Bioactive polyketides from the mangrove endophytic fungi Phoma sp. SYSU-SK-7

Journal Pre-proof Bioactive polyketides from the mangrove endophytic fungi Phoma sp. SYSU-SK-7

Yan Chen, Wencong Yang, Ge Zou, Shenyu Chen, Jiyan Pang, Zhigang She PII:

S0367-326X(19)31665-X

DOI:

https://doi.org/10.1016/j.fitote.2019.104369

Reference:

FITOTE 104369

To appear in:

Fitoterapia

Received date:

16 August 2019

Revised date:

4 October 2019

Accepted date:

4 October 2019

Please cite this article as: Y. Chen, W. Yang, G. Zou, et al., Bioactive polyketides from the mangrove endophytic fungi Phoma sp. SYSU-SK-7, Fitoterapia (2018), https://doi.org/ 10.1016/j.fitote.2019.104369

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© 2018 Published by Elsevier.

Journal Pre-proof

Bioactive polyketides from the mangrove endophytic fungi Phoma sp. SYSU-SK-7

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Correspondence author: E-mail address: [email protected] (Z. She).

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Yan Chen1,2 , Wencong Yang1 , Ge Zou1 , Shenyu Chen1 , jiyan pang1 , and Zhigang She1,2,* Affiliation 1 School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, P. R. China; 2 School of Marin Sciences, Sun Yat-sen University; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Guangzhou 510006, China.

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Abstract

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Five new polyketides, colletotric B (2), 3- hydroxy-5-methoxy-2,4,6-trimethylbenzoic acid (3), colletotric C (4), chaetochromone D (6) and 8- hydroxy-pregaliellalactone B

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(9), together with four known analogues (1, 5 and 7-8) were isolated from the mangrove endophytic fungus Phoma sp. SYSU-SK-7. Their structures were

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elucidated by analysis of extensive spectroscopic data and mass spectro metric data.

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Compounds 1-2 showed strong antimicrobial activity against the p. aeruginosa, MRSA and C. albicans with the MIC values in the range of 1.67-6.28 μg/ml.

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Furthermore, Compounds 1-5 also exhibited significant α- glucosidase inhibitory activity with the IC50 values in the range of 36.2-90.6 μM. Compound 7 was found to inhibited radical scavenging activity against DPPH with the EC50 value of 11.8 μM. Key Words: polyketides, Phoma sp., antimicrobial activity, α-glucosidase inhibitory activity, DPPH scavenging activity. 1. Introduction Mangrove endophytic fungi are endophytic microbes that inhabit the internal tissues of mangrove forest [1,2]. Due to the unique ecosystem of mangrove plants, the metabolic pathways of the endophytic fungi are easily ac tivated [3,4]. Moreover, a number of novel structure and specific bioactivity compounds have been isolated from the mangrove-derived fungi [5,6]. phoma sp. is a ubiquitous fungus commonly isolated from terrestrial and marine-derived fungi. It is reported to have antimicrobial

Journal Pre-proof [7,8], cytotoxic [7,9], anti- inflammatory [10] and anti-HIV activities [11]. In our continuing efforts on searching for natural products with novel bioactivive from mangrove-derived fungi in the South China Sea, a strain Phoma sp. SYSU-SK-7, isolated from the healthy branch of the Kandelia candel, was investigated. Five new polyketides (2-4, 6 and 9) together with four known analogues (1, 5 and 7-8) were isolated. In the in vitro bioassays, Compounds 1-2 showed strong antimicrobial activity against the p. aeruginosa, MRSA and C. albicans with the MIC values in the range of 1.67-6.28 μg/ml. In addition, compounds 1-2 also showed significant

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α-glucosidase inhibitory activity with IC 50 values of 36.2 and 35.8 μM, respectively. Compound 2 exhibited cytotoxicity against MDA-MB-435 and A549 cell lines with

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IC50 values of 16.8 and 20.7 μM, respectively. Compound 7 exhibited potent radical

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scavenging activity against DPPH with EC 50 value of 11.8 μM. Herein, the isolation, structure elucidation, and bioactivity evaluations of these compounds are presented.

