Aromatic Acids and Leucine Derivatives Produced from the Deep-Sea Actinomycetes Streptomyces chumphonensis SCSIO15079 with Antihyperlipidemic Activities

Six new aromatic acids (1–6) and three new leucine derivatives containing an unusual oxime moiety (7–9) were isolated and identified from the deep-sea-derived actinomycetes strain Streptomyces chumphonensis SCSIO15079, together with two known compounds (10–11). The structures of 1–9 including absolute configurations were determined by detailed NMR, MS, and experimental and calculated electronic circular dichroism spectroscopic analyses. Compounds 1–9 were evaluated for their antimicrobial and cytotoxicity activities, as well as their effects on intracellular lipid accumulation in HepG2 cells. Compounds 3 and 4, with the most potent inhibitory activity on intracellular lipid accumulation at 10 μM, were revealed with potential antihyperlipidemic effects, although the mechanism needs to be further studied.


Introduction
Hyperlipidemia, defined as an increase in the blood lipids levels, is a risk factor for cardiovascular diseases, including coronary atherosclerosis, cardiovascular diseases, Heart failure, and other metabolic diseases. Hyperlipidemia is also known as an aggravation of several pathological conditions such as hypothyroidism and chronic renal failure. Globally, approximately 12 million people die each year due to hyperlipidemia [1][2][3][4]. At present, drugs used to reduce the level of blood lipids mainly include statins (atorvastatin, lovastatin, etc.), fibrates (bezafibrate, lifibrate, etc.), and resin (cholestyramine, colestipol, etc.). Nevertheless, these synthetic medicines are often associated with some serious side effects such as diarrhea, nausea, gallstones, myositis, and abnormal liver function [5][6][7]. Therefore, it is urgent to discover new lipid-lowering agents with more therapeutic value and less tolerable side effects from natural products.
For the past several decades, many marine-derived active ingredients have been tested and proven with lipid-lowering or antihyperlipidemic effects, such as the seaweed polysaccharide [8], marine algal polyphenols [9], polyunsaturated fatty acids from oily fish [10,11], and fish protein [12]. Marine-derived microorganisms have shown promising potential to produce a great number of active substances including antibacterial, antiviral, antioxidant, as well as cytotoxic compounds [13][14][15][16][17][18]. Marine extremophilic microorganisms tend to produce fascinating novel types of bioactive secondary metabolites; therefore deepsea-derived microorganisms seem to be significant sources for discovering lead compounds in drug discovery [19,20].
In our search, six new aromatic acids (1-6), three new leucine derivatives containing an unusual oxime moiety (7)(8)(9), and two known compounds (10,11) were obtained from the actinomycetes strain Streptomyces chumphonensis SCSIO15079, isolated from a sediment sample of Indian Ocean, with a depth of 3386 m (Figure 1). In order to study the potential antihyperlipidemic effects of these compounds, their effects on intracellular lipid accumulation in HepG2 cells were evaluated. Herein, we describe the fermentation, isolation, structural determination, and biological activity of these compounds.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 2 of 12 antioxidant, as well as cytotoxic compounds [13][14][15][16][17][18]. Marine extremophilic microorganisms tend to produce fascinating novel types of bioactive secondary metabolites; therefore deep-sea-derived microorganisms seem to be significant sources for discovering lead compounds in drug discovery [19,20]. In our search, six new aromatic acids (1-6), three new leucine derivatives containing an unusual oxime moiety (7)(8)(9), and two known compounds (10,11) were obtained from the actinomycetes strain Streptomyces chumphonensis SCSIO15079, isolated from a sediment sample of Indian Ocean, with a depth of 3386 m (Figure 1). In order to study the potential antihyperlipidemic effects of these compounds, their effects on intracellular lipid accumulation in HepG2 cells were evaluated. Herein, we describe the fermentation, isolation, structural determination, and biological activity of these compounds.

