Isolation and Characterization of Sesquiterpenoids from Cassia Buds

DOI: 10.1021/acs.jafc.7b01294. Publication Date (Web): June 30, 2017. Copyright © 2017 American Chemical Society. *Telephone: 86-0991-3835679. Fax: 8...
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Isolation and Characterization of Sesquiterpenoids from Cassia Buds and Their Antimicrobial Activities Yindengzhi Guoruoluo,†,‡,§ Haofeng Zhou,‡ Junfei Zhou,‡ Haiqing Zhao,† Haji Akber Aisa,*,† and Guangmin Yao*,‡ †

Key Laboratory of Plant Resources and Chemistry of Arid Zone, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi, Xinjiang 830011, People’s Republic of China ‡ Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei 430030, People’s Republic of China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Cassia buds, the immature fruits of Cinnamomum cassia (Lauraceae), are widely consumed as a food spice, dietary supplements, flavoring agents, and preservatives. In this study, cassia buds were phytochemically investigated for the first time, leading to the isolation of 2 new sesquiterpenoids (1 and 2) and 10 known sesquiterpenoids (3−12). Their structures were determined by spectrometric and spectroscopic analyses, including nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, and circular dichroism. Cinnamosim A (1) represents the ninth example of the rare cyperane-type sesquiterpenoids. All of the compounds (1−12) isolated from cassia buds were evaluated for antimicrobial activities, with compounds 1−3, 5−8, 11, and 12 exhibiting strong antimicrobial activities against Candida albicans and compounds 6, 7, and 11 showing moderate antibacterial activities against Escherichia coli and Staphylococcus aureus. The present investigation indicated that sesquiterpenoids from cassia buds might be used as potential antimicrobial agents to preserve food. KEYWORDS: sesquiterpenoids, cassia buds, Cinnamomum cassia, antimicrobial activity

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The immature fruits of C. cassia are a source of cassia buds,7 which have a flavor similar to the cassia bark but possess a unique and subtle favor. Cassia buds are an ingredient of better pickles, chutneys, and ketchups and are also used in curries, poached fruit dishes, and apple desserts. However, there are no reports of the chemical components of cassia buds. To search for naturally occurring antimicrobial agents from Chinese spice, cassia buds were phytochemically investigated for the first time, leading to the isolation of 12 sesquiterpenoids (1−12) (Figure 1). Sesquiterpenoids 1−12 from cassia buds were structurally identified and evaluated for their antimicrobial activities against C. albicans, S. aureus, and E. coli.

INTRODUCTION Foodborne diseases are infections or irritations of the gastrointestinal tract caused by food or beverages contaminated with microorganisms or chemicals and are a common, costly, yet preventable, public health problem around the world. The majority of foodborne illnesses are caused by pathogenic bacteria, parasites, viruses, and chemicals.1 At present, to store food for a long time, synthetic antibacterial additives are usually used to inhibit the growth of pathogenic microorganisms.2 However, studies have shown that synthetic antibacterial compounds may not be safe for human health and the environment,3,4 and food safety, including the presence of chemical residues in food preservatives, is the common concern to consumers.5 Some spices and edible plants are not only excellent flavoring ingredients but also potential antimicrobials.6 Therefore, it is feasible to search for naturally occurring substances with antimicrobial activity as food preservatives from spices and edible plants. Cinnamomum cassia Presl. (Lauraceae), a medium-sized evergreen tree, originating in South China, is now widely cultivated in Guangxi, Hainan, Guizhou, Fujian, Guangdong, and Yunnan of China and Thailand, Indonesia, Malaysia, Laos, Vietnam, and India.7 The important spice cassia is obtained from the dried bark of C. cassia. The antimicrobial activities6 and chemical constituents of the cassia bark have been investigated,8 and the essential oil, including the major component cinnamaldehyde, of the bark of C. cassia could significantly inhibit the growth of various bacteria, such as Candida albicans, Escherichia coli (a Gram-negative bacterium), and Staphylococcus aureus (a Gram-positive bacterium).9 © 2017 American Chemical Society

