M4: Using the same
column and flow rate as those described above, chromatog-
raphy was performed starting with a mixture (70/30, v/v) of
aqueous formic acid (0.1% in water, pH 2.5) and ACN, and
increasing the ACN content up to 43% within 13 min. The
collected fraction was concentrated under reduced pressure and
freeze-dried twice, affording (2R,3R,2″R,3″R) GB-2 (4, Figure 1).
M5: Using the same column and flow rate as those described
above, chromatography was performed starting with a mixture
(68/32, v/v) of aqueous formic acid (0.1% in water, pH 2.5) and
ACN, and increasing the ACN content up to 43% within 13 min.
The collected fraction was concentrated under reduced pressure
and freeze-dried twice, affording (2R,3S,2″S) buchananiflavanone
(5, Figure 1).
Methylation of Manniflavanone. Manniflavanone (3)
(0.17 mmol) was dissolved in dry acetone (100 mL), and
methyl iodide (32 mmol) and K2CO3 (14.5 mmol) were added.
The mixture was refluxed for 24 h, with the addition of further
methyl iodide (8 mmol) and K2CO3 (7.2 mmol) after 8 h. The
mixture was evaporated to dryness and then taken up in a
water/methanol mixture (2 mL, 50:50, v/v) and purified by
solid phase extraction (SPE) (Strata Gigatube C18, Phenom-
enex, Aschaffenburg, Germany). The SPE cartridge was flushed
with water, and the methanol eluate was further purified by
means of HPLC. Monitoring the effluent (4.2 mL/min) at 290
nm, chromatography was performed using a RP column 10 ×
250 mm, Phenylhexyl, 5 μm (Phenomenex, Aschaffenburg,
Germany) as the stationary phase and starting with a mixture
(20/80, v/v) of aqueous formic acid (0.1% in water, pH 2.5)
and MeOH. The MeOH content was increased to 100% within
20 min. Collected fractions were concentrated under reduced
pressure and freeze-dried twice, affording (2R,3R,2″R,3″R)-
nonamethylmanniflavanone (3a) and a mixture of two
octamethylmanniflavanones (3b,c).
ANTIOXIDANT ASSAYS
■
Hydrogen Peroxide Scavenging Assay. Hydrogen
peroxide scavenging assay was performed in accordance with
the method of Ichikawa et al.18 Sample solutions at appropriate
concentrations were prepared using phosphate buffer (100
mM, pH 6.0). Sample solution (100 μL), phosphate buffer (30
μL, 100 mM, pH 6.0), and hydrogen peroxide solution (10 μL,
500 μM) were mixed in a 96-well clear micro plate (VWR,
Ismaning, Germany). Then peroxidase (40 μL, 150 U/mL) and
ABTS (40 μL, 0.1%) were added. The microplate was
incubated at 37 °C for 15 min. The absorbance (A) of each
well was measured at 414 nm with FLUOstar OPTIMA (BMG
LABTECH, Offenburg, Germany). The scavenging effect (E)
was calculated as shown using the formula below (blank stands
for solution without hydrogen peroxide, and control did not
include a test compound) and EC50 was calculated by the
probit method. After freeze-drying in triplicate, MPLC fractions
M1−M8 were analyzed in natural ratios.
E = [(A − A
/(A − A
)
− (A − A
× 100
)
]
blank control
blank test
)
blank control
Oxygen Radical Absorbance Capacity (ORAC) Assay.
The ORAC assay was carried out according to the method of
Ou et al.19 with some modifications. Trolox and FL were used
as a standard and a fluorescent probe, respectively. Free radicals
were produced by AAPH to oxidize FL. Different dilutions of
Trolox (200, 100, 50, 25, and 12.5 μM) and appropriate
dilutions of the tested sample were prepared with phosphate
buffer (10 mM, pH 7.4). Trolox dilution (25 μL) or sample
solution were pipetted into a well of a 96-well black microplate
(VWR), and then FL (150 μL, 10 nM) was added. The reaction
mixture was incubated at 37 °C for 30 min. Afterward,
fluorescence was measured every 90 s at the excitation of 485
nm, and the emission of 520 nm using FLUOstar OPTIMA.
After 3 cycles, AAPH (25 μL, 240 mM) was added quickly, and
then the measurement was resumed and continued up to 90
min (60 cycles in total). The background signal was determined
using the first 3 cycles. The ORAC values were calculated
according to the method of Cao et al.20 and expressed as the
Trolox equivalent (μmol TE/μmol). After freeze-drying in
triplicate, MPLC fractions M1−M8 were analyzed in natural
ratios.
Isolation and Structural Characterization of Com-
pounds with Antioxidant Activity. Fractions that showed
higher levels of antioxidant activities were subjected to the
identification and characterization of chemical compounds.