Structure Elucidation
Compound 1 was isolated as a yellowish solid. Its molecular formula of C 18  for an aromatic ring with vicinal substitution. The 1 H− 1 H COSY correlations established that C-2 to C-10 were a long-chain alkanes system ( Figure 2). The 13 C NMR spectrum, in combination with DEPT and HSQC spectra, revealed the presence of 18 carbons, including a methyl (C-7 ), six aromatic carbons (C-1 to C-6 ), nine methylenes (C-2 to C-10), and two carbonyl carbons (C-1 and C-11). The HMBC correlations from H-5 (δ H 7.27) and H-6 (δ H 7.67) to C-11 (δ C 207.4) and C-9 (δ C 26.2), respectively, established the connection of the conjugated chain to an aromatic ring. The position of the methyl group in the aromatic ring was determined by HMBC correlations observed from H-7 (δ H 2.42) to C-2 (δ C 138.6) and C-1 (δ C 139.7), which indicated a methylbenzene moiety. As only H-6 showed an HMBC correlation with the ketone signal, it was deduced that the ketone C-11 (δ C 207.4, s) must be attached directly to the benzene ring ( Figure 2). Thus, the structure of 1 was identified, as shown in Figure 1, and named 11-oxo-11-(o-tolyl) undecanoic acid (1). Compound 2 was isolated as a yellowish solid. Its molecular formula of C20H30O3 was determined by the HRESIMS (m/z 319.2268 [M − H] − , calcd for, 319.2273), which required 6 degrees of unsaturation. Its 1 H and 13 C NMR data (Tables 1 and 2) revealed that the aromatic ring with vicinal substitution of 2 was the same as 1. Compared to 1, the only Compound 2 was isolated as a yellowish solid. Its molecular formula of C 20 H 30 O 3 was determined by the HRESIMS (m/z 319.2268 [M − H] − , calcd for, 319.2273), which required 6 degrees of unsaturation. Its 1 H and 13 C NMR data (Tables 1 and 2) revealed that the aromatic ring with vicinal substitution of 2 was the same as 1. Compared to 1, the only difference of 2 was two more methylene groups in the fatty acid chains, the 1 H NMR data of which showed 11 methylene groups at δ H 1.25~1.36. Thus, the structure of 2 was identified as shown in Figure 1 and named 13-oxo-13-(o-tolyl) tridecanoic acid (2). Compound 3 was isolated as a yellowish solid. Its molecular formula of C 18 H 26 O 2 was determined by the HRESIMS (m/z 297.1825 [M + Na] + , calcd for, 297.1830), which required 6 degrees of unsaturation. It was determined as a 10,11-unsaturated analogue of 1 based on the presence of the 1 H-1 H COSY spectrum correlated two olefinic protons at δ H 6.06 (1H, dt, J = 15.6, 7.0 Hz, H-10) and 6.57 (1H, d, J = 15.6 Hz, H-11), indicating an E-disubstituted double bond, which replaced the ketone signal at C-11 of 1, as evident from the 2D NMR data in association with the HRESIMS data. Thus, the structure of 3 was identified (E)-11-(o-tolyl) undec-10-enoic acid (3).
Compound 4 was determined to be C 20 H 30 O 2 by the HRESIMS (m/z 325.2138 [M + Na] + ) data, requiring 6 degrees of unsaturation. A comparison of the NMR data between 4 and 3 (Tables 1 and 2) revealed that the side chain of 4 possessed two methylene groups, whereas the remaining NMR data of both compounds were closely similar. Thus, the structure of 4 was identified as (E)-13-(o-tolyl) undec-12-enoic acid (4).
Compound 5 was isolated as a yellowish solid. Its molecular formula of C 18 H 28 O 3 was determined by the HRESIMS (m/z 291.1969 [M − H] − , calcd for, 291.1960), which required 5 degrees of unsaturation. 1 H and 13 C NMR data (Tables 1 and 2) of 5 closely resembled those of 1. The only difference between them was that the ketone at the 11-position in 1 was replaced by an oxygenated methine group (δ H 4.86/δ C 71.1) in 5, which was corroborated by the HMBC correlation from H-11 to C-1 (δ C 144.6), 2 (δ C 135.5), and 6 (δ C 126.4). The absolute configuration of 5 was established based on a comparison of its experimental electronic of the 11R-5a model and the 11S-5b model at the B3LYP/6-31G (d,p) level in Gaussian 03, and the former was showed relatively good agreement with the experimental one ( Figure 3). Thus, the absolute structure of 5 was defined as 11R. We also tried employing Mosher's method to determine the absolute configuration of C-11 in 5. The treatment of 5 with (R)-and (S)-MTPA-Cl yielded (S)-and (R)-MTPA ester derivatives, respectively. However, the values in the 1 H-NMR spectrum of the mono-(S)-and -(R)-MTPA esters in 5 had no significant differences. This might be a result from the aliphatic chain being too long to flip. Thus, the structure of 5 was identified to be (R)-11-hydroxy-11-(o-tolyl) undecanoic acid (5).   (Tables 1 and 2), and as expected, the 13 C NMR data indicated clearly two additional methylene groups ( Table 2). Compound 6 was indicated to be Rconfiguration at C-13, and its optical rotation was ([α] 25 D +2.6 (c 0.10, MeOH)), compared with compound 5 ([α] 25 D +4.0 (c 0.10, MeOH)). Thus, the structure of 6 was identified to be (R)-11-hydroxy-11-(o-tolyl) undecanoic acid (6).