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MATERIALS AND METHODS

General Experimental Procedures. The optical rotations were measured in MeOH using a Rudolph Autopol IV automatic polarimeter, which was produced by Rudolph Research Analytical, Hackettstown, NJ, U.S.A. A Bruker AM-400 spectrometer, produced by Bruker Daltonik GmbH, Bremen, Germany, was used to acquire one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectra in methanol-d4 (Cambridge Isotope Laboratories, Inc., Andover, MA, U.S.A.). A Bruker micrOTOF II spectrometer, which was provided by Bruker Daltonik GmbH, Bremen, Germany, was used to detect high-resolution electrospray ionization mass spectrometry (HRESIMS) in the positive-ion mode. A JASCO J-810 spectrometer provided by JASCO Co., Ltd., Tokyo, Received: Revised: Accepted: Published: 5614

March 22, 2017 June 14, 2017 June 16, 2017 June 30, 2017 DOI: 10.1021/acs.jafc.7b01294 J. Agric. Food Chem. 2017, 65, 5614−5619

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of sesquiterpenoids 1−12. Japan, was used to record the electronic circular dichroism (ECD) spectra. High-performance liquid chromatography (HPLC) was conducted on an Agilent 2100 quaternary system at a flow rate of 1.5 mL/min with an ultraviolet (UV) detector, and the HPLC column is a 250 × 10.0 mm inner diameter, 5 μm, Welch Ultimate XB-C18, with a 4 × 4 mm inner diameter guard column of the same material (Welch, Shanghai, China). Column chromatography (CC) was conducted over octadecylsilyl (ODS, 50 μm, YMC Co., Ltd., Kyoto, Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), and silica gel (Qingdao Marine Chemical, Inc., Qingdao, China). Extraction and Isolation. The powered 5 kg cassia buds were extracted by soaking with 95% EtOH (40 L) 4 times at 45 °C, each for 48 h. After the solvent was removed under vacuum, the residue was extracted by soaking with petroleum ether (5 × 20 L), EtOAc (4 × 20 L), and MeOH (4 × 20 L) at 40 °C, successively. The MeOH extract (100 g) was fractionated using silica gel CC (silica gel, 200 g; column, 9 × 70 cm), eluting with a CHCl3/MeOH solvent system from 15:1, 10:1, 8:1, 5:1, and 3:1 to 2:1 (15 L for each gradient elution) to afford nine fractions (fractions 1−9), combined on the basis of the thin-layer chromatography (TLC) analysis. Fraction 1 (8.0 g) was separated by medium-pressure liquid chromatography (MPLC) over ODS (50 g; column, 3 × 30 cm) eluting with a MeOH−H2O solvent system (0:100, 10:90, 30:70, 50:50, and 60:40; 6 L for each gradient elution) to provide six subfractions (subfractions 1A−1F). The subfraction 1B (180 mg) was subjected to silica gel CC (25 × 2 cm), eluted with a gradient petroleum ether−acetone solvent system (40:1, 30:1, 20:1, 10:1, and 5:1; 1 L for each gradient elution) to give seven subfractions (subfractions 1Ba−1Bg). Subfraction 1Bg (40 mg) was purified by HPLC on a reversed-phase (RP) C18 column (62% MeOH/H2O) to afford compound 1 (tR, 33 min; 4.0 mg). Subfraction 1Bb (11 mg) was purified by HPLC on a RP C18 column (61% MeOH/H2O) to afford compound 8 (tR, 34 min; 1.4 mg). Subfraction 1C (110 mg) was fractionated using Sephadex LH-20 CC (150 × 2 cm; 100% MeOH; 1 L) and silica gel CC (20 × 1 cm; CH2Cl2−MeOH, 200:1 and 150:1; 1 L for each gradient elution) to give three subfractions (subfractions 1Ca−1Cc), and subfraction 1Cb (34 mg) was purified by HPLC on a RP C18 column (50% MeOH/H2O) to afford compound 10 (tR, 106

