New Acylated Phenolic Glycosides with ROS-Scavenging Activity from Psidium guajava Leaves

Article abstract of DOI:10.1021/acs.jafc.9b04318

Reactive oxygen species and subsequent oxidative stress are reported to play important roles in chronic metabolic diseases. Plant-derived polyphenols, especially food-derived phenolics, have attracted a lot of attention due to their potential usage against oxidative stress-related diseases. The leaf of Psidium guajava (known as guava) is regarded as a good resource of polyphenols and its products are commercially available in Japan as functional foods against multiple chronic metabolism disorders. In the course of finding novel polyphenols with antioxidative activities from guava leaf, 11 acylated phenolic glycosides (1-11), including 5 new oleuropeic acid-conjugated phenolic glycosides, named guajanosides A-E (1, 2, and 5-7), along with 17 known meroterpenoides (12-28), were isolated and identified. Their structures were determined by spectroscopic data analysis, chemical degradation, and acid hydrolysis. Compounds 1, 2, and 5-11 displayed potent reactive oxygen species-scavenging activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Western blot revealed that compound 6 markedly increased the expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO1), and the glutamate-cysteine ligase catalytic subunit. The current study revealed the presence of oleuropeic acid-derived phenolic glycosides in guava leaf and highlighted the potential usage of this type of phenolics against oxidative stress-related metabolic diseases via activation of the Nrf2 signaling pathway.

Full text of DOI:10.1021/acs.jafc.9b04318

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Bioactive Constituents, Metabolites, and Functions
New Acylated Phenolic Glycosides with ROS
Scavenging Activity from Psidium guajava Leaves
Yuanyuan Li, Dongli Li, Qi An, Hang Ma, Yu Mu, Wenjun Qiao,
Zengguang Zhang, Jingsheng Zhang, Xueshi Huang, and Liya Li
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04318 • Publication Date (Web): 11 Sep 2019
Downloaded from pubs.acs.org on September 12, 2019
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Page 1 of 37
Journal of Agricultural and Food Chemistry
New Acylated Phenolic Glycosides with ROS Scavenging Activity from Psidium
guajava Leaves
†
‡
†
‡,§
†
¶
Yuanyuan Li, Dongli Li, Qi An, Hang Ma, Yu Mu, Wenjun Qiao, Zengguang
Zhang, Jingsheng Zhang, Xueshi Huang, and Liya Li*,†
†
¶
*,†
†
Institute of Microbial Pharmaceuticals, College of Life and Health Sciences,
Northeastern University, Shenyang 110819, P. R. China
‡
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, P.
R. China
§
Bioactive Botanical Research Laboratory, Department of Biomedical and
Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston,
RI, 02881, United States
¶
Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,
Shenyang 110032, P. R. China
*
Author to whom correspondence should be addressed
Phone/Fax: 401-874-9367/5787; E-mail: huangxs@mail.neu.edu.cn
Phone/Fax: 0086-24-83656122; E-mail: lyli@mail.neu.edu.cn
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ABSTRACT: ROS and subsequent oxidative stress are reported to play important roles
in chronic metabolic diseases. Plant-derived polyphenols, especially food-derived
phenolics, have attracted a lot of attention due to their potential usage against oxidative
stress-related diseases. The leaf of Psidium guajava (known as guava) is regarded as a
good resource of polyphenols and its products are commercially available in Japan as
functional foods against multiple chronic metabolism disorders. In the course of finding
novel polyphenols with antioxidative activities from guava leaf, eleven acylated
phenolic glycosides (1-11), including five new oleuropeic acid conjugated phenolic
glycosides, named guajanosides A-E (1, 2, and 5-7), along with 17 known
meroterpenoides (12-28) were isolated and identified. Their structures were determined
by spectroscopic data analysis, chemical degradation and acid hydrolysis. Compounds
1, 2, and 5-11 displayed potent reactive oxygen species (ROS) scavenging activity in
LPS-stimulated RAW 264.7 macrophage cells. Western blot revealed that compound
6 markedly increased the expression levels of nuclear factor-erythroid 2-related
factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO1) and glutamate-
cysteine ligase catalytic subunit (GCLC). The current study revealed the presence
of oleuropeic acid-derived phenolic glycosides in guava leaf, and highlighted the
potential usage of this type of phenolics against oxidative stress-related metabolic
diseases via activation of Nrf2 signaling pathway.
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KEYWORDS: Psidium guajava, guava leaf, acylated phenolic glycosides, ROS
scavenging activity, Nrf2 signaling pathway
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INTRODUCTION
Reactive oxygen species (ROS) are a group of oxygen-containing redox-active
1
intermediates released by endogenous and exogenous processes. Although ROS have
been reported to play important roles in signaling cascade for normal physiological
functions, elevated cellular ROS levels which overcome endogenous antioxidant
capacity would cause oxidative stress condition and damage multiple biomolecules.2, 3
ROS and subsequent oxidative stress are reported to be implicated in multiple chronic
metabolic diseases, such as diabetes, cardiovascular disease, and neurodegenerative
disease.1, 4 Thus, maintaining redox homeostasis is essential for normal physiological
process in human body. Emerging evidences have demonstrated that natural
antioxidants could play positive effects against oxidative stress-induced chronic
metabolic diseases by acting as ROS scavengers.5, 6 Nuclear factor erythroid 2-related
factor 2 (Nrf2), which is expressed in a wide range of tissues and cell types, is a crucial
7
transcription factor involved in oxidation defensive system. Although Nrf2 in the
cytoplasm undergoes rapidly proteasomal degradation in the physiological condition, it
will be accumulated and translocated into nucleus under oxidative stress condition,
leading to the up-regulation of many antioxidant related enzymes, such as quinine
oxidoreductase 1 (NQO1) and γ-glutamyl cysteine synthetase catalytic subunit (GCLC)
to restore cellular redox homeostasis.8, 9 Extensive evidences demonstrate that Nrf2
signaling pathway plays a pivotal role in the prevention of cell injury resulting from
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oxidative stress. A lot of polyphenols, especially food-derived polyphenols, have been
found to active Nrf2 signaling pathway to reduce ROS production, and then exert health
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beneficial effects.9
Psidium guajava L., also known as guava, belongs to the family of Myrtaceae.