Compound 9 was obtained as a white powder, which had the molecular formula of C 6 H 12 N 2 O 2 as inferred from HRESIMS (m/z 167.0791 [M + Na] + ) and NMR data (Table 3). It had been afforded previously as a synthetic compound by the alkaline hydrolysis of aspergilliamide [21], and spectroscopic data were not reported. Thus, compound 9 was identified as (E)-2-(hydroxyimino)-4-methylpentanamide (9), and it is reported as a new natural product.
The aromatic analogues containing a linear chain were reported with lowering lipid effects [24]. For example, 2-methyl-8-hydroxybenzeneheptanoic acid, an aromatic analogue obtained from a marine-derived Streptomyces strain, remarkably decreased lipid levels including total cholesterol (TC) and triglycerides (TG) in HepG2 cells [24]. Moreover, it is revealed that the aromatic analogue containing a linear chain with seven carbons showed stronger inhibition in comparison with those bearing a linear chain with five carbons [24]. In this study, several aromatic analogues containing a linear chain with eleven or thirteen carbons have been obtained, but their lipid-lowering effect remains unknown. Thus, the obtained new compounds, including the aromatic analogues, were evaluated with their effects on intracellular lipid accumulation in HepG2 cells by oil-red O staining, together with their brief SAR discussion.
The compounds 1-9 and the positive control lovastatin were tested for cytotoxic effects toward HepG2 cells by using the MTT (Sigma Aldrich) assay, together with oleic acid (OA). The results showed there is no obvious toxicity with these compounds. HepG2 cells were incubated with OA (500 µM) and DMEM for 24 h and then treated with 10 µM of indicated compounds or lovastatin for an additional 24 h. The effects of compounds 1-9 on oleic acid-elicited intracellular lipid accumulation were showed in Figure 4. All compounds tested showed a reduction in intracellular lipid accumulation, while 3 and 4 displayed stronger inhibition than the others. As for the results, there is no clear conclusion about the difference in activities in comparison with those aromatic analogues with eleven and thirteen carbons of the linear chain in the structures. However, it was speculated that the conjugated double bond in linear chain (such as in 3 or 4) is beneficial for the intracellular lipid-lowering effect than carbonyl or hydroxyl groups in this position (Figure 4). The hypolipidemic mechanism of them needs further study. showed stronger inhibition in comparison with those bearing a linear chain with five carbons [24]. In this study, several aromatic analogues containing a linear chain with eleven or thirteen carbons have been obtained, but their lipid-lowering effect remains unknown. Thus, the obtained new compounds, including the aromatic analogues, were evaluated with their effects on intracellular lipid accumulation in HepG2 cells by oil-red O staining, together with their brief SAR discussion. The compounds 1-9 and the positive control lovastatin were tested for cytotoxic effects toward HepG2 cells by using the MTT (Sigma Aldrich) assay, together with oleic acid (OA). The results showed there is no obvious toxicity with these compounds. HepG2 cells were incubated with OA (500 μM) and DMEM for 24 h and then treated with 10 μM of indicated compounds or lovastatin for an additional 24 h. The effects of compounds 1-9 on oleic acid-elicited intracellular lipid accumulation were showed in Figure 4. All compounds tested showed a reduction in intracellular lipid accumulation, while 3 and 4 displayed stronger inhibition than the others. As for the results, there is no clear conclusion about the difference in activities in comparison with those aromatic analogues with eleven and thirteen carbons of the linear chain in the structures. However, it was speculated that the conjugated double bond in linear chain (such as in 3 or 4) is beneficial for the intracellular lipid-lowering effect than carbonyl or hydroxyl groups in this position (Figure 4). The hypolipidemic mechanism of them needs further study.