min; 4 mg). Subfraction 1D (130 mg) was separated into eight subfractions (subfractions 1 Da−1Dh) using silica gel CC (20 × 1 cm; petroleum ether−acetone, 50:1, 30:1, and 20:1; 1 L for each gradient elution). Compound 6 (tR, 24 min; 5.0 mg) was obtained from subfraction 1Db (12 mg) with RP C18 HPLC (MeOH−H2O, 68:32). Compound 3 (tR, 54 min; 1.2 mg) was obtained from subfraction 1Dc (18 mg) with RP C18 HPLC (MeOH−H2O, 68:32). Subfraction 1Dg (34 mg) was purified with RP C18 HPLC (MeOH−H2O, 65:35) to give compounds 7 (tR, 73 min; 3.0 mg) and 12 (tR, 65 min; 1.4 mg). Subfraction 1Df (31 mg) was separated over RP C18 HPLC (MeOH− H2O, 67:33) to yield compound 4 (tR, 27 min; 1.4 mg). Subfraction 1F (90 mg) was fractionated using Sephadex LH-20 CC (150 × 2 cm; MeOH; 1 L) and silica gel CC (20 × 1 cm; petroleum ether−acetone, 30:1 20:1, and 10:1; 1 L for each gradient elution) to give four subfractions (subfractions 1Fa−1Fd). Compound 11 (tR, 29 min; 1.4 mg) was isolated from subfraction 1Fc (31.6 mg) by RP C18 HPLC (MeOH−H2O, 82:18). Fraction 2 (1 g) was isolated by Sephadex LH20 (150 × 4 cm; 100% MeOH; 3 L) and then separated on silica gel CC (20 × 2 cm) eluting with petroleum ether−acetone (5:1, 4:1, and 2:1; 1.5 L for each gradient elution) to give five subfractions (subfractions 2A−2E). Subfraction 2B (61 mg) was subjected to Sephadex LH-20 CC (150 × 1 cm; MeOH; 500 mL) to give two subfractions (subfractions 2Ba and 2Bb). Subfraction 2Ba (20 mg) was purified with a HPLC RP C18 column (50% MeOH/H2O) to give compound 9 (tR, 20 min; 1.0 mg), and subfraction 2Bb (34 mg) was purified with RP C18 HPLC (53% MeOH/H2O) to give compound 5 (tR, 45 min; 1.2 mg). Fraction 3 (6 g) was separated by ODS CC (45 × 3 cm) eluting with a gradient MeOH/H2O solvent system (0:100, 10:90, 30:70, and 50:50; 10 L for each gradient elution) to give seven subfractions (subfractions 3A−3G). Then, subfraction 3E (500 mg) was subjected on silica gel CC (20 × 2 cm; CH2Cl2−MeOH, 50:1, 30:1, 20:1, and 10:1; 1.5 L for each gradient elution) to give five subfractions (subfractions 3Ea−3Ee), and subfraction 3Ec (15.2 mg) was purified with RP C18 HPLC (48% MeOH/H2O) to afford new sesquiterpenoid 2 (tR, 29 min; 3.0 mg). Cinnamosim A (1): white powder; [α]25 D , +8.5 (c 0.38, MeOH). ECD (MeOH), 237 (Δε, −5.05), 300 (Δε, 3.49) nm; 1H (400 MHz) 5615

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Journal of Agricultural and Food Chemistry Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Sesquiterpenoids 1 and 2 in CD3OD 1 δH (J in Hz)

position 1α 1β 2α 2β 3β 4 5 6 7 8α 8β 9α 9β 10 11 12 13 14 15

1.68 1.27 1.82 1.34 4.77

(m) (m) (ddd, 2.7, 7.2, 10.6, 13.8) (m, overlap) (dd, 6.8, 10.6)

5.78 (dd, 0.8, 2.2) 2.29 (dddd, 0.8, 3.1, 5.8, 17.0) 2.23 (dddd, 2.2, 5.9, 10.4, 17.0) 1.65(ddd, 5.8, 10.4, 13.6) 1.58 (ddd, 3.1, 5.9, 13.6)

1.35 1.36 0.87 2.11

(s) (s) (s) (s)

2 δC

δH (J in Hz)

34.4

1.71 1.34 1.53 2.34 3.62

31.4 75.5 213.1 65.7 118.3 149.7 22.5

(ddd, 2.8, 13.0, 13.0) (ddd, overlap) (dddd, 1.8, 3.4, 5.0, 14.0) (dddd, 2.3, 4.1, 13.0, 14.0) (dd, 2.3, 2.5)