M3 afforded (2R,3R,2″R,3″R)-manniflavanone (3, Figure 1),
and MPLC fractions (M1 and M4−M5) were further purified
by means of HPLC. M1: Chromatography was performed using
a RP column (21.2 × 250 mm, Phenylhexyl, 5 μm;
Phenomenex, Aschaffenburg, Germany) as the stationary
phase. The effluent (18 mL/min) was monitored at 290 nm.
The separation started with a mixture (83/17, v/v) of aqueous
formic acid (0.1% in water, pH 2.5) and MeOH, and the
MeOH content was increased up to 40% within 12 min.
Collected fractions were concentrated under reduced pressure
and freeze-dried twice, affording (2R,3R)-taxifolin-6-C-β-D-
(2R,3R)-Taxifolin-6-C-β-D-glucopyranoside (1, Figure 1).
Colorless powder; UV (MeOH/H2O, 5/5, v/v) λmax = 225,
290, 345 nm; (−) HRESIMS m/z 465.1035 [M − H]− (calcd
for C21H21O12, 465.1033). CD (MeOH, 0.67 mmol/L):
λmax(Δε) = 333 (+2.0), 296 (−6.5), 252 (+0.9), 222
(+6.6).1H NMR (500 MHz, DMSO-d6, COSY): 3.10 [m, 1H,
J = 9.0 Hz, H−C(4″)], 3.14 [m, 1H, J = 6.0 Hz, H−C(5″)],
3.16 [dd, 1H, J = 8.4, 8.6 Hz H−C(3″)], 3.40 [d, 1H, J = 11.1
Hz, H−C(6″α)], 3.67 [d, 1H, J = 11.1 Hz, H−C(6″β)], 3.99
[pt, 1H, J = 9.1 Hz, H−C(2″)], 4.45 [dd, 1H, J = 5.3 Hz, H−
C(3)], 4.49 [d, 1H, J = 9.8 Hz, H−C(1″)], 4.97 [d, 1H, J = 10.8
Hz, H−C(2)], 5.74 [d, 1H, J = 5.7 Hz, HO-C(3)], 5.92 [s, 1H,
H−C(8)], 6.74 [s, 2H, H−C(5′,6′)], 6.87 [s, 1H, H−C(2′)],
8.98 [2xbrs, HO-C(3′,4′)], 12.47 [s, HO-C(5)]. 13C NMR (125
MHz, DMSO-d6, HSQC, HMBC): δ 61.6 [C-6″], 70.3 [C-2″],
70.7 [C-4″], 71.6 [C-3], 73.0 [C-1″], 79.1 [C-3″], 81.6 [C-5″],
82.9 [C-2], 94.7 [C-8], 100.1 [C-4a], 106.0 [C-6], 115.2 [C-5′],
115.3 [C-2′], 119.3 [C-6′], 128.0 [C-1′], 145.0 [C-4′], 145.8 [C-
́
3], 161.3 [C-8a], 162.6 [C-5], 166.1 [C-7], 197.9 [C-4].
(2R,3R)-Aromadendrin-6-C-β-D-glucopyranoside (2, Figure 1).
Colorless powder; UV (MeOH/H2O, 5/5, v/v) λmax = 213,
228, 293, 347 nm; (−) HRESIMS m/z 449.1100 [M − H]−
(calcd for C21H21O11, 449.1084). CD (MeOH, 0.74 mmol/L):
λmax(Δε) = 329 (+1.5), 291 (−3.8), 248 (+1.3), 233 (+3.2),
218 (+7.0).1H NMR (500 MHz, DMSO-d6, COSY): 3.11 [m,
1H, J = 9.1 Hz, H−C(4″)], 3.12 [m, 1H, H−C(5″)], 3.16 [pt,
1H, J = 8.4, 8.7 Hz H−C(3″)], 3.41 [1H, H−C(6″α)], 3.66
[d, 1H, J = 10.7 Hz, H−C(6″β)], 4.00 [pt, 1H, J = 9.2 Hz,
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Figure 2. UPLC-ESI−-HRMS spectrum of 1.
H−C(2″)], 4.48 [d, 1H, J = 9.8 Hz, H−C(1″)], 4.50 [d, 1H, J =
11.0 Hz, H−C(3)], 4.99 [d, 1H, J = 11.0 Hz, H−C(2)], 5.74
[brs, HO-C(3)], 5.83 [s, 1H, H−C(8)], 6.78 [d, 2H, J = 8.6
Hz, H−C(3′,5′)], 7.29 [d, 2H, J = 8.6 Hz, H−C(2′,6′)], 9.57
[brs, HO-C(4′)], 12.50 [brs, HO-C(5)]. 13C NMR (125 MHz,
DMSO-d6, HSQC, HMBC): δ 61.5 [C-6″], 70.3 [C-2″], 70.6
[C-4″], 71.4 [C-3], 73.1 [C-1″], 79.1 [C-3″], 81.4 [C-5″], 82.7
[C-2], 95.2 [C-8], 99.5 [C-4a], 106.2 [C-6], 114.9 [C-3′,5′],
127.6 [C-1′], 129.3 [C-2′,6′], 157.7 [C-4′], 161.2 [C-8a], 162.7
[C-5], 167.5 [C-7], 197.0 [C-4].
