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Original article
01 2021
:15;
103507
doi:
10.1016/j.arabjc.2021.103507

Integrating approach to discover novel bergenin derivatives and phenolics with antioxidant and anti-inflammatory activities from bio-active fraction of Syzygium brachythyrsum

, , , , , , , ,
a Key Laboratory of Quality Evaluation of Chinese Medicine of the Guangdong Provincial Medical Products Administration, the Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
b Anhui Agricultural University, Hefei 230036, China
c Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangdong provincial Hospital of Chinese Medicine, Guangzhou 510006, China
d Guangzhou Key Laboratory of Chirality Research on Active Components of Traditional Chinese Medicine, Guangdong provincial Hospital of Chinese Medicine, Guangzhou 510006, China

⁎Corresponding authors at: Key Laboratory of Quality Evaluation of Chinese Medicine of the Guangdong Provincial Medical Products Administration, the Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou 510006, China. ginniezj@163.com (Jing Zhang), freexuwen@163.com (Wen Xu)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Syzygium brachythyrsum is an important folk medicinal and edible plant in Yunnan ethnic minority community of China, however, little is known about the chemical and bio-active properties. The present study is aimed to identify the bioactive constituents with antioxidant and anti-inflammatory properties by an integrating approach. First, two new bergenin derivatives, brachythol A (1) and brachythol B (2), together with eleven known phenolic compounds (3–13) were isolated from bioactive fractions by phytochemical method. Among these isolated chemicals, five bergenin derivatives, along with 3 phenolics were found in Syzygium genus for the first time. Then, a further chemical investigation based on ultra-high-performance liquid chromatography-Q Exactive Orbitrap mass spectrometry resulted in a total of 107 compounds characterized in the bio-active fractions, including 50 bergenin derivatives, among which 14 bergenin derivatives and 14 phenolics were potential new natural chemicals. Most of the isolated compounds showed obvious antioxidant activities, while compounds 11, 12, and 13 had favorable performance. Eight compounds (2–5, 7, and 9–11) showed good inhibitory activity on nitric oxide (NO) production in macrophage RAW 264.7 cells. The structure–activity correlation analysis indicated that the antioxidation and anti-inflammatory activities enhanced when bergenin was esterified with gallic acid, caffeic acid or ferulic acid. This is the first report of bergenins in Syzygium genus and the richness in new bio-active bergenins and gallic acid derivatives indicated that Syzygium brachythyrsum is a promising functional and medicinal resource.

Keywords

Syzygium brachythyrsum
Bergenin derivatives
Antioxidant
Anti-inflammatory
LC-MS

Abbreviations

TE

trolox equivalent

Fe2+E

Fe2+ equivalent

NO

nitric oxide

QE

Q Exactive

WF

water fraction

EAF

ethyl acetate fraction

DF

dichloromethane fraction

DPPH

1,1-diphenyl-2-picrylhydrazyl

FRAP

ferric reducing antioxidant power

CC

column chromatography

TLC

thin-layer chromatography

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

DMEM

dulbecco's modified Eagle medium

FBS

fetal bovine serum

LPS

lipopolysaccharide

DXMS

dexamethasone

DDA

data- dependent acquisition

LC

liquid chromatography

HPLC

high performance liquid chromatography

HSQC

1H detected heteronuclear singular quantum correlation

HMBC

1H detected heteronuclear multiple bond correlation

1H–1H COSY

1H–1H correlation spectroscopy

NMR

nuclear magnetic resonance

GQA

galloylquinic acid

1

1 Introduction

The genus Syzygium (Myrtaceae family) has more than 1200 to 1800 species, mostly shrubs and evergreen trees, mainly distributed in Africa, the Hawaiian Islands, India, China, Australia and New Zealand (Chua et al., 2019). Many Syzygium species with high economic value, among which S. aromaticum (clove) and S. samarangense are the most famous, were usually used as medicinal and fruits, as well as in the perfumery industry. Clove is considered as a natural food preservative and medicinal plant due to its antioxidant and antimicrobial activities (Batiha et al., 2020; Cortés-Rojas et al., 2014). The fleshy fruit of S. samarangense, known as wax apple with an aromatic flavor, is a fruit of important economical fruit in southeast Asian countries. Moreover, in the southern region of China, the leaves of this plant were consumed in a form of herbal tea as a folk medicine for the treatment of fever, eczema, and diarrhea (Sobeh et al., 2019; Yang et al., 2018). Many Syzygium species had been also used as traditional medicines worldwide and phytochemical investigations on them led to the discovery of a series of secondary metabolites such as flavonoids (Jayasinghe et al., 2007; Samy et al., 2014), terpenoids (Umehara et al., 1992; Xu et al., 2018), phenols (Li et al., 2015; Yang et al., 2018), phloroglucinols (Xu et al., 2020), essential oils (Maroyi, 2018) and with diverse bioactivities (Ryu et al., 2016), including antibacterial (Famuyide et al., 2019; Nirmala et al., 2019; Wamba et al., 2018), antibiofilm (Famuyide et al., 2019; Santos et al., 2020), antifungal (Pereira et al., 2016), anti-inflammatory (Chandran et al., 2018), antioxidant (Radünz et al., 2019), anticancer (Batiha et al., 2020), and hypoglycemic (Chua et al., 2019) activities.

Syzygium brachythyrsum, a small evergreen tree mainly distributed in the southeastern of China, is a local functional herbal tea and an important folk medicinal plant for the local Dai ethnic minority community (Li et al., 2008). It had different local names like “wild holly fruit”, “mountain syzygium”, “mali fruit”, and had the effect of relieving cough and asthma, mainly used for the treatment of cold asthma, allergic asthma, etc. (Li et al., 2008). However, the phytochemical and bio-active properties of this species still remain unexplored. Therefore, the bioactive-guided (antioxidant and anti-inflammatory) chemical investigation of S. brachythyrsum were carried out by an integrating approach, and the biological activities of the obtained compounds were evaluated. Moreover, a qualitative approach based on ultra-high-performance liquid chromatography Q Exactive Orbitrap mass spectrometry (UHPLC-QE Orbitrap MS) was developed to comprehensively investigate the chemical profile of S. brachythyrsum. This study is the first time to profile phytochemicals and evaluate biological activities of S. brachythyrsum, which will help to further develop its medicinal and functional food properties.