5.92 (d, 1.0) 1.91 1.49 1.32 1.66

34.9 40.8 74.0 29.05 29.04 28.97 30.6

1.17 1.24 1.24 1.40

δC

(ddd, (ddd, (ddd, (ddd,

(s) (s) (s) (s)

3.0, 3.6, 3.0, 3.6,

14.0, 14.0) 6.8, 14.0) 6.8, 14.0) 14.0, 14.0)

38.9 25.7 76.7 74.6 148.7 127.0 75.8 28.2 35.8 35.4 75.6 24.9 24.8 26.3 26.6

Figure 2. 1H−1H COSY, key HMBC, and NOESY correlations of sesquiterpenoid 1.

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and 13C (100 MHz) NMR data in CD3OD, Table 1; HRESIMS m/z, 275.1615 [M + Na]+, calcd for C15H24O3Na, 275.1623. 1 Cinnamosim B (2): white powder; [α]25 D , +29.0 (c 0.10, MeOH); H (400 MHz) and 13C (100 MHz) NMR data in CD3OD, Table 1; HRESIMS m/z, 293.1726 [M + Na]+, calcd for C15H26O4Na, 293.1729. Antimicrobial Activity Assay. Sesquiterpenoids 1−12 were evaluated for their antimicrobial activities against three representative microbes E. coli (a Gram-negative bacterium), C. albicans (a dimorphic fungus, growing as both yeast and filamentous cells), and S. aureus (a Gram-positive bacterium) using an agar-well diffusion method. The positive controls used were ampicillin sodium salt (an orally active broad-spectrum antibiotic) and amphotericin B (an antifungal antibiotic), and the negative control was the solvent (MeOH). Petri dishes containing 20 mL of nutrient agar medium were seeded with the test strain (a final inoculum of approximately 5 × 106 cfu/mL) and allowed to solidify. The test compound (30 μL, 10 mg/mL in methanol) was poured in the well (6 mm in diameter) and was incubated for 20 h at 37 °C. Each treatment was performed in two replicates. Antimicrobial activities of these compounds were assessed by measuring the diameter of the zone (mm), and the diameter of the inhibition zone at >7 mm is considered to be active.

RESULTS AND DISCUSSION The methanol-soluble part of the EtOH extract of the cassia buds was fractionated by silica gel CC and further purified by repeated Sephadex LH-20 CC, MPLC over silica gel and ODS, and HPLC to acquire 2 new (1 and 2) and 10 known (3−12) sesquiterpenoids, and their structures were shown in Figure 1. Cinnamosim A (1) was obtained as a white powder, [α]25 D of +8.5 (c 0.38, MeOH), and had the molecular formula of C15H24O3, determined by 13C NMR data and HRESIMS at m/z 275.1615 [M + Na]+, calcd for C15H24O3Na of 275.1623, indicating four unsaturated degrees. The 1H NMR spectrum (Table 1) displayed resonances for a singlet methyl at δH 0.87 (s, CH3-14), a gem-dimethyl in the hydroxyisopropyl group at δH 1.35 (s, CH3-12) and 1.36 (s, CH3-13), an acetyl at δH 2.11 [s, −C(O)CH3-15], an oxymethine at δH 4.77 (1H, dd, J = 6.8 and 10.6 Hz, H-3), and an olefinic methine at δH 5.78 (dd, J = 0.8 and 2.3 Hz, H-6). The 13C NMR data (Table 1) revealed the presence of 15 carbons assignable by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single-quantum correlation (HSQC) to a carbonyl (δC 5616

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Journal of Agricultural and Food Chemistry