DMSO-d6, 9/1, HSQC, HMBC): δ 48.2 [C-3], 73.0, 73.3 [C-
3″], 82.3, 82.7 [C-2], 83.8 [C-2″], 95.4, 95.5, 96.1, 96.4, 96.6
[C-6″,6,8], 100.4, 101.0 [C-4a″], 101.9, 102.0 [C-4a,8″], 115.4,
115.5, 115.6, 115.9, 116.1 [C-5‴,5′,2‴,2′], 118.1 [C-6‴], 119.2,
119.4, 119.5 [C-6‴,6′,6′], 129.0, 129.1, 129.3, 129.5 [C-1′,1‴],
145.4, 145.5, 146.2, 146.3, 146.4, 146.5 [C-3′,4′,3‴,4‴], 160.4,
161.1 [C-8a″], 162.9, 163.2 [C-5″], 163.5, 163.6 [C-8a], 164.6,
164.7 [C-5], 165.4, 166.0 [C-7″], 167.2, 167.3 [C-7], 197.3,
197.5 [C-4], 198.2 [C-4″].
(2R,3S,2″R,3″R)-GB-2 (4, Figure 1). Colorless powder; UV
(MeOH/H2O, 6/4, v/v) λmax = 204, 292, 347 nm; (−)
HRESIMS m/z 573.1037 [M − H]− (calcd for C30H21O12,
573.1033). CD (MeOH, 0.47 mmol/L): λmax(Δε)= 341 (+2.0),
320 (−1.6), 303 (−9.1), 281 (+12.0), 246 (−2.3), 237 (0.0),
(2R,3S,2″R,3″R)-Manniflavanone (3, Figure 1). Colorless
powder; UV (MeOH/H2O, 6/4, v/v) λmax = 210, 290, 346 nm;
(−) HRESIMS m/z 589.0989 [M − H]− (calcd for C30H21O13,
589.0982). CD (MeOH, 0.46 mmol/L): λmax(Δε)= 341 (+1.3),
321 (−0.7), 303 (−5.6), 283 (+7.6), 240 (−1.3), 218 (−11.7).
1H NMR (400 MHz, acetone-d6+DMSO-d6, 9/1, 8 °C,
COSY): δ 4.05 [dd, J = 5.4, 11.7 Hz, H−C(3″)], 4.30 [dd, J
= 5.4, 11.3 Hz, H−C(3″)], 4.55 [d, J = 12.1, H−C(3)], 4.71 [d,
J = 12.1, H−C(3)], 4.91 [d, J = 11.7 Hz, H−C(2″)], 5.09 [d, J
= 11.3 Hz, H−C(2″)], 5.45 [d, J = 12.1 Hz, H−C(2)], 5.64 [d,
J = 5.9 Hz, HO-C(3″)], 5.68 [d, J = 12.1 Hz, H−C(2)], 5.80 [d,
J = 5.9 Hz, HO-C(3″)], 5.81−5.94 [4xs, H−C(6″,6,8)], 6.03 [s,
H−C(6″)], 6.60 [dd, J = 1.8, 7.9 Hz, H−C(6′)], 6.66−6.71 [m,
H−C(6′,5′,5‴)], 6.77−6.80 [2xdd, J = 1.8, 8.4 Hz, H−C(6‴)],
6.83−6.89 [m, H−C(5′,5‴,2′)], 6.89−6.94 [m, H−C(2′,2‴)],
8.80−9.00 [4xbrs, HO-C(3′,4′,3‴, 4‴)], 10.64, 10.68, 11.00,
11.24 [brs, HO-C(7″,7)], 11.89, 11.97 [s, HO-C(5″)], 12.29,
1
214 (−16.4). H NMR (400 MHz, acetone-d6 + DMSO-d6, 9/
1, 8 °C, COSY): δ 4.10 [dd, J = 4.9, 11.7 Hz, H−C(3″)], 4.31
[dd, J = 4.7, 11.2 Hz, H−C(3″)], 4.61 [d, J = 12.2, H−C(3)],
4.77 [d, J = 12.2, H−C(3)], 4.93 [d, J = 11.7 Hz, H−C(2″)],
5.06 [d, J = 11.2 Hz, H−C(2″)], 5.43 [m, HO-C(3″)], 5.53 [d, J
= 12.2 Hz, H−C(2)], 5.57 [m, HO-C(3″)], 5.77 [d, J = 12.2
Hz, H−C(2)], 5.82 [s, H−C(6″)], 5.91−6.03 [4xs, H−C(6,8)],
6.73 [m, H−C(3′,5′)], 6.79 [d, J = 8.0 Hz, H−C(6‴)], 6.86−
6.90 [m, H−C(5‴)], 6.92 [s, H−C(2‴)], 7.01 [d, J = 1.5 Hz,
H−C(2‴)], 7.21 [m, J = 8.9 Hz, H−C(2′,6′)], 8.63−8.86
[4xbrs, HO-C(3‴, 4‴)], 9.33, 9.40 [brs, HO-C(4′)], 10.57,
10.91, 11.19 [brs, HO-C(7″,7)], 11.84, 11.92 [s, HO-C(5″)],
12.32, 12.39 [s, HO-C(5)]. 13C NMR (100 MHz, acetone-d6 +
DMSO-d6, 9/1, HSQC, HMBC): δ 48.1 [C-3], 72.8, 73.3
12.36 [s, HO-C(5)]. 13C NMR (100 MHz, acetone-d6
+
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Figure 3. HMBC spectrum (500 MHz, d6-DMSO) of (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (1).
