Phenolics from durian exert pronounced NO inhibitory and antioxidant activities

Article abstract of DOI:10.1021/acs.jafc.6b01580

Durian, known as the king of fruits, is native to Southeast Asia and popular in many countries. Bioactivity-guided fractionation of the peel of durian was applied to determine its bioactive constituents. Four novel phenolics, along with 16 known, were pur

Full text of DOI:10.1021/acs.jafc.6b01580

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Article
Phenolics from Durian Exert Pronounced
NO Inhibitory and Antioxidant Activities
Jianying Feng, Yihai Wang, Xiaomin Yi, Weimin Yang, and Xiangjiu He
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01580 • Publication Date (Web): 09 May 2016
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Journal of Agricultural and Food Chemistry
Phenolics from Durian Exert Pronounced NO Inhibitory and Antioxidant
Activities
Jianying Feng, Yihai Wang,* Xiaomin Yi, Weimin Yang, Xiangjiu He*
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
China
* Corresponding author:
Dr. Xiangjiu He (Tel/Fax: 86ꢀ20ꢀ3935ꢀ2132, Eꢀmail: hexiangjiu@163.com);
Dr. Yihai Wang (Tel: 86ꢀ20ꢀ3935ꢀ2140, Eꢀmail: wangyih88@163.com)
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ABSTACT
Durian, known as the king of fruits, is native to Southeast Asia and popular in many
countries. Bioactivityꢀguided fractionation of the peel of durian was applied to
determine its bioactive constituents. Four novel phenolics, along with sixteen known,
were purified and identified. Four novel phenolics were elucidated to be durianol A
(1), durianol B (2), durianol C (3) and 5′ꢀmethoxyꢀ7′ꢀepiꢀjatrorin A (4), respectively.
The novel compounds were elucidated to be durianol A (1), durianol B (2), durianol C
(3) and 5′ꢀmethoxyꢀ7′ꢀepiꢀjatrorin A (4), respectively. The antioxidant and NO
inhibitory activities were evaluated for the isolated phenolics. Some phenolics showed
significant antioxidant activity in DPPH and superoxide anion radical scavenging
capacity assay. Most of the phenolics revealed pronounced inhibitory effects on NO
production in murine RAW 264.7 cells induced by LPS, which showed more potent
NO inhibitory activity compared to indomethacin. The results strongly demonstrated
that the phenolics may be partially responsible for durian’s NO Inhibitory activity.
KEYWORDS: durian, Durio zibethinus, phenolics, antioxidant, NO inhibitory
activity
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INTRODUCTION
Known as the king of fruits, durian is native to Southeast Asia and popular in many
countries including China, Thailand, and Malaysia. Several kinds of wild Durio
species produce edible fruits, and only D. zibethinus Murr. is cultivated in large
quantities in Southeast Asia and China. The durian fruit is in round or oval shape,
about the size of a coconut. The peel of them is thorny, with a color from yellowish to
dark yellow. 1 Durian is an argumentative fruit and some people regard the durian as
having a pleasantly sweet fragrance, others find the aroma overpowering and
revolting. However, durian fruit is rich in nutrients of carbohydrates, protein and also
contains sufficient vitamins B and C. In addition to eaten raw, its attractive flesh is
frozen or dried for a popular snack. Previous chemical studies on Durio have resulted
in the identification of triterpenoids, phenolics, lignans, coumarins, flavonoids,
sulphurꢀcontaining compounds and some uncommon esters.2ꢀ5 In spite of the abundant
secondary metabolites in durian, few research has been concerned with the bioactive
constituent in durian, especially in its peel that has currently been treated as
agricultural waste.
Studies of epidemiology have revealed that high fruit consumption was beneficial
to prevent chronic diseases of atherosclerosis, cancer, diabetes and coronary heart
diseases.6 Antiꢀcytokines and antioxidants in fruits showed the potential to reduce the
probability of above diseases to improve human health.7 In the process of seeking
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effective bioactive components from medicinal plants, fruits and vegetables
,
detailed phytochemical characterization of phenolics in the peel of durian were
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conducted, and four new along with sixteen known phenolics were isolated and
identified in this study. The potent antiꢀinflammatory and antioxidant activities of the
isolated components were also evaluated in vitro.
