Next Article in Journal
New Insights into the Latest Advancement in α-Amylase Inhibitors of Plant Origin with Anti-Diabetic Effects
Next Article in Special Issue
Antioxidant and Antiproliferative Activities of Phenolic Extracts of Eriobotrya japonica (Thunb.) Lindl. Fruits and Leaves
Previous Article in Journal
Optimization of Callus Induction and Shoot Regeneration from Tomato Cotyledon Explants
Previous Article in Special Issue
Wound Healing Effect of Supercritical Carbon Dioxide Datura metel L. Leaves Extracts: An In Vitro Study of Anti-Inflammation, Cell Migration, MMP-2 Inhibition, and the Modulation of the Sonic Hedgehog Pathway in Human Fibroblasts
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 16
10.3390/plants12162943
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Chemical Constituents with Anti-Lipid Droplet Accumulation and Anti-Inflammatory Activity from Elaeagnus glabra

by
Ju-Hsin Cheng
1,2,
Ho-Cheng Wu
1,3,
Chia-Hung Yen
2,4,5,
Tsong-Long Hwang
6,7,8,
Horng-Huey Ko
1,4,5 and
Hsun-Shuo Chang
1,2,4,5,*
1
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
2
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
3
Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
4
Drug Development and Value Creation Research Center, Kaohsiung Medical University, Kaohsiung 807, Taiwan
5
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan
6
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
7
Research Center for Chinese Herbal Medicine, Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan
8
Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan
*
Author to whom correspondence should be addressed.
Plants 2023, 12(16), 2943; https://doi.org/10.3390/plants12162943
Submission received: 20 July 2023 / Revised: 8 August 2023 / Accepted: 9 August 2023 / Published: 14 August 2023
(This article belongs to the Special Issue Chemical Profiling and Biological Activity of Plant Natural Compounds II)
Browse Figures
Versions Notes

Abstract

:
Non-alcoholic fatty liver disease (NAFLD) is a type of steatosis caused by excess lipids accumulating in the liver. The prevalence of NAFLD has increased annually due to modern lifestyles and a lack of adequate medical treatment. Thus, we were motivated to investigate the bioactive components of Formosan plants that could attenuate lipid droplet (LD) accumulation. In a series of screenings of 3000 methanolic extracts from the Formosan plant extract bank for anti-LD accumulation activity, the methanolic extract of aerial parts of Elaeagnus glabra Thunb. showed excellent anti-LD accumulation activity. E. glabra is an evergreen shrub on which only a few phytochemical and biological studies have been conducted. Here, one new flavonoid (1), two new triterpenoids (2 and 3), and 35 known compounds (4–38) were isolated from the ethyl acetate layer of aerial parts of E. glabra via a bioassay-guided fractionation process. Their structures were characterized by 1D and 2D NMR, UV, IR, and MS data. Among the isolated compounds, methyl pheophorbide a (37) efficiently reduced the normalized LD content to 0.3% with a concentration of 20 μM in AML12 cell lines without significant cytotoxic effects. 3-O-(E)-Caffeoyloleanolic acid (13) and methyl pheophorbide a (37) showed inhibitory effects on superoxide anion generation or elastase release in fMLP/CB-treated human neutrophils (IC50 < 3.0 μM); they displayed effects similar to those of the positive control, namely, LY294002. These findings indicate that E. glabra can be used for developing a new botanical drug for managing LD accumulation and against inflammation-related diseases.

Graphical Abstract

1. Introduction

Non-alcoholic fatty liver disease (NAFLD) is liver steatosis caused by a build-up of fat in the liver that is not caused by alcohol use. NAFLD is defined as a spectrum of liver diseases ranging from hepatic steatosis, intermediate lesions, and non-alcoholic steatohepatitis (NASH) to cirrhosis, depending on the definition and diagnosis result [1]. It is a hepatic event in the metabolic syndrome and is prevalent in industrialized and developing countries [2]. The prevalence of NAFLD has continuously risen annually in youths and adults [3] due to sedentary lifestyles and modern Western nutrition. The prevalence of NAFLD is approximately 25% in Europe [4], ~34% in the USA [5], and 12–51% in Taiwan [5]. Except for the hepatic complications, patients with NAFLD will not only be at a higher risk for developing other cardio-metabolic diseases (type 2 diabetes mellitus, cardiovascular disease, etc. [6]), chronic kidney diseases, and hepatocellular carcinoma but will also be at an increased risk of mortality due to these disorders [1]. Currently, no effective pharmacological treatments have been approved to treat NAFLD. The primary therapy for NAFLD is limited to weight loss and exercise [7]. Thus, novel pharmacological strategies against NAFLD are urgently needed.
Hepatic steatosis (fatty liver) is the first stage of NAFLD; this refers to an intracellular accumulation of lipids and subsequent formation of lipid droplets (LDs) in the cytoplasm of hepatocytes. Excess lipids in the liver are primarily neutral lipids; they are also triglycerides and cholesterol esters. In hepatocytes and other hepatic cells (for example, hepatic stellate cells and Kupffer cells), neutral lipids are stored in dynamic organelles called lipid droplets [8]. The stored LDs as buffers for fatty acids are then utilized in times of need to generate energy, membrane components, and signaling lipids [9]. The role of LDs as buffers for fatty acid availability may even extend to lipid exchange between cells in the same tissue. The excess fatty consumption breaks an imbalance between the formation and degradation of LDs and typically develops into severe physiological consequences, including lipodystrophy, NAFLD, obesity, cardiovascular disease, and type 2 diabetes mellitus [8,10]. From the current point of view, strategies for LD homeostasis seem to be the most promising for treating abnormal lipid accumulation and for the progression of liver diseases in patients with NAFLD [8].
Natural products have been used as a medicinal agent source in previous centuries. Natural products and their derivatives have high chemical diversity, biochemical specificity, and other molecular properties that make them favorable as bioactivity screen resources. With the intention of identifying anti-LD agents, we screened over 3000 methanolic extracts from the library of Formosan plants in the Laboratory of Medicinal Botany of Kaohsiung Medical University [1]. Via high-throughput screening, extracts that inhibited LD accumulation (including LD counts, area, and intensity) by >40% without severe cytotoxicity (cell count >60% of average cell count in control wells) were considered hits in the primary screening. After that, we strengthened the inhibition criteria of LD accumulation to more than 50% and the cell count to >60% of the average cell count to obtain 22 hits. The extracts that showed a dose-dependent reduction in LD content at 50 μg/mL and a reduction of more than 50% in LD content were regarded as hits. Finally, the image-based platform was used to confirm anti-LD activity. After an evaluation, the methanolic extracts of aerial parts of Elaeagnus glabra Thunb. stood out for their potent inhibitory activity toward LD accumulation with little effect on cell viability (Figure 1).
E. glabra (Elaeagnaceae) is an evergreen shrub native to China, the Ryukyus in Japan, Korea, and Taiwan. Until now, only one antibacterial flavonoid, namely, (−)-epigallocatechin [11,12]; two steroids; and three triterpenes [13] have been identified from E. glabra. Based on the anti-LD accumulation screening results and the limited number of investigations of aerial parts from E. glabra, this study aimed to isolate components from E. glabra and evaluate their anti-LD accumulation effects.

2. Results and Discussion

In the current study, we focused on the chemical constituents from the methanolic extract of aerial parts of E. glabra and identified bioactive compounds with anti-LD accumulation activity. With the bioactivity-guided fractionation of the ethyl-acetate layer of aerial parts of E. glabra, three new compounds (1−3) and 35 known compounds (4−38) were successfully isolated (see Figure 2 and Supplementary Materials: Figure S1). The phytochemical spectra of compounds 1–3 are available in the Supplementary Materials (Figures S2–S28). In addition, some isolates were further examined for anti-LD accumulation effects in hepatic cell lines.