2

2 Materials and methods

2.1

2.1 General

Optical rotations were measured with an automatic polarimeter system (Rudolph Research Analytical, USA). NMR spectra were performed on a Bruker Avance ARX-600 spectrometer (Bruker BioSpin Group, Billerica, MA, USA) with TMS as internal standard. HR-ESI-MS were recorded on a Q-Exactive Plus hybrid Oribtrap MS system (Thermo Fisher Scientific, Rockford, IL, U.S.A.). Semi-preparative HPLC was performed on an Agilent 1200 system with a DAD detector, and an ODS silica column (10 × 250 mm, 5 µm) (YMC-Pack Ph, YMC Co., Ltd., Kyoto, Japan). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, Shandong, China), Sephadex LH-20 (Amersham, Sweden), and C18 reversed-phase silica gel (YMC ODS gel, YMC Co., Ltd., Kyoto, Japan) were used for column chromatography (CC). Thin-layer chromatography (TLC) was carried out with high-performance TLC plates pre-coated with silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, Shandong, China) and RP-18 F254S (Merck). lipopolysaccharide (LPS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and FRAP assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Cell culture medium [Dulbecco's modified Eagle medium (DMEM)], penicillin/streptomycin, fetal bovine serum (FBS), and all other materials required for culturing cells were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA).

2.2

2.2 Plant material

The leaves of S. brachythyrsum were collected in Menghai County, Xishuangbanna Dai nationality Autonomous Prefecture, Yunnan Province, China, in March 2019. The origin of the herbal material was authenticated by Prof. Guangxiong Zhou (Jinan university, Guangzhou). A voucher specimen (NO. 201903S) was deposited at Guangdong Provincial academy of Chinese medical sciences.

2.3

2.3 Extractions and isolations

The air-dried leaves of the S. brachythyrsum (10 kg) was extracted three times with 100 L of 95% ethanol at room temperature for 48 h. The combined solvent was concentrated under reduced pressure to obtained a crude extract (1.8 kg). The crude extract was suspended in distilled water (2.5 L) and extracted orderly with petroleum ether (5 × 2.5 L, boiling range: 60–90 ℃), dichloromethane (5 × 2.5 L), and ethyl acetate (5 × 2.5 L), obtained ethyl acetate fraction (EAF, 200.0 g), dichloromethane fraction (DF, 97.0 g), petroleum ether fraction (PEF, 230.0 g) and water fraction (WF, 210.0 g), respectively. Three fractions (WF, EAF, and DF) were tested in antioxidant (scavenging activities on DPPH and FRAP) and anti-inflammatory assays, and the EAF extracts were found to exhibit the strongest activities of all these fractions and then subjected to further isolation and chemical analysis.

The EAF extracts (200.0 g) were mixed with silica gel (200–300 mesh, 200.0 g) and then fractionated by a silica gel (200–300 mesh, 2.0 kg) column chromatography (CC) eluted with gradient CH2Cl2 - MeOH (100:0–0:100, v/v). 13 fractions (Fr. 1 - Fr. 13) were obtained by gradient elution with CH2Cl2 - MeOH (100:0–0:100, v/v) and combined according to similar TLC profiles. Fr. 10 (75.0 g) was isolated on silica gel (200–300 mesh) CC, isocratic elution with CH2Cl2 - MeOH (20:1–15:1, v/v), and a total of seven fractions (Fr. 10.1 - Fr. 10.7) were obtained. Fr. 10.1 (1.6 g) was separated by silica gel (100–200 mesh) CC with CH2Cl2 - MeOH (20:1, v/v) to obtained compound 6 (53.0 mg). Fr. 10.2 (100.0 mg) was isolated on silica gel (100–200 mesh) CC with CH2Cl2 - MeOH (15:1, v/v) to obtained compound 9 (18.0 mg). Fr. 10.3 (132.0 mg) was subjected to silica gel (100–200 mesh) CC with CH2Cl2 - MeOH (15:1–10:1, v/v) to give compound 10 (44.0 mg). Fr. 10.4 (120.0 mg) was separated by silica gel (100–200 mesh) CC with cyclohexane - EtOAc (3:2–1:1, v/v) to give compound 11 (24.0 mg). Fr. 10.6 (1.2 g) was recrystallized with methanol to get compound 7 (15.0 mg). The rest Fr.10.6 (1.0 g) was isolated by HPLC (MeOH - H2O, 0.1% formic acid, 9:11, 4 mL/min) to afford compound 4 (10.0 mg, tR = 12.3 min). Fr. 10.7 (33.0 g) was isolated on RP-18 silica gel CC with MeOH - H2O (10% −50%, v/v) to obtained compound 3 (35.0 mg), compound 8 (93.0 mg), and Fr. 10.7.1 fraction. Fr. 10.7.1 (3.0 g) was separated by HPLC (MeOH - H2O, 0.1% formic acid, 20:80, 4 mL/min) to give compound 2 (65.0 mg, tR = 21.0 min) and compound 1 (38.0 mg, tR = 36.1 min). Fr. 6 (1.0 g) was subjected to silica gel (200–300 mesh) CC with CH2Cl2 - MeOH (40:1–20:1, v/v) to obtained Fr. 6.1 and Fr. 6.2 fractions. Fr. 6.1 (0.5 g) was isolated by HPLC (MeOH - H2O, 0.1% formic acid, 30:70, 4 mL/min) to afford compound 12 (81.0 mg, tR = 12.0 min) and compound 13 (31.0 mg, tR = 8.0 min), Fr. 6.2 (0.12 g) was separated by HPLC (ACN - H2O, 0.1% formic acid, 30:70, 4 mL/min) to afford compound 5 (10.0 mg, tR = 19.0 min).