MATERIALS AND METHODS
General Apparatus and Chemicals. PerkinElmer 100 IR spectrometer (Perkin
Elmer Inc., Waltham, MA, USA) was used to scan IR spectra with KBr and Bruker
AVANCE IIIꢀ500 NMR spectrometer (Bruker Inc., Fällanden, Switzerland) was
applied in obtaining NMR spectra. Highꢀresolution ESIꢀMS (HRꢀESIꢀMS) data were
acquired on a Waters AQUITY UPLC/QꢀTOF mass spectrometer (Milford, MA,
USA). Preparative HPLC was performed with a Rainin HPLC system (Rainin
Instrument Co. Inc., Woburn, MA, USA), including a Rainin pump, a Rainin UV
Absorbance detector of Model UVꢀD II and a Cosmosil HPLC column (5C18ꢀMSꢀII,
10ID×250 mm, Nacalai Tesque, Kyoto, Japan). Silica gel (200ꢀ300 mesh and 300ꢀ400
mesh) was obtained from Anhui Liangchen Silicon Material Co. Ltd. (Lu′an, China).
ODS (40ꢀ60 ꢀm, Merck KGaA, Darastadt, Germany) was used for medium pressure
liquid chromatography (MPLC). Methanol for HPLC was purchased from Oceanpak
Chemical Co. (Gothenburg, Sweden). (S)ꢀ(+)ꢀ2ꢀMethylbutyric acid (Acoros organics,
Belgium) were used for HPLC analysis, which was applied with Waters 600 and
Waters 996 Photodiode Array Detector (Milford, MA, USA). Sugar reagents for
GC/MS analysis were product of Sigma (St. Louis, MO, USA). A Varian CPꢀ3800 GC
(Varian Inc., Palo Alto, CA, USA) was applied for sugar analysis.
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Plant Material. The peels of durian were collected in Sep. 2014 from the local
fruit markets of Guangzhou (Guangzhou, China) and the species was identified by
Prof. X. J. He of Guangdong Pharmaceutical University. A voucher specimen (No.
GDPUꢀNPRꢀ201403) was deposited in the Lead Compounds Laboratory, School of
Pharmacy, Guangdong Pharmaceutical University.
Extraction and Isolation Procedure. The clean, airꢀdry peels of durian (20 kg)
were extracted with 70% EtOH for four times at reflux, and the combined filtrate was
concentrated under vacuum at 55 °C. The obtained extract was suspended in 15 L
distilled water and extracted with cyclohexane, chloroform, ethyl acetate and
nꢀbutanol successively. All fractions were tested in DPPH radicalꢀscavenging capacity
assay and NO inhibitory assay. The chloroform and ethyl acetate fractions were found
to exhibit preferable activities with IC50 of 195.43±17.10 and 230.38±15.13 ꢁg/ml in
DPPH assay, and with IC50 of 32.98±2.85 and 28.70±1.35 ꢁg/ml in NO inhibitory
assay. Therefore, the fractions of chloroform and ethyl acetate were selected for
further purification. The chloroform fraction (50 g) was divided to ten fractions
(A1ꢀA10) by a preliminary isolation of a silica gel column (CHCl3/MeOH, 100:0 to
0:100, v/v). The part of A6 (8 g) was isolated by silica gel (CHCl3/MeOH, 80:20 to
0:100, v/v), ODS MPLC (MeOH/H2O, 10:90 to 100:0, v/v), Sephadex LHꢀ20
(cyclohexane/CHCl3/MeOH, 5:5:1, v/v/v) column chromatography to get compounds
1 (3 mg), 8 (23 mg) and 9 (7 mg). Fraction A9 (5 g) was subjected to an ODS MPLC
and preparative HPLC (MeOH/H2O, 60:40, v/v) to obtain compounds 2 (11 mg), 7 (6
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mg), and 14 (3 mg). Fraction A5 (8 g) was separated by silica gel (CHCl3/
CH3COCH3, 80:20 to 0:100, v/v), ODS MPLC (MeOH/H2O, 10:90 to 100:0, v/v),
Sephadex LHꢀ20 (C6H12/CHCl3/MeOH, 5:5:1, v/v/v) column chromatography, and
finally subjected to a preparative HPLC to get compound 10 (33 mg). Compounds 6
(4.3 mg, MeOH/H2O, 50:50, v/v) and 11 (5 mg, MeOH/H2O, 70:30, v/v) were
obtained from Fraction A4 through preparative HPLC. Compounds 12 (1.8 mg) and
15 (3 mg) were purified by preparative HPLC from Fraction A9 using 30% and 70%
methanol as mobile phase, respectively.