Being important food crop, guava is cultivated in the tropical and subtropical regions
worldwide, including south of China.11, 12 Besides the popular usage of its edible fruit
in food industry, guava leaf is also widely consumed as beverage in Japan, China and
Korea against multiple chronic diseases, such as diabetes and obesity.13, 14 Guava Leaf
Tea (Bansoureicha®, Yakult Honsha, Tokyo, Japan) has been approved and
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commercially available as one of the Foods for Specified Health Uses in Japan, and
regarded as a potential natural source for finding novel bioactive compounds against
metabolic diseases.10, 13 Previous phytochemical studies have revealed that abundant
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polyphenols, including flavonoids, tannins, meroterpenoides and benzophenones
are present in guava leaves. During the process of finding bioactive chemical
constituents against chronic metabolic diseases from guava leaves, five new oleuropeic
acid conjugated phenolic glycosides (1, 2, and 5-7) and six known analogues (3, 4, and
8-11), as well as 17 known meroterpenoides (12−28) were isolated and identified from
guava leaves. Oleuropeic acid conjugated phenolic glycosides are a group of phenolics
mainly found from Myrtaceous plants, especially from the genus Eucalyptus.18, 19, 20
The numbers of this type of phenolics reported are very limited (less than 40). They
usually possess a 5,7-dihydroxychromone, flavonone, or gallic acid skeleton, and are
conjugated with oleuropeic acid fragment. Previous pharmacological studies revealed
that this type of compounds possessed anti-tumor,21 anti-inflammatory,22 anti-
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melanogenesis, and antibacterial activities. To the best of our knowledge, this is the
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first report of oleuropeic acid conjugated phenolic glycosides from guava. Meanwhile,
to date, there is scarce of data about the ROS scavenging effect and mechanism of action
of this type of compounds.
Thus, we initiate the current study with following objectives: (1) to investigate the
underlying mechanisms of antioxidant action of these new acylated phenolic glycosides,
as well as (2) to better understand the relationship between the present acylated phenolic
glycosides and the health beneficial effects attributed to guava leaf. Macrophages are
crucial for the immune system and are vulnerable to ROS. Bacterial lipopolysaccharide
(LPS), an important component of the cell walls of gram-negative bacteria, can promote
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the production of ROS in macrophages and impact Nrf2 signaling pathway. LPS-
stimulated RAW 264.7 macrophage is widely accepted as a suitable model for the ROS-
scavenging activity evaluation and related mechanism study.7, 24, 25
Herein, we reported the isolation and structure elucidation of acylated phenolic
glycosides from guava leaves. In addition, ROS scavenging activities of these isolates
were evaluated with LPS-stimulated RAW 264.7 cells. We also attempted to elucidate
the underlying mechanisms of antioxidant action of these acylated phenolic glycosides
by investigating the involvement of the Nrf2 signaling pathway.
MATERIALS AND METHODS
General Experimental Procedures. Optical rotations were determined in MeOH
or CHCl using an Anton Paar MCP200 automatic polarimeter (Graz, Austria). IR
3
spectra were measured with a Bruker Tensor 27 FT–IR spectrometer (film). Ultraviolet
spectra were recorder with a Beckman Coulter DU 730 nucleic acid/protein analyzer
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(Brea, CA, USA). One- and two-dimensional NMR spectra were collected on a Bruker
Advance III-600 MHz spectrometer (Bruker Co., Rheinstetten, Germany) with
dimethyl sulfoxide (DMSO-d ) as the solvent. Semi-preparative HPLC was performed
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using an ODS column (250 mm × 10 mm, 5 μm, YMC-ODS-A, YMC Co. Ltd., Kyoto,
Japan). ESI-MS were recorded on an Agilent 1290-6420 Triple Quadrupole LC-MS
spectrometer (Santa Clara, CA, USA). HRESIMS was measured either with a Bruker
Micro TOF-Q mass spectrometer (Bruker Daltonics, Billerica, MA) or a Shimadzu
MALDI-TOF mass spectrometer (Shimadzu Corporation, Kyoto, Japan). Absorbance
and fluorescence values were analyzed using a microplate reader (BioTek Synergy H1,
BioTek Instruments, Winooski, VT, USA). Silica gel (100-200 mesh, 300-400 mesh,
Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare
Biosciences AB, Uppsala, Sweden), MCI gel (CHP-20P, Mitsubishi Chemical Corp.,
Tokyo, Japan), and ODS-A (S-50 μm, 12 nm, YMC Co., Ltd., Kyoto, Japan) were used
for column chromatography. D-glucose, L-glucose, D-galactose, and L-galactose were
got from Sinopharm Chemical Reagent Co., Ltd. (Shenyang, People’s Republic of
China). L-cysteine methyl ester hydrochloride was purchased from Maya Reagent
(Jiaxing, China). LPS, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT), and minocycline (MINO) were obtained from Sigma Chemical Co. (St. Louis,
MO, USA). All other chemicals and solvents were purchased from Sinopharm
Chemical Reagent Co., Ltd.