[C-3″], 82.1, 82.5 [C-2], 83.8 [C-2″], 95.3, 95.4, 96.0, 96.4, 96.5
[C-6″,6,8], 100.3, 100.8 [C-4a″], 101.9 [C-4a], 101.8, 102.0
[C-8″], 115.2, 115.3 [C-3′,5′], 115.4, 115.5 [C-5‴],115.6, 115.7
[C-2‴], 118.1, 119.8 [C-6‴], 128.4, 128.5 [C-1‴], 128.6, 128.8
[C-1′], 129.1 [C-2′,6′], 145.2, 145.6, 146.0, 146.5 [C-3‴,4‴],
158.5, 158.6 [C-4′], 160.3, 161.0 [C-8a″], 163.0, 163.3 [C-5″],
163.5, 163.6 [C-8a], 164.7, 164.8 [C-5], 165.5, 166.0 [C-7″],
167.1, 167.2 [C-7], 197.3, 197.4 [C-4], 198.2 [C-4″].
95.5, 96.1 [C-6″,6,8], 101.1, 101.2 [C-4a,4a″], 101.6 [C-8″],
113.4 [C-2′], 114.0 [C-2‴], 114.7, 114.9, 115.1, 115.3, 115.6,
115.7 [C-2′,5′,2‴,5‴], 116.4, 117.2 [C-6‴], 118.5, 118.9 [C-6′],
128.5, [C-1′], 129.9 [C-1‴], 144.7, 145.1, 145.3, 145.5, 145.6,
145.7 [C-3′,4′,3‴,4‴], 159.9, 160.8 [C-8a″], 162.0, 162.4 [C-5″],
162.6, 162.8 [C-8a], 163.6, 163.7 [C-5], 164.6, 165.0 [C-7″],
166.3, 166.4 [C-7], 196.1 [C-4″], 196.6 [C-4].
(2R,3R,2″R,3″R)-Nonamethylmanniflavanone (3a).
Slightly brownish powder; UV (MeOH/H2O, 9/1, v/v) λmax
= 210, 290, 346 nm; (−) HRESIMS m/z 715.2390 [M − H]−
(2R,3S,2″S)-Buchananiflavanone, (2R,3S,2″S)-2-(3,4-Dihy-
droxyphenyl)-2,2′,3,3′-tetrahydro-5,5′,7,7′-tetrahydroxy-2′-
(3,4-dihydroxyphenyl)-[3,8′-Bi-4H-1-benzopyran]-4,4′-dione
(5, Figure 1). Colorless powder; UV (MeOH/H2O, 6/4, v/v)
λmax = 215, 225, 291, 347 nm; (−) HRESIMS m/z 573.1037
[M − H]− (calcd for C30H21O12, 573.1033). CD (MeOH, 0.37
mmol/L): λmax(Δε)= 341 (+1.5), 315 (−1.2), 298 (−5.6), 282
(+5.8), 239 (−2.5), 218 (−13.1). 1H NMR (400 MHz, DMSO-
d6, COSY): δ 2.60 [dd, J = 15.2 Hz, H−C(3″α)], 2.69 [2xdd,
J = 14.7 Hz, H−C(3″αβ)], 2.96 [dd, J = 13.6, 13.8 Hz, H−
C(3″β)], 4.50 [d, J = 12.1, H−C(3)], 4.63 [d, J = 12.0, H−
C(3)], 5.29 [d, J = 12.1 Hz, H−C(2″)], 5.39 [d, J = 12.2 Hz,
H−C(2)], 5.42 [d, J = 12.1 Hz, H−C(2″)], 5.64 [d, J = 12.0
Hz, H−C(2)], 5.80, 5.87, 5.90 [3xs, H−C(6,8,6″)], 6.49 [d, J =
12.1 Hz, H−C(6′)], 6.58−6.76 [m, H−C(5′,5‴,2′,2‴)], 6.62
[m, H−C(6‴)], 6.63 [m, H−C(6′)], 6.71 [m, H−C(6‴)], 6.81
[m, J = 7.7 Hz, H−C(2′)], 6.83 [m, J = 7.7 Hz, H−C(2‴)],
8.93−9.00 [brs, HO-C(3′,4′,3‴, 4‴)], 10.83 [brs, HO-C(7″,7)],
12.05, 12.15 [s, HO-C(5″)], 12.17, 12.21 [s, HO-C(5)]. 13C
NMR (100 MHz, DMSO-d6, HSQC, HMBC): δ 43.1 [C-3″],
47.4 [C-3], 78.5, 78.6 [C-2″], 81.6, 81.9 [C-2], 94.9, 95.0, 95.2,
1
(calcd for C39H39O13, 715.2391). H NMR (500 MHz, CDCl3,