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The ethyl acetate fraction (68 g) was subjected to a silica gel column
(CHCl3/MeOH, 100:0 to 0:100, v/v) to give 8 fractions (B1ꢀB8). Fraction B6 was
isolated by ODS column (MeOH/H2O, 10:90 to 100:0, v/v), and compounds 3 (4 mg),
4 (2 mg), 18 (6 mg), and 20 (5.7 mg) were obtained by preparative HPLC
(MeOH/H2O, 80:20, v/v). Compound 5 (3 mg) was purified from fraction B3 (2.9 g).
Compounds 16 (13 mg) and 17 (22 mg) were obtained by preparative thin layer
chromatography (pTLC) (CHCl3/CH3COCH3/MeOH, 6:2:1, v:v:v) from fraction B7.
Compound 19 (3 mg) was isolated by silica gel, Sephadex LHꢀ20, ODS MPLC and
preparative HPLC from fraction B4 using 70% methanol as mobile phase.
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Durianol A (1): [α]1D5 : −13.9 (c 0.1, MeOH); UV (MeOH) λmax 216, 290, 340 nm; IR
(KBr) νmax 3483, 2975, 2940, 2883, 1724, 1616, 1569, 1510, 1454, 1425, 1380, 1276,
1
1243, 1194, 1123, 1072, 1013, 920, 881, 833, 755, 682, 624, 581, 511 cm−1; H and
13C NMR data, see Table 1; HRꢀESIꢀMS m/z 461.1407 [M+Na]+ (calcd. for
C21H26O10Na, 461.1424).
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Durianol B (2): [α]1D5 : −6.8 (c 0.1, MeOH); UV (MeOH) λmax 204, 249, 289 nm; IR
(KBr) νmax 3368, 2963, 2676, 1729, 1705, 1606, 1520, 1463, 1432, 1384, 1283, 1188,
1
1066, 1023, 880, 762, 723, 615, 519 cm−1; H and 13C NMR data, see Table 1;
HRꢀESIꢀMS m/z 437.1443 [M+Na]+ (calcd. for C19H26O10Na, 437.1424).
Durianol C (3): [α]1D5 : −7.0 (c 0.1, MeOH); UV (MeCN) λmax 240, 286 nm; IR (KBr)
νmax 3413, 2971, 1694, 1604, 1511, 1425, 1242, 1181, 1074, 1024, 832, 631 cm−1; 1H
and 13C NMR data, see Table 1; HRꢀESIꢀMS m/z 411.1684 [M + H]+ (calcd. for
C20H27O9, 411.1655).
5′ꢀMethoxyꢀ7′ꢀepiꢀjatrorin A (4): [α]1D5 : −10.2 (c 0.12, MeOH); UV (MeOH) λmax
210, 309 nm; IR (KBr) νmax 3427, 2925, 2853, 1685, 1619, 1573, 1524, 1500, 1468,
1
1425, 1384, 1325, 1208, 1207, 842, 802, 724, 602 cm−1; H and 13C NMR data, see
Table 1; HRꢀESIꢀMS m/z 425.0854 [M + Na]+ (calcd. for C20H18O9Na, 425.0849).
DPPH Radical-Scavenging Capacity Assay. DPPH assay were applied to evaluate
antioxidant activities. The procedure of which were determined by reported method.11
Briefly, DPPH solution (in methanol, 50 mg/L, 995 ꢁL) was mixed with 5 ꢁL of each
of tested sample. The samples were dissolved in DMSO. The solution system was
shaken and reacted at room temperature in the dark for 30 min. The absorbance was
measured at 517 nm. Gallic acid and ascorbic acid were used as positive control.
Superoxide Anion Radical Scavenging Capacity Assay. The antioxidant of
superoxide anion scavenging capacity of the isolated phenolics were evaluated
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according to a described procedure with some modifification.12 The 1000 ꢁL reaction
mixture contained 445 ꢁL TrisꢀHCl (pH 8.1, 50 mM), 250 ꢁL NADH (0.15 mM), 50
ꢁL PMS (0.03 mM), 250 ꢁL NBT (0.10 mM) and 5 ꢁL tested sample. All tested
samples were dissolved in TrisꢀHCl (50 mM, pH 8.1). The reaction was conducted for
5 min at 37 °C, and initiated by the addition of PMS. Gallic acid and ascorbic acid
were used as positive control. The radical scavenging capacity was calculated in the
way: scavenging activity (%) = (B1 – B2)/B1×100，B1, B2 are the absorbance of the
blank and sample at 546 nm.