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Plant Material. The leaves of P. guajava were collected from Guangdong
Province, in August 2016. The plant was identified by Professor Xiaoji Zheng
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(Guangdong Jiangmen Chinese Traditional Medicine College, Jiangmen, China). A
voucher specimen (NO 1610056) was deposited at the Institute of Microbial
Pharmaceuticals, Northeastern University.
Extraction and Isolation. The air-dried leaves (4.0 kg) of P. guajava were
powdered and defatted with petroleum ether (2 × 10 L) at room temperature. The
residue was then extracted with 75% ethanol (3 × 20 L, 3 days each) at room
temperature for three times. The supernatant was combined and evaporated under
reduced pressure to afford a crude ethanol extract (1.0 kg). The ethanol extract was then
suspended in water and partitioned successively with petroleum ether (3 × 20 L), ethyl
acetate (3 × 20 L), and n-BuOH (3 × 20 L). After solvent removal, the ethyl acetate
extract (200.0 g) was subjected to a MCI gel CHP-20P column, eluting with MeOH-
H O system (0, 20, 40, 60, 80, and 100%) to afford six fractions A-F. Fraction D (30.0
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g, 60% MeOH eluted fraction) was subjected to silica gel column chromatography
(CH Cl /MeOH 50:1, v/v) to yield three subfractions (D1-3). Subfraction D1 was
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separated by ODS column (MeOH/H O 60%, v/v) and purified by Sephadex LH-20
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column chromatography with MeOH to yield compound 1 (5.9 mg). Subfraction D2
was chromatographed over an ODS column with (MeOH/H O 60%, v/v) elution system
2
to yield compounds 2 (10.0 mg) and 10 (15.0 mg). Subfraction D3 was successively
subjected to silica gel (CH Cl /MeOH 30:1, v/v), ODS (MeOH/H O 60%, v/v) and
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Sephadex LH-20 column (MeOH) to yield compounds 3 (59.2 mg) and 11 (20.6 mg).
Fraction E (35.0 g, 80% MeOH eluted fraction) was put on a Sephadex LH-20 column
and eluted with MeOH to yield three subfractions (E1-3). Subfraction E1 was
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successively chromatographed over silica gel column (CH Cl /MeOH 30:1, v/v), ODS
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column (MeOH/H O 60%, v/v), and semi-preparative HPLC (55% MeOH/H O, v/v) to
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yield compounds 4 (20.0 mg), 6 (6.8 mg), and 7 (7.5 mg). Subfraction E2 was separated
over ODS column (MeOH/H O 65%, v/v) to afford compound 8 (100.0 mg).
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Subfraction E3 was chromatographed over a silica gel column (CH Cl /MeOH, 20:1,
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v/v), and further purified by semi-preparative HPLC (45% MeOH/H O, v/v) to yield
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compound 9 (13.6 mg). In addition, the petroleum ether extract (160.0 g) of guava
leaves was chromatographed over silica gel, ODS, Sephadex LH-20, and semi-
preparative reversed-phase HPLC to afford 17 known meroterpenoides (12-28).
Detailed isolation procedures for these known compounds are provided in the
Supporting Information.
Compound 1: yellow amorphous powder; [α]2 +8.0 (c 1.0, MeOH); UV (MeOH)
0
D
λmax (log ε) 296 (3.54), 258 (4.03), 214 (4.31) nm; IR (film) νmax 3346 , 2960, 2879,
-1 1
1702, 1661, 1620, 1591, 1424, 1390, 1357, 1259, 1178, 1079, 1025, 917 cm ; H and
1
3
-
C NMR data (in DMSO-d ) see Table 1; HRESI-MS m/z 1039.3823 [2M-H] (calcd
6
for C H O , m/z 1039.3811).
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Compound 2: yellow amorphous powder; [α]2 -27.5 (c 1.8, MeOH); UV (MeOH)
0
D
λmax (log ε) 296 (3.89), 258 (4.46), 214 (4.81) nm; IR (film) νmax 3357 , 2961, 2929,
-1
2873, 1710, 1658, 1621, 1588, 1429, 1369, 1273, 1248, 1079, 1025, 1003, 916 cm ;
1
13
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H and C NMR data (in DMSO-d ) see Table 1; HRESI-MS m/z 1123.4754 [2M-H]
6
(calcd for C H O , m/z 1123.4750).
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75 22
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Compound 5: white amorphous powder; [α] -20.0 (c 1.0, MeOH); UV (MeOH)
D
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λmax (log ε) 258 (4.13), 218 (4.78), 196 (4.75) nm; IR (film) νmax 3371, 2967, 2931,
−
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1699, 1649, 1596, 1435, 1389, 1351, 1256, 1220, 1147, 1109, 924 cm ; H and C
-
NMR data (in DMSO-d ) see Table 1; HRESI-MS m/z 511.1812 [M-H] (calcd for
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C H O , m/z 511.1816).
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31 12
Compound 6: yellow amorphous powder; [α]2 -12.0 (c 1.0, MeOH); UV (MeOH)
0
D
λmax (log ε) 354 (4.53), 258 (3.77), 192 (4.61) nm; IR (film) νmax 3274, 2924, 2855,
1701, 1652, 1604, 1496, 1443, 1358, 1300, 1269, 1201, 1170, 1069, 1022, 997, 935 cm-
1
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; H and C NMR data (in DMSO-d ) see Table 2; HRESI-MS m/z 1223.3628 [2M-
6
-
H] (calcd for C H O , m/z 1223.3608).