COSY): δ 3.70−3.93 [9xs, CH3O−C(5,7,3′,4′,3″,5″,7″,3‴,4‴)],
4.48 [d, 1H, J = 12.5, H−C(3)], 4.73 [d, 1H, J = 12.6, H−
C(3″)], 5.65 [d, 1H, J = 12.4 Hz, H−C(2)], 5.83 [d, 1H, J =
12.7 Hz, H−C(2″)], 6.15−6.19 [3xs, 3H, H−C(6,8,6″)], 6.56
[d, J = 7.9 Hz, H−C(6′)], 6.61−6.91 [H−C(2′,5′,2‴,5‴)], 6.91
[d, J = 8.2 Hz, H−C(6‴)]. 13C NMR (125 MHz, CDCl3,
HSQC, HMBC): δ 49.1 [C-3″], 51.2 [C-3], 55.6−56.3 [CH3O-
(5,7,3′,4′,3″,5″,7″,3‴,4‴)], 81.4 [C-2″], 81.6 [C-2], 93.2−93.6
[C-6″,6,8], 100.7, 100.8 [C-4a″,4a], 105.8 [C-8″], 109.4−110.8
[C-2′,5′,2‴,5‴], 119.8 [C-6′], 120.2 [C-6‴], 130.4 [C-1‴],
130.7 [C-1′], 147.7−149.4 [C-3′,4′,3‴,4‴], 162.5−170.1 [C-
5,7,8a,8a″,5″,7″], 188.0 [C-4], 188.3 [C-4″].
Octamethylmanniflavanones (3b,c). Slightly brownish
powders; UV (MeOH/H2O, 9/1, v/v) λmax = 210, 290, 346 nm;
(−) HRESIMS m/z 701.2225 [M − H]− (calcd for C38H37O13,
701.2234). 13C NMR (125 MHz, CDCl3): δ 55.5−56.1 [CH3O-
(5,7,3′,4′,3″,5″,7″,3‴,4‴)].
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Figure 4. CD-spectra of (+)-(2R,3R)-taxifolin (6), (2R,3R)-taxifolin-6-C-β-D-glucopyranoside (1), and (2R,3R)-aromadendrin-6-C-β-D-
glucopyranoside (2).
(+)-(2R,3R)-Taxifolin (6, Figure 1). CD (MeOH, c =
0.43mmol/L): λmax(Δε) = 330 (+2.5), 295 (−10.0), 252 (+2.0),
244 (+1.7), 222 (+10.6), 208 (−4.7).
highest antioxidant activity was observed both in the ORAC
and hydrogen peroxide scavenging assays for fraction M3. In
both cases, the activity of this fraction represented about the
half of the whole activity of the crude extract (Table 1). This
was followed by fraction M5, which showed the second highest
activity in both assays, and by fractions M1, M2, and M4, which
all had a similar range of antioxidative activities. Fractions M6−
M8 had the lowest activities. Therefore, fractions M1−M5 were
further purified by means of RP-HPLC as described above.
The antioxidative compound no. 1 isolated from fraction M1
was obtained as a colorless amorphous powder. This
compound showed the typical absorption maxima expected
for flavanones. Results from electrospray ionization (ESI) MS
indicated that this compound forms an [M − H]− ion with m/z
465, as well as a fragment ion with m/z 345, as expected for a
C-glycoside. As shown in Figure 2, high resolution LC-MS
analysis confirmed the target compound to have the molecular
formula C21H22O12 and the fingerprint fragment C17H14O18.