Nitric Oxide Inhibitory Assay. Macrophage RAW264.7 cells were cultured in
DMEM containing 10% FBS, 100 units/mL penicillin and 100 ꢁg/mL streptomycin at
37 °C with a humid atmosphere of 5% CO2/95% air. NO produced in the medium was
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measured by assaying the levels of NO2 via the Griess reaction.13
Acid Hydrolysis and GC Analysis of Sugars. The acid hydrolysis and GC
analysis were applied to determine the chirality of the sugars of new phenolic
glycosides. Compounds 1–3 (1.5 mg) were heated for 3 h at 90 °C in ampoule with 5
mL 2 M HCl. Then the residue was evaporated under vacuum at 55 °C after extracted
with EtOAc. The residue was dissolved in 600 ꢁL pyridine and 5 mg NH2OH·HCl
was added. Then the system was heated at 90 °C for half hour. 300 ꢁL Ac2O was
added to the mixture when the system cooled to room temperature. After
homogenized, the mixtures were heated at 90 °C for 1 h. The reaction mixtures were
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analyzed by GC using standard aldononitrile peracetates as reference samples after
cooling.
Identification of (2S)-Methyl Butanoic Acid. The unit of (2S)ꢀmethyl butanoic
acid in molecule was confirmed by a described procedure.14 Compounds 1, 2, 3 (2.0
mg) were dissolved in 2 mL 5% KOHꢀH2O and refluxed for 3 h. The reactant was
acidified to pH 4.0 with 4.0 M HCl and then extracted with CHCl3 for three times.
Then 2.0 mg N, Nꢀdimethylꢀ4ꢀaminopyridine was added after the aqueous fraction
was washed, dried in vacuum. The mixture was dissolved in 1.0 mL CHCl3. Then
10.0 µL (R)ꢀ1ꢀphenylethanol and 15.0 mg N, N′ꢀdicyclohexylcarbodiimide were
added after the mixtures cooled to 0 °C in ice bath. The system was reacted at 0 °C for
20 min and at room temperature for 15 h, and then filtrated. The filtrate was washed
with water (4 × 1 mL) and the reactant was analyzed by HPLC at following
chromatographic conditions: column RPꢀ18e (5 ꢁm), detector wavelength at 210 nm,
column temperature at 35 °C, with mobile phase of 65% MeOHꢀH2O at flow rate of
1.0 mL/min. The retention time of (R)ꢀ1ꢀphenylethylꢀ(S)ꢀ2ꢀmethylbutanoate was
17.90
min,
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(R)ꢀ1ꢀphenylethylꢀ(S)ꢀ2ꢀmethylbutanoate (tR 17.90 min). Reference substance was
obtained from (R)ꢀ1ꢀphenylethanol and (S)ꢀ2ꢀmethylbutanoic acid by the same
method.
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RESULTS AND DISCUSSION
Structural Elucidation of Phenolics. In this study, peels of durian were collected,
dried and extracted with 70% ethanol. The obtained extract was then successively
partitioned with different solvents and repeatedly chromatographed on silica gel,
Sephadex LHꢀ20, ODS MPLC and RPꢀHPLC to give four new phenolics, as well as
sixteen known congeners (Figure 1).
Compound 1 was purified as a bright white powder. The molecular formula of
C21H26O10 was deduced according to its HRꢀESIꢀMS at m/z 461.1407 [M+Na]+ (calcd.
1
for C21H26O10Na, 461.1424), which could be confirmed through its HꢀNMR and
13CꢀNMR. The maximum absorption peak at 340 and 216 nm in UV, and the strong
absorption at 1724 cm–1 in IR, indicated that compound 1 was a coumarin derivative.
The deduction was further verified by the proton signals at δH 6.32 (1H, d, J = 9.5 Hz,
1
Hꢀ3) and 7.95 (1H, d, J = 9.5 Hz, Hꢀ4) in HꢀNMR, which coincided with the
character of coumarin. Two characteristic singlet signals at δH 7.14 (1H, s) and 7.30
(1H, s) suggested the positions of Cꢀ6 and Cꢀ7 were substituted in coumarin.
Moreover, the 1HꢀNMR spectrum revealed a glucosyl anomeric proton at δH 5.18 (1H,
d, J = 7.3 Hz), a methoxyl group at δH 3.78 (3H, s) and the protons of 2ꢀmethylbutyric
acid in the higher field. The deduced fragments were linked by the correlations in
HMBC, which were showed in Figure 2. The sugars were determined as Dꢀglucose by
GC analysis of their chiral derivatives after acid hydrolysis. In the NOESY spectrum,
the main correlations approved the relative configuration of compound 1. The
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absolute configuration of (S)ꢀ2ꢀmethyl butyrate was determined by the standard
substance after derivatization according to the protocol described in the experimental
section.14 Based on above analyses, compound 1 was elucidated to be
6ꢀmethoxyꢀ7ꢀOꢀβꢀDꢀ [6ꢀ(S)ꢀ2ꢀmethylbutanoylꢀglucopyranosyl]ꢀcoumarin, and named
as durianol A. The full assignments of its NMR data were shown in Table 1.