2.3.1

2.3.1 Brachythol A (1)

white crystal; [α]D27 −78 (c 0.1, MeOH); HR-ESI-MS (negative ion mode) m/z 479.0808 [M−H]– (calcd for C21H19O13, 479.0826). 1H NMR (Methanol‑d4, 600 MHZ) and 13C NMR (Methanol‑d4, 151 MHZ) data see in Table 1.

2.3.2

2.3.2 Brachythol B (2)

white amorphous powder, [α]D27 −20 (c 0.1, MeOH); HR-ESI-MS (negative ion mode) m/z 629.0796 [M−H]−, (calcd for C28H21O17, 629.0779). 1H NMR (DMSO‑d6, 600 MHZ) and 13C NMR (DMSO‑d6, 151 MHZ) data see in Table 1.

2.4

2.4 Antioxidant analysis

2.4.1

2.4.1 DPPH radical scavenging assay

The antioxidation was performed by evaluating the ability to scavenge the DPPH radical according to previous work (Koike et al., 2015; Oh et al., 2019). The DPPH scavenging capacities were expressed as Trolox equivalent (TE) antioxidant capacity (mmol) per g of sample (Strazzullo et al., 2007). All samples were dissolved in MeOH to obtain stock solutions (50 µg/mL) and positive control (Trolox, 10 mM). The reaction mixture consisted of different concentrations (30 µL) of isolated compounds and methanolic solution (270 µL) containing DPPH radicals (6 × 10−5 mol/L) in different wells of a 96 wells microplate. The mixtures were incubated for 30 min in the dark at room temperature. The reduction of the DPPH radical was measured by monitoring the decrease of absorption at 517 nm, and each experiment was tested in triplicate.

2.4.2

2.4.2 Ferric reducing antioxidant power (FRAP)

Base on the previous study, the ferric scavenging capacity was carried out (Zhang et al., 2016). All samples were dissolve in MeOH to acquire stock solutions (50 µg/mL). The working solution was prepared by mixing TPTZ dilution, detective buffer and TPTZ solution in a ratio of 10:1:1 (v/v), and then the working solution was incubated in a water bath at 37 °C. The reaction mixture consisted of working solution (180 µL) and samples (5 µL) in different wells of a 96 well microplate. The mixture incubated for 30 min at room temperature darkly and absorbance was measured at 593 nm, and each experiment was repeated three times.

2.5

2.5 Anti-inflammatory assay

2.5.1

2.5.1 Cell viability

Cyto-toxicity was determined using the MTT colorimetric method (Hsu et al., 2010; Kumar et al., 2018). Briefly, RAW 264.7 cells were cultured in DMEM medium containing 5% inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (20 µg/mL). The cells were plated into a 96-wells plate at the density of 1.8 × 104 cells/well. After 24 h, the culture mediums were replaced with 100 µL serial dilutions of isolated compounds followed by a 24 h incubation. The final concentration of solvent was less than 0.1% in the cell culture medium. Culture mediums were removed and replaced by 100 µL of fresh basic culture medium. Afterwards, 11 µL of sterile filtered MTT solution (5 mg/mL) in phosphate buffered saline (PBS) was added to each well, reaching a final concentration of 0.5 mg/mL. After incubating for 4 h, the supernatant was removed, and the insoluble formazan crystals were dissolved in 150 µL/well of DMSO and the absorbance was measured at 490 nm. The compounds were considered to be cytotoxic if the optical density of the sample-treated group was less than 90% of that in the control (vehicle-treated) group, each experiment was tested in triplicate (Panichayupakaranant et al., 2010). Calculation of cell viability was used the following formula (1):

(1)
Cell v i a b i l i t y % = OD sample - OD blank OD control - OD blank × 100 %

2.5.2

2.5.2 Measurement of nitric oxide (NO)/Nitrite

NO production was determined by measuring the accumulation of nitrite in the culture supernatant using Griess reagent (Banskota et al., 2003; Chimento et al., 2021). Briefly, the RAW 264.7 cells were harvested and diluted to a suspension in fresh culture medium. The cells were seeded in a 24-wells plate at a density of 2 × 105 cells/well and allowed to adhere for 24 h at 37 ℃ in a humidified atmosphere containing 5% CO2. Cells were pretreated with compounds at various concentrations for 3 h and then induced with 1 µg/mL Lipopolysaccharide (LPS) for 24 h, with dexamethasone (DXMS, 2 µg/mL) as a positive control. 50 µL of each supernatant was mixed with equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 min. The concentration of nitrite was measured by optical density reading at 540 nm, each experiment was repeated three times. NO inhibition rate was calculated using the following formula (2) (Hu et al., 2014), anti-inflammatory ability is expressed by IC50 value.

(2)
NO i n h i b i t i o n % = OD LPS - OD sample OD LPS - OD blank × 100 %

2.6

2.6 Qualitative analysis of phytochemicals by LC-MS/MS

An U3000 ultra-high-performance LC (Thermo Fisher, Waltham, MA, U.S.A.) coupled with a QE Plus hybrid Orbitrap MS system via an electrospray ion source interface. The separation was performed on a Waters ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 100 mm) chromatographic column at the rate of 200 μL/min at room temperature. A gradient elution consists of acetonitrile (A) and 0.1% formic acid (B), by the programs: 17% A, 0–1 min; 17–23% A, 1–3.5 min; 23–45% A, 3.5–6 min; 45–90% A, 6–13 min; and 90% A, 13–15 min. The injection volume is 2 μL. The conditions of Mass spectrometry (MS) were set as follows: capillary temperature of 350 °C. Voltage of 3.5 kV, and heater temperature of 350 °C were used for the ESI source. As a sheath gas and aux gas of 45 and 15 psi was used. The ions were collected in a high resolution up to 37,500 and full mass scan mass range of 130–1300 m/z. Recording and integrating the chromatograms in Xcalibur software (Thermo Fisher Scientific, USA). The MS data of full scan and the fragment tandem mass spectrometry (MS/MS) data from data-dependent acquisition (DDA) were used for compounds' identification.

