Cytotoxic activity of phenolic constituents from Echinochloa crus-galli against four human cancer cell lines Academic research paper on "Chemical sciences"

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Original Article

1 Cytotoxic activity of phenolic constituents from Echinochloa crus-galli

2 against four human cancer cell lines

3 qi Sayed Gad El Molla, Amira Abdel Motaal *, Hala El Hefnawy, Ahlam El Fishawy

4 Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-El-Ainy St., Cairo 11562, Egypt

8 Article history: Echinochloa crus-galli (L.) P. Beauv., Poaceae, grains are used as a feed for birds and millet for humans.

9 Received 29 May 2015 The sulforhodamine B assay was used to assess its cytotoxicity against four human cancer cell lines. The

10 Accepted 19 July 2015 ethanolic extract (70%) proved to be most active against HCT-116 and HELA cell lines (IC50 = 11.2 ±0.11

11 Available onhne xxx and 12.0 ± 0.11 ^g/ml, respectively). On the other hand, the chloroform and ethyl acetate fractions

12 --exhibited their highest activities against HCT-116 cell lines. The chloroform and ethyl acetate frac-

13 Keywords. tions were subjected to several chromatographic separations to render pure phenolic compounds

14 HCT n"! grass C-8). Compounds 1-8 were identified as: 5,7-dihydroxy-3',4',5'-trimethoxy flavone, 5,7,4'-trihydroxy-

15 HELA 3',5'-dimethoxy flavone (tricin), quercetin, flavone, apigenin-8-C-sophoroside, 2-methoxy-4-hydroxy 17 HEPG-2 cinnamic acid, p-coumaric acid and quercetin-3-O-glucoside. All the isolated phenolic compounds exhib-

methoxylated flavonoids may be considered as lead compounds for the treatment of cancer, which supports previous claims of E. crus-galli traditional use. This is the first report of the occurrence of these phenolic compounds in E. crus-galli. © 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

20 Introduction Echinochloa crus-galli (L.) P. Beauv., Poaceae, is a problematic 38

summer weed found in rice fields and known as Barnyard grass. 39

21 Cancer is a leading cause of death in economically developed The grains are known as fodder for livestock and as millet for 40

22 countries and the second leading cause of death in developed people in many Asian countries (Boulos and El Hadidi, 1984). It 41

23 countries. Lung, stomach, liver, colon and breast cancer cause the is known traditionally to reduce body weight, blood sugar, treat 42

24 most cancer deaths each year. Breast cancer in females, and lung hypertension and help to detoxify liver and kidney. It is also used 43

25 and prostate cancer in males are the most frequently diagnosed for carbuncles, hemorrhage, sores, spleen trouble, wounds and can- 44

26 cancers and the leading causes of cancer death for each sex. About cer ('t Mannetje and Jones, 1992). It was previously reported that 45

27 30% of cancer deaths are due to the five leading behavioral and the 70% hydroalcoholic extract of the grains of E. crus-galli showed 46

28 dietary risks; high body mass index, low fruit and vegetable intake, significant antidiabetic activity in normal and alloxan induced dia- 47

29 lack of physical activity, tobacco use, and alcohol use (Jemal et al., betic rats (Devi et al., 2012), and that the methanol and aqueous 48

30 2011). extracts exhibited significant antioxidant activities (Ho etal., 2012; 49

31 Plant derived compounds have played a major role in the Mehta and Vadia, 2014). Several phenolic compounds; flavones, 50

32 development of several useful cytotoxic agents viz. vinblastine, vin- flavone glycosides, caffeoyl quinic acid derivatives were isolated 51

33 cristine, and paclitaxel (Taxol®). Other promising new agents are from other Echinochloa species such as E. utilis, E. frumentacea and 52

34 in clinical development stage, including flavopiridol and combre- E. colona (Watanabe, 1999; Kim et al., 2008; Lazaro, 2009; Gomaa 53

35 tastatin, which clarifies the urge for screening native flora in search and Abd Elgawad, 2012; Hegab et al., 2013). 54