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2. Experimental section

2.1. General experimental procedures

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Optical rotations were measured on an MCP 300 (Anton Paar shanghai china)

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polarimeter at 28 °C. IR spectra were recorded on a UV-Vis-NIR spectrophotometer (Perkin Elmer Lambda 950, USA). All NMR experiments were performed on a

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Bruker Avance 500 spectrometer (1 H 500 MHz,

13

C 125 MHz) at room temperature.

ESIMS spectra were measured on a Thermofisher TSQ Quantum Ultra mass spectrometer, and HRESIMS spectra were obtained on a Thermofisher LTQ Orbitrap Elite mass spectrometer. Column chromatography (CC) was conducted using silica gel (200-300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (Amersham Pharmacia). Thin- layer chromatography (TLC) was performed on silica gel plates (Qingdao Huang Hai Chemical Group Co., G60, F-254). ECD data were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd., UK). 2.2 Fungal material and fermentation The fungal SYSU SK-7 was isolated from healthy branch of the marine Kandelia candel, which were collected in July 2016 from Shankou Mangrove Nature Reserve in Guangxi Province, China. The isolation of the fungus was obtained using the

Journal Pre-proof standard protocol. A molecular biological protocol by DNA amplification and sequencing of ITS region were used for the identification of the fungal, as described previously [12]. A BLAST search result indicated that the sequence was the nearly similar (99%) to the sequence of Phoma sp. (compared to KU253766.1). The sequence data of the fungal strain have been deposited at Gen Bank with accession no. MN176570. The strain has been deposited in School of Chemistry, Sun Yat-Sen University, Guangzhou, China. 2.3 Extraction and isolation

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The fungus was cultured on autoclaved rice solid-substrate medium (seventy 1L Erlenmeyer flasks, each containing 60 g of rice, 1.8 g of artifical sea salts, and 60 mL

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of distilled water) for 30 days at room temperature. The mycelia and solid rice

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medium were extracted three times with EtOAc. The extract was evaporated under reduced pressure to yield 50g of residue. The residue was subjected to a silica gel

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column (80×10 cm), eluting with a gradient of petroleum ether/EtOAc from 1:0 to 0:1, to obtain 36 fractions. Fraction 6 (100 mg) was subjected to silica gel CC

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(CH2 Cl2 /MeOH v/v, 98:2) to yield compounds 1 (6.0 mg) and 3 (3.8 mg). Fraction 12

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(90 mg) was subjected to silica gel CC (petroleum ether/EtOAc v/v, 90:10) and

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purified by Sephadex LH-20 CC (100% MeOH) to give compounds 2 (5.0 mg) and 7 (3.5 mg). Fraction 16 (68 mg) was purified by Sephadex LH-20 CC (CH2 Cl2 /MeOH v/v, 1:1) and silica gel CC (petroleum ether/EtOAc v/v, 75:25) afford 4 (3.6 mg), 5 (7.6 mg) and 8 (4.2 mg). Fraction 17 (62 mg) was chromatographed on Sephadex LH-20 CC (CH2 Cl2 /MeOH v/v, 1:1) to obtain compound 6 (1.6 mg). Fraction 28 was separated by silica gel CC (petroleum ether/EtOAc v/v, 65:35) and was purified by Sephadex LH-20 CC (100% MeOH) to yield 9 (2.2 mg). colletotric B (2): amorphous powder; IR (KBr) ν max : 3440, 2925, 2860, 1728, 1661, 1450, 1314, 1255, 1173, 1098, 988 cm–1 ; 1 H and

13

C NMR data , see Table 1;

HRESIMS m/z 537.1774 [M – H]– (calcd for 537.1766, C29 H30 O10 ). 3-hydroxy-5-methoxy-2,4,6-trimethylbenzoic acid (3): white solid; IR (KBr) ν max : 3372, 2932, 1727, 1653, 1454, 1311, 1252, 1167, 1150, 1096, 1078 cm–1 ; 1 H and

