2325473 file000003 39926135

Cytotoxic Naphthoquinone and Azaanthraquinone Derivatives from an

Endophytic Fusarium solani

Nargis Sultana Chowdhury,†‡ §

Md. Hossain Sohrab,†*

Md. Sohel Rana,§ Choudhury

Mahmood Hasan,⊥⊥⊥⊥ Shirin Jamshidi,

∆ Khondaker Miraz Rahman

∆ *

† Pharmaceutical Sciences Research Division (PSRD), BCSIR Laboratories, Dhaka,

Dr. Qudrat-I-Khuda Road, Dhanmondi, Dhaka, Bangladesh

‡Department of Pharmacy, Manarat International University, Dhaka, Bangladesh

§ Department of Pharmacy, Jahangirnagar University, Savar, Dhaka, Bangladesh

⊥⊥⊥⊥Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka, Bangladesh

∆ Institute of Pharmaceutical Sciences, King's College London, 7 Trinity Street, London SE1

1DB

Supporting Information

The Plant Aponogeton undulates Roxb

In this study we investigated the fungal endophyte Fusarium sp. isolated from roots of

Aponogeton undulates Roxb growing in the deep water of Bangladesh. Aquatic plants have

adapted to life in aquatic environments (saltwater or freshwater). In Bangladesh, about 130

angiospermic, 6 pteridophytic, 3 bryophytic and several hundred algae species have been

identified as aquatic plants. The aquatic environment prevailing in deeply flooded areas in

Bangladesh has great potential regarding the propagation of aquatic plants, most of which are

untouched for the investigation of their biological activity.1 Aponogeton is a genus of 45-50

species of flowering plants, the only genus of the family Aponogetonaceae. They are found in

tropical to warm, temperate regions of Asia, Africa and Australia.2

The rootstock of Aponogeton, locally known as ghechu, constitutes an item of food

for humans during times of stress and scarcity. Traditionally ghechu is used during gestation

to remove loin pain, in the treatment of epilepsy and soothing treatment for burns, as well as

diuretics. Aponogeton undulatus Roxb. is found in India, Sri Lanka, Myanmar, Bangladesh

and China.3 The rootstock of the plant is useful as a nutrient supplement in areas where

purchasing power is limited. The nutrient composition of the rootstock of Aponogeton

undulatus shows that it can provide an adequate supply of carbohydrates (42.8 g/100 g),

protein (8.3 g/100 g), fats (0.7 g/100 g), iron (18.2 mg/100 g), calcium (37.2 mg/100 g) and

some minerals.4 The literature review revealed that the leaf pastes are used to treat cuts and

wounds, and according to Ayurveda the plant is effective against coughs, tuberculosis, acne,

cancer, diarrhea, dysentery and jaundice.5 As reported previously, the primary constituents of

Aponogeton undulatus are tannins and alkaloids which possess thrombolytic activity along

with a broad-spectrum antibacterial and cytotoxic potentiality.6

Identification of the plant material

The plant Aponogeton undulatus Roxb was identified and authenticated by Dr. Sardar Nasir

Uddin, Senior Scientific Officer, Bangladesh National Herbarium (BNH). A voucher specimen of this

collection is maintained at BNH under the accession number DACB – 32072 (Figure S1).

Figure S1: Voucher specimen of the plant Aponogeton undulatus Roxb deposited at

Bangladesh National Herbarium

Morphological Identification of fungal cultures

For the identification of endophytic fungal isolates, slides prepared from cultures were

stained with lactophenol cotton blue reagent and examined with a bright-field and phase

contrast microscope. 25

Identification was based on morphological characteristics such as

growth pattern, hyphae, the color of the colony and medium, surface texture, margin

character, aerial mycelium, sporulation and production of acervuli, coloration of the medium,

and the size and coloration of the conidia using standard identification manuals. 26

The fungi

were identified using relevant keys and taxonomic notes from various standard manuals.27

Brine Shrimp Lethality Bioassay

All the endophytic fungi were taxonomically identified on the basis of macroscopic and

microscopic morphological characters as Trichoderma sp. (AULE-1), Fusarium sp. (AURE-

1), Mucor sp. (AURE-3) and Fusarium sp. (AURE-4). The four fungal strains were cultivated

on a small scale at 28°C for 28 days in potato dextrose agar (PDA) medium. The culture

media were then extracted three times with ethyl acetate to obtain the crude extracts. On the

other hand, the powdered plant part (whole plant) of Aponogeton undulatus (AU) was

extracted using a dichloromethane-methanol (1:1) solvent system. The crude extracts of

endophytic fungi, as well as the plant, were filtered and concentrated at a low temperature

and reduced pressure.