36 for new bioactive phytochemical compounds (Reddy et al., 2003; This study was carried out in order to prove the ethnophar- 55

37 Cragg and Newman, 2005). macological use of E. crus-galli grains as a remedy for cancer 56

('t Mannetje and Jones, 1992), and to specify the compounds 57

responsible for its cytotoxic activity against four human cancer cell 58

* Corres ondin author lines; MCF-7 (breast carcinoma), HCT-116 (colon carcinoma), HELA 59

C°imlai^pao,miS1ti,mrotaai@hu.edu.eg (A.A. Motaal). (cervical carcinoma) and HEPG-2 (liver carcinoma).The use °f such 60

http://dx.doi.org/10.1016Zj.bjp.2015.07.026

0102-695X/© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

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2 S.G. El Molla etal./ Revista Brasileira de Farmacognosia xxx (2015) xxx-xxx

relatively cheap millets in the Egyptian's daily diet could contribute to the prevention of cancer in the overgrowing number of cancer patients.

Materials and methods

Plant material

Grains of E. crus-galli (L.) P. Beauv., Poaceae, were collected between July and September, 2010 from plants grown in the Food Technology Research Institute, Faculty of Agriculture, Cairo University, Giza. They were identified by Prof. Dr. Osama El Kopacy, Professor of Botany, Faculty of Agriculture, Cairo University. A voucher specimen number 9010 was placed at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

General

Silica gel H (Merck, Darmstadt, Germany) was used for vacuum liquid chromatography (VLC), silica gel 60 (70-230 mesh ASTM; Fluka, Steinheim, Germany), sephadex LH-20 (Pharmacia, Stockholm, Sweden), polyamide and cellulose (Merck, Darmstadt, Germany) were used for column chromatogra-phy (CC). Thin-layer chromatography (TLC) was performed on silica gel GF254 pre-coated plates (Fluka) using the following solvent systems: S1, chloroform:methanol (9:1, v/v); S2, petroleum ether:ethyl acetate:formic acid (45:16:3.6, v/v/v); S3, chloroform:acetone:formic acid (65:15:1.5, v/v/v); S4, chloroform:acetone:formic acid (75:16.5:8.5 v/v/v); S5, ethyl acetate:methanol:water (100:16.5:13.5, v/v/v); S6, chloro-form:methanol (8:2, v/v). The chromatograms were visualized under UV light (at 254 and 366 nm) before and after exposure to ammonia vapor and spraying with AlCl3, as well as after spraying with p-anisaldehyde/sulphuric acid spray reagent. Melting points (uncorrected) were determined on a D. Electrothermal 9100 instrument (Labe-quip, Markham, Ontario, Canada). UV spectra were recorded using a Shimadzu UV 240 (P/N 204-58000) spectrophotometer (Kyoto,Japan). A Varian Mercury-VX-300 NMR instrument (Palo Alto, CA, USA) was used for 1H NMR (300 MHz) and 13C NMR (75 or 125 MHz). The NMR spectra were recorded in DMSO-d6 and chemical shifts were given in 1 (ppm) relative to tetramethylsilane as an internal standard.

Extraction, fractionation and isolation

The air-dried powdered grains of E. crus-galli (1 kg) were percolated with ethanol 70% till exhaustion to yield 115 g of the alcoholic extract (AlEx). The residue was suspended in distilled water and partitioned successively using n-hexane, chloroform, ethyl acetate, and n-butanol saturated with water giving the fractions HxFr, ClFr, EtFr, and BuFr, respectively. The fractions were separately concentrated under reduced pressure to yield 58 g, 7.6 g, 5.8 g and 4.3 g, respectively. The ClFr (5 g) was chromatographed over a VLC column (5 cm x 20 cm, silica gel H, 250 g). Gradient elution was carried out using n-hexane/methylene chloride mixtures, methylene chloride/ethyl acetate mixtures, and ethyl acetate/methanol mixtures. Fractions of 200 ml each were collected and monitored by TLC to yield seventeen fractions (Ac - Qc). Fraction Gc (60% methylene chloride in ethyl acetate) was re-chromatographed over silica gel 60 column using methylene chloride in ethyl acetate (9:1, v/v) as an eluent to give compound 1 (22 mg, yellow powder, Rf 0.72 in S2, m.p. 251-253 °C) and compound 2 (17 mg, yellow powder, Rf 0.70 in S2, m.p. 249-251 °C). Fraction lc (90% ethyl acetate in methanol) was purified using several sephadex LH-20 column to yield compound 3 (23 mg, yellow powder, Rf 0.60