2.1. Structure Elucidation of Compounds 1−3

Compound 1 was isolated as a pale yellow amorphous solid with a negative optical rotation. Its molecular formula was determined to be C18H14O7 based on the results from HRESIMS, which is consistent with 12 hydrogen deficiencies. The hydroxy (3275 cm−1), carbonyl groups (1686 cm−1), and aromatic ring(s) (1606, 1519 cm−1) were observed in the IR spectrum. The UV absorption at 286 and 330 nm suggested the presence of a flavan moiety in 1 [14]. The characteristic flavan aromatic rings [penta-substituted aromatic ring: δH 6.35 (1H, s, H-8) and δC 153.4 (C-5), 103.4 (C-6), 156.2 (C-7), 95.7 (C-8), 163.1 (C-9), 105.6 (C-10); ABX system aromatic ring: δH 6.86 (1H, dd, J = 8.3, 2.3 Hz, H-6′), 6.80 (1H, d, J = 8.3 Hz, H-5′), 7.04 (1H, d, J = 2.3 Hz, H-2′) and δC 131.4 (C-1′), 115.2 (C-2′), 146.2 (C-3′), 146,1 (C-4′), 116.1 (C-5′), 119.3 (C-6′)] and a flavan 2H-pyran [ring C: δH 5.02 (1H, br s, H-2), 4.30 (1H, br dd, J = 4.2, 2.7 Hz, H-3), 2.87 (1H, dd, J = 17.0, 2.7 Hz, H-4b), 2.96 (1H, dd, J = 17.0, 4.2 Hz, H-4a) and δC 80.7 (C-2), 66.6 (C-3), 29.4 (C-4)] could also be found in the NMR spectra (Table 1). Further HMBC correlations between H-2/C-1′, C-2′ revealed that the ABX system aromatic ring occupied ring B, and the HMBC correlations between H-8/C-6, C-7, C-9, C-10, H-4/C-5, C-10, and H-2/C-9 indicated that the penta-substituted aromatic ring was located at ring A (Figure 3). Apart from the flavan moiety, the 1H- and 13C-NMR spectral data indicated the existence of a lactone ring [δH 6.07 (1H, d, J = 9.6 Hz, H-3″), 8.14 (1H, d, J = 9.6 Hz, H-4″); δC 103.4 (C-6), 156.2 (C-7), 164.5 (C-2″), 109.4 (C-3″), 141.2 (C-4″)]. Based on the HMBC correlation (Figure 3) between H-3″/C-6, H-4″/C-2″, C-5, and C-7, the lactone ring and flavan were connected with C-6 and C-7 to become the flavanocoumarin. For now, the above elucidations suggested the molecular formula of 1 as C18H14O3, showing losses of 64 atomic mass units as four oxygen atoms compared with its HRESIMS. Finally, based on the chemical shifts of C-3 (δC 66.6), C-5 (153.4), C-3′ (146.2), and C-4′ (146,1), four hydroxy groups were attached to C-3, C-5, C-3′, and C-4′, respectively. The small coupling constant of H-2/H-3 (J2,3 = 2.7 Hz) in 1 indicates that the relative configuration of C-2/C-3 was a 2,3-cis-configuration [15]. In accordance with the optical rotation rule [15,16], C-2/C-3 was in the (2R,3R)-form with a negative optical rotation. Accordingly, the structure of 1 was determined and named elaeagncoumarin.
Compound 2, a whitish powder, was established with its molecular formula of C39H52O7 using HRESIMS, accounting for 14 degrees of unsaturation. Compound 2 showed IR absorptions at 3385 (hydroxyl group(s)), 1768, and 1700 (carbonyl groups). The spectroscopic data of 2 (Table 2) are comparable with those of the literature compound 11α,12α-epoxy-3β-hydroxy-olean-13β,28-olide [17], except for the 3-OH group in 11α,12α-epoxy-3β-hydroxy-olean-13β,28-olide, which was changed to a p-E-coumaroyl moiety in 2. Based on the 1H NMR data, the down-field shift in H-3 (δH 4.62) demonstrated that the p-E-coumaroyl moiety was located at C-3. A further HMBC correlation between H-3/C-9′ verified the junction between C-3 and the p-E-coumaroyl moiety (Figure 3). The NOESY plot of H-3/H-5, H-5/H-9, and H-9/H3-27 indicated that H-3, H-5, H-9, and H3-27 were in an α-axial configuration (Figure 4). H2-19 was assumed to be in an α-axial form due to the NOESY correlation between H3-27/H2-19, while H-18 was in the β-equatorial position in ring D. Therefore, H-13 was in the β-equatorial configuration based on the NOESY correlation between H-13/H3-18. In the NOESY spectra, the correlations between H3-24/H3-25, H3-25/H-11 and H3-26 suggested that H-11, H3-24, H3-25, and H3-26 occupied the β-axial configuration (Figure 4). The above NOESY correlations illustrated that compound 2 had the same relative configuration as the triterpene compound 11α,12α-epoxy-3β-hydroxy-olean-13β,28-olide [17]. Based on the above evidence, the structure of 2 was identified and named elaeagterpene A.
Compound 3 was obtained as a whitish powder. The analysis of the HRESIMS of 3 indicated the molecular formula C39H52O6, representing 14 degrees of unsaturation. IR absorptions at 3358, 1741, and 1704 cm−1 supported the presence of hydroxy and carbonyl groups. The physical data and NMR spectroscopic data of 3 and 2 (Table 2) implied their similar structure, except for differences in the substituents on C-11/C-12 (2: epoxide group; 3: double bond), C-19 (2: H2; 3: methyl group), and C-20 (2: dimethyl group; 3: methyl group). The above signals indicated the ursane-type triterpenoid of 3. The HMBC plots (Figure 3) of H3-25/C-1, C-5, C-9, and C-10; H3-26/C-7, C-8, and C-9; and H3-27/C-13, C-14, and C-15 could be used to assign the positions of C-8, C-9, C-13, and C-14. A further COSY correlation between H-9/H-11 and HMBC correlations between H-12/C-9 and C-14 verified that the double bond was located at C-11/C-12 (Figure 3). The doublet methyl groups [δH 1.04 (3H, d, J = 6.3 Hz, H-29), 0.96 (1H, J = 6.3 Hz, H-30)] were attached to C-19 and C-20 based on the HMBC correlations between H-29/C-18, C-19, C-20, H-30/C-20, and C-21. The relative configurations of 3 were indicated by the NOESY plots of H-3/H-5, H-5/H-9, and H-9/H3-27 (Figure 4) and a comparison with 3β,13-dihyroxyurs-11-en-28-oic acid γ-lactone [18]. Thus, the structure of compound 3 was assigned and named elaeagterpene B.
Through a comparison of the experiments and reported spectroscopic data ( [ α ] D , UV, IR, NMR, and MS), 35 known compounds were identified as 11 triterpenoids: arjunolic acid (4) [19], alphitolic acid (5) [20], betulinic acid (6) [21], cleistocalyxic acid E (7), cleistocalyxic acid G (8) [22], lupeol (9) [23], pomolic acid (10) [24], ursolic acid (11) [25], 2α,3β,23-trihydroxy-11α,12α-epoxyolean-28,13β-olide (12) [19], 3-O-(E)-caffeoyloleanolic acid (13) [26], and (+)-3β-O-trans-caffeoyl betulinic acid (14) [27]; eight flavonoids: plumbocatechins B (15) [28,29], (−)-catechin (16) [30], dihydromyricetin (17) [31], (−)-epicatechin (18) [32], (−)-epigallocatechin (19) [33], (−)-gallocatechin (20) [34], naringenin (21) [35], and plumbocatechin A (22) [28]; five benzenoids: methyl galloate (23) [36], protocatechualdehyde (24) [37], syringaldehyde (25) [38], 4-(2-hydroxyethyl)benzoic acid (26) [39], and 4-hydroxy-3-methoxyphenyl)propane-1,2-diol (27) [40]; two α-tocopherol derivatives: α-tocopherol (28) [41] and 5-formyl-7,8-dimethyl tocol (29) [42]; three steroids: β-sitosterol (30) [43], β-sitosterone (31) [44], and ergosterol peroxide (32) [45]; two lignans: pinoresinol (33) [46] and syringaresinol (34) [47]; one alkanoid: 2,3-dihydroxypropyl hexadecanoate (35) [48]; one apocarotenoid: vomifoliol (36) [49]; one chlorophyll: methyl pheophorbide a (37) [50]; and one polyisoprenoid: ficarprenol-10 (38) [51].

2.2. Anti-LD Accumulation Activity of Compounds Isolated from Aerial Parts of E. glabra

In this study, twelve compounds present in sufficient amounts (9, 11, 15, 16, 18–20, 22, 25, 30, 31, and 37) were evaluated for anti-LD accumulation activity (Table 3). To assess the anti-LD formation activity, the average LD counts/cell of bovine serum albumin (BSA)-conjugated oleic acid (OA) + drug vehicle (DMSO)-treated wells (hereinafter referred to as OA) were used as the standard for 100% fat loading, and triacsin C (TC) was used as the reference control. Compared with the vehicle control, methyl pheophorbide a (37) in 100 μg/mL did not affect the cell viability of the AML12 cell line, thus ensuring that the concentrations of methyl pheophorbide a (37) were safe. However, methyl pheophorbide a (37) significantly decreased the relative LD count, leaving only 0.3 ± 0.1%. Next, the representative images revealed the anti-LD accumulation activity directly. In Figure 5A, the blue pseudocolor indicates the nuclei area, and the green ones depict the LD area. Methyl pheophorbide a (37) at a concentration of 20 μM exhibited significant LD-accumulation-reducing effects (Figure 5A), with an LD content reduction of more than 90% without cytotoxicity (Figure 5B).