13

C

Journal Pre-proof NMR data , see Table 2; HRESIMS m/z 209.0819 [M – H]– (calcd for 209.0892, C11 H13 O 4). colletotric C (4): white solid; IR (KBr) ν max : 3346, 2866, 1783, 1672, 1638, 1445, 1311, 1237, 1150, 1065, 981 cm–1 ; 1 H and 13 C NMR data , see Table 3; HRESIMS m/z 389.1595 [M + H]+ (calcd for 389.1522, C21 H26 O7). chaetochromone D (6): white solid; IR (KBr) ν max : 3160, 2925, 1685, 1430, 1388, 1160 cm–1 ; 1 H and

13

C NMR data , see Table 2; HRESIMS m/z 247.0615 [M – H]–

(calcd for 247.0684, C13 H11 O 5).

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8-hydroxy-pregaliellalactone B (9): colourless oil; [α] 25 D 33.5 (с 0.23, CDCl3 ); IR (KBr) ν max : 3432, 3165, 2910, 2830, 1610 cm–1 ; 1 H and

13

C NMR data , see Table 2;

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HRESIMS m/z 195.1028 [M – H]– (calcd for 105.1099, C11 H15 O3).

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2.4. Antimicrobial activity

Preliminary antimicrobial tests were used for the disc diffusion method [13],

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Muller Hinton ager (MHA) and Sabouraud agar (SA) were used for antibacterial and antifungal tests, respectively. Two Gram-positive methicillin-resistent Staphylococcus

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aureus (A7983, clinical isolate donated by Zhijun Yu in Dalian Friendship Hospital,

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Dalian, China), Bacillus subtilis (ATCC 6633), two Gram-negative pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 6539), and one fungus

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Candida albicans (ATCC 10231) were used. The MIC (minimum inhibition concentration) was determined as the concentration which the growth was inhibited 80% of bacterial. 100 μL bacterial suspension were added to the solutions in 96-well to achieve a final volume of 200 μL and final sample concentrations from 50 to 0.125 μg mL–1 . Bank well was also incubated with only medium under the same condition. OD measurement was record at 595 nm. The experiments were carried out in three duplicate and the positive control for antifungal and antifungal tests were used by ampicillin and ketoconazole, respectively. 2.5. α-glucosidase assay A colorimetric α-glucosidase (Sigma-Aldrich Co. CAS number: 9001-42-7, E.C 3.2.1.20) assay was performed according to the previously described method [14]. 1-deoxynojirimycin (St. Louis, MO, USA) used as a positive control.

Journal Pre-proof 2.6. Cytotoxicity Assay Standard MTT assays employing MDA-MB-435 and A549 cell lines were performed as described previously [15]. The IC50 was determined by a 50% reduction of the absorbance in the control assay and the results were performed in triplicates in three independent experiment. The positive control was used by Epirubicin (EPI). 2.7. DPPH radical scavenging activity assay The DPPH radical scavenging assay was conducted with 96-well plates using a revised method [16]. A range of 50 μL of different concentrations for the test

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compounds were added to 50 μL of 0.16 mM DPPH (Sigma-Aldrich, St. Louis, MO, USA) in each well. After incubated in darkness of the reaction mixture for 30 min, the

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absorbance was measured on the microplate reader at 517 nm. The positive control

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was used by Vitamin C. 3. Results and Discussion

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Compound 2 was obtained as an amorphous powder. The molecular formula was determined as C 29 H30O10 based on the HRESIMS at 537.1774 [M – H]– , indicating 15

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degrees of unsaturation. The 1 H NMR spectrum (Table 1), displayed two chelated

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hydroxyl group 2-OH (δH 11.49) and 2''-OH (δH 10.80), three aromatic protons at δH 6.35 (s, H-3 and H-5) and 6.75 (s, H- 3''), signals of six methyl groups at δH 2.68 (s,