in S2, m.p. 310-312°C). Fraction Ec (20% methylene chloride in ethyl acetate) was re-chromatographed on a silica gel 60 column, using n-hexane/ethyl acetate (8:2 v/v) as an eluent to give compound 4 (15 mg, white powder, Rf 0.91 in S2, m.p. 349-351 °C).The EtFr (5 g) was chromatographed over polyamide column (250 g, 5 cm x 120 cm). Gradient elution was carried out with water, followed by increasing amount of methanol starting with 5% up to 90% methanol. Fractions of 250 ml each were collected and monitored by TLC to yield five main fractions (AEt -EEt). Fraction BEt (40% methanol in water) was re-chromatographed over cellulose column (50 g, 3.5 cm x 120 cm) using 10% methanol in water as an eluent to give compound 5(15 mg, yellowish brown powder, Rf 0.15 in S4, m.p. 216-218 °C). Fraction CEt (60% methanol in water) was purified several times over cellulose columns (50 g, 3.5 x 10 cm) using 15% methanol in water to yield compound 8 (12 mg, yellow powder, Rf 0.42 in S4, m.p. 239-240°C). FractionDEt (70% methanol in water) was purified using several sephadex LH-20 columns to yield compound 6 (11 mg, yellowish white powder, Rf 0.71in S4, m.p. 168-170 °C) and compound 7(15 mg, yellowish white powder, Rf 0.73in S4, m.p. 169-171 °C).

Assessment of cytotoxic activity

The cytotoxicities of the AlEx, the four fractions (HxFr, ClFr, EtFr and BuFr) and the eight isolated compounds from ClFr, and EtFr were assessed using the sulforhodamine B assay (Skehan et al., 1990) against the four human cancer cell lines; colon (HCT-116), cervical (HELA), liver (HEPG-2) and breast (MCF-7) adenocarci-noma using Doxorubicin® as a reference standard. Active fractions and compounds were assessed against normal human fibroblast cell lines (HFB4). Dose-dependent activities were studied for all samples using concentrations from 5 to 50 |ig/ml, and the 1C50 values (concentration which reduced survival to 50%) were calculated. Three separate experiments, each with three replicates, were performed for each sample.

Results and discussion

The cytotoxic effects of the extracts of E. crus-galli grains at concentrations up to 50 |ig/ml and 48 h of exposure showed that the AlEx exhibited 1C50 values of 12.0 ±0.11, 11.2 ±0.11, 18.9 ±0.12, and 14.2 ±0.11 |g/ml against HELA, HCT-116, MCF-7, and HEPG-2 cells, respectively (Table 1). According to the US NC1 plant screening program a crude extract is considered to possess an in vitro cytotoxic activity if its 1C50 value is less than 20 |ig/ml, following an incubation period of 48 and 72 h (Boik,

Table 1

Cytotoxic activities of alcoholic extract, fractions and the isolated compounds of Echinochloa crus-galli (1C50 values are given in |g/ml).