Compound 2 was obtained as bright needle crystals. The molecular formula of
C19H26O10 was deduced on the basis of HRꢀESIꢀMS m/z 437.1443 [M+Na]+ (calcd.
for C19H26O10Na, 437.1424). The IR spectrum suggested the presence of hydroxyl
(3368 cm−1), alkyl (2963 cm−1), carbonyl (1705 cm−1), and aromatic ring (1606 and
1
1520 cmꢀ1) functionalities. The HꢀNMR spectrum showed typical signals for 1, 3,
4ꢀtrisubstituted benzene at δH 7.48 (1H, d, J = 9.1 Hz), 7.47 (1H, br. s) and 7.13 (1H,
d, J = 9.1 Hz) with a typical ABX spin system. The signal at δH 3.80 (3H, s) was a
methoxyl linked to an aromatic ring. The NMR spectrum of 2 showed an anomeric
proton signal at δH 5.08 (1H, d, J = 7.5 Hz), corresponding to an anomeric carbon at
δC 99.2 in the spectrum of HSQC. That indicated the presence of a sugar moiety
which was identified as Dꢀglucose by above GC analysis. And its coupling constants
1
(J = 7.5 Hz) exposed the βꢀanomeric configuration. In the higher filed of HꢀNMR,
the isobutyl signal was evident, which was further inferred as 2ꢀmethyl butyric acid
on the basis of signal of carbonyl in the 13CꢀNMR. Therefore, the molecule of
compound 2 was consisted of three parts, a βꢀglucopyranosyl, a 3ꢀhydroxyanisic acid
and a 2ꢀmethyl butyric acid. The 2DꢀNMR, including HSQC and HMBC, were
applied to elucidate the structure of 2. In HMBC, the correlations from Hꢀ1′ to Cꢀ4,
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from the two protons of Hꢀ6′ to Cꢀ1′′ provided the mutual connection positions of
three fragments, which the key correlations were shown in Figure 2. In the NOESY
spectrum, the main correlations approved the relative configuration of compound 2.
The absolute configurations of Dꢀglucose and (S)ꢀ2ꢀmethyl butyrate were determined
by GC and HPLC analyses of their relevant derivatives. Combined with the above
analyses, compound 2 was elucidated to be 3ꢀmethoxyꢀ4ꢀOꢀβꢀDꢀ[6ꢀ(S)ꢀ2ꢀ
methylbutanoylꢀglucopyranosl] benzoic acid, and named as durianol B. The full
assignments of its NMR data were shown in Table 1.
The IR spectrum compound 3 suggested the presence of hydroxyl (3413 cm−1),
alkyl (2971 cm−1), carbonyl (1694 cm−1), and aromatic ring (1604 and 1511 cmꢀ1)
functionalities. The molecular formula of C20H26O9 was verified on the basis of
1
HRꢀESIꢀMS m/z 411.1684 [M+H]+ (calcd. for C20H27O9, 411.1655). The HꢀNMR
spectrum showed characteristic proton signals for 1, 4ꢀdisubstitued benzene at δH 7.61
(2H, d, J = 8.3 Hz) and 7.03 (2H, d, J = 8.3 Hz) in a typical A2B2 spinning system.
The signals at δH 7.54 (H, d, J = 15.9 Hz) and 6.40 (H, d, J = 15.9 Hz) were a pair of
oleafic group in trans configuration. The sugar obtained from the hydrolysate was
identified as Dꢀglucose by GC analysis of their chiral derivatives. The anomeric
configuration of glucose was determined to be βꢀconfiguration by the coupling
constants of the anomeric proton at δH 4.99 (1H, d, J = 7.4 Hz). In the high filed of
1HꢀNMR, the isobutyl signal was evident and it was further inferred as 2ꢀmethyl
butyric acid on the basis of signal of carbonyl in the 13CꢀNMR. Therefore, three
fragments could be deduced in the molecule, which were βꢀDꢀglucopyranosyl, a
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transꢀcinnamic acid and a 2ꢀmethyl butyric acid. The 2DꢀNMR including HSQC and
HMBC data were applied to prove the connections of the deduced fragments. The
correlations from Hꢀ1′ to Cꢀ4, from the two protons of Hꢀ6′ to Cꢀ1′′ confirmed the
mutual connection position of three fragments in HMBC. Other key correlations in
HMBC were showed in Figure 2. In the NOESY spectrum, the main correlations
approved the relative configuration of compound 3. The fragment of (S)ꢀ2ꢀmethyl
butyrate was confirmed by standard substance after derivatives. From these results,
compound 3 was established as 4ꢀOꢀβꢀDꢀ[6ꢀ(S)ꢀ2ꢀmethylbutanoyl]ꢀglucopyranosyl
cinnamic acid, and named as durianol C.