2.3. Anti-Inflammatory Activity of Compounds Isolated from Aerial Parts of E. glabra

NAFLD is accompanied by inflammation. In the NASH stage, the liver is inflamed. As the NAFLD progresses, persistent inflammation causes scar tissue around the liver; this is called the fibrosis stage. After years of inflammation, liver damage occurs and becomes cirrhosis. To explore the potential of compounds for managing inflammation, eight compounds were preliminarily evaluated for their effects on neutrophil pro-inflammatory responses by suppressing fMLP/CB-induced superoxide anion generation and elastase release (Figure 6 and Table 4). Among them, compounds 13 and 37 showed significant inhibitory activity toward superoxide anion generation or elastase release (Figure 6). These two compounds were further evaluated for their anti-inflammatory activity IC50 values (Table 4). 3-O-(E)-Caffeoyloleanolic acid (13) showed superoxide anion inhibition with an IC50 value of 3.01 ± 0.58 μM, and methyl pheophorbide a (37) showed potent anti-inflammatory activity on both superoxide anion generation and elastase release with IC50 values of 1.29 ± 0.27 and 2.35 ± 0.12 μM, respectively.
There are approximately 40 Elaeagnus species in the world. Most isolates from Elaeagnus species are triterpenoids and flavonoids. The current study found 13 triterpenoids and 10 flavonoids in the aerial parts of E. glabra. Our chemical findings were consistent with the previous reports and can contribute to the chemotaxonomy of the Elaeagnus species. Furthermore, compounds 2 (oleanan-13β,28-olide-type) and 3 (urs-13β,28-olide-type) were triterpenes connected with caffeic acid, which has rarely been seen in Elaeagnus, even in natural sources. This finding not only sheds light on the structural diversity of E. glabra but also uncovers different types of compounds from the natural source. Furthermore, twenty compounds from aerial parts of E. glabra were found in Elaeagnus genus, including compounds 4, 5, 7, 8, 12−14, 15, 22−27, 29, 33−35, 37, and 38, thus providing another natural source of the above compounds for further application.
Our results from two different bioactivity models demonstrated that methyl pheophorbide a (37) can not only inhibit LD accumulation but also simultaneously display superoxide anion generation and elastase release inhibition. Methyl pheophorbide a (37) is a chlorophyll with several bioactivities in the literature, including antioxidant [52], cytotoxicity [52,53], phototoxicity [54], and iron-binding capacity [52]. To the best of our knowledge, this study demonstrated the first finding on the anti-LD accumulation activity of methyl pheophorbide a (37). It may be possible to further explore the anti-LD accumulation activity of chlorophylls accompanied by anti-inflammatory activity.

3. Materials and Methods

3.1. General Experiment Procedures

Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Kyoto, Japan), and IR spectra (ATR) were acquired with a Jasco FT/IR-4600 spectrometer. We recorded 1D (1H, 13C, DEPT) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectra on a Varian Germini-2000 spectrometer (Varian, Inc. Vacuum Technologies, Lexington, MA, USA) operated at 200 (1H) and 50 MHz (13C), a Varian Unityplus-400 spectrometer (Varian, Inc. Vacuum Technologies, Lexington, MA, USA) operated at 400 (1H) and 100 MHz (13C), a Varian Mercuryplus-400 spectrometer (Varian, Inc. Vacuum Technologies, Lexington, MA, USA) operated at 400 (1H) and 100 MHz (13C), and a Varian VNMRS-600 spectrometer (Varian, Inc. Vacuum Technologies, Lexington, MA, USA) operated at 600 (1H) and 150 MHz (13C). Low-resolution mass spectra were obtained with POLARIS Q Thermo Finnigan (Thermo Fisher Scientific, Chicago, IL, USA), Waters ZQ 4000 (Waters, Milford, MA, USA), and VG Quattro GC/MS/MS/DS (Waters, Milford, MA, USA) mass spectrometers. EIMS was taken on a JEOL JMS-700 mass spectrometer (JEOL, Tokyo, Japan). High-resolution electrospray ionization mass spectroscopy (HRESIMS) was recorded on a Bruker APEX II mass spectrometer (Bruker, Karlsruhe, Germany) and VARIAN 901-MS (Varian, CA, USA). Silica gel (70–230 and 230–400 mesh; Silicycle, Quebec, QC, Canada) was used for column chromatography (CC), and silica gel 60 F254 (Merck, Darmstadt, Germany) and RP-18 F254S (Merck, Darmstadt, Germany) were used for thin layer chromatography (TLC) and preparative TLC, respectively, and visualized with a Ce2(SO4)3 aqueous solution. Further purification was performed using medium-performance liquid chromatography (MPLC; ceramic pump: VSP-3050; EYELA, Kyoto, Japan).

3.2. Plant Material

Aerial parts of Elaeagnus glabra Thunb. were collected in February 2018 in Sandimen Pingtung County, Taiwan, and identified by Prof. Ih-Sheng Chen. A voucher specimen (Chen 6318) was deposited with the herbarium of the College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.

