Odisolane, a Novel Oxolane Derivative, and Antiangiogenic

Apr 26, 2016 - ... (R*)-2-((2S*,3R*)-tetrahydro-2-hydroxy-2-methylfuran-3-yl)propanoic acid (1), namely, odisolane, along with five known heterocyclic...
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Odisolane, a Novel Oxolane Derivative, and Antiangiogenic Constituents from the Fruits of Mulberry (Morus alba L.) Seoung Rak Lee,† Jun Yeon Park,‡ Jae Sik Yu,† Sung Ok Lee,§ Ja-Young Ryu,# Sang-Zin Choi,# Ki Sung Kang,‡ Noriko Yamabe,*,‡ and Ki Hyun Kim*,† †

School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea College of Korean Medicine, Gachon University, Seongnam 461-701, Republic of Korea § College of Life Sciences, Kyung Hee University, Yongin 17104, Republic of Korea # Dong-A ST Research Center, Yongin 446-905, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Mulberry, the fruit of Morus alba L., is known as an edible fruit and commonly used in Chinese medicines as a warming agent and as a sedative, tonic, laxative, odontalgic, expectorant, anthelmintic, and emetic. Systemic investigation of the chemical constituents of M. alba fruits led to the identification of a novel oxolane derivative, (R*)-2-((2S*,3R*)-tetrahydro-2hydroxy-2-methylfuran-3-yl)propanoic acid (1), namely, odisolane, along with five known heterocyclic compounds (2−6). The structure of the new compound was elucidated on the basis of HR-MS, 1D and 2D NMR (1H−1H COSY, HSQC, HMBC, and NOESY) data analysis. Compound 1 has a novel skeleton that consists of 8 carbon units with an oxolane ring, which until now has never been identified in natural products. The isolated compounds were subjected to several activity tests to verify their biological function. Among them, compounds 1, 3, and 5 significantly inhibited cord formation in HUVECs. The action mechanism of compound 3, which had the strongest antiangiogenic activity, was mediated by decreasing VEGF, p-Akt, and pERK protein expression. These results suggest that compounds isolated from M. alba fruits might be beneficial in antiangiogenesis therapy for cancer treatment. KEYWORDS: Morus alba, Moraceae, oxolane derivative, angiogenesis, VEGF

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INTRODUCTION Mulberry (Morus alba L., Moraceae) is mostly found in subtropical regions and has been harvested in East Asia, especially in Korea and China. Its leaves are the solitary food supply for the silkworm (Bombyx mori L.), and it plays a key factor in the economical and ecological mulberry-growing countries.1 Mulberry is used in traditional Chinese medicine, and almost all tree parts have been used for medicinal purposes all around the countries.2 The leaves have been reported to show antidiabetic, diuretic, and hypotensive activity, whereas the root bark of the mulberry tree has been known for its antiinflammatory, antitussive, and antipyretic effects in past decades.2 The fruit of M. alba is consumed as a fresh fruit and is commonly used in Chinese medicines as a warming agent and as a laxative, tonic, anthelmintic, sedative, emetic, expectorant, and odontalgic.2,3 In Korea, the fruit known as “Odi” is a famous herbal medicine known as Mori Fructus that is used for a variety of illnesses including arthritis, rheumatism, and diabetes.4,5 Mori Fructus is also utilized for therapy of hypertension, dental diseases, and anemia.6 The medicinal value of the mulberry tree is derived from its many important secondary metabolites.2,3,5−7 The secondary metabolites of the M. alba fruit include a number of lignans, anthocyanins, flavonoids, benzofuran derivatives, and phenolic constituents,8−12 which show anti-inflammatory, antioxidant, and neuroprotective effects.9,10 However, despite intensive chemical investigations of M. alba fruits, to the best of our knowledge, there have been rarely reports on bioactive heterocyclic © XXXX American Chemical Society

compounds from the fruits, with the exception of a few benzofuran derivatives.10 In the search for structurally new secondary metabolites in natural Korean sources, a systemic investigation of the chemical constituents of MeOH extracts of M. alba fruit was carried out, which led to the isolation of one novel oxolane derivative (1) together with five known heterocyclic compounds (2−6). The structure of the new compound was verified by analyzing spectroscopic data, distinctly by extensive 1D and 2D NMR experiments and HR-MS data. All compounds were tested for antioxidant, anticancer, antiangiogenesis, and kidney protection effects. Based on the functional activity of screening results, we report herein the isolation and structural elucidation of compounds 1−6 (Figure 1), and their antiangiogenic effects along with their mechanism of action.