Compound 4 was isolated as a paleꢀyellow oily matter. The molecular formula of
C20H18O9 was verified on the basis of HRꢀESIꢀMS at m/z 425.0854 [M+Na]+ (calcd.
for C20H18O9Na, 425.0849), which could also be confirmed through 1Hꢀ and
13CꢀNMR. The UV maximum absorption at 309 and 210 nm, and the absorption of IR
at 1685 cm–1 suggested that it was a coumarin derivative. The characteristic was
confirmed by the obvious signals δH 6.27 (Hꢀ3) and 7.91 (Hꢀ4) with coupling
1
constants of 9.5 Hz in the HꢀNMR spectrum. Meanwhile, there was found two
coincident proton signals at δH 6.76 indicating a symmetric benzene ring when
combined its 13CꢀNMR data. The deduction was determined by the longꢀrange
correlations between Hꢀ2′/6′ and Cꢀ3′/5′, Cꢀ1′, Cꢀ4′ in spectrum of HMBC. The other
signals were a fragment with three Oꢀbearing carbons. The above data were
coincident with the reported data of cleomiscosins.15 The structure of compound 4
1
was deduced by its Hꢀ1H COSY and HMBC experiment (Figure 2). Its relative
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configuration was determined using the NOESY spectrum. The correlations in
NOESY (Figure 3) were observed for Hꢀ7′/Hꢀ8′ with coupling constants of 7.9 Hz in
1
the HꢀNMR spectrum suggested that these protons lie on one face of the molecule
with a trans configuration. In view of the optical activity [α]1D5 = –10.2 (c 0.12, MeOH)
of compound 4 , it was concluded to be a single enantiomer and its absolute
configuration was opposite with 8′ꢀepiꢀcleomiscosin.16 Owing to these observations
and 1HꢀNMR, 13CꢀNMR data shown in the Table 1, compound 4 was elucidated to be
5′ꢀmethoxyꢀ7′ꢀepiꢀjatrorin A. The longꢀrange correlations between Hꢀ7′ and Cꢀ7 were
extreme weak in HMBC. Fortunately, it existed a pattern that Cꢀ7 shifted to high field
and Cꢀ8 shifted to low field when the connection mode converted from Cꢀ7′– Cꢀ7 to
Cꢀ8′–Cꢀ7.17 Based on above analyses, compound 4 was elucidated to be
5′ꢀmethoxyꢀ7′ꢀepiꢀjatrorin A, which was a novel compound as far as we knew.
The sixteen known compounds were confirmed as propacin (5),18 jatrocin A (6),
cleomiscosin A (7),19 cleomiscosin B (8),19 propacin isomer (9),20 7ꢀhydroxycoumarin
(10),21 scopoletin (11),21 fraxetin (12),22 fraxidin (13),22 3,4ꢀdihydroxybenzoic acid
(14),23 4ꢀhydroxyꢀ3ꢀmethoxybenzoic acid (15),23 ethyl protocatechuate (16),24
3,4ꢀdihydroxybenzaldehyde
(17),25
evofolinꢀB
(18),26
2ꢀhydroxyꢀ8αꢀ
28
hydroxycalamenene (19),27 αꢀconidendrin (20) by comparing spectroscopic data
with the reported compounds in literature.
Antioxidant Activity. The DPPH and PMS/NADHꢀNBT assays were applied to
evaluate the antioxidant activities. Ascorbic acid and gallic acid were used as positive
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292
293
294
295
296
297
298
299
300
control for their efficient antioxidant and widely used in food and medicinal industry.
In the DPPH assays (Table 2), compound 13 exhibited stronger activity with IC50 of
7.83±1.00 ꢀM than Gallic acid (19.10±2.23 ꢀM) and ascorbic acid (10.21±1.45 ꢀM).
Other phenolics showed weaker antioxidant activity by comparing with the controls.
It was noteworthy that compound 16 (ethyl protocatechuate), showed more potent
antioxidant capacity. Only few compounds (such as 13) showed pronounced activity
in the PMS/NADHꢀNBT assay (Table 2).