3.3. Extraction and Isolation

Dried aerial parts (8.4 kg) of Elaeagnus glabra were extracted at room temperature with methanol (MeOH) (60 L) three times to yield a MeOH extract (610 g). The MeOH extract was suspended in water and partitioned with ethyl acetate (EtOAc) to yield a water layer (394 g) and an EtOAc layer (90 g). The EtOAc layer (90 g) was subjected to column chromatography (silica gel; n-hexane/acetone 95/5 to 33/67, then washed with 100% methanol) to yield ten fractions (Fr. 1–10). Fr. 2 (2.4 g) was subjected to column chromatography (silica gel, n-hexane/CH2Cl2 4/1 to 3/2, column size: 2 × 30 cm) to yield seven fractions (Fr. 2-1~2-7). Fr. 2-1 was subjected to MPLC (silica gel, n-hexane/EtOAc 10/1, column size: 1.5 × 30 cm) to give eight fractions (Fr. 2-1-1~2-1-8). Fr. 2-1-2 was subjected to MPLC (RP-18; methanol/acetone 1:1; column size: 1.5 × 30 cm) to afford 11 fractions (Fr. 2-1-2-1~2-1-2-11) and compound 28 (34.8 mg). Fr. 2-1-2-11 was separated with prep. TLC (n-hexane/CH2Cl2/acetone 10/0.5/0.5) to afford compound 29 (0.5 mg). Fr. 2-6 was subjected to MPLC (silica gel, n-hexane/acetone 20/1, column size: 1 × 30 cm) to afford four fractions (Fr. 2-6-1~2-6-4). Fr. 2-6-2 was subjected to MPLC (RP-18, water/acetone 1/10, column size: 1 × 30 cm) to give five fractions (Fr. 2-6-2-1~2-6-2-5). Fr. 2-6-2-3 was separated with prep. TLC (n-hexane/acetone 8:1) to afford compound 38 (2.2 mg). Fr. 2-9 was subjected to MPLC (RP-18, water/methanol 1/10, column size: 1 × 30 cm) to afford nine fractions (Fr. 2-9-1~2-9-9) and compound 9 (73.7 mg). Fr. 2-11 was subjected to MPLC (RP-18, water/acetone 1/8, column size: 2 × 30 cm) to give five fractions (Fr. 2-11-1~2-11-5). Fr. 2-11-4 was subjected to MPLC (silica gel; n-hexane/acetone 20:1; column size: 1 × 30 cm) to afford compound 31 (34.8 mg). Fr. 3 was recrystallized from MeOH to give compound 30 (118.9 mg). Fr. 6 (1.7 g) was subjected to MPLC (RP-18; water/acetone 1/3; column size: 2 × 30 cm) to yield six fractions (Fr. 6-1~6-6). Fr. 6-2 was subjected to MPLC (silica gel, CH2Cl2/acetone 12/1, column size: 1.5 × 30 cm) to yield 15 fractions (Fr. 6-2-1~6-2-15). Fr. 6-2-10 was subjected to MPLC (silica gel, n-hexane/acetone 4/1, column size: 1 × 30 cm) to produce ten fractions (Fr. 6-2-10-1~6-2-10-10). Fr. 6-2-10-5 was subjected to MPLC (silica gel, n-hexane/EtOAc 3/1, column size: 1 × 30 cm) to afford eight fractions (Fr. 6-2-10-5-1~6-2-10-5-8). Fr. 6-2-10-5-4 was subjected to MPLC (silica gel, n-hexane/EtOAc 2:1, column size: 1 × 30 cm) to obtain six fractions (Fr. 6-2-10-5-4-1~6-2-10-5-4-6). Fr. 6-2-10-5-4-2 was further separated with prep. TLC (n-hexane/EtOAc 2/1) to give compound 10 (0.9 mg). Fr. 6-2-13 was subjected to MPLC (RP-18, water/acetonitrile 1/6, column size: 1 × 30 cm) to produce ten fractions (Fr. 6-2-13-1~6-2-13-10). Fr. 6-2-13-8 was separated with prep. TLC (CH2Cl2/Ethanol (EtOH) 12/1) to give compound 4 (2.1 mg). Fr. 6-3 was subjected to MPLC (silica gel, CH2Cl2/EtOAc 10/1, column size: 1.5 × 30 cm) to afford eight fractions (Fr. 6-3-1~6-3-8). Fr. 6-3-1 was separated with prep. TLC (n-hexane/acetone 4/1) to give compound 37 (7.1 mg). Fr. 6-3-2 was subjected to MPLC (silica gel, n-hexane/EtOAc 4/1, column size: 1 × 30 cm) to obtain seven fractions (Fr. 6-3-2-1~6-3-2-7). Fr. 6-3-2-1 was subjected to MPLC (silica gel, n-hexane/EtOAc 5/1, column size: 1 × 30 cm) to produce compound 6 (1.9 mg). Fr. 6-3-3 was subjected to MPLC (silica gel, n-hexane/CH2Cl2/acetone 7/1/1, column size: 1 × 30 cm) to obtain 11 fractions (Fr. 6-3-3-1~6-3-3-11). Fr. 6-3-3-4 was subjected to MPLC (RP-18, water/MeOH 1/6, column size: 1 × 30 cm) to afford compound 32 (3.2 mg). Fr. 6-3-4 was separated with Sephadex LH-20 (column size: 3 × 70 cm) and eluted with methanol to provide six fractions (6-3-4-1~6-3-4-6). Fr. 6-3-4-5 was subjected to MPLC (silica gel, n-hexane/acetone 6/1, column size: 1 × 30 cm) to obtain ten fractions (Fr. 6-3-4-5-1~6-3-4-5-10). Fr. 6-3-4-5-6 was subjected to MPLC (silica gel, n-hexane/EtOAc 2/1, column size: 1 × 30 cm) to give compound 35 (3.9 mg). Fr. 6-3-5 was subjected to MPLC (silica gel, n-hexane/EtOAc 4/1, column size: 1 × 30 cm) to produce compound 11 (1.7 mg). Fr. 8 (6.8 g) was subjected to column chromatography (silica gel, CH2Cl2/MeOH 35/1 to 20/1) to yield 11 fractions (Fr. 8-1~8-11). Fr. 8-4 was subjected to MPLC (silica gel, n-hexane/CH2Cl2/acetone 6/1/1, column size: 1.5 × 30 cm) to obtain 11 fractions (Fr. 8-4-1~8-4-11). Fr. 8-4-8 was subjected to prep. RP-18 TLC (water/acetone 1/4) to afford compound 3 (0.6 mg). Fr. 8-4-9 was subjected to prep. RP-18 TLC (water/acetone 1/4) to yield three fractions (Fr. 8-4-9-A~8-4-9-C). Fr. 8-4-9-B was subjected to MPLC (RP-18, water/acetonitrile 1/4, column size: 1 × 30 cm) to produce compound 2 (0.9 mg). Fr. 8-4-10 was subjected to MPLC (RP-18, water/acetone 2/1, column size: 1 × 30 cm) to afford compound 33 (1.0 mg). Fr. 8-4-11 was subjected to MPLC (RP-18, water/acetone 2/1, column size: 1 × 30 cm) to afford compound 34 (1.7 mg). Fr. 8-6 was subjected to MPLC (silica gel, n-hexane/EtOAc 2/1, column size: 1.5 × 30 cm) to yield six fractions (Fr. 8-6-1~8-6-6). Fr. 8-6-3 was subjected to MPLC (silica gel, n-hexane/CH2Cl2/EtOAc 2/1/1, column size: 1.5 × 30 cm) to afford compound 14 (9.6 mg). Fr. 8-6-3-3 was subjected to MPLC (silica gel, n-hexane/EtOAc 2/1, column size: 1 × 30 cm) to give compound 21 (0.6 mg) and compound 24 (0.7 mg). Fr. 8-6-4 was subjected to MPLC (RP-18, water/acetonitrile 1/5, column size: 1.5 × 30 cm) to produce compound 13 (6.6 mg). Fr. 8-6-6 was subjected to MPLC (RP-18, water/acetonitrile 2/1, column size: 1 × 30 cm) to obtain eleven fractions (Fr. 8-6-6-1~8-6-6-11). Fr. 8-6-6-2 was subjected to prep. RP-18 TLC (water/acetone 2/1) to yield three fractions (Fr. 8-6-6-2-A~8-6-6-2-C). Fr. 8-8 was subjected to MPLC (silica gel, CH2Cl2/EtOAc 2/3, column size: 2 × 30 cm) to obtain four fractions (Fr. 8-8-1~8-8-4). Fr. 8-8-3 was subjected to MPLC (RP-18, water/MeOH 1/3, column size: 1.5 × 30 cm) to yield nine fractions (Fr. 8-8-3-1~8-8-3-9). Fr. 8-8-3-2 was subjected to prep. TLC (CH2Cl2/EtOAc 1/1) to produce three fractions (Fr. 8-8-3-2-A~8-8-3-2-C) and compound 26 (2.5 mg). Fr. 8-8-3-2-C was further separated with MPLC (silica gel, CH2Cl2/EtOAc 1/3, column size: 1 × 30 cm) to give compound 36 (2.4 mg). Fr. 8-8-3-8 was subjected to prep. TLC (n-hexane/EtOAc 2/3) to produce compound 5 (6.5 mg). Fr. 9 (11.2 g) was subjected to column chromatography (silica gel, CH2Cl2/MeOH 12/1 to 6/1) to yield 13 fractions (Fr. 9-1~9-13). Fr. 9-5 was subjected to MPLC (silica gel, n-hexane/acetone 2/1, column size: 1.5 × 30 cm) to obtain 13 fractions (Fr. 9-5-1~9-5-13). Fr. 9-5-7 was subjected to MPLC (RP-18, water/MeOH 2/1, column size: 1 × 30 cm) to produce compound 23 (2.7 mg). Fr. 9-5-10 was subjected to MPLC (RP-18, water/MeOH 2/1, column size: 1 × 30 cm) to obtain 11 fractions (Fr. 9-5-10-1~9-5-10-11). Fr. 9-5-10-1 was subjected to MPLC (silica gel, n-hexane/CH2Cl2/MeOH 1/1/0.1, column size: 1 × 30 cm) to afford compound 27 (0.5 mg). Fr. 9-5-11 was subjected to MPLC (silica gel, n-hexane/CH2Cl2/MeOH 1/1/0.1, column size: 1.5 × 30 cm) to produce compound 12 (4.3 mg). Fr. 9-5-11-19 was subjected to column chromatography (silica gel, n-hexane/acetone 1.5/1) to yield nine fractions (Fr. 9-5-11-19-1~9-5-11-19-9). Fr. 9-5-11-19-7 was further separated with MPLC (RP-18, water/MeOH 1/1.5, column size: 1 × 30 cm) to give compound 7 (4.6 mg) and compound 8 (1.6 mg). Fr. 9-7 was subjected to MPLC (silica gel, n-hexane/acetone 1/1, column size: 1.5 × 30 cm) to obtain eight fractions (Fr. 9-7-1~9-7-8). Fr. 9-7-6 was subjected to MPLC (RP-18, water/MeOH 1.5/1, column size: 1 × 30 cm) to yield 21 fractions (Fr. 9-7-6-1~9-7-6-21). Fr. 9-7-6-18 was subjected to MPLC (silica gel, CH2Cl2/acetone 2/1, column size: 1 × 30 cm) to afford compound 1 (0.9 mg). Fr. 9-9 was subjected to MPLC (silica gel, CH2Cl2/EtOAc 1/1, column size: 1.5 × 30 cm) to obtain ten fractions (Fr. 9-9-1~9-9-10). Fr. 9-9-3 was subjected to MPLC (RP-18, water/acetonitrile 3/1, column size: 1 × 30 cm) to yield eight fractions (Fr. 9-9-3-1~9-9-3-8). Fr. 9-9-3-3 was further separated with prep. RP-18 TLC (water/MeOH 1.5/1) to give compound 15 (3.6 mg) and compound 17 (1.3 mg). Fr. 9-9-4 was subjected to prep. RP-18 TLC (water/MeOH 1.5/1) to afford compound 25 (1.5 mg). Fr. 9-10 was subjected to MPLC (silica gel, CH2Cl2/MeOH 7/1, column size: 2.5 × 30 cm) to obtain nine fractions (Fr. 9-10-1~9-10-9). Fr. 9-10-2 was subjected to MPLC (RP-18, water/MeOH 2/1) to yield two fractions (Fr. 9-10-2-1~9-10-2-2). Fr. 9-10-2-1 was further separated with prep. RP-18 TLC (water/MeOH 1/1) to yield six fractions (Fr. 9-10-2-1-A~9-10-2-1-F). Fr. 9-10-2-1-D was subjected to MPLC (silica gel, CH2Cl2/EtOAc 1/1, column size: 1 × 30 cm) to afford compound 22 (4.6 mg). Fr. 9-10-3 was subjected to prep. RP-18 TLC (water/MeOH 2/1) to afford compound 18 (4.1 mg). Fr. 9-10-5 was subjected to MPLC (silica gel, CH2Cl2/MeOH 6/1) to yield ten fractions (Fr. 9-10-5-1~9-10-5-10). Fr. 9-10-5-5 was further separated with prep. RP-18 TLC (water/acetonitrile 3/1) to afford compound 16 (1.6 mg). Fr. 9-10-5-7 was subjected to prep. RP-18 TLC (water/acetonitrile 3/1) to afford compound 19 (12.9 mg) and compound 20 (1.2 mg).