Extracts of the plant Aponogeton undulatus (AU) and its associated endophytic fungal strains

AULE-1, AURE-1, AURE-3 and AURE-4 were screened for probable cytotoxic activities

using a brine shrimp lethality bioassay, with vincristine sulphate as the positive control. The

result of the assay is shown in Figure S1. The ethyl acetate extract of Fusarium sp. (AURE-4)

was found to be most active with an LC50 value of 10.18 µg /mL, which was comparable to

that observed for the crude extract of the plant (AU). The positive control vincristine sulphate

showed an LC50 value of 0.25 µg/mL. The extracts of other fungal strains did not show

notable activity (LC50>85 µg/mL) in the assay, and it was decided not to pursue further with

these strains. The endophytic fungus Fusarium sp. was selected for further investigation,

based on the preliminary bioactivity data, and was cultured at a large scale to isolate bioactive

secondary metabolites.

Figure S1: Preliminary cytotoxicity screening of isolated endophytic fungal strains from

Aponogeton undulatus by Brine Shrimp Lethality Bioassay.

Procedure for the preparation of the slide

After four days of the incubation at 28º C on potato dextrose agar media, a small portion of

the colony was taken into lacto-phenol cotton blue solution (0.05 gram cotton blue in 100 ml

lacto-phenol). A drop of the sample was poured on a glass slide and spread with the help of a

sterilized needle – it was then covered with a cover slip.

It was then examined for a characteristic arrangement of spores under 10X, 40X and 100X

objective lenses of a compound microscope (Kruss Optronic, Germany).

Sequence Data (Genbank accession number KY511422)

For identification and differentiation, the Internal Transcript Spacer regions (ITS4 and ITS5)

and the intervening 5.8S rRNA region was amplified and sequenced using electrophoretic

sequencing on an ABI 3730 x l DNA analyzer (Applied Biosystems, USA) using Big Dye

Terminator v 3.1 cycle sequencing kit. The ITS regions of the fungus were amplified using

PCR (Hot Start Green Master Mix, Promega, USA) and the universal ITS primers, ITS4 (5′-

TCC GTA GGT GAA CCT GCG G-3′) and ITS5 (5′- GGA AGT AAA AGT CGT AAC

AAG G -3′). The PCR products were purified and desalted using the Hot Start Green Master

Mix (Cat: M7432, Promega, USA.) and sequenced on an ABI 3730 x l DNA analyzer

(Applied Biosystems, USA). The sequences were aligned and prepared with the software

Chromas (V 2.6.2) and matched against the nucleotide-nucleotide database (BLASTn) of the

U.S. National Center for Biotechnology Information (NCBI) for final identification of the

endophytic isolate.

>Seq1 [organism= Fusarium solani] 5.8s rRNA

CTGGGAATTGTTATACTGATTCGAGGTCACATTCAGAAGTTGGGTGTTTTACGGC

GTGGCCGCGCCGCTCTCCAGTTGCGAGGTGTTAGCTACTACGCAATGGAAGCTGC

GGCGGGACCGCCACTGTATTTGGGGGACGGCGTTGTGCCCGCAGGGGGCTTCCG

CCGATCCCCAACGCCAGGCCCGGGGGCCTGAGGGTTGTAATGACGCTCGAACAG

GCATGCCCGCCAGAATACTGGCGGGCGCAATGTGCGTTCAAAGATTCGATGATT

CACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATG

CCAGAGCCAAGAGATCCGTTGTTGAAAGTTTTAATTTATTTGCTTGTTTACTCAG

AAGAAACATTATAGAAACAGAGTTAGGGGGTCCTCTGGCGGGGGCGGCCCGTGT

TACGGGGCCGTCTGTTCCCGCCGAGGCAACGTTTTAGGTATGTTCACAGGGTTGA

TGAGTTGTATAACTCGGTAATGATCCCTCCGCTGGTTCACCAACGGAGACCTTGT

TACGACTTTTACTTCCAAATATT

HPLC method for purity determination

HPLC analyses were performed on a Waters Alliance 2695 system, eluting in gradient with according

to the condition reported herein:

1) 10 minutes method: flow 0.5 mL/min

Solvents: A) water + 0.1 % formic acid

B) acetonitrile + 0.1% formic acid

Time (min) 0 2 5 6 7.5 9 10

A (%) 95 95 50 50 5 95 95

B (%) 5 5 50 50 95 5 5

2) 5 minutes method: flow 1 mL/min

Solvents: A) water + 0.1 % formic acid

B) acetonitrile + 0.1% formic acid

Time (min) 0 3 3.5 4.5 5

A (%) 95 10 5 5 95

B (%) 5 90 95 95 5

The analyses were performed on a Monolithic C18 50 X 4.60 mm column by Phenomenex. UV

detection was performed on a Waters 2996 Photodiode Array Detector.

Figure S3: Figures showing electron density surfaces painted according to the value of the

electrostatic potential for compounds 2 and 3.

Figure S4: MTT dose-response curve of 1 - 3, and doxorubicin against MDA MB 231 cell

line.

Spectral Data for the Known Compounds (4-7)

Fusarubin (4)

Red solid; UV (MeOH): λmax (log ε) = 239 (2.39), 293 (2.37), 548 (2.10) nm; ESI-MS; [M +

Na]+ m/z 329.0631 (calculated for C15H14O7Na, 329.0631);

1H NMR (400 MHz, CDCl3): δ

12.93 (1H, s, 5-OH), 12.66 (1H, s, 10-OH), 6.17 (1H, s, H-8), 4.88 (2H, s, H-1), 3.93 (3H, s,

7-OCH3), 3.02 (1H, d, J = 17.9, Ha-4), 2.70 (1H, bd, J = 18.4, Hb-4), 2.25 (1H, bs, 3-OH),

1.64 (3H, s, 3-CH3) ; 13

C NMR (100 MHz, CDCl3): δ 184.9 (C, C-9), 178.3 (C, C-6), 160.6

(C, C-10), 160.4 (C, C-7), 156.8 (C, C-5), 137.2 (C, C-10a), 137.2 (C, C-4a), 109.6 (C, C-5a),

109.6 (C, C-8), 107.5 (C, C-9a), 93.8 (C, C-3), 32.5 (C, C-4), 58.3 (CH2, C-1), 56.7 (CH3, C-

7-OCH3), 22.6 (CH3, C-3-CH3); 1H NMR and

13C NMR data were consistent with reported

data.19

Anhydrofusarubin (5)

Violet solid; UV (MeOH): λmax (log ε) = 235 (2.88), 303 (2.68), 502 (2.54) nm; ESI-MS; [M +

H]+

m/z 289.0707 (calculated for C15H13O6, 289.0707); 1H NMR (400 MHz, CDCl3): δ 13.05

(1H, s, 5-OH), 12.66 (1H, s, 10-OH), 6.17 (1H, s, H-8), 5.99 (1H, s, H-4), 5.21 (2H, s, H-1), 3.91

(3H, s, 7-OCH3), 2.25 (1H, br.s, 3-OH), 1.64 (3H, s, 3-CH3); 13

C NMR (100 MHz, CDCl3): δ

182.9 (C, C-9), 177.9 (C, C-6), 161.6 (C, C-3), 160.0 (C, C-7), 157.9 (C, C-5), 133.1 (C, C-4a),

122.8 (C, C-10a), 111.0 (C, C-5a), 110.0 (C, C-8), 108.0 (C, C-9a), 94.9 (C, C-4), 63.0 (CH2, C-

1), 56.7 (CH3, C-7-OCH3), 20.1 (CH3, C-3-CH3); 1H NMR and

13C NMR data were consistent

with reported data.11,7

Javanicin (6)

Red solid; 1H NMR (400 MHz, CDCl3): δ 13.25 (1H, s, 5-OH), 12.85 (1H, s, 8-OH), 6.20 (1H, s,

H-6), 4.02 (3H, s, 6-OCH3), 3.92 (2H, s, 3-OH), 2.32 (3H, s, H-11), 2.28 (3H, s, H-10).

NMR data were consistent with reported data.19

Cerevesterol (7)

White needle like solid; 1H NMR (400 MHz, CDCl3): δ 5.34 (1H, br.s, H-7), 5.22 (1H, dd, J =

15.2, 7.4, H-23), 5.14 (1H, dd, J = 15.2, 7.4, H-22), 4.07 (1H, m, H-3), 3.61 (1H, br.s, H-6 1),

2.13 (1H, q, J = 12.2, Hax-4), 1.76 (1H, dd, J = 16.4, 3.6, Heq-4), 1.07 (3H, s, H-19), 1.01; (3H,

d, J = 6.4, H-21), 0.90 (3H, d, J = 6.8, H-28), 0.82 (3H, d, J = 6.4, H-26/27), 0.81 (3H, d, J = 6.4,

H-26/27), 0.58 (3H, s, H-18); 13

C NMR (100 MHz, CDCl3): δ 144.0 (C, C-8), 135.3 (C, C-22),

132.2 (C, C-23), 117.5 (C, C-7), 75.9 (C, C-6), 73.7 (C, C-5), 67.7 (C, C-3), ESI-MS: [M + Na]+

m/z 453.3349 (calculated for C28H46O3Na, 4 53.3349,); 1H NMR and

13C NMR data were

consistent with reported data.20

Figures S5 to S12: 1H NMR (400 MHz, CDCl3),

13C NMR (100 MHz, CDCl3), DEPT 90, DEPT

135 and NOESY spectra of 9-desmethylherbarine (1).

Figure S5

Figure S6

Figure S7

Figure S8

Figure S9

Figure S10

Figure S11

Figure S12

Figures S13 to S16: HRMS of 9-desmethylherbarine (1).

Figure S13

C:\EXACTIVE DATA\...\8490 02-Jul-15 3:50:30 PM AU-16

8490 #86-129 RT: 1.25-1.87 AV: 44 NL: 1.82E6T: FTMS + p ESI Full ms [100.00-2000.00]

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

273.0755

299.0888

362.2412

475.3251

149.0120 603.1470

909.1910665.1176994.1681 1207.2639 1901.53791622.14751453.0968 1781.7723

Figure S14



260 280 300 320 340 360 380 400 420 440 460 480

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

273.0755

299.0888

313.0680

362.2412

475.3251

413.2660259.0963340.2592329.0629 393.0759 453.3433

357.0280

277.1068

375.0385

291.0862

441.2973 493.3498458.9447

481.2922

Figure S15



550 600 650 700 750 800 850 900 950

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

603.1470

588.4093

619.1121909.1910

665.1176

575.1886

634.0766566.4274

701.4933 924.1554

673.1106

895.2151745.1781525.2880 803.5428 953.1543831.5733

Figure S16



560 570 580 590 600 610 620 630 640 650 660 670 680

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

603.1470

605.1232588.4093

619.1121

665.1176

575.1886

634.0766566.4274617.1628 651.1384

590.1712

625.1292 671.1346

608.0704

641.0941569.1021 601.1016 661.1058583.0909 679.1333


13C NMR (100 MHz, CDCl3),

13C NMR (176

MHz, CDCl3), DEPT 90 and DEPT 135 spectra of 7-desmethylscorpinone (2)

Figure S17

Figure S18

Figure S19

Figure S20

Figure S21

Figure S22

Figure S23

Figure S24

Figure S25

Figure S26

Figure S27

Figures S28 to S29: HRMS of 7-desmethylscorpinone (2)

Figure S28



100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

270.0758

181.0287149.0120125.0388

301.1409162.9913 362.2414139.0545 319.2243195.0176 211.0941 249.1574 384.1934335.2192

265.1048

274.9315

286.0709

Figure S29



340 360 380 400 420 440 460 480 500 520 540 560 580 600

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

362.2414

413.2661

475.3253

384.1934

353.2298

340.2594

588.4094441.2975

453.3435371.1012

393.2975566.4275430.3891407.3131 458.9449 493.3500 599.1161523.3243

481.3135418.8800

549.0066 573.3426


13C NMR (100 MHz, CDCl3), DEPT 135 and

DEPT 90 spectra of 7-desmethyl-6-methylbostrycoidin (3)

Figure S30

Figure S31

Figure S32

Figure S33

Figure S34

Figure S35

Figure S36

Figures S37 to S39: HRMS of 7-desmethyl-6-methylbostrycoidin (3)

Figure S37



200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

286.0707

181.0286

362.2412475.3251

588.4093 701.4932 859.5373 1243.9720 1343.96561123.5640 1543.9535 1923.89291797.9149

Figure S38



100 150 200 250 300 350 400 450

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

286.0707

181.0286149.0119

125.0388362.2412301.1407162.9913 475.3251413.2660249.1572195.0175 340.2592226.9514 384.1932 453.3433

274.9318

493.3499

Figure S39



440 460 480 500 520 540 560 580 600 620 640 660 680

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

475.3251

588.4093453.3433

599.1158441.2973

566.4274 615.4228

577.1338

631.1419458.9447 661.3301544.3216493.3499 683.3432522.3085

481.3134


13C NMR (100 MHz, CDCl3) and DEPT 135

spectra of fusarubin (4)

Figure S40

Figure S41

Figure S42

Figure S43

Figure S44

Figures S45 to S47: HRMS of fusarubin (4),

Figure S45



200 400 600 800 1000 1200 1400 1600 1800 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

335.2191

286.0708

235.0939

377.2296

181.0286481.2921

635.1369

689.4595 957.1756 1191.7325 1307.8153 1580.8362 1982.43651871.1319

Figure S46



100 150 200 250 300 350 400 450 500

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

335.2191

286.0708

235.0939

329.0631

377.2296

181.0286353.2296 481.2921295.2266149.0120 195.1015 277.2161 463.2817393.2245125.0388 162.9913 437.2507

249.1572

227.1277495.2716

Figure S47



500 550 600 650 700 750 800 850 900 950

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abundance

635.1369

547.3239 647.4490

588.4093

600.1475507.3290 563.2885

666.0662 689.4595525.1334 793.5223

705.4540

627.4228

957.1756863.2067755.3766 835.5335 907.1659

Figures S48 to S51: 1H NMR (400 MHz, CDCl3) and

13C NMR (100 MHz, CDCl3) spectra of

anhydrofusarubin (5)

Figure S48

Figure S49

Figure S50

Figure S51

Figures S52 to S53: 1H NMR (400 MHz, CDCl3) spectrum of javanicin (6)

Figure 52

Figure 53

Figure S54: 1H NMR (400 MHz, CDCl3) spectrum of cerevesterol (7).

Figure S54

Figure S55: HRMS of cerevesterol (7).

C:\Users\...\Rashedul Islam\101353 11/30/2015 3:49:06 PM AU-25


200 300 400 500 600 700 800 900 1000

m/z

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

453.3349

413.2673

377.3213 883.6812317.1732

179.0006 299.1626481.2938

551.3331 706.6337214.0064 803.5455 951.6680

855.7445

1049.6695

C:\Users\...\Rashedul Islam\101353 11/30/2015 3:49:06 PM AU-25

450 452 454 456 458 460

m/z

0

20

40

60

80

100

0

20

40

60

80

100

Relative Abundance

Observed Data

453.3349

454.3383

451.3196455.3407449.3042 452.3231 459.2523456.3421 460.2576458.2996

Theoretical Isotope Model: [M+Na]+

453.3339

454.3373

455.3404456.3434 458.3491 459.3519

NL:2.71E6

101353#338-403 RT: 1.20-1.42 AV: 66 T: FTMS + p ESI Full ms [150.00-2000.00]

NL:1.71E4

C 28 H46 O3 Na: C 28 H46 O3 Na 1p (gss, s /p:40) Chrg 1R: 30000 Res .Pwr . @FWHM

Figure S56: Mode of interaction of 1, 2 and 3 after SMINA molecular docking with mixed AT/GC

DNA sequence 5’-TAGCTAGCTAGCTAGCG-3’

References

(1) Chong, K. Y.; Tan, H. T.; Corlett, W.; Richard, T. Raffles Museum of Biodiversity Research,

National University of Singapore 2009, 273.

(2) Islam, Q. Hydrobiologia 1996, 340, 317-321.

(3) Watve, A. The IUCN red list of threatened species 2011, e.T177309A7410517

(4) Islam, M. R.; Alam, M. B.; Tamima, U.; Jenny, S. I. Asian. Pac. J. Trop. Dis. 2015, 8, 431-7.

(5) Biswas, S. K. Indian J. Med. Sci. 1977, 31, 68-71.

(6) Chowdhury, N. S.; Alam, M. B.; Haque, A. S. M. T.; Zahan, R.; Mazumder, M. E. H. Global

J. Pharmacol. 2011, 5, 27-32.

(7) Robert, A. B.; James, H. T.; Stanley, N. J. Physiol. Biochem. 1981, 71, 951-954.

2325473 file000003 39926135

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