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Compound 7: yellow amorphous powder; [α] +20.0 (c 0.6, MeOH); UV (MeOH)
D
λmax (log ε) 354 (3.96), 258 (4.17), 192 (4.73) nm; IR (film) νmax 3339, 2923, 2854,
-1
1698, 1651, 1604, 1507, 1445, 1359 1303, 1263, 1201, 1169, 1088, 1024, 999, 935 cm ;
1
13
-
H and C NMR data (in DMSO-d ) see Table 2; HRESI-MS m/z 1223.3604 [2M-H]
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(calcd for C H O , m/z 1223.3608).
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Acid Hydrolysis and Monosaccharide Determination of Compounds 5-7. Acid
hydrolysis of compounds 5-7 were performed according to a previously reported
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method. Briefly, compounds 5-7 (each 2 mg) were dissolved in 0.5 mL of MeOH and
then hydrolyzed with 2 M HCl (2 mL) at 80 °C for 4 h. After cooling, the yield mixture
was washed with CH Cl (2 mL) for three times, and the aqueous layer was dried with
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evaporator in vacuo. The dried powder was then dissolved in pyridine (1 mL) and
reacted with L-cysteine methyl ester hydrochloride (2 mg) at 80 °C with stirring for 2
h. Then N-trimethylsilylimidazole (0.3 mL) was added and reacted at 80 °C for another
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2 h. After that, 1 M HCl (5 mL) was added to the mixture and then extracted with
hexane (5 mL) for three times. The hexane extract was combined and submitted for
GC-MS analysis. Gas chromatography was performed with a Shimadzu GCMS-
QP2020 gas chromatograph mass spectrometer (Shimadzu Corporation, Kyoto, Japan)
equipped with an AOC-20i auto injector (Shimadzu Corporation). The separation was
conducted over a SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm × 0.25 μm,
Shimadzu Corporation) at a flow rate of 1 mL/min with a split ratio of 12:1. Helium
was used as carrier gas and the injection volume was 1 μL. The inlet temperature was
set as 270 °C. The initial oven temperature was 180 °C and raised to 280 °C by 5 °C/min,
hold for 3 min, and then reached 300 °C by 20 °C/min, and hold for 4 min. The retention
time of derivatives of standard D-glucose, L-glucose, D-galactose, and L-galactose
were 13.45, 13.74, 13.84 and 14.21 min, respectively. The configurations of D-glucose
for compounds 5 and 6, and D-galactose for compound 7 were determined based on
comparison of the retention time of corresponding derivatives with those of authentic
sugars derivatives prepared in the same method.
Methanolysis of Compound 8. A solution of compound 8 (6.7 mg) in MeOH (1
mL) containing 1% NaOMe was stirred at room temperature for 12 h. Then two drops
of AcOH was added and the yield mixture was dried under vacuum. The residue was
subjected to preparative thin layer chromatography (CH Cl /MeOH 15:1, v/v) to yield
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(+)-oleuropeic acid methyl ester 1b (2.6 mg): [α] +76.0 (c 1.0, CHCl ). H-NMR (in
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CDCl , 600 MHz): δ 7.01 (m, 1H, H-2), 3.75 (s, 3H, -OCH ), 2.56 (m, 1H, H-6a), 2.36
3
H
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(m, 1H, H-3a), 2.20 (m, 1H, H-6b), 2.04 (m, 1H, H-3b), 2.01 (m, 1H, H-5a), 1.57 (m,
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1H, H-5b), 1.57 (m, 1H, H-4), 1.24 (s, 3H, H-10), 1.23 (s, 3H, H-9). C-NMR (in
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CDCl , 150 MHz): δ 130.1 (C-1), 139.4 (C-2), 27.3 (C-3), 44.1 (C-4), 23.3 (C-5), 25.2
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C
(C-6), 167.8 (C-7), 72.4 (C-8), 26.6 (C-9), 27.4 (C-10), 51.6 (-OCH3).
DPPH Assay. The DPPH free radical scavenging assay was performed according
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to previously reported method. Briefly, a series of different concentrations of the
tested samples and the positive control ascorbic acid (6.25 to 200 μM) were mixed with
freshly prepared DPPH (0.2 mM) in a 96-well microplate with total volume of 150 μL.
After leaving the mixture reacted in the dark for 30 min, its absorbance was determined
at 520 nm. All the wells with sample only were set as blank to obtain an absorbance
value which was subtracted from the test sample readings. The free radical scavenging
Acontrol -
(
Asample - Ablank sample
)
capacity was calculated using the following equation: % SC =
Acontrol
× 100%. The IC values were calculated by SPSS 17.0 statistic software. Results were
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expressed as the mean ± SD of three replicates.
Reactive Oxygen Species Scavenging Assay. ROS scavenging activity of
compounds 1-11 were evaluated with LPS-induced RAW 264.7 cell model.
Intracellular ROS were determined by using a fluorescent probe 2′,7′-dichlorofluoresin
diacetate (DCFH-DA) (Beyotime Biotechnology, Shanghai, China), according to the
manufacturer’s instructions. Briefly, RAW 264.7 cells cultured in DMEM medium
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were seeded in 96-well-plates with 3 × 10 cells/well. After incubation for 24 h, LPS (1
μg/mL) and the tested samples (final concentrations at 30 μM) were added and co-
incubated for another 6 h. Then the supernatant of each well was removed and serum-
free medium with DCFH-DA (10 μM) were added into wells for 60 min treatment in
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the dark. After that, cells were washed with phosphate buffered saline and the
fluorescence of each well was measured by using a microplate reader at an emission
wavelength of 525 nm and an excitation wavelength of 488 nm. Minocycline (MINO)
was used as the positive control. All samples were tested in triplicate and showed no
cell toxicity against RAW 264.7 cells at 30 μM as shown in MTT assay (See Supporting
Information Figure S51).