3.4. Spectroscopic Data of New Compounds

3.4.1. Elaeagncoumarin (1)

[(7R,8R)-8-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one]

Pale yellow amorphous solid; [ α ] D 24 − 24.8 (c 0.04, MeOH); UV λmax (MeOH) (log ε): 286 (3.07), 330 (4.00) nm; UV λmax (MeOH + KOH) (log ε): 383 (4.05) nm; IR vmax (ATR): 3275 (OH), 1686 (carbonyl group), 1606, 1519 (aromatic ring) cm−1; ESIMS m/z 365 [M + Na]+; HRESIMS m/z 365.06340 [M + Na]+ (calcd. for C18H14NaO7, 365.06317); 1H-NMR and 13C-NMR (Table 1).

3.4.2. Elaeagterpene A (2)

[(3S,4aR,6aR,6bS,8aS,12aR,12bS,12cS,13aS,13bR,13cS)-4,4,6a,6b,11,11,13c-Heptamethyl-15-oxooctadecahydro-1H,9H-12b,8a-(epoxymethano)piceno [13,14-b]oxiren-3-yl(E)-3-(3,4-dihydroxyphenyl)acrylate]

Whitish powder; [ α ] D 23 + 21.8 (c 0.25, MeOH); UV λmax (MeOH) (log ε): 200 (3.99), 238 (3.70), 290 (3.74), 328 (3.88) nm; UV λmax (MeOH + KOH) (log ε): 202 (4.82), 257 (4.67), 398 (3.81) nm; IR vmax (ATR): 3385 (OH), 1768, 1700 (carbonyl group) cm−1; ESIMS m/z 655 [M + Na]+; HRESIMS m/z 655.36022 [M + Na]+ (calcd. for C39H52NaO7, 655.36053); 1H-NMR and 13C-NMR (Table 2).

3.4.3. Elaeagterpene B (3)

[(3S,4aR,6aR,6bS,8aS,11R,12S,12aR,12bS,14aR,14bS)-4,4,6a,6b,11,12,14b-Heptamethyl-16-oxo-2,3,4,4a,5,6,6a,6b,7,8,10,11,12,12a,14a,14b-hexadecahydro-1H,9H-12b,8a-(epoxymethano)picen-3-yl(E)-3-(3,4-dihydroxyphenyl)acrylate]

Whitish powder; [ α ] D 23 + 42.3 (c 0.09, MeOH); UV λmax (MeOH) (log ε): 202 (4.47), 250 (3.51), 298 (4.24), 326 (4.34) nm; UV λmax (MeOH + KOH) (log ε): 206 (5.05), 257 (4.08), 310 (3.96), 372 (4.46) nm; IR vmax (ATR): 3358 (OH), 1741, 1704 (carbonyl group) cm−1; ESIMS m/z 639 [M + Na]+; HRESIMS m/z 639.36548 [M + Na]+ (calcd. for C39H52NaO6, 639.36561); 1H-NMR and 13C-NMR (Table 2).

3.5. Cell Line

Cells were cultured as described previously [55]. The medium for AML12 (BCRC 60326; Bioresources Collection and Research Center (BCRC), Hsinchu, Taiwan) was a 1:1 mixture of DMEM and Ham’s F12 medium with 10% FBS and 1× ITS-A supplement (Thermo Fisher Scientific).

3.6. LD Assay

The LD accumulation of the test compounds was evaluated according to the studies published by co-author Professor Chia-Hung Yen [1]. The LD accumulation was detected by BODIPY® 493/503 dye (Thermo Fisher Scientific). LD accumulation was achieved by treating cells with OA conjugated to BSA. All data were analyzed with GraphPad Prism 6.01 software (La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s comparison test was used to compare differences between multiple groups. A p-value < 0.05 was considered statistically significant.

3.7. Superoxide Anion and Elastase Release Assays

Neutrophils were collected from healthy adults aged 20–35 and isolated using Ficoll-Paque density separation. The study was approved by Chang Gung Memorial Hospital’s Institutional Review Board (IRB No. 201902217A3) following the Declaration of Helsinki guidelines. The methods for testing the effects of isolates on superoxide anion generation and elastase release in neutrophils were based on studies published by Professor Tsong-Long Hwang [56,57]. Ferricytochrome c (0.6 mg/mL) was used to measure superoxide anion. Elastase substrate (methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, 100 µM; Merck) was employed to detect the elastase release. The elastase level was detected using a spectrophotometer at OD 405 nm. A PI3K inhibitor, namely, LY29002, was used as a positive control in the neutrophil assays. All assays were repeated at least three times. Results are presented as the mean ± standard error of the mean (S.E.M.). Student′s t-test was used to compare the test isolates with a DMSO (0.1%) control. A probability of less than 0.05 was considered significant.

4. Conclusions

This study used 3000 Formosan plant extracts as a natural product library for a high throughput screening for anti-NAFLD drug discoveries. The aerial parts of E. glabra were selected as the research material due to their significant anti-LD accumulation activity without severe cytotoxicity. In the current study, we uncovered three new compounds and 35 known compounds from aerial parts of E. glabra. The skeletons of triterpenoids and flavonoids were a major part of this study, which was consistent with the chemotaxonomy of Elaeagnaceae. The bioactivity results indicated that chlorophyll (compound 37) could reduce LD accumulation. This is the first report on the anti-LD accumulation activity of aerial parts of E. glabra. Furthermore, 3-O-(E)-caffeoyloleanolic acid (13) and methyl pheophorbide a (37) showed potent inhibitory activities on superoxide anion or elastase release in human neutrophils, displaying effects similar to those of the positive control, namely, LY294002. Our findings from the current study support the idea that methyl pheophorbide a (37) can both reduce LD accumulation and show anti-inflammatory activity, thus helping to manage NAFLD progression and related liver inflammation. Taken together, this study revealed the chemical characteristics and bioactivities of E. glabra, providing substantive evidence that E. glabra could be used for the development of anti-NAFLD drugs.

Supplementary Materials

The following supporting information can be downloaded from https://www.mdpi.com/article/10.3390/plants12162943/s1, Figure S1: Structures of known compounds 4–38; Figures S2–S28: The phytochemical spectra of compounds 1–3.

Author Contributions

Conceptualization, H.-S.C.; methodology, H.-S.C., C.-H.Y. and T.-L.H.; formal analysis, J.-H.C., C.-H.Y. and T.-L.H.; investigation, J.-H.C.; resources, H.-S.C.; data curation, H.-S.C. and H.-C.W.; writing—original draft preparation, J.-H.C. and H.-C.W.; writing—review and editing, H.-S.C., C.-H.Y., T.-L.H. and H.-H.K.; visualization, J.-H.C. and H.-C.W.; supervision, H.-S.C. and H.-H.K.; project administration, H.-S.C.; funding acquisition, H.-S.C. and T.-L.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Science and Technology Council [(NSTC), formerly known as Ministry of Science and Technology (MOST)], Taiwan (MOST 111-2320-B-037-017-MY3; MOST 112-2321-B-255-001).

Data Availability Statement

The data presented in this study are available in the Supplementary Materials.