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H-8), 2.12 (s, H-8'), 2.16 (s, H-9'), 2.38 (s, H-10'), 2.20 (s, H-8''), 2.51 (s, H-9''), two oxygenated methyl at δH 3.88 (s, OMe-5') and δH 3.98 (s, OMe-7''). There are 29 carbon signals in the

13

C NMR data (Table 1), indicating the presence of eight

methyls (two oxygenated), three sp 2 methines, 18 nonprotonated carbons (three carbonyl and 15 olefinic). Comprehensive analysis of the NMR data implied that 2 was similar to 1, expect for the appearance of an additional methoxy group OCH3 -7'' (δH 3.98, δC 52.3) in compound 2, which was located at C-7'' based on the HMBC correlation from OMe-7'' to the carbonyl carbon C-7''. The deduction further confirmed by the HMBC correlations (Figure 2). Thus, the planar structure of compound 2 was established as shown (Figure 1) and it may be an artefact product obtained from the purification of 1 through methanol. Compound 3, white solid, had a molecular formula of C 11 H14 O4 based on HRESIMS

Journal Pre-proof analysis, indicating five degrees of unsaturation. The 1 H NMR spectrum (Table 2) revealed the presence three methyls at δH 2.17 (s, H-8'), 2.18 (s, H-9') and 2.29 (s, H-10'), one methoxy group at δH 3.78 (s, OMe-5').

13

C NMR data resolved 11 carbon

resonances composed of three sp3 methyl carbons (one oxygenated), one carbonyl carbon and six aromatic carbons. The HMBC correlations (Figure 2) from H3 -8' to C-1', C-2' and C-3', from H3 -9' to C-3', C-4' and C-5', and from H3 -10' to C-1', C-5' and C-6', indicated a 2, 4, 6- trimethyl aromatic unit. The cross-peaks of H3 -8' and H3 -10' to C-7' supporting the carboxyl group was located at C-1'. Thereafter, the

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methoxyl was linked to C-5' based on the correlation from OMe-5' to C-5'. Thus, compound 3 was assigned as 3-hydroxy-5-methoxy-2,4,6-trimethylbenzoic acid.

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Comound 4, isolated as a white solid, exhibited a negative HRESIMS ion peak at m/z

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389.1595 [M +H]+, indicating its molecular formula as C 21 H25O7 . The NMR data (Table 3) of 4 have a closely resemble to those of 3, except for the presence of three

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methyl groups ( one oxygenated), one carbonyl carbon and six aromatic carbons. The HMBC correlations from H-3'' to C-2'' and C-4'', from OH-2'' to C-1'', from H-8'' to

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C-4'' and C-5'', from H-9'' to C-5'', C-6'' and C-1'', confirmed the presence of a

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4,6-dihydroxy-2,3-dimethylbenzoic moiety. The HMBC correlation from OMe-7'' to the carbonyl carbon C-7'' revealed that the methoxyl was lacated at C-7''. Thus, the

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structure of 4 was determined and named as colletotric C. The HRESIMS data of compound 6 suggested a molecular formula of C 13 H12O5 . The 1

H NMR data of 6 (Table 2) displayed signals of two aromatic protons [δH 7.39 (s,

H-5); 6.84 (s, H-8)], one conjugated olefin proton [7.98 (s, H-2) ], two methylenes [δ H 2.64 (t, J =7.2 Hz); 2.78 (t, J = 7.2 Hz)], and one methyl group at δ H 2.14 (s) . The

13

C

NMR data of 6 (Table 2) exhibited 13 carbon resonances assignable to one methyl, two methylenes, three sp2 methines, five quaternary carbons and two carbonyl carbons. Comparing of the NMR data of 6 with chaetochromone B [17] revealed that they possessed the same 6,7-dihydroxy-4H-chromen-4-one moiety. The spin system H2 -9/H2 -10, obtained from the 1 H- 1 H COSY spectrum, together with the correlations (Figure 2) from H2 -9 to C-2, C-3 and C-4, from H3 -12 to C-10 and C-11, suggested the side chain of butan-2-one moiety located at C-3. Thus, the constitution of 6 was