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were calculated using a Jasco P-1020 polarimeter (Jasco, Easton, MD, USA). IR spectra were recorded using a Bruker IFS-66/S FT-IR spectrometer (Bruker, Karlsruhe, Germany). Electrospray ionization (ESI) and HR-ESI mass spectra were obtained using a Waters Micromass Q-Tof Ultima ESI-TOF mass spectrometer (Waters, New

Received: March 30, 2016 Revised: April 24, 2016 Accepted: April 26, 2016

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DOI: 10.1021/acs.jafc.6b01461 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Structures of compounds 1−6. ESIMS (positive-ion mode) m/z: 197.0787 [M + Na]+ (calcd for C8H14O4Na, 197.0790).

York, NY, USA). Nuclear magnetic resonance (NMR) spectra, including 1H−1H COSY, HSQC, HMBC, and NOESY experiments, were documented using a Bruker AVANCE III 700 NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C) (Bruker), with chemical shifts given in ppm (δ) using tetramethylsilane (TMS) as an internal standard (0 ppm) for 1H and 13C NMR analysis. Preparative high performance liquid chromatography (HPLC) was performed using a Waters 1525 Binary HPLC pump with Waters 996 photodiode array detector (Waters Corporation, Milford, CT, USA). Semipreparative HPLC was conducted with a Shimadzu Prominence HPLC System with SPD-20A/20AV Series Prominence HPLC UV−vis detectors (Shimadzu, Tokyo, Japan). Silica gel 60 (Merck, 230−400 mesh) and RP-C18 silica gel (Merck, 40−63 μm) were used for column chromatography. The packing material for molecular sieve column chromatography was Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Merck precoated silica gel F254 plates and RP-18 F254s plates (Merck, Darmstadt, Germany) were used for TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde−sulfuric acid. Plant Material. The fruits of M. alba were obtained at the Kyungdong Market (Woori Herb), Seoul, Korea, in January, 2014. A voucher specimen (MA 1414) of the material was verified by one of the authors (S.-Z.C.) and was stored in laboratory 306 of the Dong-A ST Research Center, Yongin, Korea. Extraction and Isolation. Dried and mashed M. alba fruits (9.7 kg) underwent extraction by mixing of the material with 70% aqueous MeOH three times at room temperature and filtration. After evaporation of the filtrate in vacuo, the resultant residue (1.4 kg) was dissolved in deionized water and then solvent-partitioned with hexane, CHCl3, EtOAc, and n-BuOH (800 mL × 3), affording 27.8, 85.3, 32.9, and 138.8 g, respectively. The CHCl3-soluble fraction (85.3 g) was loaded onto a silica gel (230−400 mesh) column and separated with CHCl3−MeOH (40:1−1:1, gradient system) to yield five fractions (A−E). Fraction B (4.3 g) was subjected to an RP-C18 silica gel (230−400 mesh) column chromatography eluted with 70% MeOH/H2O to give 11 fractions (B1−B11). Fraction B1 (226 mg) was fractionated with Sephadex LH-20 column chromatography eluted with 100% MeOH to give six subfractions (B1-1−B1-6). Subfraction B1-3 (33 mg) was separated by semipreparative reversed-phase HPLC using 4% MeOH/H2O (flow rate: 2 mL/min) to yield compounds 4 (0.4 mg, tR = 25.0 min) and 5 (4.4 mg, tR = 37.2 min). Fraction B2 (753 mg) was separated using silica gel (230−400 mesh) column chromatography eluted with CHCl3−MeOH (40:1−5:1, gradient system) to afford nine subfractions (B2-1−B2-9). Subfraction B2-2 (176 mg) was purified by semipreparative reversed-phase HPLC using a 250 mm × 10 mm i.d., 10 μm, Luna C-18 column using 29% MeOH/H2O (flow rate: 2 mL/min) to isolate compounds 2 (1.6 mg, tR = 65.1 min) and 3 (2.1 mg, tR = 72.0 min). Subfraction B2-3 (84 mg) also underwent semipreparative reversed-phase HPLC using 18% MeOH/H2O (flow rate: 2 mL/min) to afford compound 1 (1.3 mg, tR = 40.5 min). Finally, fraction B4 was separated on silica gel (230−400 mesh) column chromatography using CHCl3−MeOH (40:1−5:1, gradient system) to give seven subfractions (B4-1−B4-7). Subfraction B4-2 (20.6 mg) was separated by semipreparative reversed-phase HPLC with 58% MeOH/H2O (flow rate: 2 mL/min) to afford compound 6 (1.8 mg, tR = 26.1 min). Odisolane (1). Amorphous powder. [α]25 D : −16.2 (c 0.65, MeOH). IR (KBr) νmax: 3375, 2947, 2833, 1720, 1628, 1610, 1505, 1450, 1355, 1033, 670 cm−1. 1H (700 MHz) and 13C (175 MHz) NMR data: see Table 1. ESIMS (positive-ion mode) m/z: 197.08 [M + Na]+. HR-