Acknowledgments

We thank the Center for Research Resources and Development of Kaohsiung Medical University for providing a nuclear magnetic resonance (NMR) spectrometer and senior technician Chyi-Jia Wang for measuring the 2D NMR data. We thank the staff from the Natural Product Libraries and High-Throughput Screening Core Facility (NPS core lab) at Kaohsiung Medical University for their technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Yen, C.H.; Chang, H.S.; Yang, T.H.; Wang, S.F.; Wu, H.C.; Chen, Y.C.; Lin, K.J.; Wang, S. High-content screening of a Taiwanese indigenous plant extract library identifies Syzygium simile leaf extract as an inhibitor of fatty acid uptake. Int. J. Mol. Sci. 2018, 19, 2130. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Ashtari, S.; Pourhoseingholi, M.A.; Zali, M.R. Non-alcohol fatty liver disease in Asia: Prevention and planning. World J. Hepatol. 2015, 7, 1788–1796. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Simon, T.G.; Roelstraete, B.; Hartjes, K.; Shah, U.; Khalili, H.; Arnell, H.; Ludvigsson, J.F. Non-alcoholic fatty liver disease in children and young adults is associated with increased long-term mortality. J. Hepatol. 2021, 75, 1034–1041. [Google Scholar] [CrossRef] [PubMed]
  4. Cholongitas, E.; Pavlopoulou, I.; Papatheodoridi, M.; Markakis, G.E.; Bouras, E.; Haidich, A.B.; Papatheodoridis, G. Epidemiology of nonalcoholic fatty liver disease in Europe: A systematic review and meta-analysis. Ann. Gastroenterol. 2021, 34, 404–414. [Google Scholar] [CrossRef] [PubMed]
  5. Mitra, S.; De, A.; Chowdhury, A. Epidemiology of non-alcoholic and alcoholic fatty liver diseases. Transl. Gastroenterol. Hepatol. 2020, 5, 16. [Google Scholar] [CrossRef] [PubMed]
  6. Chalasani, N.; Younossi, Z.; Lavine, J.E.; Diehl, A.M.; Brunt, E.M.; Cusi, K.; Charlton, M.; Sanyal, A.J. The diagnosis and management of non-alcoholic fatty liver disease: Practice guideline by the American association for the study of liver diseases, American college of gastroenterology, and the American gastroenterological association. Hepatology 2012, 55, 2005–2023. [Google Scholar] [CrossRef] [PubMed]
  7. Cigrovski Berkovic, M.; Bilic-Curcic, I.; Mrzljak, A.; Cigrovski, V. NAFLD and physical exercise: Ready, steady, go! Front. Nutr. 2021, 8, 734859. [Google Scholar] [CrossRef]
  8. Gluchowski, N.L.; Becuwe, M.; Walther, T.C.; Farese, R.V., Jr. Lipid droplets and liver disease: From basic biology to clinical implications. Nat. Rev. Gastroenterol. Hepatol. 2017, 14, 343–355. [Google Scholar] [CrossRef]
  9. Welte, M.A. Expanding roles for lipid droplets. Curr. Biol. 2015, 25, R470–R481. [Google Scholar] [CrossRef] [Green Version]
  10. Walther, T.C.; Farese, R.V., Jr. Lipid droplets and cellular lipid metabolism. Annu. Rev. Biochem. 2012, 81, 687–714. [Google Scholar] [CrossRef] [Green Version]
  11. Nishino, C.; Enoki, N.; Tawata, S.; Mori, A.; Kobayashi, K.; Fukushima, M. Antibacterial activity of flavonoids against Staphylococcus epidermidis, a skin bacterium. J. Agric. Food Chem. 1987, 51, 139–143. [Google Scholar] [CrossRef]
  12. Mori, A.; Nishino, C.; Enoki, N.; Tawata, S. Antibacterial activity and mode of action of plant flavonoids against Proteus vulgaris and Staphylococcus aureus. Phytochemistry 1987, 26, 2231–2234. [Google Scholar] [CrossRef]
  13. Tagahara, K.; Kato, A.; Hashimoto, Y.; Suzuta, Y. Constituents of the genus Elaeagnus (III): On the constituents of Elaeagnus glabra Thunb. Shoyakugaku Zasshi 1984, 3, 131. [Google Scholar]
  14. Sisa, M.; Bonnet, S.L.; Ferreira, D.; Van der Westhuizen, J.H. Photochemistry of flavonoids. Molecules 2010, 15, 5196–5245. [Google Scholar] [CrossRef] [PubMed]
  15. Salim, F.; Zain, M.M.; Ridzuan, M.S.M.; Langat, M.K.; Mulholland, D.A.; Ahmad, R. Flavan-3-ols from the leaves of Malaysian Uncaria longiflora var. pteropoda (Miq.) Ridsd. Phytochem. Lett. 2013, 6, 236–240. [Google Scholar] [CrossRef] [Green Version]
  16. Tobiasona, F.L.; Fronczek, F.R.; Steynberg, J.P.; Steynberg, E.C.; Hemingway, R.W. Crystal structure, conformational analyses, and charge density distributions for ent-epifisetinidol: An explanation for regiospecific electrophilic aromatic substitution of 5-deoxyflavans. Tetrahedron 1993, 49, 5927–5940. [Google Scholar] [CrossRef]
  17. Abdel-Monem, A.R.; Kandil, Z.A.; Abdel-Naim, A.B.; Abdel-Sattar, E. A new triterpene and protective effect of Periploca somaliensis Browicz fruits against CCl4-induced injury on human hepatoma cell line (Huh7). Nat. Prod. Res. 2015, 29, 423–429. [Google Scholar] [CrossRef]
  18. Tkachev, A.V.; Denisov, A.Y.; Gatilov, Y.V.; Bagryanskaya, I.Y.; Shevtsov, S.A.; Rybalova, T.V. Stereochemistry of hydrogen peroxide—Acetic acid oxidation of ursolic acid and related compounds. Tetrahedron 1994, 50, 11459–11488. [Google Scholar] [CrossRef]
  19. Lalith, J.G.; Wannigama, P.; Macleod, J.K. Triterpenoids from Anamirta cocculus. Phytochemistry 1993, 34, 1111–1116. [Google Scholar] [CrossRef]
  20. Raju, R.; Gunawardena, D.; Ahktar, M.A.; Low, M.; Reddell, P.; Münch, G. Anti-inflammatory chemical profiling of the Australian rainforest tree Alphitonia petriei (Rhamnaceae). Molecules 2016, 21, 1521. [Google Scholar] [CrossRef] [Green Version]
  21. Kahnt, M.; Heller, L.; Al-Harrasi, A.; Schäfer, R.; Kluge, R.; Wagner, C.; Otgonbayar, C.; Csuk, R. Platanic acid-derived methyl 20-amino-30-norlupan-28-oates are potent cytotoxic agents acting by apoptosis. Med. Chem. Res. 2018, 27, 1757–1769. [Google Scholar] [CrossRef]
  22. Wang, C.; Wu, P.; Tian, S.; Xue, J.; Xu, L.; Li, H.; Wei, X. Bioactive Pentacyclic Triterpenoids from the Leaves of Cleistocalyx operculatus. J. Nat. Prod. 2016, 79, 2912–2923. [Google Scholar] [CrossRef]
  23. Fomogne-Fodjo, M.C.Y.; Ndinteh, D.T.; Olivier, D.K.; Kempgens, P.; van Vuuren, S.; Krause, R.W.M. Secondary metabolites from Tetracera potatoria stem bark with anti-mycobacterial activity. J. Ethnopharmacol. 2017, 195, 238–245. [Google Scholar] [CrossRef] [PubMed]
  24. Cheng, D.L.; Cao, X.P. Pomolic acid derivatives from the root of Sanguisorba officinalis. Phytochemistry 1992, 31, 1317–1320. [Google Scholar] [CrossRef]
  25. Wu, C.; Cui, X.; Yu, P.; Yang, M.; Zhang, Y.; Liu, X.; Qu, G. Triterpenic Acids from Sorbaria sorbifolia. Chem. Nat. Compd. 2019, 55, 580–582. [Google Scholar] [CrossRef]
  26. Fuchino, H.; Satoh, T.; Tanaka, N. Chemical evaluation of Betula species in Japan. I.Constituents of Betula ermanii. Chem. Pharm. Bull. 1995, 43, 1937–1942. [Google Scholar] [CrossRef] [Green Version]
  27. Tanachatchairatana, T.; Bremner, J.B.; Chokchaisiri, R.; Suksamrarn, A. Antimycobacterial activity of cinnamate-based esters of the triterpenes betulinic, oleanolic and ursolic acids. Chem. Pharm. Bull. 2008, 56, 194–198. [Google Scholar] [CrossRef] [Green Version]
  28. Van Der westhuizen, J.H.; Steenkamp, J.A.; Ferreira, D. An unusual reaction of flavan-3-ols with acetone of relevance to the formation of the tetracyclic ring system in peltogynoids. Tetrahedron 1990, 46, 7849–7854. [Google Scholar] [CrossRef]
  29. Yue, J.M.; Zhao, Y.; Zhao, Q.S.; Lin, Z.W.; Sun, H.D.; Wu, H.M.; Xu, J.F. Phenolics from Ceratostigma minus. J. Integr. Plant Biol. 1998, 40, 1035–1039. [Google Scholar]
  30. Huang, A.C.; Wilde, A.; Ebmeyer, J.; Skouroumounis, G.K.; Taylor, D.K. Examination of the phenolic profile and antioxidant activity of the leaves of the Australian native plant Smilax glyciphylla. J. Nat. Prod. 2013, 76, 1930–1936. [Google Scholar] [CrossRef]