Journal Pre-proof established as shown (Figure 1). Compound 9 was obtained as a colourless oil. The molecular formula was determined as C11 H16 O3 by HRESIMS with four degrees of unsaturation. The 1 H and

13

C NMR

spectra for 9 (Table 2) were similar to those of pregaliellalactone B [18], except for an oxygenated methine (δC 68.9, C-8; δH 4.44, m, H-8) in 9, which replaced the methylene (δC 28.1, C-8; δH 2.22, overlap, H-8) in pregaliellalactone B. Thus, compound 9 is the 8-hydroxy derivative of pregaliellalactone B. The deduction was supported by the 1 H- 1 H COSY correlations of H-8/H2 -9 as well as the HMBC

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correlstions from H-8 to C-6 and C-7. The 5R configuration at C-5 was determined by the positive cotton effects at 211 nm [19]. But, the failed MTPA esters experiment of 9

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using Mosher’s method make the absolute configuration of the hydroxyl group was not assigned. Finally, the structure of 9 was named as 8-hydroxy-pregaliellalactone B.

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The other known compounds were identified as colletotric A (1)[20], orsellinic

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acid (5) [21], citromycin (7) [22] and (–)-2,3-dihydrocitromycin (8) [23] by comparison with NMR data in the literature. Antimicrobial activities

Staphylococcus

aureus,

isolated

compounds

Bacillus

subtilis,

1-8

against

pseudomonas

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methicillin-resistent

of the

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The

aeruginosa, Salmonella typhimurium and Candida albicans were tested (Table 4).

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Compound 1 showed strong antimicrobial activity against the B. subtilis, B. aeruginosa, MRSA and C. albicans with the MIC values of 6.55, 3.27, 6.28 and 3.27 μg/ml, respectively. Compound 2 showed strong antibacterial activity against the P. aeruginosa and MRSA with the MIC values of 1.67 and 3.36 μg/ml, respectively. Compound 3 exhibited significant antifungal activity against C. albicans with the MIC value of 2.62 μg/ml, and antibacterial activity against B. subtilis with the MIC value of 5.25 μg/ml. Compound 5 showed potent antimicrobial activity against P. aeruginosa and C. albicans with the MIC value of 2.10 μg/ml. The results indicated that the methylester displayed improved biological activity and showed a selective antibacterial activity against P. aeruginosa and MRSA. Compounds 1-2 exhibited more strong antimicrobial activity than compounds 3-5. While, other compounds did not exhibit obvious activity at 50 μg/ml.

Journal Pre-proof In addition, the α-glucosidase inhibitory activity of compounds 1-8 have evaluated. Compounds 1-5 showed significant α-glucosidase inhibitory activity with IC50 values ranging from 36.2 to 90.6 μM (Table 5) compared to 62.8 μM for the positive control (1-deoxynojirimycin). Moreover, compounds 1-2 also showed cytotoxicity against MAD-MB-435 and A549 human cancer cell lines (Table 5), with IC50 values ranging from 16.82 to 37.73 μM. Compounds 6-8 exhibited radical scavenging activity against DPPH with EC50 values of 68.1, 11.8 and 30.6 μM, respectively (Table 5). It is noteworthy that the α-glucosidase inhibitory activity of 1 and antioxidant activity of 7

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was the first reported.

In summary, five new polyketides (2-4, 6 and 9) together with four known

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analogues (1, 5 and 7-8) were isolated from the mangrove endophytic fungus Phoma

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sp. SYSU-SK-7. Structurely, compounds 1-2 were tridepsides and 4 was depsides, which were also the typical compounds of lichens, like deleseic acid [24],

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2,4-di-O-methylgyrophoric acid [25], gyrophoric [26], 3-hydroxyumbilicaric acid [27], cytonic acid [28], lasallic acid [29], salvianolic acid [30] and jaboticabin [31]. The

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depsides were reported to possessing antiviral [32], antioxidant [33], antimicrobial

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[34-35] and anti- inflammatory activity [36]. To this work, compounds 1-5 exhibited significant antimicrobial activity, including compound 2 against MRSA and P.