Western Blotting. Compound 6 was selected for antioxidative mechanism study
due to its superior activity in ROS scavenging assay. The effects of compound 6 on
Nrf2 as well as its target downstream genes GCLC and NQO1 were investigated by
6
western blot experiment. RAW 264.7 cells were seeded at a 1 × 10 cells per well in a
6-well plate. After 24 h incubation, the cells were pretreated with different
concentrations of compound 6 (3.3, 10 or 30 μM) for 2 h, followed by exposure to LPS
(1 μg/mL) for additional 6 h. After that, cells were lysed with a RIPA buffer and then
centrifuged at 15 000 g for 15 min at 4 °C. The protein lysates were resolved by 10%
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and
electrophoretically transferred to PVDF membrane. The membrane was then blocked
with 5% (w/v) non-fat dry milk in TBST, and then incubated with specific primary
antibodies to Nrf2, GCLC, and NQO1 (Cell Signaling Technology, Danvers, MA, USA)
at 4 °C overnight. Β-actin was used as a loading control. Next day, the membrane was
incubated with goat anti-rabbit IgG-HRP secondary antibody (Cell Signaling
Technology, Danvers, MA, USA) at room temperature for 1 h. Chemiluminescence
using ECL detection kit (GE Healthcare, Piscataway, NJ, USA) was detected by using
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Bio-Rad ChemiDoc™ XRS+ System (Bio-Rad, Hercules, CA, USA). The experiment
was repeated for three times.
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Statistical Analysis. Quantitative data was presented as mean ± S.E. The
statistical analysis was performed by GraphPad Prism 4.0 (GraphPad Software Inc.,
USA) and SPSS 17.0 (IBM., USA). The statistical significance between groups was
interpreted by one-way ANOVA followed by Tukey’s test. All statistical tests with p <
0.05 were considered significantly different.
RESULTS AND DISCUSSION
The ethyl acetate extract of P. guajava leaves was subject to column
chromatography over MCI gel, silica gel, Sephadex LH-20 and semi-preparative HPLC
to yield 28 phenolics, including 11 acylated phenolics glycosides (1-11) and 17
meroterpenoides (12-28). Their structures were determined on the basis of
spectroscopic data analysis, chemical degradation, acid hydrolysis, as well as by
comparison of these to literature data (Figure 1). Among these isolates, five oleuropeic
acid conjugated phenolic glycosides (1, 2, and 5-7) are new compounds, and another
14 compounds (3, 4, 8-10, and 12-20) are reported from this plant for the first time.
Structure Elucidation of Isolated Phenolics. Compound 1 was obtained as
yellow amorphous powder, [α]2 +8.0 (c 1.0, MeOH). The negative HR-ESIMS of 1
0
D
-
gave a quasimolecular peak at m/z 1039.3823 [2M-H] , corresponding to the molecular
formula of C H O with 11 indices of hydrogen deficiency. The IR spectrum of 1
2
6
32 11
-1
-1
showed absorption bands of hydroxyl (3346 cm ), carbonyl (1702 and 1661 cm ), and
-1
aromatic ring (1620 and 1591 cm ) functionalities. The UV spectrum of 1 showed
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absorption maximum at 292 and 256 nm, indicating its 5,7-dihydroxychromone
2
7
1
skeleton. This conclusion was supported by H NMR data of 1 (in DMSO-d , 600
6
MHz, Table 1) which showed the presence of a hydroxyl group (δ 13.00, s, 1H, OH-
H
5), two singlet aromatic protons (δ 6.14, s, 1H, H-3; δ 6.18, s, 1H, H-6), and a singlet
H
H
1
methyl group at δ 2.31 (s, 3H, 2-Me). H NMR spectrum of 1 also revealed the
H
presence of an olefinic proton (δ 6.90, m, H-2′′), two singlet methyl groups (δ 1.05,
H
H
s, 6H, H -9′′ and H -10′′), and an anomeric proton of a glycosyl group (δ 4.64, d, 1H,
3
3
H
1
3
J = 10.2 Hz, H-1′). The C NMR and DEPT spectra of 1 displayed 26 resonances
ascribed to two carbonyl carbons, two olefinic carbons, eight aromatic carbons, one
oxygenated tertiary carbon, six methines (five oxygenated), four methylenes (one
oxygenated), and three methyls. All of the proton signals were assigned to the
corresponding carbons by HSQC spectrum analysis. The NMR data of 1 resembled
those of the known compound 8-β-C-glucopyranosyl-5,7-dihydroxy-2-
methylchromone (isobiflorin), which was isolated from Myrtaceae family previously,27
indicating compound 1 also possessed a 5,7-dihydroxy-2-methylchromone skeleton
and C-glucosyl residue. The linkage of the glucosyl moiety to chromone skeleton
through C-8 was deduced based on HMBC correlation signals from anomeric proton of
glucose (H-1′) to C-7 (δ 165.1), C-8 (δ 104.6) and C-9 (δ 157.1). The β-configuration
C
C
C
2
8
of glucose was determined by the large J value of H-1′ (10.2 Hz). Further comparison
of NMR data of 1 with isobiflorin revealed additional signals of a monoterpene, which
was deduced as oleuropeic acid based on COSY and HMBC experiments. The linkage
of oleuropeic acid moiety to β-glucose at C-6′ was established by the HMBC
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3
3
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correlations from H-6′a (δ 4.30, m, 1H) to carbonyl carbon at δ 166.8 (C-7′′), and
H
C
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01
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06
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supported by the remarkable downfield shift of C-6′ (ca. 3 ppm), as well as upfield shift
2
7
of C-5′ (ca. 3 ppm) when compared to isobiflorin. Although determination of the
absolute stereochemistry of oleuropeic acid unit in 1 with chemical degradation was
difficult due to its limited quantity, the presence of I-oleuropeoyl was deduced based
2
0
on the co-isolation of (+)-I-oleuropeic acid (1a) ([α] +76.0, CHCl ) from guava leaves
D
3
in the current project. This assumption was further supported by the methanolysis of
compound 8, another oleuropeic acid derivative isolated in the current project, which
2
0
19
gave (+)-I-oleuropeic acid methyl ester (1b) ([α] +76.0, CHCl ). On the basis of
D
3
above evidence, the structure of compound 1 was determined as 8-β-C-(6′-O-
oleuropeoyl)-glucopyranosyl-5,7-dihydroxy-2-methylchromone and named as
guajanoside A.