Table 1. 1H (700 MHz) and 13C (175 MHz) NMR Data of Compound 1 in CD3ODa δH

position 2 3 4α 4β 5α 5β 6 1′ 2′ 3′

2.36 1.82 2.19 3.78 3.96 1.56

m dd (12.5, 5.0) m m m s

2.37 m 1.27 d (7.0)

δC

HMBC (H→C)

97.0 52.9 34.3

C-6, C-1′, C-3′ C-2, C-3, C-5, C-2′

67.6

C-2, C-3, C-4

26.7 181.0 45.3 17.9

C-2, C-3 C-3, C-4, C-1′, C-3′ C-3, C-1′, C-2′

a

The assignments were based on 1H−1H COSY, HSQC, and HMBC experiments. Coupling constants (in Hz) are given in parentheses.

Measurements of Cell Viability in Human Umbilical Vein Vascular Endothelial Cells (HUVECs). HUVECs were used to measure antiangiogenic potential of the isolated compounds. HUVECs were purchased from American Type Culture Collection (Manassas, VA, USA), and were maintained using the Clonetics EGM-2 MV BulletKit (Takara Bio Inc., Shiga, Japan) in an atmosphere of 5% CO2 at 37 °C. The cells were subcultured daily and plated at the appropriate density according to each experimental design when the cells were approximately 80% confluent. The cytotoxicity of compounds 1−6 to HUVECs was tested using the MTT as reported previously.13 Measurements of Tube Formation in HUVECs. The tube formation assay was conducted following a reported method with minor modification.13 In brief, cells were seeded (3 × 102 cells/well) onto the Matrigel-coated plate, and incubated at 37 °C for 24 h with compounds 1−6. After 24 h, cellular images were captured and the degree of tube formation was measured by quantifying tube length. Western Blot Analysis. Western blotting was performed following a reported method.14 Amounts of proteins were quantified by the Lowry and Bradford methods. Proteins were separated by electrophoresis, blotted onto polyvinylidene difluoride membranes, and analyzed with each antibody (VEGF, phosphorylated-Akt, Akt, phosphorylated-ERK, and ERK). Statistical Analysis. Statistical significance was determined through analysis of variance, followed by a multiple comparison test with a Bonferroni adjustment. A p value of less than 0.05 was considered statistically significant.

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RESULTS AND DISCUSSION Isolation of Compounds 1−6 from Morus alba. Chemical investigation of the 70% aqueous MeOH extracts of M. alba fruits resulted in the identification of a novel oxolane derivative, odisolane (1), together with five known heterocyclic compounds (2−6) (Figure 1). Heterocyclic compounds have been rarely reported from the fruits of M. alba, and the five heterocyclic compounds were identified as 3-benzofurancarboxaldehyde (2),15 loliolide (3),16 (R)-5-hydroxypyrrolidin-2-one

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in this study, although it has been reported as a synthetic product.15 Structural Elucidation of the New Oxolane Derivative. Compound 1 was obtained as an amorphous powder with the molecular formula C8H14O4 on the basis of the [M + Na]+ peak at m/z 197.0787 (calcd for C8H14O4Na, 197.0790) in the HRESIMS. The IR absorption bands of 1 were characteristic of hydroxy (3375 cm−1) and carbonyl (1720 cm−1) functional groups. The 1H NMR data (Table 1) of 1 showed signals for two methyl groups at δH 1.56 (3H, s) and 1.27 (3H, d, J = 7.0 Hz), an oxygenated methylene at δH 3.96 and 3.78 (each 1H, m), a methylene at δH 2.19 (1H, m) and 1.82 (1H, dd, J = 12.5, 5.0 Hz), and two methines at δH 2.37 (1H, m) and 2.36 (1H, m), which were classified by HSQC data analysis. The 13C NMR (Table 1) of 1 exhibited 8 carbon signals in total for one

Figure 2. (A) Key 1H−1H COSY (bold line) and HMBCs (→) of 1 and (B) proposed Newman projection at the C-2′ to C-3 bond of 1.