RESULTS AND DISCUSSION
■
In a first antioxidative screening, the aqueous ethanolic extract
of Garcinia buchananii was analyzed by means of ORAC and
H2O2 assays (Table 1). The ethanolic extract of Garcinia
buchananii revealed an extraordinarily high antioxidant value of
1359 μmol TE/100 mg. This is higher than or comparable to
that of natural product extracts known to have high
antioxidative activities, such as bilberry, elderberry, red wine
extract, and grape seed extract. These have ORAC values of
265, 222, 694, and 1189 μmol TE/100 mg,19 respectively.
To get a deeper insight into the chemistry of this highly
antioxidative aqueous ethanolic extract of Garcinia buchananii,
as well as to focus on the highly antioxidative compounds, the
sample was fractionated using medium pressure liquid RP-18
chromatography. Eight fractions (M1−M8) were obtained and
then analyzed in their natural ratios for antioxidant activities
using the ORAC and H2O2 scavenging assays. By far, the
1
The H NMR spectrum of compound 1 showed an aromatic
singlet for H−C(8) at 5.92 ppm and three aromatic protons
H−C (5′,6′, 2′) resonating at 6.74 and 6.87 ppm. In addition,
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Figure 5. COSY-NMR (400 MHz, d6-acetone + d6-DMSO, 9/1, v/v) of (2R,3S,2″R,3″R)-manniflavanone (3).
the two aliphatic protons H−C(3) and H−C(2) were observed
coupling with each other at 4.45 and 4.97 ppm suggesting a
taxifolin aglycone. Besides the signals of the flavanone aglycone,
D-glucopyranoside,26 isolated also from fraction M1 and
detected as the major constituent in fraction M2.
To clarify the configuration of the carbon atoms C(2) and
C(3) present in the aglycone taxifolin and aromadendrin of
compounds 1 and 2, circular dichroism (CD) spectroscopic
measurements were performed using the commercially available
reference isomer (+)-(2R,3R)-taxifolin (6) as well as the
isolated C-glycosides 1 and 2 (Figure 4). The CD spectra of
(+)-(2R,3R)-taxifolin (6) was well in line with literature
data.27,28 The data obtained clearly demonstrated that the
spectra of C-glycosides 1 and 2 isolated from fraction M1 were
similar to the spectrum of (+)-(2R,3R)-taxifolin; therefore, the
stereochemistry could be deduced as (2R,3R)-taxifolin-6-C-β-D-
glucopyranoside (1) and (2R,3R)-aromadendrin-6-C-β-D-glu-
copyranoside (2).
1
the H NMR spectrum also exhibited seven aliphatic protons
resonating at 3.10 ppm [H−C(4″)], 3.14 ppm [H−C(5″)], 3.16
ppm [H−C(3″)], 3.40 ppm [H−C(6″α)], 3.67 ppm [H−
C(6″β)], 3.99 ppm [H−C(2″)], and 4.49 ppm [H−C(1″)] as
expected for a hexose unit.
Considering all the coupling constants of the sugar moiety in
the molecule, and, in particular, the coupling constant of J ∼9
Hz observed for the protons H−C(1″) and H−C(2″), and
comparing these values with the H−C(1)/H−C(2) coupling
constants reported for β-D-glucopyranosides and α-D-glucopyr-
anosides,21−24 the D-glucopyranose moiety was proposed and
the β-linkage undoubtedly identified.
A comparison of the 13C NMR spectrum, in which 21 signals
appeared, with the results of the heteronuclear single-quantum
correlation spectroscopy (HSQC) experiment showing 12
signals, revealed 9 signals corresponding to quaternary carbon
atoms. Unequivocal assignment of these quaternary carbon
atoms and the hydrogen-substituted carbon atoms, respectively,
could be successfully achieved by means of heteronuclear
multiple bond correlation spectroscopy (HMBC) and HSQC.
The typical 13C chemical shifts of the sugar part confirmed the
D-glucopyranose. Additionally, the HMBC experiment revealed
a correlation between the sugar proton H−C(1″) resonating at
4.49 ppm and neighboring carbon atoms C(5), C(7) and C(6),
as well as no correlation to C(8a), thus demonstrating clearly
the intramolecular 6-C-linkage of the β-D-glucopyranose to its
aglycone (Figure 3). The characteristic chemical shift of the
carbon atom C(1″) at 73.0 ppm confirmed the C-linkage of
the sugar part and the taxifolin-6-C-β-D-glucopyranoside.25 The
same strategy resulted in the structure of aromadendrin-6-C-β-
The antioxidative compounds 3−5 isolated from fractions
M3−5 showed the typical absorption maxima expected for
flavanones, and high resolution LC-MS analysis confirmed the
target compound to have the molecular formula C30H22O13 for
1
3 and C30H22O12 for 4 and 5, respectively. The H NMR
measurements of 3−5 in d3-MeOD (RT), DMSO-d6 (RT, 45
°C), and acetone-d6 (RT) as well as mixtures of acetone-d6 +
DMSO-d6 (9/1, v/v, RT, 8 °C) showed two series of signals
typical for biflavanoids and the duplication of nearly all signals,
indicating the presence of two main rotational isomers.28,29 The
1
sharpest signals were obtained at 8 °C (Figure 5). The H
NMR spectrum of compound 3 showed four sharp
exchangeable signals at 12.36 and 12.29 for HO-C(5) and
11.97 and 11.89 ppm for HO-C(5″), four broad exchangeable
singlets from 10.64 to 11.24 ppm for the OH-groups of 7 and
7″, several broad exchangeable signals between 8.80 and 9.00
ppm for HO-C(3′,4′,3‴,4‴), and two exchangeable doublets for
the protons HO-C(3″) at 5.64 and 5.80 ppm. Typical A-ring
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Figure 6. CD-spectra of (2R,3S,2″R,3″R)-manniflavanone (3), (2R,3S,2″R,3″R)-GB-2 (4), and (2R,3S,2″S)-buchananiflavanone (5).