Compound 2 was obtained as yellow amorphous powder, [α]2 -27.5, (c 1.8,
0
D
MeOH). The molecular formula of 2 was deduced as C H O based on the
2
9
38 11
-
13
quasimolecular peak at m/z 1123.4754 [2M-H] in negative HRESIMS and C NMR
data. The IR spectrum of 2 highly resembled to that of 1. Comparison of the 1D NMR
data with those of compound 1 suggested the structure of compound 2 was also
composed of oleuropeoyl and 8-β-C-glucopyranosyl-5,7-dihydroxychromone moieties.
However, a group of proton signals for isobutyl group (δ 2.52, d, 2H, J = 7.2 Hz, H -
H
2
1′′′; δ 2.26, m, 1H, H-2′′′; δ 1.00, d, 6H, J = 6.6 Hz, H -3′′′ and H -4′′′), which was
H
H
3
3
1
1
deduced based on the H- H COSY experiment (as shown in Figure 2), rather than
methyl group were observed in 2, indicating the substitute at C-2 in 2 was replaced by
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isobutyl group. This conclusion was confirmed by the HMBC correlations from H-1′′′
to C-2 (δ 170.0), C-3 (δ 108.5), C-2′′′ (δ 26.9), C-3′′′ (δ 22.7) and C-4′′′ (δ 22.7),
C
C
C
C
C
and supported by HRESIMS data. In addition, HMBC correlation signal from glucosyl
H-2′ (δ 5.42, t, 1H, J = 10.2 Hz) to oleuropeoyl carboxylic carbon at δ 166.1
H
C
suggested the C-2′ linkage of oleuropeoyl group. This conclusion was supported by
observed upfield chemical shifts of C-1′, C-3′, C-6′, as well as downfield chemical
shifts of C-2′ and C-5′ in the glucosyl moiety relative to 1 due to esterification effect
(Table 1). The absolute stereochemistry of oleuropeoyl group in 2 was also assigned as
I like 1 as discussed above. Consequently, the structure of compound 2 was determined
as 8-β-C-(2′-O-oleuropeoyl)-glucopyranosyl-5,7-dihydroxy-2-isobutylchromone and
named as guajanoside B.
Compound 5 was obtained as a white amorphous powder. Its molecular formula,
-
C H O , was established on the negative HR-ESIMS (m/z 511.1812 [M-H] , calcd
2
4
32 12
1
3
511.1816) and C NMR data. The IR spectrum of 5 suggested the presence of hydroxyl
-1
-1
-1
group (3371 cm ), carbonyl (1699 and 1649 cm ), and benzene ring (1597 cm )
1
13
functionalities. Analysis of the H and C NMR spectra of 5 revealed it also had β-
1
glucopyranosyl and oleuropeoyl groups. In addition, H NMR spectrum of 5 showed
the presence of two singlet aromatic protons (δ 7.04, H-2; δ 7.08, H-6) and a
H
H
1
3
methoxyl group (δ 3.72, s, 3H). The C NMR spectrum of 5 showed six characteristic
H
aromatic carbon signals at δ 105.3, 111.1, 136.9 × 2, 150.4, and 152.9. The 1D NMR
C
1
9
data of 5 resembled to known compound eucalmaidin B, which was an oleuropeic
acid derivative possessed a gallic acid unit in the structure. By comparison of 1D NMR
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data of 5 with those of eucalmaidin B, an extra methoxyl group (δ 3.72, s; δ 56.5)
H
C
31
32
33
34
35
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38
39
40
41
was observed, which suggested one of the 17ethane17 groups on gallic acid fragment
in eucalmaidin B was methylated. In the HMBC spectrum, the correlation signals from
methoxyl protons to C-3 (δ 152.9) indicated methoxyl group was connected to C-3 of
C
gallic acid. Additionally, the β-glucopyranose moiety located at C-4 was determined
based on weak but detectable HMBC correlation between the aromatic proton (δ 4.78,
H
d, 1H, J = 7.2 Hz, H-1′) and C-4 (δ 136.9). Acid hydrolysis of 5 with 2 M HCl liberated
C
D-glucose, which was determined by subsequent GC-MS analysis following
derivatization to trimethylsilylated L-cysteine adduct. The presence of R-oleuropeoyl
unit was determined based on the co-isolation of 1a and methanolysis result of known
compound 8. Therefore, the structure of 5 was determined as 4-β-O-(6′-O-oleuropeoyl)-
glucopyranosyl-5-hydroxy-3-methoxybenzoate and named as guajanoside C.
