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

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.


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

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-S31). In addition, some isolates were further examined for anti-LD accumulation effects in hepatic cell lines.

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.

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 proinflammatory responses by suppressing fMLP/CB-induced superoxide anion generation

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 IC 50 values (Table 4) an IC 50 value of 3.01 ± 0.58 µM, and methyl pheophorbide a (37) showed potent antiinflammatory activity on both superoxide anion generation and elastase release with IC 50 values of 1.29 ± 0.27 and 2.35 ± 0.12 µM, respectively. 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.  The results are presented as the mean ± S.E.M. (n = 3-4). The compound was not considered antiinflammatory when the IC50 value was >10 µM. # A phosphatidylinositol-3-kinase inhibitor (LY294002) was used as a positive control.
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  The results are presented as the mean ± S.E.M. (n = 3-4). The compound was not considered anti-inflammatory when the IC 50 value was >10 µM. # A phosphatidylinositol-3-kinase inhibitor (LY294002) was used as a positive control.
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.

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.

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.

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.

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.