3

3 Results and discussion

3.1

3.1 Identification of isolated compounds

In this study, the leaves of S. brachythyrsum were extracted and divided into four fractions. The EAF extract was subjected to repeated CC and semi-preparative HPLC, 13 compounds were obtained (1–13, Fig. 1), including 2 new (1–2) and 11 known (3–13) compounds, which were classified into 6 bergenin derivatives (1–6), 2 flavonoids (9 and 10), and 5 gallic acid derivatives (7–8 and 11–13). Eight compounds (1–5, 8, 12, 13) were isolated from Syzygium genus for the first time, including 5 bergenin derivatives and 3 gallic acid derivatives. New compounds were elucidated by extensive spectroscopic data, while known compounds were identified by comparing their spectroscopic data with related literatures. The antioxidation and anti-inflammatory activities of all compounds were detected, respectively.

Structures of compounds 1–13 and key HMBC and 1H–1H COSY correlations of compounds 1–2.
Fig. 1
Structures of compounds 1–13 and key HMBC and 1H–1H COSY correlations of compounds 1–2.

Compound 1 (Fig. 1) was a white crystal, possessed a molecular formula of C21H20O13, which was determined by its HR-ESI-MS (m/z 479.0808 [M−H]−, calcd. for C21H19O13 479.0826), corresponding to 12 degrees of unsaturation. The 1H NMR spectrum of 1 (Table 1) suggested the presence of three aromatic protons at δH 7.12 (s, 2H) and 7.11 (s, 1H); one methoxy single at δH 3.92 (s,3H); one methylene signal at δH 3.75 (dd, J = 12.4, 1.7 HZ, 1H) and δH 3.63 (dd, J = 12.4, 7.1 Hz, 1H); and five oxymethine signals at δH 3.97 (m, 1H), 4.17 (t, J = 8.9 Hz, 1H), 4.22 (t, J = 10.0 Hz, 1H), 5.06 (d, J = 10.2 Hz, 1H), and δH 5.09 (dd, J = 10.3, 8.9 Hz, 1H). The 13C NMR spectrum (Table 1) showed the skeleton carbons of compound 1 composing of two aromatic rings (δC 152.38, 149.35, 146.50, 146.50, 142.35, 140.19, 120.73, 119.38, 117.00, 111.20, 110.40, and 110.40), two carbonyl groups (δC 167.43 and 165.57), five oxymethines (δC 81.26, 81.11, 74.27, 73.43 and 72.39), one oxymethene (δC 62.26) and one methoxyl (δC 60.93). The 1H NMR and 13C NMR spectra of compound 1 were similar to those of compound 3 (11-O-galloylbergenin) (Feng et al., 2011), (Table S1) indicating it was a galloyl bergenin. Thus, the signals of aromatic protons at δH 7.11 (H-7) and the seven oxygenated protons at δH 3.97 (H-2), 5.09 (H-3), 4.17 (H-4), 4.22 (H-4a), 5.06 (H-10b), 3.75 (H-11), and 3.63 (H-11) belong to bergenin group, and the signals of the two aromatic protons at δH 7.12 × 2 (H-2′, 6′) belong to the galloyl moiety.

Table 1 1H and 13C NMR Data for Compounds 1 and 2.
1 (Methanol‑d4) 2 (DMSO‑d6)
1H 13C 1H 13C
2 3.97 m 81.11 4.02 m 72.92
3 5.09 m 72.39 4.72 m 80.88
4 4.17 (t 8.9) 73.43 4.11 m 72.13
4a 4.22 (t 10.0) 81.26 4.09 m 78.91
6 165.57 163.51
6a 119.38 118.58
7 7.11 s 111.20 7.01 s 109.48
8 152.38 150.89
9 142.35 141.04
10 149.35 148.27
10a 117.00 116.12
10b 5.06 (d 10.2) 74.27 4.95 m 70.81
11 3.75 (dd 12.4, 1.7)
3.63 (dd 12.4, 7.1)		 62.26
 4.70 m
3.35 (t 10.3)		 64.59
9-OMe 3.92 s 60.93 3.75 s 59.90
1′ 120.73 121.63
2′ 7.12 s 110.40 6.69 s 107.34
3′ 146.50 144.29
4′ 140.19 136.12
5′ 146.50 145.37
6′ 7.12 s 110.40 116.38
7′ 167.43 167.36
1′' 120.91
2′' 6.89 s 106.74
3′' 143.26
4′' 134.74
5′' 145.44
6′' 116.20
7′' 168.07

δ in ppm, J in HZ, 600 MHZ and 151 MHZ for 1H and 13C, respectively.

The linkage between galloyl and bergenin moieties at C-3 site was determined on the basis of the HMBC (Fig. 1) correlation of H-3 (δH) with C-7′ (δC). The methoxy group was fixed at C-9 site by the HMBC correlation from δH 3.92 to δC 142.35 (C-9). The assignment of compound 1 was confirmed by the 1H–1H COSY (Fig. 1) and HSQC correlations confirmed the above assignment, so compound 1 was determined as 3-O-galloylbergenin and was named as brachythol A.