0
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Guajanoside D (6) was obtained as a yellow amorphous powder with [α]2 -12.0
D
(c 1.0, MeOH). Its molecular formula C H O was elucidated from the HRESIMS
3
1
32 13
-
13
(m/z 1223.3628 [2M-H] ) and C NMR data (Table 2). The IR spectrum of 6 showed
-1
-1
absorption bands of hydroxyl (3274 cm ), carbonyl (1701 and 1652 cm ), and aromatic
-1
1
ring (1604 and 1496 cm ) functionalities. The H NMR spectrum of 6 showed the
presence of a hydroxyl group at δ 12.65 (5-OH), an ABX system at δ 6.82 (d, 1H, J
H
H
= 8.4 Hz, H-5′), 7.50 (s, 1H, H-2′), and 7.52 (d, 1H, J = 8.4 Hz, H-6′), as well as two
singlet aromatic protons at δ 6.17 (s, 1H, H-6) and 6.38 (s, 1H, H-8), suggesting the
H
1
3
presence of a quercetin moiety. This conclusion was supported by UV and C NMR
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spectral data. In addition, 1D NMR data (Table 2) revealed the presence of β-glucosyl
with anomeric proton at δ 5.53 (d, 1H, J = 7.2 Hz, H-1′′) and anomeric carbon at δ
H
C
100.7 (C-1′′). Acid hydrolysis of 6 liberated D-glucose residue. Apart from these two
moieties, the 13C NMR and DEPT data of 6 showed another 10 carbon signals,
including one carbonyl carbon (δ 166.3), four olefinic carbons (δ 149.0, 139.3, 129.6,
C
C
109.7), one 18ethane (δ 39.5), two methylenes (δ 30.7, 26.8, 24.4), and one methyl
C
C
(δ 21.1). The 1D NMR data of 6 highly resembled to those of known compound
C
2
9
8, .except that the signals for isopropanol unit in 8 were replaced by isopropenyl group
in 6. This indicated that the oleuropeic acid unit in 8 was changed to perillic acid and
compound 6 was the dehydration derivative of 8. This conclusion was confirmed by
HMBC correlation signals from H -9′′′ (δ 4.67, s, 1H; 4.72, s, 1H) to C-4′′′ (δ 39.5)
2
H
C
and C-10′′′ (δ 21.1), and from H -10′′′ (δ 1.69, s, 3H) to C-4′′′ and C-9′′′ (δ 109.7).
C
3
H
C
The absolute stereochemistry of C-4′′′ in 6 was also assigned as I based on biogenesis
consideration. Thus, the structure of guajanoside D (6) was established as shown in
Figure 1.
Compound 7, a yellow amorphous powder, had the same molecular formula of 6
-
as C H O deduced from its HRESIMS data (m/z 1223.3604 [2M-H] , calcd
3
1
32 13
1223.3608). The IR spectrum of 7 closely resembled to those of 6. Analysis of 1D and
2D NMR data suggested high similarities between the structures of 6 and 7 except for
the sugar moiety. The monosaccharide in 7 was identified as β-D-galactopyranose by
comparison of its NMR data with literature,30 along with acid hydrolysis and
subsequent GC-MS analysis. The HMBC correlations from H -6′′ (δ 4.05, m, 2H) to
2
H
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C-7′′′ (δ 166.4) inferred that the monoterpene also located at C-6′′ of β-D-galactose.
C
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The absolute stereochemistry of C-4′′′ in 7 was also assigned as I like 6 based on
biogenesis consideration. Thus, the structure of guajanoside E (7) was determined as
depicted in Figure 1.
Besides guajanosides A-E, six known acylated phenolic glycosides, including
3
1
31
29
29
cypellocarpin C (3), eucamalduside A (4), cypellogin A (8), cypellogin B (9),
quercetin-3-O-β-D-(6’’-O-p-coumaroyl)-galactopyranoside (10),30 and guavaric A
(11)32 were also isolated and identified from guava leaves. Compounds 1-4 were
glycosides bearing 5,7-dihydroxychromone, 5 was glycoside bearing galloyl group,
whereas 6-11 were quercetin glycosides. All of the above phenol glycosides were
esterified with (+)-oleuropeic acid except compounds 10 and 11. In addition, 17
3
3
33
33
meroterpenoides, including euglobal B1-1 (12), euglobal Ib (13), euglobal Ic (14),
euglobal III (15),34 euglobal Iib (16),34 euglobal-Iva (17),35 euglobal Ivb (18),35
euglobal V (19),36 ecalrobusone E (20),37 guajadial C (21),38 guajadial D (22),38
guajadial E (23),38 guajudial (24),39 macrocarpal A (25),40 psiguajadial H (26),41
4
1
41
psiguajadial I (27), psiguajadial J (28) were isolated and identified from the leaves
89 of guava. Among these known isolates, 14 compounds (3, 4, 8-10, and 12-20) are
90
91
92
93
94
reported from this plant for the first time. Although Myrtaceous plants were reported to
be rich in phenol glycosides esterified with oleuropeic acid, to the best of our
knowledge, this is the first report of this type of compounds from P. guajava.