(4),17 methyl (R)-pyroglutamate (5),18 and indole (6)19 based on NMR spectroscopic data compared with previously published literature values. These known compounds 2−6 from M. alba were isolated for the first time. In particular, compound 2 is reported for the first time as a natural product

Figure 3. (A) The effect of compounds 1−6 on HUVEC proliferation and assessment of cytotoxicity. Cells were treated with a series of compound concentrations (3.125−100 μM) or the DMSO vehicle (control) for 24 h, and then cell viability was evaluated by the MTT assay. (B) Representative photographs of tube formation of HUVECs on Matrigel after incubation with or without the compounds 1−6 at 24 h. (C) Effects of compounds 1−6 on tube formation of HUVECs on Matrigel. The relative length of tubes was measured using ImageJ software. C

DOI: 10.1021/acs.jafc.6b01461 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. (A) Western blot results show the levels of VEGF (21 kDa), phosphorylated-Akt, Akt (60 kDa), phosphorylated-ERK, and ERK (42/44 kDa) in HUVEC cells treated with compound 3 at different concentrations for 24 h. A representative immunoblot of three independent experiments is shown. (B) Quantitative graphs for the protein expressions of VEGF, phosphorylated-Akt, and phosphorylated-ERK. Density ratios over GAPDH (37 kDa) were measured by densitometer. *p < 0.05 compared with the control group.

carboxyl carbon at δC 181.0, one hemiacetal quaternary carbon at δC 97.0, one oxygenated methylene carbon at δC 67.6, two methine carbons at δC 52.9 and 45.3, one methylene carbon at δC 34.3, and two methyl carbons at δC 26.7 and 17.9, which were assigned by HSQC experiment. The two degrees of unsaturation deduced from the molecular formula of 1, together with the NMR data demonstrating one carboxyl carbon, indicated that compound 1 should be a cyclic compound with a one-ring system. Analysis of 2D NMR spectroscopic data (1H−1H COSY and HMBC) allowed the gross structure of 1 to be established (Figure 2). The oxolane system from C-2 to C-5 via oxygen was established through the 1 H−1H COSY correlations for H-3/H-4/H-5 and HMBC correlations from H3-6 to C-2/C-3 and from H2-5 to C-2 (Figure 2). The HMBC correlations from H3-3′ to C-1′ (δC 181.0), C-2′ (δC 45.3), and C-3 (δC 52.9) enabled us to determine the linkage between propionic acid and the oxolane unit in compound 1, which finally established the planar structure of 1. The relative configurations of C-2, C-3, and C-2′ were established by interpretation of the NOESY spectrum. The NOESY correlations of H-3/H-4β/H-5β suggested that H3 is on the same side of H-4β and H-5β in the oxolane ring system, indicating a β-orientation of H-3. The stereochemistry

of C-2 was determined by the NOESY correlations from H3-6 to H-4α/H-5α, indicating the anti relationship between H-3 and H3-6. In addition, NOESY correlation was observed between H-3 and H3-3′ (Figure 2). This NOESY data, coupled with the relative configuration of C-3 described above, demonstrated that H-3 and H3-3′ have a syn relationship in compound 1. Therefore, the stereochemistry of 1 could either be 2S,3R,2′R or 2S,3R,2′S depending on the conformation of the C-2′/C-3. The MM2 calculation of the energy minimizing process in Chem3D Ultra 10.0 suggested that compound 1 has the 2S*,3R*,2′R*. Thus, the structure of 1 was established as (R*)-2-((2S*,3R*)-tetrahydro-2-hydroxy-2-methylfuran-3-yl)propanoic acid, namely, odisolane. To the extent of our knowledge, the structure of 1 has a unique skeleton that has not been reported in natural products. Effect of Compounds 1−6 in Biological Activity Tests. Based on the M. alba fruit’s ability to exert multiple biological activities,2,3 several biological activity tests were performed on the isolated compounds. The isolated compounds 1−6 were tested for antioxidant, anticancer, antiangiogenesis, and kidney protection effects. Their free radical scavenging activity was measured using DPPH.20 The cytotoxicity of compounds 1−6 to human gastric cancer AGS cells was tested using the MTT D