Cyperane-type sesquiterpenoids are very rare in nature. Previously, only eight cyperane-type sesquiterpenoids have been reported.10−16 Five of these were isolated from the Asteraceae family,10,12,14−16 and only two were isolated from the Cyperaceae family.11,13 Cinnamosim A (1) represents the ninth example of the cyperane-type sesquiterpenoids, and this is the first time to report the presence of cyperane sesquiterpenoid in the Lauraceae family. Cinnamosim B (2) was obtained as a white powder, [α]25 D of +29.0 (c 0.10, MeOH). The molecular formula, C15H24O4, was established by the 13C NMR data and HRESIMS at m/z 293.1726 [M + Na]+, calcd for C15H26O4Na of 293.1729, suggesting three degrees of unsaturation. Analyses of the 1H NMR spectrum (Table 1) of compound 2 suggested the presence of four methyls at δH 1.17 (s, CH3-12), 1.24 (6H, s, CH3-13, CH3-14), 1.40 (s, CH3-15), an oxymethine at δH 3.62 (dd, J = 2.3 and 2.5 Hz, H-3), and an olefinic proton at δH 5.92 (d, J = 1.0 Hz, H-6). As shown in Table 1, the 13C NMR spectrum of compound 2 exhibited a total of 15 resonances, identified by HSQC and DEPT spectra to be four methyls at δC 24.8 (C-13), 24.9 (C-12), 26.6 (C-15), and 26.3 (C-14), four methylene carbons at δC 25.7 (C-2), 28.2 (C-8), 35.8 (C-9), and 38.9 (C-1), an olefinic carbon δC 127.0 (C-6), an oxygenated methine carbon at δC 76.7 (C-3), an olefinic quaternary carbon at δC 148.7 (C-5), three oxygenated quaternary carbons at δC 74.6 (C-4), 75.6 (C-11), and 75.8 (C-7), and a quaternary carbon at δC 35.4 (C-10). A double bond accounts for unsaturated degrees, and the remaining two unsaturated degrees indicate the occurrence of two rings in compound 2, which must also be a bicyclic sesquiterpenoid. The 1H−1H COSY spectrum suggested the existence of two partial structures of “CH2-1−CH2-2−CH(OH)-3” and “CH28−CH2-9” (Figure 3). HMBCs from H3-14 (δH 1.24) to C-1,

213.1, C-4), an olefinic quaternary carbon (δC 149.7, C-7), an olefinic methine (δC 118.3, C-6), an oxygenated methine (δC 75.5, C-3), three quaternary carbons, including one oxygenated (δC 74.0, C-11), four methyls, and four methylenes. The double bond and carbonyl group account for two unsaturated degrees, and the remaining two unsaturated degrees suggest that compound 1 had two additional rings. Thus, compound 1 is a bicyclic sesquiterpenoid. HSQC, 1H−1H correlation spectroscopy (COSY), and heteronuclear multiple-bond correlation (HMBC) data analyses constructed the planar structure of compound 1 (Figure 2). HSQC and 1H−1H COSY spectra revealed the presence of two partial structures (bold lines in Figure 2): CH2-1−CH2-2− CH(OH)-3 and CH2-8−CH2-9. The cross peaks of H3-14 to C-1, C-9, and C-10 in the HMBC spectrum of compound 1 (arrow lines in Figure 2) revealed the connection of C-14 to C1 and C-9 through the quaternary carbon C-10. The cross peaks of H-3 to C-10, C-5, and C-6, of H-6 to C-3, C-5, and C10, and of H3-14 to C-10 and C-5 in the HMBC spectrum suggested the construction of C-3, C-6, and C-10 through C-5 and the existence of the five-membered ring of C-1/C-2/C-3/ C-5/C-10. The connection of C-8 to C-7 of the double bond was suggested by the chemical shift of H2-8, 1H−1H COSY correlation of H2-8 and H-6, and HMBCs of H-8 to C-7 and C6 and of H-6 to C-8. Thus, a six-membered ring of C-10/C-9/ C-8/C-7/C6/C-5 was formed. The location of the hydroxyisopropyl group to C-7 of the double bond was suggested by the cross peaks of H3-12/13 to C-11 and C-7 in the HMBC spectrum. HMBCs from H3-15 to C-5 and from H-3/H3-15 to carbonyl C-4 fixed the location of the acetyl group at C-5. Therefore, the structure of compound 1 was identified to be a rare cyperane sesquiterpenoid.10 NMR data of cinnamosim A (1) were similar to those of 4-oxo-cyperan-3α,7α,11-triol, a known cyperane sesquiterpenoid isolated from Achillea clypeolata.10 The most noticeable difference was the occurrence of an additional double bond (δC 118.3, C-6; 149.7, C-7) in compound 1, instead of an oxygenated quaternary carbon (δC 73.58, C-7) and a methylene (δC 29.58, C-6) in 4-oxo-cyperan3α,7α,11-triol. Therefore, cinnamosim A (1) is a dehydration derivative of 4-oxo-cyperan-3α,7α,11-triol. Nuclear Overhauser effect spectroscopy (NOESY) spectrum analysis was used to establish the relative configuration of cinnamosim A (1) (Figure 2). The NOESY correlation between H3-14 and H3-15 suggested that these two methyls are on the same side and in β orientations, which are in agreement with the cis fusion of the two rings in the cyperane sesquiterpenoid.10 Consequently, the orientation of 3-OH was determined to be α by the NOESY correlations between H-3 and H3-14. However, there is no NOESY correlation between H-3 and H3-15. The reason may be the presence of a stable hydrogen bond of the 3-OH group and C-4 carbonyl, resulting in a long distance from H-3 to H3-15. The stable conformation is shown in Figure 2. The large coupling constants (J = 6.8 and 10.6 Hz) of H-3 with H2-2 revealed the axial orientation of H-3 and further confirmed the relative configuration. The ECD spectrum of compound 1 exhibited a positive Cotton effect at 300 (Δε, 3.49) nm, which is similar to that of a known cyperane sesquiterpenoid cyperolone;11 thus, the absolute configuration of C-5 and C-10 was established to be the same as that of cyperolone.11 The structure of compound 1 was therefore established as (3R,5R,10S)-3,11-dihydroxycyperan-6(7)-ene-4-one and named cinnamosim A.