Guava Leaf Phenolics Scavenged Free Radicals in the DPPH Assay and ROS
in LPS-induced RAW264.7 Macrophages. Since guava leaf tea has been widely used
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09
10
11
12
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15
to treat oxidative stress related diseases,42 antioxidative activities of these isolates were
evaluated with diphenylpicrylhydrazyl (DPPH) free radical scavenging assay. As
shown in Table 3, acylated phenolic glycosides (1-11) showed superior antioxidant
activity (IC < 200 µM except compound 3) compared with the meroterpenoides (IC
5
0
50
> 200 µM, see Supporting Information Table S1). It was noteworthy that the antioxidant
activities of compounds 1, 2, and 5-11 (IC values ranged from 84 to 131 µM) were
5
0
comparable to that of positive control ascorbic acid (IC = 108.60 µM). The DPPH
5
0
free radical scavenging activity difference among compounds 1-11 might be due to the
hydroxyl group numbers, which was in agreement with previous report.25 ROS
overproduction would break the balance of oxidative and antioxidative system, thereby
2
causing damage to healthy tissue cells. To further confirm the antioxidant activities of
these acylated phenolic glycosides, ROS scavenging activity of them were evaluated in
RAW 264.7 macrophages. As shown in Figure 3, upon exposure to LPS for 6 h, ROS
levels were significantly elevated (around 2-folds) in LPS-treated negative control
group when compared to the non-treated control group. All of the tested compounds (1-
11), as well as positive control minocycline, displayed potent ROS scavenging activity
at 30 µM in LPS-induced RAW 264.7 cells. Moreover, compounds 5-11 exhibited
comparable ROS scavenging activity to the positive control minocycline (See support
information Table S2). The above results suggested that acylated phenolic glycosides
in guava leaf could reduce oxidative stress in LPS-stimulated RAW 264.7 macrophages,
which was in agreement with the data obtained from the DPPH free radical scavenging
16 assay. The aforementioned results highlighted the potential usage of these acylated
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phenolic glycosides from guava leaves against oxidative stress-related chronic
metabolic diseases.
18
19
20
21
22
Effects of Guava Leaf Phenolics on Nrf2 Signaling Pathway. Nrf2 is a major
redox-sensitive transcription factor involved in modulating the expression of multiple
antioxidant target genes, and plays a crucial role in protecting macrophages from
damage due to elevated ROS.24, 43 NQO1, a NAD(P)H-dependent reductase capable of
23 reducing a broad range of substrates, is an important downstream gene of Nrf2.44
Besides NQO1, GCLC is also known as a target gene of Nrf2 and plays a key role in
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
4
5
the synthesis of glutathione, as well as related cellular redox process. To understand
the antioxidant mechanism of acylated phenolic glycosides in guava leaf, the effect of
compound 6 (which showed superior ROS-scavenging activity) on Nrf2 signaling
pathway was investigated. As showed in Figure 4, compound 6 significantly enhanced
the total protein levels of Nrf2, NQO1 and GCLC in LPS-stimulated RAW264.7 cells
at 10 and 30 μM. Our results indicated that compound 6 could act as an Nrf2 activator
to up-regulate the expression of its target antioxidant genes, and then reduce the
production of ROS in marcophages, thereby exerting antioxidant effects.
In summary, 28 phenolics, including 11 acylated phenolic glycosides and 17
meroterpenoides were isolated and identified from P. guava leaves. Among these
isolates, five are new compounds, and 14 are being reported from P. guava for the first
time. Isolated acylated phenolic glycosides display potent antioxidative activities in
both DPPH and ROS scavenging assay. Preliminary molecular mechanism
investigation reveals that acylated phenolic glycosides could active Nrf2 signaling
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pathway, which reduce ROS level and exert antioxidative effects. The current study
highlighted bioactive polyphenols present in guava leaf supporting its nutraceutical and
functional food applications. Further in vivo studies are warranted to confirm the
potential of these bioactive phenolics as dietary agents for the prevention and/or therapy
of oxidative stress-related chronic metabolic diseases.
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ASSOCIATED CONTENT
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Supporting Information
IR, HRMS, and NMR spectra of compounds 1, 2, 5-7, detailed information for isolation
procedure and DPPH free radical scavenging activities of known compounds 12-28, as
well as cell proliferation assay result and ROS scavenging ratio of compounds 1-11 are
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
* Tel/Fax: 0086-24-83656122; E-mail: lyli@mail.neu.edu.cn (L.-Y. Li)
* Tel /Fax: 0086-24-83656106; E-mail: huangxs@mail.neu.edu.cn (X.-S. Huang)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support was provided by the Youth Foundation of Wuyi University (No:
2017td01), Jiangmen Program for Innovative Research Team (No:
2018630100180019806) and Foundation from Department of Education of Guangdong
Province (No: 2016KCXTD005 and 2017KSYS010).
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FIGURE CAPTIONS
Figure 1. Structures of compounds 1-11.
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Figure 2. Key H- H COSY and HMBC correlations of compounds 2 and 5
Figure 3. Effect of compounds 1-11 on the production of ROS in LPS-stimulated RAW
264.7 macrophages. Data are the mean ± SD for three independent experiments. ### p
< 0.001 vs. control group; ** p < 0.01, *** p < 0.001 vs. LPS group.
Figure 4. Effects of compound 6 on the protein expression of Nrf2, GCLC and NQO1
in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells were pretreated with
different concentrations of compound 6 (3.3, 10 or 30 μM) for 2 h, followed by exposure
to LPS (1 μg/mL) for additional 6 h. The cells were lysed with RIPA buffer and the
protein levels for total Nrf2, GCLC and NQO1were measured by using immunoblot
analysis. β-actin was used as a loading control. MINO was used as positive control.
And all the experiments have been repeated three times independently. Data presented
as mean ± S.E., n = 3. # p < 0.05, ## p < 0.01 as compared with the control group; * p
< 0.05, ** p < 0.01, *** p < 0.001 as compared with the LPS group.
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