Compound 2 (Fig. 1) was a white amorphous powder, and its molecular formula was C28H22O17, which was determined by HR-ESI-MS (m/z 629.0796 [M−H]−, calcd. for C28H21O17 629.0779), corresponding to 18 degrees of unsaturation. The 1H NMR spectrum of 2 (Table 1) indicated the presence of three aromatic protons at δH 7.01 (s, 1H), 6.69 (s, 1H) and 6.89 (s, 1H); one methoxyl singlet at δH 3.75 (s,3H); oxymethene signals at δH 3.35 (t, J = 10.3 HZ, 1H) and 4.70 (m, 1H); and five oxymethine signals at δH 4.02 (m, 1H), 4.09 (m, 1H), 4.11 (m, 1H), 4.72 (m, 1H) and 4.95 (m, 1H). Its 13C NMR spectrum (Table 1) showed 18 signals made up of three aromatic rings (δC 150.89, 148.27, 145.44, 145.37, 144.29, 143.26, 141.04, 136.12, 134.74, 121.63, 120.91, 118.58, 116.38, 116.20, 116.12, 109.48, 107.34, and 106.74), three carbonyl groups (δC 168.07, 167.36 and 163.51), five oxymethines signals (δC 80.88, 78.91, 72.92, 72.13 and 70.81), one oxymethene signal (δC 64.59) and one methoxy signal (δC 59.90). The C-H direct correlations were confirmed by analysis of its HSQC data. The NMR spectra data of compound 2 was similar to compounds 1, 3 and 6, (Figure S1) except for two galloyl groups linked to the bergenin moiety. The signals of aromatic protons at δH 7.01 (H-7), and the seven oxygenated protons (δH 4.02 (H-2), 4.72 (H-3), 4.11 (H-4), 4.09 (H-4a), 4.95 (H-10b), 3.35 (H-11), and 4.70 (H-11)) were easily assigned to the bergenin moiety, and the signals of remaining two aromatic protons at δH 6.69 (H-2′) and δH 6.89 (H-2′') belonged to the galloyl moiety, respectively.

The linkages of the two galloyl groups to the bergenin moiety at C-11 and C-3 were confirmed on the basis of the HMBC correlation of H-11 with C-7′ and H-3 with C-7′', respectively. The methoxylation at C-9 was also assigned due to the HMBC cross signal at δH 3.75 and δC 141.04 (C-9). In summary, the two galloyl groups only have two aromatic proton signals, δH 6.69 and 6.89, respectively. Combining the MS data and the aromatic atom signals of the two galloyl groups, it can be inferred that the two gallic acid groups are directly connected by C-6′ and C-6′', the HSQC, 1H–1H COSY and HMBC (Fig. 1) correlations further proved the structure. Thus, compound 2 was determined as 3, 11-O-diphenicbergenin and was named as brachythol B.

Other known compounds were identified as 11-O-galloylbergenin (3) (Feng et al., 2011), 11-O-caffeoylbergenin (4) (Sanogo et al., 2009), bergenin 11-O-(E)-ferulate (5) (Ito et al., 2012), bergenin (6) (Li et al., 2018), ellagic acid (7) (Yang and Guo, 2007), valoneaic acid dilactone (8) (Nawwar et al., 1997), kaempferol (9) (Fang et al., 2008), quercetin (10) (Li et al., 2006), gallic acid (11) (Yang and Guo, 2007), ethyl gallate (12) (Zhou et al., 2007), and gentisic acid (13) (Tan et al., 2013) by comparing their NMR, HR-ESI-MS data with the literatures. Eight compounds (1–5, 8, 12, 13) were isolated from Syzygium genus for the first time.

3.2

3.2 Chemical profile of EAF by UHPLC-QE Orbitrap MS

QE Orbitrap MS was used to further explore potential new phytochemicals from S. brachythyrsum in the present study. The molecular formula was confirmed by combination of high resolution precursor ions in negative mode. Preliminary identification was conducted by comparison of them to isolated compounds and literatures. Fragmentation patterns, diagnostic fragment ions and characteristic neutral losses were summarized based on the analysis of the isolated components. Besides, nitrogen rule, isotope pattern, and chromatographic elution sequence order were also employed for structural elucidation of isomeric components. A total of 107 compounds were characterized in the EAF extract of S. brachythyrsum, including 9 flavonoids, 48 phenolic acids, and 50 bergenin derivatives, among which 14 bergenins and 14 phenolics were potential new natural chemicals. Essential information on each component was shown in Table 2, including the retention time, high accurate precursor ions, and characteristic fragment ions. These identified compounds covered most of the major and medium peaks in negative mode.