aromatic singlets for H−C(6,8,6″) from 5.81 to 5.94 ppm as
well as characteristic C-ring aromatic protons resonating at
6.03−6.94 ppm could be observed. In addition, the two sets of
aliphatic protons H−C(3,3″) and H−C(2,2″) coupling with
each other were observed suggesting a GB-type biflavanoid
(Figure 5). The vicinal coupling constants of 11.3−12.1 Hz
indicated their trans-diaxial relative configuration.
Unequivocal assignment of carbon atoms was successfully
achieved by means of HMBC and HSQC. The HMBC
experiment revealed a correlation between the proton H−C(3)
resonating at 4.55 and 4.71 ppm and neighboring carbon atom
C(8a″) and C(7″), as well as no correlation to C(5″), thus
demonstrating clearly the intramolecular C-3/C-8″-linkage of
the two flavanone monomers. Additionally, methylation of 3
was performed,30 whereas after HPLC cleanup a mixture of two
8-fold methylated manniflavanone derivatives (3b,c) and
nonamethylmanniflavanone (3a) was obtained. It is known
from the literature27,31 that the 13C chemical shifts of sterically
hindered OMe substituents of flavonoids occur between 59 and
61 ppm as compared with 55−57 ppm for non ortho-
disubstituted methoxyl groups. In 3a−c, all aromatic OMe
substituents were observed between 55.5 and 56.3 ppm. If the
intramolecular linkage had been through C-3/C-6″, then C(5″)-
OMe would have been sterically hindered and consequently
deshielded. NMR data of compounds 3 and 3a are in line with
partially described data for manniflavanone.30
The 1/2D-NMR data of compound 4 was very similar to
manniflavanone (3), besides the substitution pattern of the
naringenin B-ring giving doublets (J = 8.9) H−C(2′/6′) and
H−C(3′/5′) at 7.21 and 6.73 ppm. Compound 4 was identified
as GB-2, naringenin-C-3/C-8″ dihydroquercetin linked biflava-
none.29,32
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manniflavanone (3), showing typical CD bands with corre-
sponding signs for 3−8″-biflavanones28 and/or mono flava-
nones like naringenin.34 In combination with Gaffield's rule,35
the results from Duddeck et al.27 and the detailed investigations
from Ferrari et al.28 for n → π* and π → π* transitions of 3−8″-
biflavanones consisting of either flavanones and hydroxyflava-
nones or flavanones and benzofuranones, the stereochemistry
of buchananiflavanone (5) could be deduced as (2R,3S,2″S).
Antioxidative Activity of Isolated Compounds 1−5.
Compounds 1−5 as well as known reference compounds with
high antioxidative activity,19,33 quercetin, rutin, (−)-epicatechin,
ascorbic acid, and ( )-naringenin, were analyzed by means of
ORAC and hydrogen peroxide scavenging assays (Table 2). In
comparison to known very antioxidative single compounds,
generally compounds 1−5 revealed relative high activity. By far,
the highest activity in both assays was observed for
(2R,3S,2″R,3″R)-manniflavanone (3) and (2R,3S,2″R,3″R)-GB-
2 (4), which showed outstanding activity in comparison to that
of quercetin, rutin, (−)-epicatechin, ascorbic acid, and
( )-naringenin as well as available literature data.19,33 Also,
all EC50 values of the hydrogen peroxide scavenging activity of
compounds 1−5 are lower than that of ascorbic acid. The
newly isolated compound (2R,3S,2″S)-buchananiflavanone (5)
revealed high activity in the hydrogen peroxide scavenging
assay and also a very strong activity in the ORAC assay.