Strain Material
The strain SCSIO15079 used in this investigation was isolated from the deep-sea sediment sample of the Indian Ocean

Cultivation and Extraction
A few loops of cells of the strain were inoculated into a 500 mL Erlenmeyer flask containing 150 mL of seed medium (malt extract 1%, yeast extract 0.4%, glucose 0.4%, pH 7.2) and then cultivated on a rotary shaker at 180 rpm, 28 • C for 48 h as seed culture. Then, 2 L of seed culture was inoculated into a 65 L fermenter containing 40 L medium (soybean meal 1%, corn steep liquor 0.5%, glucose 0.5%, yeast extract 0.1%, glycerol 2%, meat extract 0.2%, CaCO3 0.2%, MgSO 4 0.01%, pH7.2). After cultivation at 180 rpm and 28 • C for 80 h, the bacterial culture broth was centrifuged at 4000 rpm. Then, the mycelium and supernatant were broken using an ultrasonic treatment apparatus for 15 min and extracted three times with an equal volume of ethyl acetate, respectively. The organic extract was then concentrated under a vacuum to provide the crude extract. EtOAc was concentrated in vacuo to yield an organic extract (about 51 g).

Isolation and Purification
The EtOAc crude extract (51 g) was subjected to silica gel vacuum liquid chromatography using a step gradient elution of petroleum ether (PE)−EtOA (0-100%, V/V), EtOAc−MeOH (0-100%, V/V) to yield nine fractions according to TLC profiles (Fr.  HepG2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator with 5% CO 2 at 37 • C, and cells in the logarithmic phase were taken for the experiments [26]. The oleic acid (OA) was prepared with 10% bovine serum albumin (BSA) and added to the culture medium at a final concentration of 1%, as previously described. Blank control cells were treated with 1% BSA. After reaching 70% confluence, HepG2 cells were serum-starved for18 h and exposed to 0.5 µM OA, with or without compound/positive, for 24 h [27].

Cell Viability Assay
Cell viability was measured by an MTT assay. In brief, HepG2 cells were plated at a density of 2 × 10 4 cells/well in 96-well plates. After the cells were attached to the plate, they were treated with the OA (0.25, 0.5, 1 mM) or compounds/lovastatin (5, 10, 20 µM). After 24 h, 20 µL of an MTT solution (5 mg/mL) was added to each well, followed by incubation for 4 h. The medium was removed, and the formazan crystals formed in living cells were dissolved in 150 µL of DMSO. Cell viability was measured at 490 nm using an iMak microplate reader (Molecular Devices, Bio-Rad, Hercules, CA, USA). Each treatment was performed in triplicate.

Oil Red O Staining
Cells were fixed with 4% paraformaldehyde, and Oil Red O staining was performed [24]. Oil droplets were observed using microscopy (Olympus, Tokyo, Japan). Next, cells were treated with isopropanol and lipid accumulation were measured using a microplate reader and recording the absorbance at 510 nm [28].

Cell Culture and Cytotoxic Bioassay
Human cancer cell lines, HeLa, HCT-116, and A549, were purchased from ATCC. The HCT-116 cells were grown and maintained in a RPMI-1640 medium with 10% FBS, while the other cells were grown in a DMEM medium with 10% FBS. The cell viability was determined using a MTT assay with 50 µM of compounds 1-9.

Conclusions
In this study, nine new compounds, including six aromatic analogues (1-6) and three leucine derivatives containing an unusual oxime moiety (7-9), were obtained from the deep-sea-derived actinomycete strain Streptomyces chumphonensis SCSIO15079. The new structures including absolute configurations were determined by spectroscopic methods coupled with experimental and calculated ECD. All of those new compounds showed inhibitory activities with intracellular lipid accumulation in HepG2 cells, while compound 3 and 4, with a conjugated double bond in linear chain, displayed stronger inhibition than others. It will provide a new type of potential lead compounds for the development of antihyperlipidemic therapeutics.