DOI: 10.1021/acs.jafc.6b01461 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry assay.21 The kidney protection effect against oxidative renal cell damage was measured in LLC-PK1 cells.22 A tube formation assay was conducted following reported methods using HUVECs.23 Compounds 1−6 exerted no antioxidant, anticancer, and kidney protection effects (Figures S7−S9), whereas significant antiangiogenic effects were noted after cotreatment with some of the isolates 1−6. Cytotoxicity of Compounds 1−6 in HUVECs. Biologically harmful substances may affect changes in morphology, cell growth, and death.23 Therefore, monitoring of cell viability was performed for every compound of potential interest in the present study. We examined dose response studies on the proliferation of HUVECs in vitro to determine the effects of compounds 1−6 on endothelial cell growth. The cell viability of HUVECs after treatment with compounds 1−6 is shown in Figure 3A. Compounds 1, 4, and 6 exhibited no toxic effects on HUVECs up to 100 μM. In contrast, treatment with compounds 2, 3, and 5 exerted concentration dependent inhibition of HUVEC cell viability (Figure 3A). A maximum dose of each compound showing no toxic effects was selected for further tube formation assays. Comparison of Effects of Compounds 1−6 on Tube Formation of HUVECs in Matrigel. Angiogenesis is controlled by various types of activators and inhibitors that regulate the proliferation or migration of cells.24,25 The effect of nontoxic doses of compounds 1−6 on HUVEC tube formation is shown in Figure 3B. Compounds 2, 4, and 6 exerted no effect on tube formation in HUVEC. In contrast, compounds 1, 3, and 5 inhibited tube formation of HUVECs (Figure 3C). A molecular mechanistic study was further conducted with compound 3, which showed the most potent inhibitory effect on tube formation (Figure 3C). Effect of Compound 3 on Vascular Endothelial Growth Factor (VEGF), Akt, and Extracellular SignalRegulated Protein Kinase (ERK) Protein Expression in HUVECs. VEGF is known as an important endothelial cell mitogen that plays major roles in pathological conditions related to aingiogenesis.26,27 PI3K/Akt signaling is known to be involved in the overactivated cancers, and it induces secretion of VEGF and plays a crucial role in the modulation of cancer cell growth.28 Also, VEGF is a strong activator of ERK1 and ERK2 in cell proliferation.29 As shown in Figure 4, compound 3 significantly decreased the protein expression of VEGF, phosphorylated Akt, and ERK1/2. VEGF is important target for antiangiogenic therapy.30 Recently, brucine, an indole alkaloid from Strychnos nux-vomica, was reported to attenuate VEGF-induced angiogenesis by reducing VEGFR2 activity in HUVECs.31 Indole-3-carbinol, a major component of cruciferous vegetables, also suppressed phorbol myristate acetate activated capillary-like tube formation in endothelial EA.hy926 cells via inhibiting VEGF.32 Compound 3, the most potent inhibitor of tube formation, is a monoterpene lactone, and, to the best of our knowledge, this kind of natural product has not been investigated for antiangiogenic effects. The present finding suggests for the first time that the monoterpene lactone, compound 3, is a potential inhibitor of tube formation in HUVECs through the VEGF-mediated mechanistic pathway. In conclusion, we isolated and structurally identified six heterocyclic compounds including a novel oxolane derivative, (R*)-2-((2S*,3R*)-tetrahydro-2-hydroxy-2-methylfuran-3-yl)propanoic acid (1), namely, odisolane, together with five known heterocyclic compounds 2−6 from the fruits of M. alba. This particular investigation describes the first evidence that a

monoterpene lactone (3) inhibited angiogenesis through the molecular mechanisms involving inhibition of VEGF, PI3-K/ Akt, and MEK/ERK signaling pathways in HUVECs. In the near future, studies will bring attention to the effect of monoterpene lactones on unexplored areas like involvement of the epithelial−mesenchymal transition.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01461. 1 H and 13C NMR, HSQC, HMBC, 1H-1H COSY, and NOESY spectra of 1 and comparison of activities and effects of 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(N.Y.) Phone: +82-31-750-5402. Fax: +82-31-750-5416. Email: [email protected]. *(K.H.K.) Phone: +82-31-290-7700. Fax: +82-31-290-7730. Email: [email protected]. Author Contributions

S.R.L., J.S.Y., and J.Y.P. performed most of the experimental work. S.-Z.C., S.O.L., and J.-Y.R. conceived the project and designed the experiments. S.-Z.C. provided the needed materials. K.H.K. designed and implemented the separation and purification protocols. K.S.K. and N.Y. designed and implemented the biological test protocols. K.S.K., N.Y., and K.H.K. drafted and revised the manuscript. All authors read and approved the final manuscript. Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2A10005315). This research was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1C1A1A02037383). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b01461 J. Agric. Food Chem. XXXX, XXX, XXX−XXX