Table 2 Summary of Identified Compounds by UHPLC − QE Orbitrap MS.
Name RT Molecular Formula [M−H]− Error(ppm) Fragment [negative]
Flavonoids
methoxymyricetin-O-glucuronic acid 7.06 C22H20O14 507.0780 0.222 331.0454 [-gluc]a, 317.0252
quercetin-O-pantose 7.35/7.64 C20H18O11 433.0772 −0.455 301.0343 [-pan]a
dihydromyricetin 7.49 C15H12O8 319.0455 −0.441 273.0040, 245.0090, 217.0132
myricetin 8.18 C15H10O8 317.0298 −0.540 273.0406, 245.0454, 178.9975, 151.0024, 137.0230
morin 8.31 C15H10O8 317.0298 −0.540 273.0406, 245.0454, 178.9975, 151.0024, 137.0230
quercetin 9.23 C15H10O7 301.0353 −0.126 273.0403, 257.0455, 245.0456, 229.0500, 193.0137, 178.9975, 151.0024
kaemferol 10.05 C15H10O6 285.0402 −0.211 257.0456, 241.0490, 229.0492, 213.0553, 185.0604, 169.0122, 151.0024
isorhamnetin 10.20 C16H12O7 315.0506 −0.396 301.0308
Phenolic acids
quinic acid 1.28 C7H12O6 191.0552 −0.951 173.0451, 127.0387, 111.0436
quinic acid-O-galloyl 1.35/1.81/2.34 C14H16O10 343.0661 0.097 191.0551 [-galloyl]a, 169.0132
ginnalin C 1.37 C12H14O10 317.0542 2.780 169.0134
gallic acid 1.81 C7H6O5 169.0131 −1.127 125.0230, 97.0280
4-O-methylgallic acid glucuronide 2.32 C14H16O11 359.0615 −0.494 183.0281 [-gluc]a, 169.0130, 151.0024
methyl ellagic acid glucuronide 2.35 C21H16O14 491.0469 0.152 300.9984, 275.0197, 169.0130, 143.0336
gentisic acid-O-pantose 2.36 C12H14O8 285.0615 −0.251 153.0181 [-pan]a, 109.0285, 149.0079, 133.0281
chlorogenic acid 2.79/3.65 C16H18O9 353.0873 −0.675 191.0551
digallic acid 2.82/2.97 C14H10O9 321.0251 −0.605 169.0131
ellagic acid-O-gallic acid 4.11/5.64/7.14 C21H10O13 469.0049 0.017 300.9984, 285.0047, 169.0131
monodecarboxy-O-valoneic acid dilactone 4.15/7.61/8.00 C20H10O11 425.0148 −0.234 300.9981, 270.9879
gentisic acid 4.16 C7H6O4 153.0182 −1.132 109.0281, 95.0123, 85.0280
quinic acid-O-coumaroyl 4.29/5.44 C16H18O8 337.0925 −0.391 191.0552 [-coumaroyl]a, 173.0445, 155.0336
ellagic acid-O- pantose 5.55 C19H14O12 433.0410 −0.259 300.9985 [-pan]a
ferulic acid 4-O-glucuronide 5.56 C16H18O10 369.0820 −0.690 207.0289, 193.0128 [-gluc]a
flavellagic acid 6.19/6.35 C14H6O9 316.9935 −0.405 169.0134, 197.0449, 241.0352
ethyl gallate 6.36 C9H10O5 197.0447 −0.897 169.0131, 125.0233
ellagic acid 6.78 C14H6O8 300.9987 0.030 257.0096, 229.0132
2-O-methylellagic acid 8.17 C15H8O8 315.0143 −0.300 300.9945
3-O-methylellagic acid 8.38 C15H8O8 315.0144 −0.270 300.9943
ellagic acid-O-digalloylquinic acid 7.49/7.51 C35H24O21 779.0736 −0.121 300.9985, 273.0041, 245.0089, 229.0135, 169.0132
digallic acid ethyl ester 8.29/8.57 C16H14*O9 349.0561 −0.445 197.0446, 169.0132
ellagic acid-O-galloylquinic acid b 4.74/7.38/7.81 C28H20O17 627.0626 −0.152 300.9988, 273.0044, 229.0136, 193.0132
luteic acid-O-galloylquinic acid b 5.90/6.40/6.88/7.44 C28H22O18 645.0731 −0.217 300.9985, 273.0043, 247.0242, 235.0244, 229.0138, 207.02881, 193.0132
luteic acid derivative 1 b 6.06 C35H26O22 797.0838 1.219 318.0015, 300.9985, 273.0047, 207.0281, 193.0129
luteic acid derivative 2 b 6.84/6.91 C28H30O25 765.1001 0.819 300.9990, 273.0046, 247.0251, 169.0133
ellagic acid -O-syringyl glucose b 7.76 C29H26O18 661.1044 −0.207 300.9987, 275.0197, 257.0090, 209.0082
methoxy Luteic acid-O-galloylquinic acid b 7.84 C29H24O18 659.0891 0.123 331.0090, 300.9989, 298.9830, 273.0043
luteic acid derivative 3 b 7.84 C29H24O18 659.0888 −0.237 300.9989, 287.0194, 207.0287, 193.0131
luteic acid derivative 4 b 7.84 C26H18 O16 585.0524 0.278 300.9988, 283.0461, 169.0130
Bergenin derivatives
norbergenin 1.38/1.82 C13H14O9 313.0565 0.075 235.0240, 207.0289, 193.0131
bergenin 1.38/2.31 C14H16O9 327.0726 0.445 312.0482, 294.0376, 276.0268, 249.0401, 234.0163, 222.0161, 207.0288, 193.0125
norbergenin-O-galloyl 1.4/2.72/3.17 C20H18O13 465.0671 −0.384 313.0558 [-galloyl]a, 295.0457, 277.0337, 235.0241, 207.0289, 193.0131, 169.0129, 151.0021
norbergenin derivative 1 b 1.81/2.26/2.62 C20H20O14 483.0776 −0.418 465.0680, 313.0561, 287.0771, 271.0460, 169.0130, 125.0229
methoxybergenin 4.69 C15H18O9 341.0871 −0.735 327.0672, 249.0374, 207.0286, 193.0133
diacetyl bergenin 4.71/5.17 C18H22O11 413.1085 −0.445 327.0715, 294.0380, 249.0404, 234.0164, 207.0291, 193.0127
brachythol B 4.84 C28H22O17 629.0783 −0.112 300.9987, 275.0186, 249.0409, 192.0046
bergenin-O-galloyl 5.10/5.67 C21H20O13 479.0832 0.046 327.0714, 313.0566, 295.0474, 271.0459, 193.0133, 169.0131
norbergenin-derivative 2 b 5.85/6.62 C28H24O18 647.0888 −0.237 313.0564, 275.0199, 247.0244, 231.0292, 203.0341, 193.0133
bergenin-O-hexahydroxy diphenic acid 6.03 C28H22O17 629.0783 −0.112 300.9987, 275.0186, 249.0409, 192.0046
norbergenin-O-salicylic acid 6.36 C20H18O11 433.0771 −0.545 313.0545, 235.0243, 207.0288, 193.0132
norbergenin-O-caffeoyl b 6.65/7.03 C22H20O12 475.0880 −0.189 313.0561, 295.0465, 235.0243, 207.0291, 193.0132
bergenin-O-methoxygallic acid 7.09/7.50 C22H22O13 493.0988 0.056 327.0718, 207.0289, 192.0054
di-O-galloylbergenin 7.31/7.70/7.90 C28H24O17 631.0935 −0.502 479.0836, 327.0704, 249.0397, 193.0133, 169.0131
norbergenin-O-flavogallonic acid b 7.47 C34H22O21 765.0580 −0.101 450.9940, 432.9833, 407.0045
brachythol B-O-galloyl b 7.52/7.91 C35H26O21 781.0892 −0.151 517.6822, 479.0827, 300.9990, 275.0200
bergenin-O-caffeoyl 7.91/8.10/8.24 C23H22O12 489.1038 −0.029 327.0716, 313.0559, 281.0672, 249.0406, 235.0239, 221.0445, 207.0290, 193.0132, 161.0233
bergenin-O-syringyl 3.42/3.89/5.05/5.86/8.25 C23H24O13 507.1145 0.086 327.0723, 299.0768, 271.0464, 207.0290, 192.0054
norbergenin derivative 3 b 8.04 C19H26O10 413.1449 −0.420 313.0565, 271.0460, 253.0359, 169.0132
bergenin-O-flavogallonic acid b 8.28 C35H24O21 779.0737 −0.061 450.9946, 432.9837, 407.0039, 379.0090, 351.0141, 285.0039
bergenin-O-coumaric acid 8.68/8.79 C23H22O11 473.1089 −0.075 458.0890, 327.0724, 312.0482, 265.0718, 207.0289, 192.0054
bergenin-O-sinapinic acid 8.71/8.81 C25H26O13 533.1303 0.246 327.0707, 207.0290, 192.0054
bergenin-O-feruloyl 8.79/8.91 C24H24O12 503.1198 0.341 327.0705, 249.0406, 207.0290, 192.0055, 175.0390
bergenin-O-hydroxybenzoic acid 7.63/8.13 C21H20O11 447.0931 −0.205 327.0507, 284.0327, 207.0290, 192.0055
bergenin derivative b 9.36/9.68 C26H30O12 533.1665 0.051 327.0715, 249.0402, 234.0163, 207.0289, 192.0054
a -gluc, -pan, -galloyl, and -coumaroyl denote moieties of glucuronic acid, pantose, galloyl, and coumaroyl groups, respectively.
b denotes potential new chemical.