Conclusions. The application of the newly developed
method for screening of natural antioxidative compounds by
means of the hydrogen peroxide scavenging assay dilution
analysis in combination with the ORAC assay on Garcinia
buchananii extracts, revealed (2R,3R)-taxifolin-6-C-β-D-gluco-
pyranoside (1), (2R,3R)-aromadendrin-6-C-β-D-glucopyrano-
side (2), (2R,3S,2″R,3″R)-manniflavanone (3), (2R,3S,2″R,3″R)-
GB-2 (4), and the previously unreported compound (2R,3S,2″S)-
buchananiflavanone (5) as highly antioxidative active constituents.
When taken together, our findings indicate that G. buchananii
bark extract is a rich natural source of antioxidants with the
potential to be utilized as food supplements in the future.
Table 2. Antioxidant Activities of Isolated Compounds 1−5
and Reference Compounds
ORAC
a,
b
c
,
d
e,f,g
H2O2 assay
assay
literature
(μmol
TE/
μmol)
EC50 (μM)
g
(2R,3R)-taxifolin-6-C-β-
D-glucopyranoside (1)
11.0 (9.0−11.4)
9.57
( 0.50)
n.r.
g
(2R,3R)-aromadendrin-6-
C-β-D-glucopyranoside
(2)
10.9 (9.5−12.9)
4.23
( 0.08)
n.r.
g
(2R,3S,2″R,3″R)-
manniflavanone (3)
2.8 (2.4−3.2)
2.2 (1.9−2.6)
14.4 (12.8−16.5)
11.3 (9.7−13.2)
16.5 (15.0−18.3)
6.9 (5.9−8.0)
6.1 (5.3−7.1)
4.1 (3.7−4.6)
8.6 (6.8−11.9)
13.73
( 0.43)
n.r.
g
(2R,3S,2″R″3R)-GB-2 (4)
12.10
( 0.26)
n.r.
g
(2R,3S,2″S)-
buchananiflavanone (5)
10.50
( 0.43)
n.r.
f
(+)-taxifolin (6)
ascorbic acid
rutin
7.63
( 0.68)
9.74
e
0.34
( 0.10)
0.95
e
f
6.45
( 0.28)
6.01 ;13.70
e
f
quercetin
5.61
( 0.07)
7.28 ;8.04
f
(−)-epicatechin
( )-naringenin
9.65
( 0.53)
9.14
f
3.96
( 0.19)
9.23
a
Each sample was analyzed by means of the H2O2 assay by triplicate
b
studies. The range in parentheses represents the 95% confidence
interval. Each sample was analyzed by means of the ORAC assay by
quadruplicate studies. The numerical value in parentheses represents
c
d
e
f
the SD. Values are from Ou et al. Values are from Wolfe and Liu in
which the stereochemistry of naringenin and taxifolin is not stated.
g
n.r.: not reported.
In comparison to manniflavanone (3), 1/2D-NMR measure-
ments of compound 5 revealed a methylene group at carbon
atom C(3″) resonating at 43.1 ppm and therefore diaster-
eotopic protons H−C(3″αβ). Again, unequivocal assignment of
carbon atoms could be successfully achieved by means of
HMBC and HSQC, and the linkage between the eridictyol
monomers was confirmed via HMBC experiment, revealing a
correlation between the proton H−C(3) resonating at 4.50 and
4.63 ppm and neighboring carbon atom C(8a″) and C(7″), as
well as no correlation to C(5″), thus demonstrating clearly the
intramolecular C-3/C-8″-linkage of the two eridictyol mono-
mers. To the best of our knowledge, compound 5, which we
have named buchananiflavanone, 3′,3‴,4′,4‴,5,5″,7,7″-octahy-
droxy-3,8″-biflavanone, has never been described before.
To clarify the configuration of the carbon atoms C(2) and
C(3) in compounds 3−5, the following CD spectroscopic
measurements were performed with the commercially available
(+)-(2R,3R)-taxifolin (6) as well as compounds 3−5. The CD
spectrum of GB-2 (4) (Figure 6) was well in line with literature
data.28 Consequently, the stereochemistry of GB-2 (4) could be
deduced as (2R,3S,2″R,3″R). Since the CD spectrum of
manniflavanone (3) was identical to GB-2 (4), the absolute
configurations must be the same, and thus, the absolute
configurations of manniflavanone must be revised to
(2R,3S,2″R,3″R)-manniflavanone (3) (Figure 6). The CD
spectrum of buchananiflavanone (5) (Figure 6) was also very
similar to (2R,3S,2″R,3″R)-GB-2 (4) and (2R,3S,2″R,3″R)-
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49-8161-71-2911. Fax: +49-8161-71-2949. E-mail:
timo.stark@tum.de.
Funding
S.P.P. and O.B.B. are supported by the University of Idaho
College of Science.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the NMR team of Lehrstuhl fur Lebensmittelchemie
und Molekulare Sensorik for performing the NMR measure-
̈
ments and Evelyn Eggenstein, Lehrstuhl fur Biologische
̈
Chemie, TU Munchen, for help with the CD spectrometer.
̈
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