  31. Xu, J.; Wang, X.; Yue, J.; Sun, Y.; Zhang, X.; Zhao, Y. Polyphenols from acorn aeaves (Quercus liaotungensis) protect pancreatic beta cells and their inhibitory activity against α-glucosidase and protein tyrosine phosphatase 1B. Molecules 2018, 23, 2167. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Lage, G.A.; Medeiros, F.d.S.; Furtado, W.d.L.; Takahashi, J.A.; Filho, J.D.d.S.; Pimenta, L.P.S. The first report on flavonoid isolation from Annona crassiflora Mart. Nat. Prod. Res. 2014, 28, 808–811. [Google Scholar] [CrossRef]
  33. Lin, G.; Chang, L.; Liu, Y.; Xiang, Z.; Chen, J.; Yang, Z. Enantioselective total syntheses of (+)-gallocatechin, (−)-epigallocatechin, and 8-C-ascorbyl-(−)-epigallocatechin. Asian J. Chem. 2013, 8, 700–704. [Google Scholar] [CrossRef]
  34. Terfassi, S.; Dauvergne, X.; Cérantola, S.; Lemoine, C.; Bensouici, C.; Fadila, B.; Christian, M.; Marchioni, E.; Benayache, S. First report on phytochemical investigation, antioxidant and antidiabetic activities of Helianthemum getulum. Nat. Prod. Res. 2022, 36, 2806–2813. [Google Scholar] [CrossRef] [PubMed]
  35. Meena, H.; Hnamte, S.; Siddhardha, B. Secondary metabolites from endophytic fungi: Chemical diversity and application. In Advances in Endophytic Fungal Research: Present Status and Future Challenges; Singh, B.P., Ed.; Springer International Publishing: Cham, Switzerland, 2019; pp. 145–169. [Google Scholar]
  36. Madikizela, B.; Aderogba, M.A.; Finnie, J.F.; Van Staden, J. Isolation and characterization of antimicrobial compounds from Terminalia phanerophlebia Engl. & Diels leaf extracts. J. Ethnopharmacol. 2014, 156, 228–234. [Google Scholar] [CrossRef]
  37. Li, H.; Zhang, Y.; Liu, Q.; Sun, C.; Li, J.; Yang, P.; Wang, X. Preparative separation of phenolic compounds from Chimonanthus praecox flowers by high-speed counter-current chromatography using a stepwise elution mode. Molecules 2016, 21, 1016. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Niu, C.; Zhang, Z.Z.; Yang, L.P.; Zhai, Y.Y.; Li, S.N.; Hao, S.Y.; Chen, X.Y.; Wang, J.H.; Wang, Z.H. Chemical constituents of Curculigo orchioides. Chem. Nat. Compd. 2020, 56, 957–959. [Google Scholar] [CrossRef]
  39. Li, A.; Mishra, Y.; Malik, M.; Wang, Q.; Li, S.; Taylor, M.; Reichert, D.E.; Luedtke, R.R.; Mach, R.H. Evaluation of N-phenyl homopiperazine analogs as potential dopamine D3 receptor selective ligands. Bioorg. Med. Chem. 2013, 21, 2988–2998. [Google Scholar] [CrossRef] [Green Version]
  40. Shu, P.; Sun, M.; Li, J.; Zhang, L.; Xu, H.; Lou, Y.; Ju, Z.; Wei, X.; Wu, W.; Sun, N. Chemical constituents from Ginkgo biloba leaves and their cytotoxicity activity. J. Nat. Med. 2020, 74, 269–274. [Google Scholar] [CrossRef]
  41. Tan, S.P.; Ali, A.M.; Nafiah, M.A.; Amna, U.; Ramli, S.A.; Ahmad, K. Terpenes and phenolic compounds of Murraya koenigii. Chem. Nat. Compd. 2017, 53, 980–981. [Google Scholar] [CrossRef]
  42. Tang, C.; Tao, G.; Wang, Y.; Liu, Y.; Li, J. Identification of α-tocopherol and its oxidation products by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. J. Agric. Food Chem. 2020, 68, 669–677. [Google Scholar] [CrossRef] [PubMed]
  43. Liao, P.C.; Lai, M.H.; Hsu, K.P.; Kuo, Y.H.; Chen, J.; Tsai, M.C.; Li, C.X.; Yin, X.J.; Jeyashoke, N.; Chao, L.K.P. Identification of β-sitosterol as in vitro anti-inflammatory constituent in Moringa oleifera. J. Agric. Food. Chem. 2018, 66, 10748–10759. [Google Scholar] [CrossRef]
  44. Huang, C.M.; Sung, P.J.; Kuo, Y.H.; Chang, T.H.; Chen, C.L.; Cheng, M.J.; Chen, J.J. A new dihydroagarofuranoid sesquiterpene and cytotoxic constituents of Microtropis fokienensis. Chem. Nat. Compd. 2020, 56, 440–444. [Google Scholar] [CrossRef]
  45. Yodsing, N.; Lekphrom, R.; Sangsopha, W.; Aimi, T.; Boonlue, S. Secondary metabolites and their biological activity from Aspergillus aculeatus KKU-CT2. Curr. Microbiol. 2018, 75, 513–518. [Google Scholar] [CrossRef]
  46. Sribuhom, T.; Sriphana, U.; Thongsri, Y.; Yenjai, C. Chemical constituents from the stems of Alyxia schlechteri. Phytochem. Lett. 2015, 11, 80–84. [Google Scholar] [CrossRef]
  47. Zou, G.A.; Wang, Y.; Zou, Z.M.; Chen, S. Lignans from stems of Croton caudatus var. tomentosus. Chem. Nat. Compd. 2013, 49, 93–94. [Google Scholar] [CrossRef]
  48. Gudmundsson, H.G.; Linderborg, K.M.; Kallio, H.; Yang, B.; Haraldsson, G.G. Synthesis of enantiopure ABC-type triacylglycerols. Tetrahedron 2020, 76, 130813. [Google Scholar] [CrossRef]
  49. Xiao, W.L.; Chen, W.H.; Zhang, J.Y.; Song, X.P.; Chen, G.Y.; Han, C.R. Ionone-type sesquiterpenoids from the stems of Ficus pumila. Chem. Nat. Compd. 2016, 52, 531–533. [Google Scholar] [CrossRef]
  50. Lin, H.Y.; Chiu, H.L.; Lu, T.L.; Tzeng, C.Y.; Lee, T.H.; Lee, C.K.; Shao, Y.Y.; Chen, C.R.; Chang, C.I.; Kuo, Y.H. Ficusmicrochlorin A—C, two new methoxy lactone chlorins and an anhydride chlorin from the leaves of Ficus microcarpa. Chem. Pharm. Bull. 2011, 59, 113–116. [Google Scholar] [CrossRef] [Green Version]
  51. Hurtado-Díaz, I.; Sánchez-Carranza, J.N.; Romero-Estrada, A.; González-Maya, L.; González-Christen, J.; Herrera-Ruiz, M.; Alvarez, L. 16-Hydroxy-lycopersene, a polyisoprenoid alcohol isolated from Tournefortia hirsutissima, inhibits nitric oxide production in RAW 264.7 cells and induces apoptosis in Hep3B Cells. Molecules 2019, 24, 2366. [Google Scholar] [CrossRef] [Green Version]
  52. Das, P.; Mandal, S.; Gangopadhyay, S.; Das, K.; Mitra, A.G.; Dasgupta, S.; Mukhopadhyay, S.; Mukhopadhyay, A. Antioxidative and anticarcinogenic activities of methylpheophorbide a, isolated from wheat grass (Triticum aestivum Linn). Nat. Prod. Res. 2016, 30, 474–477. [Google Scholar] [CrossRef]
  53. Wongsinkongman, P.; Brossi, A.; Wang, H.K.; Bastow, K.F.; Lee, K.H. Antitumor agents. Part 209: Pheophorbide-a derivatives as photo-independent cytotoxic agents. Bioorg. Med. Chem. 2002, 10, 583–591. [Google Scholar] [CrossRef] [PubMed]
  54. Rushdi, M.I.; Abdel-Rahman, I.A.M.; Saber, H.; Attia, E.Z.; Abdelraheem, W.M.; Madkour, H.A.; Abdelmohsen, U.R. The genus Turbinaria: Chemical and pharmacological diversity. Nat. Prod. Res. 2021, 35, 4560–4578. [Google Scholar] [CrossRef] [PubMed]
  55. Yen, C.H.; Lai, C.C.; Shia, T.H.; Chen, M.; Yu, H.C.; Liu, Y.P.; Chang, F.R. Gynura divaricata attenuates tumor growth and tumor relapse after cisplatin therapy in HCC xenograft model through suppression of cancer stem cell growth and Wnt/β-catenin signalling. J. Ethnopharmacol. 2018, 213, 366–375. [Google Scholar] [CrossRef]
  56. Kao, T.I.; Chen, P.J.; Wang, Y.H.; Tseng, H.H.; Chang, S.H.; Wu, T.S.; Yang, S.H.; Lee, Y.T.; Hwang, T.L. Bletinib ameliorates neutrophilic inflammation and lung injury by inhibiting Src family kinase phosphorylation and activity. Br. J. Pharmacol. 2021, 178, 4069–4084. [Google Scholar] [CrossRef] [PubMed]
  57. Korinek, M.; Hsieh, P.S.; Chen, Y.L.; Hsieh, P.W.; Chang, S.H.; Wu, Y.H.; Hwang, T.L. Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo. Biochem. Pharmacol. 2021, 190, 114596. [Google Scholar] [CrossRef]
Plants 12 02943 g001
Figure 1. Use of the high-throughput screening platform for anti-LD candidate discovery from the Formosan methanolic extract bank and the results.
Figure 1. Use of the high-throughput screening platform for anti-LD candidate discovery from the Formosan methanolic extract bank and the results.