As show in the Table 2, abundant bergenin derivatives were found in the extract of S. brachythyrsum, which were rarely reported from Syzygium genus. According to distinct MS/MS fragmentation behaviors, they could be mainly divided into the following subtypes: bergenin/norbergenin aglycones, O-galloyl, O-caffeoyl, O-syringyl, O-feruloyl, O-coumaric acids, O-sinapinic acids, and O-hexahydroxy diphenic acids etc. The molecular weight of the basic structure of bergenin is 328 Da (C14H16O9), and the molecular weight of the basic structure of norbergenin is 314 Da (C13H14O9). For bergenin derivatives, the number of galloyl groups could be calculated by adding n × C7H4O4 (152 Da), the number of caffeoyl groups could be calculated by adding n × C9H6O3 (162 Da), the number of O-hexahydroxy diphenic acid groups could be calculated by adding C14H8O8 (304 Da), the number of hydroxyl groups could be calculated by adding n × O (16 Da), and the number of methoxyl groups could be calculated by adding n × CH2 (14 Da). In the MS/MS spectra of negative ion modes, bergenin derivatives showed characteristic fragment ions, such as [M−14 Da], [M−18 Da], [M−152 Da], and/or [M−162 Da], denoting homolytic cleavage of CH3, OH groups, galloyl, and caffeoyl groups. These ions could be used as diagnostic ions for identification of bergenin derivatives. Herein, norbergenin-O-caffeoyl, taken as an example, produced [M−H]– at m/z 475.0880 (C22H19O12), further fragmented into m/z 313.0561 (C13H13O9), 295.0465 (C13H11O8), 235.0243 (C11H7O6), 207.0291 (C10H7O5), and 193.0132 (C9H5O5), which represented the neutral losses of [caffeic acid–H2O], [H2O], [C2H4O2], [CO], and [CH2].

The phenolic acids determined in S. brachythyrsum were mainly gallic acid, quinic acid, ferulic acid, and syringic acid derivatives, usually combined with glycosides. The molecular weight of the substitution group of galloyl is 152 Da (C7H4O4), while that of quinic acid group is 174 Da (C7H10O5), ferulic acid group is 176 Da (C10H8O3), syringic acid group is 180 Da (C9H8O4), caffeoyl group is 162 Da (C9H6O3), and galloylquinic acid (GQA) group is 344 Da (C14H16O10). These ions could be used as diagnostic ions for identification of phenolic acids derivatives. Herein ellagic acid-O-galloylquinic acid was taken as an example, it produced [M−H]– at m/z 627.0626 (C28H19O17), and further fragmented into m/z 300.9985 (C14H5O8), 273.0043(C13H5O7), and 229.0138 (C12H5O5), which represented the neutral losses of [GQA–H2O], [CO], and [CO2]. It is noteworthy that many potential compounds contain the m/z 300.9989 fragment ion, including the isolated compounds 2 and 7. The common substructure is the lactone structure composed of two gallic acids, indicating that the m/z 300.9989 refers to the corresponding moiety. In the present study, many new compounds were referred to as bergenin and gallic acid derivatives, confirming that the S. brachythyrsum contains a large amount of bergenin and phenolic acid compounds, which provides a reference for the research and development of the S. brachythyrsum as a functional plant.