NO Inhibitory Activity. NO is a signal transmission molecule that act as a crucial
role in immune and inflammatory responses and neuronal transmission for brain. The
inhibitors of NO production had potential to turn into antiꢀinflammatory agents. The
isolates were tested on Abelson murine leukemia virusꢀinduced tumor (RAW 264.7)
where NO production originated in the stimulation of LPS. And the Griess reaction
was applied to quantify the effect of evaluation. Indomethacin (IC50 47.4 ꢁM), a
typical and common nonꢀsteroidal antiꢀinflammatory agent, was selected as positive
control. Most of the tested compounds showed obvious inhibitory effects on NO
production compared to indomethacin, and the result were exhibited in Table 3.
Among them, Compounds 8, 13, and 16 showed significant inhibitory activity with
IC50 values below 10 ꢁM, with the values of 3.56, 3.70 and 7.29 ꢁM, respectively. It
was noteworthy that compound 8 possessed pronounced inhibitory activity, while
compound 7 was weak. It could infer that the coumarinoligan with an aromatic moiety
close to the Cꢀ8 position, showed more potent effects on NO inhibitory activity.
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321
322
Meanwhile, the most of the tested compounds from durian had less IC50 values than
positive control. The MTT assay was applied to evaluated cytotoxicity of the isolated
phenolics to RAW 264.7 cells. The results showed that most of the tested isolates
exhibited no cytotoxic effect at effective concentration.
In conclusion, twenty phenolics, including four novels, were purified and identified
from durian. Some phenolics showed potent antioxidant and NO inhibitory activities.
The results strongly demonstrated the phenolics isolated from durian may be partially
responsible for its NO inhibitory activity.
Funding
This research was supported by Guangdong natural science foundation (No.
2014A030313588), the project of Guangdong provincial department of science and
technology (2015A020211027) and the national natural science foundation of China
(No. 81573303).
Notes
The authors declare no competing financial interest.
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Table 1. NMR data of compounds 1–4 (δ in ppm and J in Hz) a, b
1
2
3
4
no.
δH
δC
δH
δC
δH
δC
δH
δC
1
124.2
112.7
148.5
150.0
114.3
122.5
167.0
128.0
129.6
116.4
158.7
116.4
129.6
143.4
117.1
167.6
2
160.4
113.4
144.2
109.7
145.9
149.6
103.0
148.8
112.3
99.1
7.47 s
7.61 d (8.3)
7.03 d (8.3)
160.2
113.1
144.8
104.0
143.1
136.6
131.8
136.8
111.5
125.9
105.6
148.0
136.1
148.0
3
6.34 d (9.5)
7.97 d (9.5)
7.30 s
6.27 d (9.5)
7.91 d (9.5)
6.67 s
4
5
7.13 d (9.1)
7.48 d (9.1)
7.03 d (8.3)
7.61 d (8.3)
7.54 d (15.9)
6.40 d (15.9)
6
7
8
7.14 s
9
10
1′
2′
3′
4′
5′
6′
5.18 d (7.3)
3.30ꢀ3.36
3.30ꢀ3.36
3.16 m
5.08 d (7.5)
3.20ꢀ3.32
3.20ꢀ3.32
3.16 m
99.2
76.5
72.9
70.0
73.9
5.00 d (7.4)
3.20ꢀ3.40
3.20ꢀ3.40
3.16 m
99.7
76.2
73.0
70.0
73.8
76.4
6.76 s
72.9
70.1
3.77 m
73.8
3.65 m
3.66 m
4.03 dd (11.8, 8.0)
4.33 dd (11.8, 1.8)
4.02 dd (11.7, 7.6)
4.31 dd (11.7, 1.8)
4.04 dd (11.6, 7.3)
4.33 d (11.6)
63.6
63.5
6.76 s
105.6
76.4
77.8
59.9
7′
8′
4.97 d (7.9)
4.33 m
9′
3.65 m, 3.27 m
1″
175.6
40.1
26.1
11.1
16.1
56.0
175.4
40.2
26.2
11.3
16.1
55.6
175.4
40.1
26.1
11.2
16.1
2″
2.35 m
2.32 m
2.35 m
3″
1.32 m,1.49 m
0.68 t (7.4)
1.02 d (7.0)
3.78 s
1.34 m, 1.51 m
0.76 t (7.4)
1.03 d (7.0)
3.80 s
1.38 m, 1.52 m
0.77 t (7.4)
1.05 d (6.9)
4″
5″
ꢀOMe
3.77 s
56.1
a Measured in DMSOꢀd6.
b Assignments were based on HSQC, HMBC, and 1Hꢀ1H COSY experiments
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Table 2. Antioxidant activities of the phenolics from durian a
IC50 b (µM)
PMS/NADH-NBT
Compd.