Figure 3. 1H−1H COSY and key HMBCs of sesquiterpenoid 2.

C-10, and C-9 revealed the connections of these two partial structures with CH3-14 through quaternary carbon C-10. The formation of the six-membered ring C-10/C-5/C-4/C-3/C-2/ C-1 was deduced from the HMBCs from methyl H3-15 (δH 1.40) to C-4 (δC 74.6), C-3 (δC 76.7), and C-5 (δC 148.7) and from H-3 and H3-14 to C-5 (δC 148.7). HMBCs from H-6 to C-10, C-7, and C-8 and from H2-8 to C-7 implied the presence of another six-membered ring of C-10/C-9/C-8/C-7/C6/C-5. The location of oxygenated isopropyl at C-7 was concluded by the HMBCs of H3-12/H3-13 to C-11 (δC 75.6) and C-7 (δC 75.8). Thus, the planar structure of compound 2 was 5617

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Journal of Agricultural and Food Chemistry determined, which belongs to a eudesmane-type sesquiterpenoid. The relative configuration of compound 2 was established on the basis of the coupling constants and the NOESY spectrum analysis. In the eudesmane-type sesquiterpenoid, CH3-14 is usually in a β orientation. Thus, CH3-14 in compound 2 should be in an axial bond in the chair conformation of the sixmembered ring of C-10/C-5/C-4/C-3/C-2/C-1 (Figure 3). The smaller coupling constants of H-3 with H2-2, J = 2.5 Hz, assigned H-3 to be in an equatorial bond in the chair conformation of the six-membered ring; thus, H-3 is βorientated. The lack of NOESY correlation between H3-14 and H3-15 suggested the α orientation of H3-15. The β orientation of 7-OH was deduced from the NOESY correlations of H3-12/ H3-13 and H2-8 and the lack of NOESY correlations of H3-12/ H3-13 and H3-14 or H2-9. Therefore, compound 2 was determined as eudesm-5-ene-3α,4α,7β,11-tetraol, named cinnamosim B. A total of 10 known sesquiterpenoids were identified to be 1β,7-dihydroxyl opposit-4(15)-ene (3),17 1β,11-dihydroxyl opposit-4(15)-ene (4),18 4(15)-eudesmene-1β,7,11-triol (5),19 1β,6α-dihydroxyeudesm-4(15)-ene (6),20 caryolane-1,9β-diol (7),21 4α-10α-dihydroxy-5β-H-guaja-6-ene (8),22 1β,4β,11trihydroxyl-6β-gorgonane (9),23 aromadendrane-4β,10α-diol (10),24 aromadendrane-4α,10α-diol (11),24 and 1-epimeraromadendrane-4β,10α-diol (12),25 by the spectroscopic data analyses, including NMR and HRESIMS, and a comparison to those in the literature. These compounds 1−12 isolated from cassia bud were evaluated for their antimicrobial activities against three representative microbes C. albicans, E. coli, and S. aureus, which are a dimorphic fungus (because it grows as both yeast and filamentous cells), Gram-negative bacterium, and Grampositive bacterium, respectively. The positive controls used were ampicillin sodium salt (an orally active broad-spectrum antibiotic) and amphotericin B (an antifungal antibiotic). The preliminary antimicrobial activities are shown in Table 2. New compound 1 and known compound 5 selectively inhibited the proliferation of C. albicans with inhibitory zone diameters of 11 and 9 mm, respectively, while, compounds 4 and 10 showed