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aeruginosa with the MIC values of 3.36 and 1.67 μg/ml, respectively, and compound 3 against B. subtilis and C. albicans with the MIC values of 5.25 and 2.62 μg/ml, respectively. Moreover, compounds 1-2 also showed potent α-glucosidase inhibitory activity comparing to 1-deoxynojirimycin (positive control). Conflict of Interest The authors declare no conflict of interest. Acknowledgements We thank the National Natural Science Foundation of China (2187713), Guangdong Special Fund for Marine Economic Development (GDME-2018C004), Guangdong MEPP Fund (GDOE-2019A21), the Key Project of Natural Science Foundation of Guangdong Province (2016A040403091), the Special Promotion Program for Guangdong Provincial Ocean and Fishery Technology (A201701C06) and the open

Journal Pre-proof foundation of Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education (rdyw2018001 ) for generous support. Appendix A. Supplementary Data Supplementary data to this article can be found online. References [1] F.L. de Souza Sebastianes, A.S. Romao-Dumaresq, P.T. Lacava. Curr. Genet. 59 (2013) 153-166. [2] K. Chowdhary, N. Kaushik, A.G. Coloma, C.M. Raimundo. Phytochem. Rev 11

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(2012) 467-485.

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6268 - 6271.

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Hasegawa, T. Tsubogo, H. Uchiro. Chem. Asian J. 12 (2017) 628 - 632.

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[5] M. Isaka, N. Rugseree, P. Maithip, P. Kongsaeree, S. Prabpai, Y. Thebtaranonth. Tetrahedron 61 (2005) 5577 - 5583.

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[8] Y.S. Che, J.B. Gloer, D.T. Wicklow. J. Nat. Prod. 65 (2002) 399-402. [9] L.W. Wang, B.G. Xu, J.Y. Wang, Z.Z. Su, F.C. Lin, C.L. Zhang. Appl. Microb. Cell Physiol. 93 (2012) 1231-1239. [10] E.L. Kim, J.L. Li, B. Xiao, J. Hong, E.S. Yoon, J.H. Jung. Chem. Pharm. Bull. 60 (2012) 1590-1593. [11] J.H. Pan, J.J. Deng, Y.G. Chen, J.P. Gao, Y.C. Lin, Z.G. She, Y.C. Lin. Helv. Chim. Acta 93 (2010) 1369-1374. [12] Y. Chen, Z.M. Liu, H. J. Liu, Y. H. Pan, J. Li, L. Liu, Z.G. She, Mar. Drugs 16 (2018) 54. [13] S.K. Santhosh, A. Venugopal, M.C. Radhakrishnan, Indian J. Biol. Sci. 2 (2016) 119-124

Journal Pre-proof [14] Y.Y. Liu, Q. Yang, G.P. Xia, H.B. Huang, H.X. Li, L. Ma, Y.J. Lu, X.K. Xia, Z.G. She. J. Nat. Prod 78 (2015) 1816-1822. [15] Y. Chen, Z.M. Liu, Y. Huang, L. Liu, J.G. He, L. Wang, J. Yuan, Z.G. She. J. Nat. Prod. 82 (2019) 1752-1758. [16] W.C. Yang, H.Y. Bao, Y.Y. Liu, Y.Y. Nie, J.M. Yang, P.Z. Hong, Y. Zhang. Molecules 23 (2018) 2245. [17] D. Kumla, J. Pereira, T. Dethoup, L. Gales, N. Sekeroglu, M.M. Pinto, A. Kijjoa. Mar. Drugs 16 (2018) 289.

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[18] S.J. Li, X. Zhang, X.H. Wang, C.Q. Zhao. Eur. J. Med. Chem 156 (2018) 316-343.

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[19] M. Braun, A. Hohmann, J. Rahematpura, C. Buhne, S. Grimme. Chem. Eur. J. 10

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[20] W.X. Zou, J.C. Meng, H. Lu, G.X. Chen, G.X. Shi, T.Y. Zhang, R.X. Tan. J. Nat.

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Prod. 63 (2000) 1529-1530.

[21] J.F. Sanchez, Y.M. Chiang, E. Szewczyk, A.D. Davidson, M. Ahuja, C.E. Oakley,

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J.W. Bok, N. Keller, B.R. Oakley, and C.C.C. Wang, Mol. BioSyst. 6 (2010) 587-593.

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[22] Y. Kusakabe, Y. Yamauchi, C. Nagatsu. J. Antibiotics 22 (1969) 112-118.

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[23] R.J. Capon, M. Stewart, R. Ratnayake, E. Lacey, J.H. Gill. J. Nat. Prod. 70 (2007)

[24] T. Narui, K. Sawada, S. Takatsuki. Phytochemistry. 48 (1998) 815-822. [25] J.A. Elix, V.K. Jayanthi, C.C. Leznoff. Aust. J. Chem. 34 (1981) 1757-1761. [26] KC.S. Kumar, K. Müller. J. Nat. Prod. 62 (1999) 821-823. [27] J.A. Elix, Y. Jin, M.T. Adler. Aust. J. Chem. 42 (1989) 765-770. [28] B. Guo, J.R. Dai, S. Ng. J. Nat. Prod. 63 (2000) 602-604. [29] T. Narui, S. Takatsuki, K. Sawada. Phytochemistry. 42 (1996) 839-842. [30] C. Ai, L. Li. Planta Med. 58 (1992) 197-199. [31] K.A. Reynertson, A.M. Wallace, S. Adachi. J. Nat. Prod. 69 (2006) 1228-1230. [32] N. Neamati, H. Hong, A. Mazumder. J. Med. Chem. 40 (1997) 942-951. [33] M.E. Hidalgo, E. Fernandez, W. Quilhot. Phytochemistry. 37 (1994) 1585-1587.

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[36] A.L.P. de Miranda, J.R.A. Silva, C.M. Rezende. Planta Med. 66 (2000) 284-286.

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Figure 1. The structures of compounds 1-9.

Figure 2. Key HMBC and COSY correlations of compounds 2–4, 6 and 9.

Journal Pre-proof Table 1. 1 H and

13

C NMR data of 2 (500 and 125 MHz) in CDCl3. no.

δ C, type

δ H (J in Hz)

104.1, C

9'

10.2, CH3

2.16, s

2

161.4, C

10'

16.9, CH 3

2.38, s

3

101.6, CH

1''

112.2, C

4

166.3, C

2''

160.4, C

5

112.1, CH

3''

108.6, CH

6

144.6, C

4''

153.4, C

7

169.6, C

5''

121.9, C

8

24.9, CH 3

6''

141.8, C

1'

133.2, C

7''

171.8, C

2'

125.7, C

8''

f

2

3'

149.4, C

9''

4'

121.9, C

OM e-5'

5'

154.2, C

OM e-7''

6'

126.6, C

OH-2

11.49, s

7'

166.7, C

OH-2''

10.80, s

8'

13.0, CH 3

Table 2. 1 H and

13

6.35, s

2.68, s

2.12, s

no. 1'

131.9, C

2'

119.7, C

3'

155.5, C

4'

115.4, C

5'

154.1, C

6'

12.6, CH3

2.20, s

19.1, CH3

2.51, s

62.3, CH 3

3.88, s

52.3, CH 3

3.98, s

C NMR data of 3, 6 and 9 (500 and 125 MHz). 3

δ Ca,

6.75, s

oo

6.35, s

pr

1

δ H (J in Hz)

e-

δ C, type

Pr

no.

6

δ H (J in Hz) a

no.

al

type

δ Ca,

type

9 δ H (J in Hz)

no.