DPPH
568.50±6.28
268.37±11.02
807.22±18.66
260.26±5.44
7.83±1.00
6
−
394.34±71.33
7
8
−
12
−
13
11.4±1.44
56.61±3.09
15
−
23.82±0.54
870.07±63.28
16
17
58.13±0.72
494.85±14.91
1201.88±30.76
90.97±2.10
19
−
−
20
372.08±73.02 c
195.43±17.10 c
230.38±15.13 c
392.06±85.04 c
10.21±1.45
70% Ethanol exact of durian
Chloroform fraction
Ethyl acetate fraction
n-Butanol fraction
Ascorbic acid
Gallic acid
54.12±8.31
19.10±2.23
83.29±14.22
a Data are represented as the mean value ± SD, n=3; b The test concentrations ranged from 0 to
1500 ꢁM; c The concentration units are ꢁg/ml.
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Table 3. Inhibitory effects of the phenolics on NO production induced by LPS in
RAW 264.7
IC50 (µM)
IC50 (µM)
Compd.
Compd.
NO Inhibitory Assay MTT Assay
NO Inhibitory Assay MTT Assay
36.32±1.39
38.07±2.40
41.26±0.44
35.23±1.79
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
17.87±2.79
31.53±2.24
7.29±1.00
16.03±2.98
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
1
2
3
4
5
6
7
14
15
16
17
18
19
21.70±2.35
28.88±2.08
32.91±0.74
30.82±3.17
20
70% Ethanol exact of
durian
3.56±0.49
>50.00
21.29±2.54 b
>50.00 b
8
>50.00
>50.00
>50.00
>50.00
>50.00
>50.00
Chloroform fraction
Ethyl acetate fraction
n-Butanol fraction
Indo a
32.98±2.85 b
28.70±1.35 b
33.06±1.12 b
47.40±4.50
>50.00 b
>50.00 b
>50.00 b
>50.0
9
30.28±3.56
26.01±3.37
28.15±3.94
3.70±1.75
10
11
12
13
a Indomethacin, positive control;
b The concentration units are ꢁg/ml.
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Figure Captions
Figure 1. Chemical structures of compounds 1ꢀ20 isolated from durian
Figure 2. The selected key 1Hꢀ1H COSY and HMBC correlations of compounds 1
Figure 3. The key NOESY correlations of compound 4
−4
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Figure 1
O
9
4''
4''
4''
3''
3''
COOH
3''
3
4
8
2
O
7
6
2''
2''
6
3
2''
9
5''
5''
O
O
1
O
5''
10
COOH
1
5
8
5
7
1''
O
1''
O
1''
O
6'
4
6'
7
6'
4
2
5
2
O
O
O
3
6
5O' H
5O' H 2'
5O' H
O
O
O
4'
4'
4'
O
O
2'
1'
2'
1'
1'
OH3'
OH 3'
OH 3'
OH
OH
OH
3
2
1
5
4
R2
R2
6
HO
O
3
10
9
2
O
O
O
7
O
O
O
O
7
O
8
2'
O
7'
1'
O
8'
O
O
R1
3'
6'
HO
R1
9'
4'
HO
HO
5'
O
O
O
OH
R1=CH2OH R2=OMe
R1=CH3 R2=OMe
5
6
7
R1=CH3
R1=CH2OH R2=OH
R1=CH2OH R2=OMe
R2=OMe
8
9
4
O
R2
R1
R2
O
OH
HO
O
O
O
R1
R3
O
OH
OH
10 R1=H
R2=OH
R2=OH
R2=OMe
R2=OH
R3=H
R3=H
R3=OH
R3=OH
14 R1=OH R2=OH
15 R1=OMe R2=OH
16 R1=OH R2=OC2H5
17 R1=OH R2=H
18
11 R1=OMe
12 R1=OMe
13 R1=OMe
O
O
O
HO
OH
HO
O
OH
20
19
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Figure 2
O
O
O
O
COOH
O
O
O
O
O
O
OH
OH
OH
OH
O
O
OH
OH
1
2
COOH
HO
O
O
O
O
O
O
O
O
O
OH
OH
HO
OH
OH
O
4
3
1H-1H COSY
HMBC
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Figure 3
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TOC Graphic
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