selective inhibitory activities against S. aureus with inhibitory zone diameters of 7.5 and 8 mm, respectively. Compounds 2, 3, 8, and 12 not only inhibited the proliferation of C. albicans but also inhibited the proliferation of S. aureus with inhibitory zone diameters of 10, 9, 8, and 7 mm and 9, 7.5, 10, and 9 mm, respectively. Compounds 3, 5−8, 11, and 12 also exhibited moderate antibacterial activities against C. albicans. Compound 2−4, 6−8, 11, and 12 showed moderate antibacterial activities against S. aureus. Interestingly, compounds 6, 7, and 11 exhibited inhibitory effects against three test microbes C. albicans, S. aureus, and E. coli, with compound 6 showing stronger inhibition than that of the other isolated compounds, with inhibitory zones of 11, 11, and 8.5 mm, respectively. The positive control, ampicillin sodium salt, showed antibacterial activities against S. aureus and E. coli with diameters of the inhibitory zone of 19 and 15 mm, respectively, at the concentration of 50 μg/disk, and another positive control, amphotericin B, showed selective inhibition against C. albicans with an inhibitory zone diameter of 16 mm at the concentration of 100 μg/disk. Although none of the sesquiterpenoids 1−12 showed stronger inhibitory activities against three test microbes than the positive controls, cassia buds are widely consumed as a food spice and dietary supplements, and sesquiterpenoids from cassia buds might be used as potential antimicrobial agents for food preservatives.

Table 2. Antimicrobial Activities of Sesquiterpenoids 1−12

Haji Akber Aisa: 0000-0003-4652-6879 Guangmin Yao: 0000-0002-8893-8743

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01294. HRESIMS and NMR spectra of compounds 1 and 2 (PDF)

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amount (μg/disk)

C. albicans

1 2 3 4 5 6 7 8 9 10 11 12 ASSa ASSa ABb

300 300 300 300 300 300 300 300 300 300 300 300 50 5 100

11 10 8 9 11 10 9

10 10

E. coli

*Telephone: 86-0991-3835679. Fax: 86-0991-3835679. E-mail: [email protected]. *Telephone: 86-27-83657802. Fax: 86-27-83692762. E-mail: [email protected]. ORCID

Funding

S. aureus

This study was supported by the Foundation of Xinjiang Technical Institute of Physics and Chemistry, the Key Laboratory of Plant Resources and Chemistry in Arid Regions, Chinese Academy of Sciences (to Guangmin Yao, 2008DP173091-2016-01), and the Fundamental Research Funds for the Central Universities [to Guangmin Yao, 2016YXMS148, Huazhong University of Science and Technology (HUST)].

9 7 7.5 8.5 7

7 15

AUTHOR INFORMATION

Corresponding Authors

inhibitory zone diameter (mm) compound

ASSOCIATED CONTENT

S Supporting Information *

11 8.5 7.5

Notes

The authors declare no competing financial interest.

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8 10 8 >40 19

ACKNOWLEDGMENTS The authors are grateful to the Analytical and Testing Center at HUST for the ECD data collection.

16

a

ASS = ampicillin sodium salt, used as a positive control. bAB = amphotericin B, used as a positive control.

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DOI: 10.1021/acs.jafc.7b01294 J. Agric. Food Chem. 2017, 65, 5614−5619

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DOI: 10.1021/acs.jafc.7b01294 J. Agric. Food Chem. 2017, 65, 5614−5619