7.98, s

1

116.5, CH 2

5.05, m

a

δ Cb,

type

δ Hb (J in Hz)

154.6, CH

3

122.9, C

2

136.6, CH

5.80, m

4

179.0, C

3

29.3, CH 2

2.23, m

4a

112.2, CH

4a

32.7, CH 2

1.85, m

5

117.6, C

119.9, C

6

146.0, C

5

81.5, CH

4.99, m

7'

167.7, C

7

153.9, C

6

148.3, CH

7.20, s

8'

10.8, CH3

2.17, s

7a

103.6, CH

7

136.4, C

9'

8.3, CH3

2.18, s

8

154.2, C

6.84, s

8

68.9, CH

4.44, m

10'

15.7, CH3

2.29, s

9

21.2, CH 2

2.64, t (7.2)

9a

28.6, CH 2

1.85, m

OM e-5'

61.1, CH3

3.78, s

10

42.8, CH 2

2.78, t (7.2)

9b

11

210.7, C

12

29.8, CH 3

α

Jo u

rn

2

b

M easured in M eOH-d4. M easured in CDCl3.

7.39, s

4b

10 2.14, s

1.74, m

1.74, m 9.6, CH 3

0.98, t (7.4)

Journal Pre-proof Table 3. 1 H and

13

C NMR data of 4 (500 and 125 MHz) in CDCl3. no.

δ C, type

132.9, C

2''

160.4, C

2'

118.3, C

3''

108.6, CH

3'

154.3, C

4''

153.7, C

4'

114.2, C

5''

121.9, C

5'

154.3, C

6''

141.4, C

6'

120.8, C

7''

171.8, C

7'

166.9, C

8''

12.6, CH3

2.20, s

8'

11.7, CH 3

2.20, s

9''

29.7, CH 3

2.52, s

9'

8.90, CH 3

2.23, s

OM e-5'

62.3, CH 3

3.84, s

10'

16.8, CH 3

2.35, s

OM e-7''

f

4

1'

δ H (J in Hz)

1''

112.1, C

OH-2''

ePr al rn Jo u

52.3, CH 3

oo

δ C, type

pr

no.

δ H (J in Hz)

6.72, s

4.0, s 10.81, s

Journal Pre-proof Table 4. Antimicrobial activity of compounds 1-9 (MIC, μg/ml). Compounds

B. subtilis

M RSA

P. aeruginosa

salmonella

C. albicans

1

6.55

6.28

3.27

26.2

3.27

2

26.9

3.36

1.67

>50.0

>50.0

3

5.25

>50.0

>50.0

>50.0

2.62

4

9.70

>50.0

>50.0

>50.0

>50.0

5

>50.0

8.40

2.10

>50.0

2.10

6

>50.0

>50.0

>50.0

>50.0

>50.0

7

>50.0

>50.0

>50.0

>50.0

>50.0

8

>50.0

>50.0

>50.0

>50.0

>50.0

-

-

-

-

-

-

-

-

-

0.10

Ampicillin

0.07

0.15

0.15

0.31

-

oo

f

9 Ketoconazole

-: not tested.

α-glucosidase inhibitory activity

A549

>100

37.01

37.73

>100 >100 >100 >100

16.82 >50 >50 >50

20.75 >50 >50 >50

>100 >100

68.1 11.8

>50 >50

>50 >50

>100 -

30.6 -

>50 -

>50 -

62.8

-

-

-

-

22.3 -

0.26

5.60

2 3 4 5

35.8 53.3 60.2 90.6

al

36.2

rn Jo u

8 9

1-deoxynojirimycina Ascorbic acid a

a

-: not tested. a: positive control

μM)

activity

MAD-MB-435

1

6 7

Cytotoxic activity (IC50 ,

EC50 (μM)

Pr

IC50 (μM)

DPPH scavenging

e-

compounds

EPI

pr

Table 5. α-glucosidase inhibitory activity, DPPH free radical scavenging activity, and cytotoxic activity of compounds 1-9.

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

Graphical Abstract