3.3

3.3 Antioxidant activity

All the isolated compounds were evaluated for antioxidant activity using DPPH and FRAP assays (Koike et al., 2015; Zhang et al., 2016; Oh et al., 2019). Trolox solutions of different concentrations (0.02, 0.04, 0.08, 0.12, 0.16, 0.24 mM) constitute a standard curve (R2 = 0.9992) in DPPH assay. FeSO4 solutions of different concentrations (0.075, 0.15, 0.3, 0.6, 0.9, 1.2, 1.5 mM) constitute a standard curve (R2 = 0.9990). All the compounds showed certain antioxidant activity with TE and Fe2+E values ranging from 0.430 to 5.074 mmol (trolox)/g (sample) and 0.474 to 27.608 mmol (FeSO4)/g (sample) (Fig. 2). Nine compounds (1–4, 7–8, 10–13) showed comparable antioxidant activity to positive control Trolox, all compounds (except compound 1) showed comparable antioxidant activity to positive control FeSO4, as shown in Fig. 2. Generally, different structures of compounds have various biological activities (Grande et al., 2016). It's noting that in DPPH and FRAP assays, gallic acid (11) showed the highest, while bergenin (6) was the one with lower antioxidant activity. However, when the hydroxyl group of bergenin was esterified with gallic acid, the antioxidant activity of the products, such as compounds (1–5), was enhanced. This might be due to the addition of galloyl, caffeoyl, and feruloyl groups in bergenin, which increased the number of hydroxyl groups in bergenin and led to the increase of hydrogen and/or electron (Mainini et al., 2013) (Fig. 4). The result was also observed that valoneaic acid dilactone (8) showed higher antioxidant activity compared to ellagic acid (7) in DPPH assays, indicating that there was a structure–activity relationship, which could provide insights for the research of natural antioxidants.

(A) DPPH radical scavenging activities of compounds 1–13, expressed as mmol of trolox equivalents (TE) per g of sample. (B) Ferric reducing antioxidant activities of compounds 1–13, expressed as mmol of FeSO4 equivalents (Fe2+E) per g of sample. All data were expressed as mean ± SD (n = 3).
Fig. 2
(A) DPPH radical scavenging activities of compounds 1–13, expressed as mmol of trolox equivalents (TE) per g of sample. (B) Ferric reducing antioxidant activities of compounds 1–13, expressed as mmol of FeSO4 equivalents (Fe2+E) per g of sample. All data were expressed as mean ± SD (n = 3).

3.4

3.4 Anti-inflammatory activity

MTT colorimetric assays were carried out to evaluate the toxic effect of the isolated compounds on RAW 264.7 macrophage cell line. As shown in Figure. S2, there was no evidence of cytotoxicity of all compounds at concentration at 5–50 µM except compound 7.

Compounds 1–13 were evaluated for anti-inflammatory activities by using RAW 264.7 macrophage cell line model. Nitric oxide is a signaling molecule that plays key roles in immune and inflammatory responses and neuronal transmission in the brain (Karpuzoglu and Ahmed, 2006; Francomano et al., 2019). Under normal conditions, NO has neuroprotective and antioxidant effects. However, the exceeding release of NO from activated microglia causes a number of neurodegenerative diseases (Hämäläinen et al., 2008; Paesano et al., 2005). Inhibitors of NO production can be considered as potential anti-inflammatory agents. Anti-inflammatory ability of compounds 1–13 on NO production decreased in a turn of ellagic acid (IC50 = 3.190 μM), 11-O-galloylbergenin (IC50 = 4.421 μM), bergenin 11-O-(E)-ferulate (IC50 = 5.456 μM), quercetin (IC50 = 8.268 μM), brachythol B (IC50 = 9.215 μM), kaempferol (IC50 = 11.857 μM), 11-O-caffeoylbergenin (IC50 = 19.840 μM), gallic acid (IC50 = 20.203 μM), valoneaic acid dilactone (IC50 = 30.417 μM), brachythol A (IC50 = 30.450 μM), ethyl gallate (IC50 = 31.437 μM), gentisic acid (IC50 = 55.340 μM), and bergenin (IC50 = 130.933 μM), respectively (Fig. 3). In the bioactivity assay, 6 compounds (2–3, 5, 7, and 9–10) exhibited potential anti-inflammatory activities with IC50 values of 9.215, 4.421, 5.456, 3.190, 11.857 and 8.268 μM, respectively, compared with 2.076 μM of dexamethasone as the positive control. It is worth noting that the observed NO inhibitory activities appear to be somewhat correlated to their structures. For example, with regard to the results for compounds 1–5 and 7, it appeared that phenolic acid groups in the form of ester bonds might be important for the higher activity. Interestingly, comparing the structures and inhibitory activities of compounds 6 and 11 with those of compounds 1–3, it appeared that the increasing number of galloyl groups may increase the inhibition of NO production and the position of the galloyl groups will also affect the inhibition of NO production. It suggests that there may be a structure–activity relationship. (Fig. 4)

Anti-inflammatory activities of compounds 1–13 and dexamethasone, expressed as IC50 value. All data were expressed as mean ± SD (n = 3).
Fig. 3
Anti-inflammatory activities of compounds 1–13 and dexamethasone, expressed as IC50 value. All data were expressed as mean ± SD (n = 3).
The potential structure–activity relationship of bergenin derivatives in antioxidant and anti-inflammatory activities.
Fig. 4
The potential structure–activity relationship of bergenin derivatives in antioxidant and anti-inflammatory activities.

4

4 Conclusions

In conclusion, this is the first report for isolating and characterizing the chemical constituents from folk herbal tea S. brachythyrsum by phytochemical means and LC-MS analysis methods. Two new bergenin derivatives were isolated and a large number of potential new bergenin derivatives were characterized, which proved S. brachythyrsum is a rich resource for bergenin derivatives and phenolic acid. Amongst the isolates, most of the compounds showed comparable antioxidant and anti-inflammatory activities. Interestingly, there is a structure–activity relationship between the biological activities of bergenin derivatives and the substituent groups. It appeared that the increase in the number and position of galloyl, caffeoyl, and feruloyl groups may enhance their anti-inflammatory activities. The antioxidant and anti-inflammatory activities for S. brachythyrsum and its constituents were studied for the first time, and the results presented herein confirmed its health promise as a medicinal and edible herb.

Funding sources

This work was supported by the special foundation of Guangzhou key laboratory (No. 202002010004), Pearl River S&T Nova Program of Guangzhou (201806010048), and Special Subject of TCM Science and Technology Research of Guangdong Provincial Hospital of Chinese Medicine (YN2018QJ07, YN2016QJ01).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103507.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

Supplementary data 1


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