Plants 12 02943 g001
Plants 12 02943 g002
Figure 2. Structures of compounds 1−3.
Figure 2. Structures of compounds 1−3.
Plants 12 02943 g002
Plants 12 02943 g003
Figure 3. Key HMBC and COSY correlations of compounds 1−3.
Figure 3. Key HMBC and COSY correlations of compounds 1−3.
Plants 12 02943 g003
Plants 12 02943 g004
Figure 4. Key NOESY correlations of compounds 2 and 3.
Figure 4. Key NOESY correlations of compounds 2 and 3.
Plants 12 02943 g004
Plants 12 02943 g005
Figure 5. Effect of methyl pheophorbide a (37) on LD accumulation. (A) Representative images of the anti-LD formation activity of methyl pheophorbide a (37). (B) Quantification results of the LD assay and cell viability. AML12 cells were treated with BSA or OA (125 µM) with 20 µM methyl pheophorbide a (37) for 24 h. AML12 cells were used as a cell model for lipid accumulation—they were treated with 125 μM oleic acid (OA) for 24 h. Nuclei and LD were stained with Hoechst 33342 (blue) and BODIPY® 493/503 (green), respectively. The asterisk indicates a significant difference from the solvent control cells (*** p < 0.001, one-way ANOVA).
Figure 5. Effect of methyl pheophorbide a (37) on LD accumulation. (A) Representative images of the anti-LD formation activity of methyl pheophorbide a (37). (B) Quantification results of the LD assay and cell viability. AML12 cells were treated with BSA or OA (125 µM) with 20 µM methyl pheophorbide a (37) for 24 h. AML12 cells were used as a cell model for lipid accumulation—they were treated with 125 μM oleic acid (OA) for 24 h. Nuclei and LD were stained with Hoechst 33342 (blue) and BODIPY® 493/503 (green), respectively. The asterisk indicates a significant difference from the solvent control cells (*** p < 0.001, one-way ANOVA).
Plants 12 02943 g005
Plants 12 02943 g006
Figure 6. Preliminary screening of the inhibitory activities toward superoxide anion and elastase release of isolates from aerial parts of E. glabra. Percentage of inhibition (Inh%) at 10 μM. The results are presented as the mean ± S.E.M. (n = 3–5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control (DMSO).
Figure 6. Preliminary screening of the inhibitory activities toward superoxide anion and elastase release of isolates from aerial parts of E. glabra. Percentage of inhibition (Inh%) at 10 μM. The results are presented as the mean ± S.E.M. (n = 3–5). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control (DMSO).
Plants 12 02943 g006
Table 1. 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data of compound 1.
Table 1. 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data of compound 1.
Position1
δH (J in Hz)δC
1––
25.02, br s80.7
34.30, br dd (4.2, 2.7)66.6
4a2.96, dd (17.0, 4.2)29.4
4b2.87, dd (17.0, 2.7)
5–153.4
6–103.4
7–156.2
86.35, s95.7
9–163.1
10–105.6
1′–131.4
2′7.04, d (2.3)115.2
3′–146.2
4′–146.1
5′6.80, d (8.3)116.1
6′6.86, dd (8.3, 2.3)119.3
2″–164.5
3″6.07, d (9.6)109.4
4″8.14, d (9.6)141.2
Table 2. 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data of compounds 2 and 3.
Table 2. 1H NMR (600 MHz, CD3OD) and 13C NMR (150 MHz, CD3OD) data of compounds 2 and 3.
Position23
δH (J in Hz)δCδH (J in Hz)δC
11.60, m38.91.12, m/1.93, m39.0
21.60, m/1.81, m24.41.50, m/1.76, m32.5
34.62, dd (11.4, 4.8)82.04.60, dd (11.4, 4.8)82.0
4–39.1–39.3
50.94, m56.00.99, m56.0
61.60, m18.61.67, m18.7
71.20, m/1.41, m32.21.30, m32.3
8–42.6–43.2
91.66, br s52.02.11, br s54.3
10–37.7–37.5
113.12, dd (3.9, 1.8)53.85.60, dd (10.5, 3.3)130.0
123.06, d (3.9)58.26.06, dd (10.5, 1.5)134.8
13–89.4–91.9
14–41.8–43.0
151.15, m27.81.67, m26.6
161.29, m/2.28, m22.31.30, m/2.30, ddd (18.9, 13.5, 5.7)23.9
17–45.4–46.6
182.40, dd (13.8, 3.0)50.81.70, d (11.4)61.8
191.29, m/1.99, m38.91.12, m39.2
20–32.30.95, m41.5
211.29, m/1.47, ddd (18.6, 14.0, 4.4)35.21.35, m/1.59, m31.8
221.60, m/1.70, dd (14.0, 4.4)28.21.76, m/1.81, m24.5
230.92, s16.90.92, s16.7
240.99, s28.30.98, s28.3
251.14, s17.71.01, s15.8
261.08, s20.71.06, s19.6
271.17, s19.31.25, s16.6
28–181.6–182.6
290.96, s24.01.04, d (6.3)18.3
301.02, s33.50.96, d (6.3)19.4
1′–127.7–127.7
2′7.04, d (2.3) 115.17.04, d (2.4) 115.1
3′–146.85–146.85
4′–149.6–149.6
5′6.78, d (8.6)116.56.78, d (8.4)116.5
6′6.95, dd (8.6, 2.3)122.96.95, dd (8.4, 2.4)122.9
7′7.54, d (15.9)146.787.53, d (15.9)146.75
8′6.26, d (15.9)115.56.25, d (15.9)115.5
9′–169.1–169.2
Table 3. Anti-lipid droplet accumulation activity of compounds isolated from E. glabra.
Table 3. Anti-lipid droplet accumulation activity of compounds isolated from E. glabra.
Compound (100 μg/mL)Relative Lipid Droplet Count (%) aCell Viability (%, 24h, DAPI Image) b
lupeol (9)95.2 ± 1.8113.7 ± 3.6
ursolic acid (11)99.2 ± 4146.5 ± 4.5
plumbocatechins B (15)105.9 ± 7.8132.5 ± 11.8
(−)-catechin (16)110.7 ± 5.1154 ± 12.3
(−)-epicatechin (18)115.9 ± 6145.9 ± 10.3
(−)-epigallocatechin (19)93.5 ± 6.6127.2 ± 22.6
(−)-gallocatechin (20)116.7 ± 7.8153.9 ± 6.2
plumbocatechin A (22)118.2 ± 3.1147.8 ± 9.6
syringaldehyde (25)102 ± 5137.8 ± 5.7
β-sitosterol (30)112.7 ± 7.6128.5 ± 9.1
β-sitosterone (31)121.5 ± 4.3135.3 ± 7.3
methyl pheophorbide a (37) 0.3 ± 0.199.5 ± 5.4
TC c60.6 ± 0.2126.1 ± 6.1
a Relative LD counts—the average LD counts/cells of oleic-acid-treated groups were used as 100% fat-loading in the AML12 cell line. b Cell viability—the average nucleus counts of DMSO were used as 100% cell viability in the AML12 cell line. c Triacsin C (TC) is an inhibitor of long fatty acyl CoA synthetase and was used as a positive control for LD inhibition. The drug concentration was 8 nM.
Table 4. Inhibitory effects of the active compounds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.
Table 4. Inhibitory effects of the active compounds on superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils.
CompoundSuperoxide Anion
IC50 (μM)		Elastase Release
IC50 (μM)
elaeagterpene A (2)>10>10
cleistocalyxic acid E (7)>10>10
2α,3β,23-trihydroxy-11α,12α-epoxyolean-28,13β-olide (12)>10>10
3-O-(E)-caffeoyloleanolic acid (13)3.01 ± 0.58>10
(+)-3β-O-trans-caffeoyl betulinic acid (14)>10>10
syringaldehyde (25)>10>10
4-(2-hydroxyethyl)benzoic acid (26)>10>10
methyl pheophorbide a (37)1.29 ± 0.272.35 ± 0.12
#LY2940021.29 ± 0.522.61 ± 0.72
The results are presented as the mean ± S.E.M. (n = 3–4). The compound was not considered anti-inflammatory when the IC50 value was >10 µM. # A phosphatidylinositol-3-kinase inhibitor (LY294002) was used as a positive control.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Cheng, J.-H.; Wu, H.-C.; Yen, C.-H.; Hwang, T.-L.; Ko, H.-H.; Chang, H.-S. Chemical Constituents with Anti-Lipid Droplet Accumulation and Anti-Inflammatory Activity from Elaeagnus glabra. Plants 2023, 12, 2943. https://doi.org/10.3390/plants12162943

AMA Style

Cheng J-H, Wu H-C, Yen C-H, Hwang T-L, Ko H-H, Chang H-S. Chemical Constituents with Anti-Lipid Droplet Accumulation and Anti-Inflammatory Activity from Elaeagnus glabra. Plants. 2023; 12(16):2943. https://doi.org/10.3390/plants12162943

Chicago/Turabian Style

Cheng, Ju-Hsin, Ho-Cheng Wu, Chia-Hung Yen, Tsong-Long Hwang, Horng-Huey Ko, and Hsun-Shuo Chang. 2023. "Chemical Constituents with Anti-Lipid Droplet Accumulation and Anti-Inflammatory Activity from Elaeagnus glabra" Plants 12, no. 16: 2943. https://doi.org/10.3390/plants12162943

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop