Flavonoids from Erythrina schliebenii - Journal of Natural Products

Publication Date (Web): January 23, 2017. Copyright © 2017 The American Chemical Society and American Society of Pharmacognosy. *Tel: +255-754-206560...
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Flavonoids from Erythrina schliebenii Stephen S. Nyandoro,*,†,∥ Joan J. E. Munissi,†,∥ Msim Kombo,† Clarence A. Mgina,† Fangfang Pan,‡ Amra Gruhonjic,§ Paul Fitzpatrick,§ Yu Lu,⊥ Bin Wang,⊥ Kari Rissanen,‡ and Máté Erdélyi*,∥,# †

Chemistry Department, College of Natural and Applied Sciences, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania ‡ Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box. 35, FI-40014 University of Jyvaskyla, Finland § Sahlgrenska Cancer Centre, University of Gothenburg, Gothenburg SE-405 30, Sweden ⊥ Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumour Research Institute, Beijing 101149, People’s Republic of China ∥ Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg SE-412 96, Sweden # Swedish NMR Centre, University of Gothenburg, Gothenburg SE-405 30, Sweden S Supporting Information *

ABSTRACT: Prenylated and O-methylflavonoids including one new pterocarpan (1), three new isoflavones (2−4), and nineteen known natural products (5−23) were isolated and identified from the root, stem bark, and leaf extracts of Erythrina schliebenii. The crude extracts and their constituents were evaluated for antitubercular activity against Mycobacterium tuberculosis (H37Rv strain), showing MICs of 32−64 μg mL−1 and 36.9−101.8 μM, respectively. Evaluation of their toxicity against the aggressive human breast cancer cell line MDA-MB-231 indicated EC50 values of 13.0−290.6 μM (pure compounds) and 38.3 to >100 μg mL−1 (crude extracts). Erythrina schliebenii Harms (Fabaceae) is a rare coral tree endemic to Tanzania. It was twice reported to be extinct, but survives in a restricted distribution in the Kilwa district, southeastern Tanzania.1−4 Its existence is threatened by habitat loss due to land clearance for agricultural practices. Traditionally, this plant is used for the treatment of stomachache and diarrhea, for prevention of jaundice of newborn babies, and as an abortive agent.5 Erythrina species have previously been reported to accumulate prenylated and O-methyl (iso)flavonoids;6−15 however, E. schliebenii has not yet been phytochemically investigated. Herein the isolation, identification, and evaluation of the antitubercular and cytotoxic activities of four new (1−4) and nineteen known (5−23) natural products from E. schliebenii are reported.

along with the six known compounds, 5,7-dihydroxy-4′methoxy-3′-(2,3-dihydroxy-3-methylbutyl)isoflavone (5),16 4′O-methylabyssinone V (6),7,8 5′-methoxy-3′-(3-methylbut-2enyl)biochanin A (piscerythrinetin, 7),17 3′-(3-methylbut-2enyl)biochanin A (8),6 burttinone (9),15 and 5,7-dihydroxy3′,4′,5′-trimethoxyisoflavone (panchovillin, 10),18 and the ubiquitous lupeol19 and stigmasterol.20 Isoflavone 5 has previously been isolated only from the family Moraceae,16 and hence this is the first report of its occurrence in the family Leguminosae. Chromatographic separation of the CH2Cl2 extract of the root bark of E. schliebenii yielded four known compounds, the pterocarpans orientanol B (11) 21 and neurautenol (12),8,9 the flavanone abyssinone V (13),7,8,10,11,22 and the isoflavone 5′-(3-methylbut-2-enyl)pratensein (14).6 By chromatographic purification of the MeOH extract of the root

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RESULTS AND DISCUSSION Repeated column chromatographic separation of the MeOH extract of the stem bark of E. schliebenii over silica gel and Sephadex LH-20 yielded three new natural products (1, 2, and 4) © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 13, 2016 Published: January 23, 2017 377

DOI: 10.1021/acs.jnatprod.6b00839 J. Nat. Prod. 2017, 80, 377−383

Journal of Natural Products

Article

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for 1 Acquired in Methanol-d4 at 25 °C [δH, Multiplicity (J in Hz)]

bark one new natural product (3) and nine known compounds, flavanone 6 and the isoflavonoids 7, 8, 10, 14, 3′-Omethylpratensein (15),23 parvisoflavone B (16),24 2,3-dihydro7-de-O-methyltrobustigenin (17),25 and (R)-saclenone (18),26 were isolated. From the MeOH extract of the leaves, the Omethylflavonoids 6-hydroxy-5,7,4′-trimethoxyflavanone (hamiltone A, 19),26 6-methoxyhamiltone A (20),27 5,6,7,4′tetramethoxyflavone (tetramethylisoscutellarein, 21), 28 3′,4′,5,6,7-pentamethoxyflavone (sinensetin, 22),29,30 and 6hydroxy-2,3,4,4-tetramethoxychalcone (23)31 were obtained by silica gel column chromatographic separation, followed by purification on Sephadex LH-20 or by recrystallization. The Omethylflavonoids 19−22 and the chalcone 23 are reported for the first time from the genus Erythrina. The structures of the four new secondary metabolites (1−4) were determined by NMR spectroscopic and mass spectrometric analyses, whereas those of the known compounds (5−23) were confirmed by comparison of their observed and reported spectroscopic and physical data (Supporting Information) and single-crystal X-ray crystallography.

δC, type

δH

(J in Hz)

1 2 3 4 4a 6

131.9, CH 109.3, CH 158.6, C−O 102.6, CH 156.6, C−O 66.1, CH2

7.35 6.51

d (8.5) dd (8.5, 2.4)

6.31

d (2.4)

6a 6b 7 8 9 10 10a 11a 11b 1′

39.9, CH 119.4, C 102.8, CH 122.0, CH 159.0, C−O 110.7, C 158.8, C−O 78.3, CH 111.9, C 26.0, CH2

4.22 3.61 3.56

dd (10.1, 4.1) d (10.1) dd (6.6, 4.1)

6.51 7.13

d (8.2) d (8.2)

5.50

d (6.6)

2.73 2.87 3.61

m dd (13.6, 3.2) m

1.19 1.23 3.82

s s s

position

2′ 3′ 4′ 5′ 9-OCH3

77.7, C−O 72.0, C−O 24.3, CH3 23.6, CH3 55.0, C−O

1′b (δH 2.87) to C-9 (δC 159.0) and C-10 (δC 110.7) defined the position of the dihydroxyisopentyl substituent attached to ring D. TOCSY correlations (Figure S7, Supporting Information) revealed the ABX spin system H-1′a (δH 2.73), H-1′b (δH 2.87), and H-2′ (δH 3.61), whereas the position of the Me-4′ and Me-5′ groups was indicated by the HMBC cross-peaks of their protons (δH 1.19 and δH 1.23) to C-3′ (δC 72.0) and C-2′ (δH 77.7). The absolute configuration of C-2′ was not assigned. On the basis of the above spectroscopic features, the new compound 1, schliebenin A, was characterized as 3-hydroxy-10-(2,3-dihydroxy-3-methylbutyl)-9-methoxypterocarpan. Compound 2 was obtained as a pale yellow, amorphous solid. It was assigned the molecular formula C21H20O6 based on HRESIMS ([M + H]+ m/z 369.1355, calcd 369.1338) and NMR analyses (Table 2, Figures S9−S12, Supporting Information). The NMR signals at δH 8.07 and δC 155.1 were diagnostic for an H-2 and C-2 of an isoflavone.25 The presence of five oxygenated carbons was indicated by the signals at δC 155.1 (C-2), δC 163.9 (C-5), δC 166.3 (C-7), δC 159.8 (C-8a), and δC 159.3 (C-4′) (Table 2, Figure S10, Supporting Information). In line with the biosynthetically viable substitution pattern,37 ring A was dihydroxylated at C-5 (δC 163.9) and C-7 (δC 166.3), as confirmed by the meta coupling (J = 2.1 Hz) of H-6 (δH 6.24) and H-8 (δH 6.36). Assignment of ring C was based on the HMBC cross-peaks of H-2 (δH 8.07) with C-3 (δC 124.6), C-4 (δC 181.8), and C-8a (δC 159.8). The substitution pattern of ring B was elucidated based on the scalar couplings of H-2′ (δH 7.32), H-5′ (δH 7.41), and H-6′ (δH 7.01) (Table 2) with a methoxy substituent attached to C-4′ (δC 159.3) indicated by the HMBC cross-peaks of Me-4′ (δH 3.83) to C-4′. Attachment of a 2hydroxyl-3-methylbuten-3-yl functionality at C-3′ was revealed by the HMBC cross-peaks of H-1″ (δH 2.85) to C-2′ (δC 132.9), C-3′ (δC 127.8), and C-4′ (δC 159.3) (Table S1, Figure S13, Supporting Information). The absolute configuration of C-2′

Compound 1 was obtained as an amorphous powder, whose molecular formula C21H25O6 was determined based on HRESIMS ([M + Na]+ m/z 395.1467, calcd 395.1471) and NMR (Table 1) analyses. Its NMR spectra (Figures S1−S4, Supporting Information) showed signals characteristic for a pterocarpan (δH 5.50, 4.22, 3.61, and 3.56) skeleton32−35 and closely resembled those reported for 3-hydroxy-10-(3-hydroxy3-methylbutyl)-9-methoxypterocarpan,36 except for the signals of the C-10 substituent. Based on their HMBC cross-peaks (Table S1, Figure S5, Supporting Information), the five aromatic protons were deduced to belong to rings A (δH 7.35, 6.51, and 6.31) and D (δH 7.13 and 6.51) of the pterocarpan skeleton, with their multiplicities (Table 1) revealing their regiochemistry. The HMBC cross-peak of H-1 (δH 7.35) to C-4a (δC 156.6) and C11a (δC 78.3) allowed the assignment of the H-1, H-2, H-4 spin system to ring A, whereas the HMBC cross-peaks of H-7 (δH 6.51) to C-6b (δC 119.4) and C-10a (δC 158.8) allowed the assignment of the H-7, H-8 spin system to ring D. The position of H-11a (δH 5.50) was determined based on its HMBC cross-peaks to C-1 (δC 131.9), C-6 (δC 66.1), C-10a (δC 158.8), and C-11b (δC 111.9). The relative configuration of the C-11a−C-6a junction was determined upon observation of the NOE correlation of H-11a (δH 5.50) with H-6a (δH 3.56) (Figure S6, Supporting Information) and the 3JH6a,H11a = 6.6 Hz value indicative of the 6a,11a-cis orientation of these protons.21 Moreover, the HMBC cross-peaks of H-1′a (δH 2.73) and H378

DOI: 10.1021/acs.jnatprod.6b00839 J. Nat. Prod. 2017, 80, 377−383

Journal of Natural Products

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Table 2. 1H (500 MHz, 2; 799.88 MHz, 3) and 13C (125 MHz, 2; 201.15 MHz, 3) NMR Spectroscopic Data for 2 and 3 Acquired in Methanol-d4 and CDCl3, Respectively, at 25 °C [δH, Multiplicity (J in Hz)] 2 position 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 4′-OCH3 5-OH 7-OH 5′-OH

3

δC, type

δH

155.1, CH 124.6, C 181.8, CO 106.3, C 163.9, C−O 100.4, CH 166.3, C−O 95.0, CH 159.8, C−O 124.2, C 132.9, CH 127.8, C 159.3, C−O 129.8, CH 115.1, CH 33.5, CH2 90.0, C−O 145.8, C 17.5, CH3 114.0, CH2

8.07

s

6.24

(2.1)

6.36

d (2.1)

7.32

d (2.3)

7.41 7.01 2.85 4.56

dd (8.5, 2.3) d (8.5) m m

1.8 4.82 3.88

56.1, C−O

δC, type

δH

J in Hz)

7.89

s

6.30

d (2.4)

6.37

d (2.4)

7. 02

d (2.4)

7.20 6.73 6.94

d (2.4) d (16.0) d (16.0)

s m

153.3, CH 123.8, C 180.7, CO 106.4, C 163.2, C−O 99.7, CH 162.7, C−O 94.1, CH 158.1, C−O 127.5, C 115.1, CH 149.2, C−O 145.0, C−O 131.0, C 119.0, CH 122.4, CH 134.2, CH 142.2, C 18.7, CH3 118.5, CH2

s

61.8, C−O

(J in Hz)

2.00 5.16 5.12 3.83 12.87 5.81 5.73

s s s s s s s

group (H-4″), which were deduced to constitute a prenyl unit (3methyl-1,3-butadien-1-yl). The HMBC cross-peaks confirmed the attachment of this prenyl unit at C-3′ of ring B. On the basis of the above spectroscopic data this new isoflavone (3), schliebenone B, was characterized as 5,7-dihydroxy-3-[3hydroxy-4-methoxy-5[(E)-3-methylbuta-1,3-dienyl]phenyl-1benzopyran-4-one. Compound 4 was obtained as an amorphous solid and was assigned the molecular formula C21H20O7 based on HRESIMS ([M + H]+ m/z 385.1291, calcd 385.1287) and NMR (Table 3) analyses. Its NMR data (Table 3, Figures S23−S26, Supporting Information) were in agreement with an isoflavone skeleton with a singlet at δH 8.12 being characteristic for H-2 of isoflavones. The 5,7-dioxygenation pattern of its ring A, analogous to that of 2 and 3, was confirmed by the meta-coupling (J = 2.2 Hz) of H-6 (δH 6.24) and H-8 (δH 6.36). The HMBC cross-peaks (Table S2, Figure S27, Supporting Information) of H-2 (δH 8.12) to C-3 (δC 124.3), C-4 (δC 181.7), C-8a (δC 159.5), and C-1′ (δC128.3) further confirmed that the compound is an isoflavone and provided the basis for the assignment of the constitution of the C-ring. The regiochemistry of ring B was assigned based on the J = 2.1 Hz coupling of H-2′ (δH 6.98) and H-6′ (δH 7.15), indicating their meta orientation, and the HMBC cross-peaks (Figure S27, Supporting Information) of H-2′ (δH 6.98) to C-4′ (δC 146.6) and C-6′ (δC 118.8), of H-6′ (δH 7.15) to C-6′ (δC 117.1), of H-1″ (δH 6.87) to C-4′ (δC 146.6) and C-6′ (δC 118.8), and of H-2‴ (δH 3.81) to C-4′ (δC 146.6). The E configuration of the Δ1″(2″) double bond was indicated by their 16.2 Hz coupling constant. This new compound (4), schliebenone C, was therefore characterized as 5,7,3′-trihydroxy-4′-methoxy-5′[(E)-3-hydroxy-3-methylbuten-1-yl]isoflavanone.

was not assigned. This new compound (2), schliebenone A, was therefore characterized as 3′-(2-hydroxy-3-methylbuten-3-yl)biochanin A. Compound 3 was isolated as a yellow, amorphous solid and was assigned the molecular formula C21H20O6 based on HRESIMS ([M + H]+ m/z 369.1336, calcd 369.1346) and NMR (Table 2) analyses. Its NMR spectra (Figures S15−18, Supporting Information) displayed features indicative of an isoflavone backbone, similar to those of 2, i.e., a singlet at δH 7.89 typical for H-2 and a singlet at δH 12.87 (OH-5) diagnostic for a hydrogen-bonded phenolic group. Ring A of 3 showed similar features to those of 2, i.e., two meta-coupled (J = 2.4 Hz) protons at δH 6.30 (H-6) and δH 6.37 (H-8) suggesting a metadisubstituted ring. The HMBC cross-peaks (Table S2, Figure S19, Supporting Information) of OH-5 (δH 12.87) to C-4a (δC 106.4) and C-6 (δC 99.7), those of H-6 (δH 6.30) to C-4a (δC 106.4) and C-8 (δC 94.1), and those of H-8 (δH 6.37) to C-4a (δC 106.4), C-6 (δC 99.7), C-7 (δC 162.7), and C-8a (δC 158.1) confirmed the substitution pattern of ring A, whereas those of H2 (δH 7.89) to C-3 (δC 123.8), C-4 (δC 180.7), C-8a (δC 158.1), and C-1′ (δC 127.5) permitted the assignment of ring C. The trisubstituted B-ring was revealed by the meta-coupled (J = 2.4 Hz) H-6′ (δH 7.20) and H-2′ (δH 7.02) and the HMBC crosspeaks of OH-3′ (δH 5.73) to C-4′ (δC 145.0) and C-3′ (δC 115.1), H-6′ (δH 7.20) to C-3 (δC 123.8), C-4′ (δC 145.0), C-2′ (δC 115.1), and C-1″ (δC 122.4), and H-2′ (δH 7.02) to C-3 (δC 123.8), C-6′ (δC 119.0), C-4′ (δC 145.0), and C-3′ (δC 149.2), which allowed the assignment of the constitution of this ring. Additionally, the 1H NMR spectrum of 3 comprised two mutually coupled (J = 16 Hz) trans olefinic protons at δH 6.94 (H-2″) and 6.73 (H-1″), two terminal olefinic protons at δH 5.16 (H-5″a) and 5.12 (H-5″b), and a singlet at δH 2.00 for a methyl 379

DOI: 10.1021/acs.jnatprod.6b00839 J. Nat. Prod. 2017, 80, 377−383

Journal of Natural Products

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Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for 4 Acquired in Methanol-d4 at 25 °C [δH, Multiplicity (J in Hz)] position

δC, type

δH

2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 4′-OCH3

155.2, CH 124.3, C 181.7, CO 106.0, C 163.7, C−O 100.2, CH 166.2, C−O 94.8, CH 159.5, C−O 128.3, C 117.1, CH 151.2, C−O 146.6, C−O 132.3, C 118.8, CH 121.4, CH 140.3, CH 71.5, C−O 29.8, CH3 29.8, CH3 61.3, C−O

8.12

s

6.24

d (2.2)

6.36

d (2.2)

6.98

d (2.1)

Erythrina species possess metabolites with considerable cytotoxic activity.36 As part of an ongoing investigation of East African medicinal plants in a search for novel cytotoxic and antitubercular natural products, we have studied the crude extracts of E. schliebenii and their isolated constituents for in vitro activity against Mycobacterium tuberculosis (H37Rv strain) and the aggressive human breast cancer cells (MDA-MB-231). The crude extracts showed low toxicity against cancer cells with EC50 = 38.3 to >100 μg mL−1, whereas their isolated constituents showed EC50 = 13.0−290.6 μM, with schliebenone B (3) being the most potent (13.0 μM) (Table 4). Moreover, the crude

(J in Hz)

Table 4. Antimycobacterial (MIC, μM) and Cytotoxic (EC50, μM) Activities of the Crude Extracts of E. schliebenii and Some of their Isolated Constituents sample a

7.15 6.87 6.45

d (2.1) d (16.2) d (16.2)

1.41 1.41 3.81

s s s

CH2Cl2 extract of roots MeOH extract of rootsa MeOH extract of stema MeOH extract of leafa 3 6 7 8 10 11 13 14 15 16 17 18 19 20 21 22 23 lupeol isonicotinylhydrazine (control) rifampicin (control)

Compounds 6−8, 20, 21, and 23 were crystallized and analyzed by single-crystal X-ray diffraction, confirming their structures, including their stereochemical configurations (Figure 1). Compounds 20 and 23 were obtained as single stereoisomers, whereas 6−8 and 21 were obtained as racemates.

MIC (μM)

EC50 (μM)

32−64 32−64 32−64 32−64 87.4 49.5 77.2 36.9 93.0 90.8 78.3 86.9 101.8 90.9 92.5 67.6 96.9 92.9 93.5 85.9 92.9

>100 >100 38.3 >100 13.0 21.6 49.4 27.9 29.0 n.d n.d 77.0 31.8 16.9 275.0 58.0 200.6 290.6 292.3 268.7 290.6 208.9

0.3 0.09

a MIC and EC50 are given in μg/mL for the crude extracts; n.d. = not determined.

extracts exhibited antitubercular activities of EC50 = 32−64 μg mL−1 (minimum inhibitory concentrations, MIC), whereas their constituents showed EC50 = 36.9−101.8 μM, with 3′-(3methylbut-2-enyl)biochanin A (8) being the most active (36.9 μM) among the isolates. Overall, 23 flavonoids were isolated from the root, stem bark, and leaf extracts of E. schliebenii, among which four were identified as new natural products. The crude extracts and their isolated constituents exhibited moderate antitubercular and cytotoxic activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. Column chromatography was carried out using silica gel 60 (230−400 mesh). NMR spectra were acquired on Bruker 400, 500, and 800 MHz spectrometers and were processed using MestReNova-9.0. The structural assignment was based on 1H and 13C NMR, gCOSY, gNOESY, gHSQC, and gHMBC spectra. The residual solvent peaks were used for chemical shift referencing (CDCl3, δH 7.26 and δC 77.16; CD3OD, δH 3.30 and δC 49.00). LC-MS (ESI) chromatograms were acquired on a PerkinElmer PE SCIEX API

Figure 1. X-ray crystal structures of (a) 4-O′-methylabyssinone V (6), (b) piscerythrinetin (7), (c) 3′-(3-methylbut-2-enyl)biochanin A (8), (d) 6-methoxyhamiltone A (20), (e) 5,6,7,4′-tetramethoxyflavone(tetra-O-methylisoscutellarein), 21), and (f) 6-hydroxy-2,3,4,4-tetramethoxychalcone (23). 380

DOI: 10.1021/acs.jnatprod.6b00839 J. Nat. Prod. 2017, 80, 377−383

Journal of Natural Products

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150EX instrument equipped with a Turbolon spray ion source and a Gemini 5 mm RPC18 110 Å column, applying a H2O/CH3CN 80:20− 20:80 gradient solvent system with a separation time of 8 min. HRESIMS was obtained with a Q-TOF-LC/MS spectrometer (StenhagenAnalyslab AB, Gothenburg, Sweden) using a 2.1 × 30 mm 1.7 μm RPC18 column and a H2O/CH3CN gradient system (5:95−95:5 gradient and 0.2% formic acid). Analytical TLC was performed on silica gel 60 F254 (Merck, Darmstadt, Germany) precoated aluminum plates, which after development with an appropriate solvent system were evaluated under UV light (254 and 366 nm), then sprayed with anisaldehyde reagent (prepared by mixing 3.5 mL of anisaldehyde with 2.5 mL of concentrated H2SO4, 4 mL of glacial HOAc, and 90 mL of MeOH) followed by heating for identification of UV-negative compounds and detection of a color change of the UV-positive spots. Gel filtration was carried out over Sephadex LH-20 (Pharmacia, Uppsala, Sweden) suspended in CH2Cl2/CH3OH (1:1) or 100% CH3OH. Preparative HPLC was performed on a Waters 600E system using the Chromulan (Pikron Ltd.) software and an RP-C8 Kromasil column (250 mm × 25 mm) with the solvent system H2O/CH3OH (gradient, 70:30 to 100% CH3OH, for 20−40 min, flow rate of 8−15 mL/min). Plant Materials. The root bark of E. schliebenii was collected on October 6, 2012, from Mchakama village, in the Kilwa district, Lindi region, Tanzania. The plant was identified and authenticated by Mr. Frank M. Mbago, a senior taxonomist at the Herbarium of the Department of Botany, University of Dar es Salaam, where a voucher specimen is deposited with reference number FMM T8:3611. The leaves, stem, and root barks of E. schliebenii were collected from the edge of the Namatimbili Forest Reserve in the same village on March 19, 2013, by one of the authors (S.S. N.) with the assistance of Mr. Yahaya S. Abeid, a part-time botanist at the Herbarium of the Department of Botany of the University of Dar es Salaam. The sample specimen Y.SA 3653 was matched with authentic FMM T8:3611 and deposited at the Herbarium of the Department of Botany, University of Dar es Salaam. Extraction and Isolation. The air-dried and pulverized stem bark of E. schliebenii was soaked twice in MeOH at room temperature for 48 h, in each case yielding 30 g of crude extract after evaporation, of which 20 g was adsorbed on silica gel and loaded on a silica gel column. It was eluted with increasing amounts of EtOAc in isohexane, giving 40 fractions. On the basis of TLC analysis the fractions were combined and subjected to further purification. Fraction 3 was purified over silica gel, eluting with 10% EtOAc in isohexane, giving lupeol (10 mg). Combined fractions 6− 8 were passed through silica gel, eluting with 10% EtOAc in isohexane, yielding 30 subfractions. Further purification of the combined subfractions 19−30 over Sephadex LH-20, eluting with 1:1 CH2Cl2/ MeOH, afforded abyssinone V methyl ether (6, 10 mg) as white crystals. Purification of the combined subfractions 13−18 over silica gel, eluting with 100% CH2Cl2, yielded stigmasterol (12 mg). Gel permeation of the combined fractions 11−13 over Sephadex LH-20, eluting with 1:1 CH2Cl2/MeOH, yielded 35 subfractions. Combined subfractions 23− 35 were subjected to chromatography over silica gel, eluting with 20% EtOAc in isohexane, yielding 3′-(3-methylbut-2-enyl)biochanin A (8, 8 mg), schliebenone A (2, 2 mg), and 5,7-dihydroxy-4′-methoxy-3′-(2,3dihydroxy-3-methylbutyl)isoflavone (5, 3 mg). Fractions 14 and 15 were combined and purified over Sephadex LH-20, eluting with 1:1 CH2Cl2/MeOH, and gave 5′-methoxy-3′-(3-methylbut-2-enyl)biochanin A (piscerythrinetin (8), 10 mg). Fractions 24 and 25 were combined and purified over silica gel, eluting with 20% EtOAc in isohexane, yielding 65 subfractions. Repeated column chromatographic separation of the combined subfractions 35−65 over silica gel, eluting with 30% EtOAc in isohexane, followed by purification on Sephadex LH-20, eluting with MeOH, afforded burttinone (9, 5 mg). Fraction 26 was purified over Sephadex LH-20, yielding panchovillin (10, 95 mg). Combined fractions 29−33 were subjected to column chromatography over silica gel, eluting with 40% EtOAc in isohexane, to yield 40 subfractions. Combined subfractions 21−35 were purified over Sephadex LH-20, eluting with 1:1 CH2Cl2/MeOH, and afforded schliebenin A (1, 2.6 mg). Column chromatographic purification of the combined subfractions 36−40 over Sephadex LH-20, eluting with MeOH, gave schliebenone C (4, 1.8 mg).

Column chromatographic separation of the CH2Cl2 extract of the roots of E. schliebenii over silica gel, eluting with petroleum ether and EtOAc with increasing polarity (20−100% EtOAc), yielded 51 fractions. The second fraction was subjected to gel filtration on Sephadex LH-20 (1:1 MeOH/CH2Cl2), giving a mixture of two compounds in equal ratio (36 mg), which was further purified by HPLC (40−100% MeOH in H2O), yielding orientanol B (11, 8 mg). Fraction 4 was subjected to silica gel column chromatography, using 20−75% EtOAc in petroleum ether, affording 42 subfractions, of which 2−5 gave 5′-(3-methylbut-2enyl)pratensein (14, 5 mg) upon isolation on Sephadex LH-20 with MeOH/CH2Cl2 (1:1). Abyssinone-V (13, 10 mg) was obtained from the combined subfractions 9−15 upon silica gel column chromatography with 30−50% EtOAc in petroleum ether. Repeated chromatographic purification over Sephadex LH-20 with a MeOH/CH2Cl2 (1:1) eluent of the combined subfractions 23−42 yielded neurautenol (12, 16 mg). The air-dried and pulverized root bark of E. schliebenii was soaked twice in MeOH at room temperature for 48 h, yielding 38 g of crude extract, following evaporation, of which 18 g was adsorbed on silica gel and loaded on a column of silica gel. It was eluted with increasing amounts of EtOAc in isohexane, giving 22 fractions, of which the similar fractions were combined based on TLC analysis and subjected to further purification. Fraction 12 contained some solids that were washed with 50% EtOAc/isohexane, yielding compound 10 (23 mg). Fractions 7 and 8 were combined and purified on Sephadex LH-20 using MeOH/ CH2Cl2 (1:1) and were further purified by repeated silica gel (30% EtOAc in isohexane) and Sephadex LH-20 (MeOH/CH2Cl2 (1:1)) chromatography, yielding schliebenone B (3, 12 mg), piscerythrinetin (8, 8 mg), and the isoflavone 5′-(3-methylbut-2-enyl)pratensein (14, 6 mg). Fractions 9−11 were combined and purified on silica gel using 40− 50% EtOAc in isohexane, to yield 18 subfractions. Subfractions 14 and 15 contained solids that were washed with 20% EtOAc in isohexane, redissolved in MeOH, and purified on Sephadex LH-20 with MeOH as eluent to afford 2,3-dihydro-7-de-O-methyltrobustigenin (17, 15 mg) and schliebenone B (3, 6 mg). From the Sephadex LH-20 purification of subfractions 14 and 15 also 3′-O-methylpratensein (15, 8 mg) was obtained. Subfractions 8 and 9 were combined and purified on silica gel using 40% EtOAc in isohexane to give subfractions. Subfractions 4 and 5 were further purified by HPLC using 40−100% MeOH in H2O to give parvisoflavone B (16, 10 mg). The HPLC fractions 4−6 were combined, based on TLC analysis, and purified over silica gel eluting with 20% EtOAc in isohexane, to yield 18 subfractions. Repeated column chromatographic separation of the combined subfractions 8−11 over Sephadex LH-20 with MeOH as eluent afforded (R)-saclenone (18, 22 mg) and 3′-(3-methylbut-2-enyl)biochanin A (8, 15 mg). 4-O′Methylabyssinone V 4′ (7, 24 mg) was obtained from purification of fraction 3 of the main column, with purification over silica gel using 20% EtOAc in isohexane, followed by repeated Sephadex LH-20 gel permeation using MeOH as eluent. This also afforded 3′-(3methylbut-2-enyl)biochanin A (9, 8 mg). The air-dried and pulverized leaves of E. schliebenii were soaked twice in MeOH at room temperature for 48 h, yielding 83 g of crude extract following evaporation of the solvent. The extract (43 g) was adsorbed on silica gel, loaded on a silica gel column, and eluted with 30−100% EtOAc in isohexane and subsequently with 10−20% MeOH in EtOAc, obtaining 21 fractions. Fraction 7, eluted with 50% EtOAc in isohexane, yielded red crystals, which were purified from chlorophyll using 20% EtOAc in isohexane to yield 6-hydroxy-2,3,4,4-tetramethoxychalcone (23, 109 mg). Fractions 9−11, obtained via elution with 75% EtOAc in isohexane, formed white crystals, which were separated from chlorophyll using 30% EtOAc in isohexane, to yield 6-methoxyhamiltone A (20, 500 mg). The residual filtrates were concentrated on a rotatory evaporator, precipitated with isohexane, and the precipitate was further purified on silica using 30% EtOAc in isohexane to give a white powder, which was recrystallized from 50% EtOAc in isohexane to afford 6-methoxyhamiltone A (20, 3 g). A similar procedure was adopted for fractions 14− 20, yielding tetra-O-methylisoscutellarein as white crystals (21, 4 g). Fractions 12 and 13 were combined and purified on silica gel using 50% EtOAc in isohexane to give 14 subfractions, of which subfractions 3−6 gave crystalline 6-methoxyhamiltone A (20, 30 mg). Subfractions 10− 381

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14 were combined and repeatedly purified on Sephadex LH-20 using MeOH as eluent, to give hamiltone A (19, 75 mg). Sinensetin (22, 65 mg) was obtained from fraction 21, by purification on silica gel using 75% EtOAc in isohexane, followed by purification on Sephadex LH-20 with MeOH eluent. 3-Hydroxy-10-(2,3-dihydroxy-3-methylbutyl)-9-methoxypterocarpan (schliebenin A, 1): pale yellow, amorphous powder; [α]D −34 (MeOH, c 0.002); HRESIMS [M + Na]+ m/z 395.1467 (calcd for C21H25O6Na, 395.1471); 1H and 13C NMR data, see Table 1. 3′-(2-Hydroxy-3-methylbuten-3-yl)biochanin A (schliebenone A, 2): pale yellow, amorphous powder; [α]D −3.2 (MeOH, c 0.002); HRESIMS [M + H]+ m/z 369.1355 (calcd for C21H21O6, 369.1338); 1H and 13C NMR data, see Table 2. 5,7-Dihydroxy-3-[3-hydroxy-4-methoxy-5{(E)-3-methylbuta-1,3dienyl}phenyl-1-benzopyra-4-one (schliebenone B, 3): yellow, amorphous solid; HRESIMS [M + H]+ m/z 369.1336 (calcd for C21H20O6,369.1346); 1H and 13C NMR data, see Table 2. 5,7,3′-Trihydroxy-4′-methoxy-5′-(3-hydroxy-3-methylbuten-1-yl)isoflavanone (schliebenone C, 4): pale yellow, amorphous powder; [α]D −10.8 (MeOH, c 0.001); HRESIMS [M + H]+ m/z 385.1291 (calcd for C21H21O7, 385.1287); 1H and 13C NMR data, see Table 3. Cytotoxicity Assays. The cytotoxic activity of the crude extracts and the isolated compounds was evaluated against the human breast cancer cell line MDA-MB-231, as described previously by Nyandoro et al.38 Antitubercular Assays. Antitubercular activity of the methanol extract and the isolated compounds was evaluated against Mycobacterium tuberculosis (H37Rv strain) following the protocol described by Nyandoro et al.38

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR, mass spectra, X-ray crystallography. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00839. Crystallographic data (CIF) (CIF) (CIF) (CIF) (CIF) (CIF) Additional figures and tables (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +255-754-206560. E-mail: [email protected] (S.S. Nyandoro). *Tel: +46-31-786-9033. E-mail: [email protected] (M. Erdelyi). ORCID

Kari Rissanen: 0000-0002-7282-8419 Máté Erdélyi: 0000-0003-0359-5970 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Swedish Research Council (Swedish Research Links, 2012-6074) and the Academy of Finland (KR, grant nos. 263256, 265328, and 292746) is gratefully acknowledged. We thank Mr. F. M. Mbago, the curator at the Herbarium of the Department of Botany, University of Dar es Salaam, for locating and identifying the investigated plant species.

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REFERENCES

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