Tested sample IC50 ±SD(^g/ml)

HELA HCT- 116 MCF-7 HEPG-2

AlEx 12.0 ± 0.11 11.2 ± 0.11 18.9 ± 0.12 14.2 ± 0.11

HxFr 29.5 ± 0.01 20.5 ± 0.03 17.1 ± 0.01 15.5 ± 0.04

ClFr 27.3 ± 0.21 17.1 ± 0.01 21.7 ± 0.01 23.9 ± 0.02

EtFr 21.3 ± 0.01 3.8 ± 0.01 16.8 ± 0.01 22.4 ± 0.04

BuFr 18.5 ± 0.01 4.2 ± 0.03 13.5 ± 0.01 14.3 ± 0.03

1 4.5 ± 0.03 18.5 ± 0.04 11.5 ± 0.01 4.5 ± 0.03

2 2.8 ± 0.01 3.6 ± 0.03 6.6 ± 0.03 2.4 ± 0.02

3 13.8 ± 0.01 20.4 ± 0.02 12.7 ± 0.02 11.3 ± 0.02

4 11.0 ± 0.03 27.0 ± 0.01 14.0 ± 0.02 9.3 ± 0.03

5 11.8 ± 0.03 14.5 ± 0.04 6.3 ± 0.01 7.4 ± 0.03

6 5.1 ± 0.01 2.7 ± 0.03 12.4 ± 0.03 17.3 ± 0.02

7 6.5 ± 0.01 4.5 ± 0.02 15.6 ± 0.02 13.5 ± 0.02

8 11.5 ± 0.03 13.5 ± 0.01 5.3 ± 0.02 11.3 ± 0.03

Doxorubicin 4.5 ± 0.03 4.5 ± 0.53 4.3 ± 0.03 4.2 ± 0.03

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Table 2

Cytotoxic activities of the isolated compounds of Echinochloa crus-galli (IC50 values are given in |xM).

Tested

IC50 iSD(^M)

compound HELA HCT-116 MCF-7 HEPG-2

1 3.0 i 0.04 53.7 i 0.03 33.3 i 0.02 3.0 i 0.01

г S.6 i 0.02 10.S i 0.02 19.9 i 0.04 7.2 i 0.01

З 45.6 i 0.03 67.4 i 0.01 42.0 i 0.02 37.3 i 0.01

4 49.4 i 0.02 121.4 i 0.01 62.9 i 0.02 41.S i 0.05

5 19.3 i 0.03 23.7 i 0.04 10.3 i 0.01 12.1 i 0.03

б 26.5 i 0.03 13.9 i 0.03 63.S i i 0.03 S9.0 i 0.01

l 39.5 i 0.03 27.5 i 0.02 95.0 i 0.02 S2.2 i 0.02

s 24.7 i 0.02 29.0 i 0.03 11.4 i 0.01 24.2 i 0.03

Doxorubicin S.2 i 0.03 S.2 i 0.05 9.3 i 0.03 7.7 i 0.03

2001). The ClFr exhibited its highest activity against the HCT-116 cell lines (17.1 ±0.01 |g/ml), while the HxFr against HEPG-2 (15.5 ± 0.04 |ig/ml). The EtFr and BuFr showed significantly higher cytotoxic activities against HCT-116 cell line with 1C50 values of 3.8 ±0.01 and 4.2 ±0.03 |g/ml, respectively, comparable to the standard Doxorubicin (1C50 4.5 ±0.53 |g/ml) (Table 1). The two fractions, ClFr and EtFr, were subjected to further purification with

the aim of identifying the corresponding cytotoxic compounds. Further studies are still running on the BuFr. The ClFr yielded four flavonoids (1-4) and the EtFr yielded four phenolic compounds (5-8).

The UV spectral data of compounds 1, 2, 4 and 5 indicated a flavone, while that of compounds 3 and 8 indicated the presence of a 3-substituted flavonol nucleus (Tables 3 and 4). The

NMR (Tables 5 and 7) and 13C NMR (Tables 6 and 8) spectral data of compounds 1-5 and 8 were in accordance with those reported for 5,7-dihydroxy-3',4',5'-trimethoxy flavone, 5,7,4'-trihydroxy-3',5'-dimethoxy flavone (tricin), quercetin, flavone, apigenin-8-C-sophoroside and quercetin-3-O-glucoside, respectively (Mabry et al., 1970; Bhattacharyya et al., 1978; Agrawal, 1989; Nickavar et al., 2003; Moon et al., 2005; Gangwar and Saxena, 2010; Khanam et al., 2011; El-Sayed et al., 2013). While data of compounds 6 and 7 (Tables 4, 7 and 8) were similar to those reported for 2-methoxy-4-hydroxy cinnamic acid and p-coumaric acid, respectively (Kort et al., 1996; Sajjadi et al., 2012). All the compounds were isolated for the first time from genus Echinochloa, except for tricin (2) and p-coumaric acid (7) which were isolated for the first time from E. crus-galli. Tricin has been previously isolated from three other species of Echinochloa namely Echinochloa

180 181 182

Table З

UV spectral data (Xmax in nm) for compounds 1-4.

Shift reagent 1 г З 4

MeOH 270,309sh,332 244,269,309sh,350 256,269sh,301sh,370 250,294,307sh,370

NaOMe 287,300sh,364 263,275sh,330,416 249sh,321,394 250,295,309sh

AlCl3 252sh,278,300,347,381sh 258sh,277,303,366sh,393 272,304sh,331,455 251,292,306sh

AlCl3 /HCl 280,295sh,340,282sh 259sh,277,302,360,386 265,301sh,359,428 250,293,309sh

NaOAC 273,299sh,359 264,276sh,321,414 257sh,274,329,390 24S,292,307

NaOAC/H3 BO3 272,313sh,330 270,304sh,350,422sh 261,303sh,385 255sh,294,307sh

Table 4

UV spectral data (Xmax in nm) for compounds 5-S.

Shift reagent 5 б l 8

MeOH 270,304sh,336 332,277sh 327,275sh 257,26Ssh,291sh,361

NaOMe 279,327,394sh - - 272,328,411

AlCl3 276,305,350,3S4 - - 276,305sh,332sh,43S

AlCl3 /HCl 27S,303,343,3S0 - - 26S,295sh,357sh,405

NaOAC 2S0,300,379 - - 274,326,3S1

NaOAC/H3 BO3 271,329sh,344 - - 261,296sh,376

Table 5

1H NMR chemical shifts (1 in ppm) for compounds 1-4 (DMSO, 300 MHz, J in Hz).

H 1 г З 4

3 6.53 6.56 - 6.89

- 1H,S 1H,S - 1H,S

5 - - - 7.43-7.53 4H, m

6 6.19 6.21 6.18 7.43-7.53

- 1H, d, J =1.8 1H, d, J =1.8 1H, d, J =1.8 4H, m

7 - - - 7.43-7.53 4H, m

S 6.90 6.93 6.41 7.43-7.53

- 1H, d, J =1.8 1H, d, J =1.8 1H, d, J =1.8 4H, m

2' 7.2S 7.30 7.52 8.06

- 2H, d, J = 2.1 2H, d, J =2.1 1H, d,J =9.3 2H, m

3' - - - 7.81 2H, m

4' - - - -

5' - - 6.89 7.81

- - - 1H, d,J =9.3 2H, m

6' 7.28 7.30 7.66 8.06

- 2H, d, J = 2.1 2H, d, J =2.1 1H, dd, J =1.8 and 9.3 2H, m

OCH3-3', 5' 3.9

- 3.7 6H, s

OCH3-3', 4', 5' 9H, m

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Table б

13C NMR chemical shifts (1 in ppm) for compounds 1-4 (DMSO, 75 MHz, J in Hz).

C 1 2 З 4

2 164.19 14e.72 1e2.47

3 103.70 103.50 135.e2 10e.87

4 181.77 181.e7 175.72 m.99

5 1e1.41 1e1.29 1e0.59 125.39

e 98.88 98.81 98.15 124.70

7 1e3.e4 1e3.53 1e3.89 134.15

8 94.21 94.12 93.30 118.40

9 157.35 157.23 15e.0e 155.59

10 103.70 103.50 102.90 123.2e

1' 120 120.34 121.89 131.e8

2' 104.39 104.33 114.99 m.24

3' 148.21 148.11 144.89 129.01

4' 139.89 13e.79 147.e2 131.e8

5' 148.21 148.11 115.55 129.01

e' 104.39 104.33 119.92 120.59

OCH3-3' 5e.38 5e.29 - -

OCH3-4' 48.e4 - - -

OCH3-5' 5e.e4 5e.29 - -

191 utilis, Echinochloa frumentacea and Echinochloa colona and proved

192 to possess strong antioxidant and phytotoxic activities (Watanabe,

193 1999; Kim et al., 2008; Gomaa and AbdElgawad, 2012; Hegabetal., 2013).

10.8 ± 0.02 and 19.9 ± 0.04 |M) against the four cancer cell lines; 201

HEPG-2, HELA, HCT-116 and MCF-7, respectively, compared to 202

7.7 ± 0.03,8.2 ± 0.03,8.2 ± 0.05 and 9.3 ± 0.03 |M for Doxorubicin® 203

(Tables 1 and 2). While compound 1 showed significantly high 204

activities against HELA and HEPG-2 cell lines (1C50 = 4.5 ± 0.03 and 205

4.5 ±0.03 |g/ml corresponding to 3.0 ±0.04 and 3.0 ±0.04 |M, 206

respectively). It was previously reported that tricin isolated from 207

the leaves of Sasa senanensis showed high cytotoxic activity against 208

oral squamous carcinoma cell lines, whereas luteolin glycosides 209

showed no cytotoxicity up to 0.8mg/ml (Matsuta et al., 2011). 210

Methoxylated flavones (8-hydroxy-7,3',4',5'-tetramethoxyflavone 211

and 8,4'-dihydroxy-7,3',5'-trimethoxyflavone) formerly isolated 212

from the stem bark of Muntingia calabura exhibited effective cyto- 213

toxicities (ED50 values 3.56 and 3.71 | g/ml, respectively) against 214

the P-388 cell line in vitro (Chen et al., 2004). Also methoxylated 215

flavonols isolated from Cleome droserifolia herb showed highly sig- 216

nificant cytotoxic activities against HCT-116 and MCF-7 cell lines 217

(Ezzat and Abdel Motaal, 2012). 218

Compounds 6 and 7 exhibited highest cytotoxic activi- 219

ties against HCT-116 (1C50 2.70 ±0.034 and 4.53 ±0.021 |g/ml; 220

13.9±0.03 and 27.5 ±0.02 |M), and HELA (1C50 5.15 ±0.013 221

and 6.50 ±0.013 |g/ml; 26.5 ±0.03 and 39.5 ±0.03 |M) cell 222

lines, respectively (Tables 1 and 2). 1n a previous report, 223

1 R1—R2—H; R3—R4—R5—OCH3

2 R1—R2—H; R3—R5—OCH3; R4—OH

3 R-i—OH; R2—H; R3—R4—OH; R5—H

5 Rt—H; R2—C-sophoroside; R3—R5—H; R4—OH В Rt—O-ß-D-glucopyranoside; R2—H; R3—R4—OH; R5—H

6 R—OCH3

7 R—H

According to the US NCI plant screening program, pure compounds are considered to possess an in vitro cytotoxic activity if their IC50 values in cancer cells are less than 4 ^g/ml (Boik, 2001). The methoxylated flavone tricin (2) was the most active as it possessed the lowest 1C50 values (7.2 ±0.01, 8.6 ±0.02,

4-hydroxy-3-methoxycinnamic acid inhibited proliferation and 224

induced apoptosis in human breast cancer cells (Hamdan et al., 225

2013). 226

Thus the activity of the crude ethanolic extract of E. crus- 227

galli could be directly correlated to its phenolic content, and 228

Table У

1H NMR chemical shifts (1 in ppm) for compounds 5-8 (DMSO, 300 MHz, J in Hz).

H Б S H б У

3 e.74 - 2 - 7.5e

- 1H,S - - - 2H, d, J =9.3 Hz

e e.22 e.20 3 7.27 e.82

- 1H,S 1H, d, J = 1.8 Hz - 1H,S 2H, d, J =9.3 Hz

8 - e.40 5 e.81 e.82

- - 1H, d, J = 1.8 Hz - 1H, d, J = 8.3 Hz 2H, d, J =9.3 Hz

2 8.02 7.e0 e 7.09 7.5e

-' 2H, d, J = 8.4 Hz 1H, d, J = 9.3 Hz - 1H, d, J = 8.3 Hz 2H, d, J =9.3 Hz

3 e.91 - 7 7.53 7.51

-' 2H, d, J = 8.4 Hz - - 1H, d, J= 15.8 Hz 1H, d, J= 15.8 Hz

5 e.91 e.90 8 e.34 e.33

-' 2H, d, J = 8.4 Hz 1H, d, J = 9.3 Hz - 1H, d, J= 15.8 Hz 1H, d, J= 15.8 Hz

e 8.02 7.70

- 2H, d, J = 8.4 Hz 1H, dd, J =1.8 and 9.3 Hz

- 5.45

1 '' 5.1 1H, d, J =7.5 Hz

- 1H, d, J = 20.9 Hz -

1 ''' 5.3

1H, d, J=3 Hz

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Table 8

13 C NMR chemical shifts (1 in ppm) for compounds 5-8 (DMSO, 75 MHz, J in Hz).

C 5 8 C 6 7

2 163.60 156.28 1 144.59 144.17

3 103.70 133.31 2 147.99 125.25

4 181.90 177.40 3 115.73 129.99

5 160.40 161.20 4 149.15 159.54

6 98.10 98.60 5 122.86 129.99

7 162.40 164.04 6 125.89 125.25

8 103.80 93.44 7 115.73 115.78

9 156.10 156.15 8 111.26 115.29

10 103.70 103.96 9 168.9 167.97

1' 121.60 121.55 OCH3 55.80 -

2' 128.80 115.16

3' 115.80 144.75

4' 161.2 148.40

5' 115.80 116.17

6' 128.80 121.14

1 '' 71.40 100.87

2'' 81.80 74.06

3'' 78.65 77.50

4'' 59.91 69.91

5'' 81.72 76.48

6'' 58.15 60.95

1 ''' 106.03

2''' 71.78

3''' 78.10

4''' 66.65

5''' 74.02

6''' 60.02

229 methoxylation is the key for enhancing the cytotoxic activity

230 when considering structure-activity relationship of the isolated

231 compounds. In general methoxylated compounds (1, 2 and 6)

232 exhibited significantly higher cytotoxic activities compared to the

233 other phenolic compounds and the anticancer drug Doxorubicin®

234 (Tables 1 and 2). All the tested active fractions and compounds

235 showed no cytotoxicity against the normal melanocytes HFB4.

236 Conclusion

237 Here we reported the cytotoxic activity of the ethanolic extract

238 of the edible grains of E. crus-galli against four human cancer cell

239 lines, which supports previous claims of E. crus-galli traditional use.

240 Eight phenolic constituents were isolated for the first time from E.

241 crus-galli, and six of them were reported for the first time in the

242 genus. Two methoxylated flavones and one methoxylated cinnamic

243 acid exhibited highly significant cytotoxic activities compared to

244 Doxorubicin®.

245 Author contributions

246 AA, HE and AE participated in study concept and design, acqui-

247 sition of data, analysis and interpretation of data, and critical

248 revision of the manuscript for important intellectual content. AA

249 drafted the manuscript. SG carried out the extraction, fractionation

250 and isolation of pure compounds, and participated in drafting the

251 manuscript.

252 Conflicts of interest

253 The authors declare no conflicts of interest.

254 Acknowledgments

255 The authors are thankful to the researchers at the National Can-

256 cer Institute, Cairo, Egypt (NCI) for carrying out the cytotoxicity assays.

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