Skip to main content
PMC home page
African Journal of Traditional, Complementary, and Alternative Medicines logoLink to African Journal of Traditional, Complementary, and Alternative Medicines
. 2016 Sep 29;13(6):135–143. doi: 10.21010/ajtcam.v13i6.19

PHENOLIC PROFILE OF Centaurea aegyptiaca L. GROWING IN EGYPT AND ITS CYTOTOXIC AND ANTIVIRAL ACTIVITIES

Riham Omar Bakr 1,*, Shaza Abd El Halim Mohamed 2, Nahla Ayoub 3
PMCID: PMC5412184  PMID: 28480370

Abstract

Background:

Centaurea aegyptiaca L (Asteraceae), is one of the most attractive plants growing wildly in Sinai, and is not well investigated for its phytochemical constituents. This study represents the first in-depth characterization of the phenolic profile of the aerial parts of C. aegyptiaca methanolic extract utilizing liquid chromatography (LC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS).

Material and Methods:

Phenolic profile was researched utilizing LC-HRESI-MS-MS. Assessment of cytotoxic activity against four human cancer cell lines (Hep-G2; hepatocellular carcinoma cells, MCF-7; breast adenocarcinoma cells, and HCT-116; colon carcinoma and HELA; cervical carcinoma cells) was performed using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Antiviral activity was surveyed utilizing cytopathic effect inhibition assay.

Results:

A total of sixty-one compounds were tentatively distinguished (twenty-one phenolic acids and their derivatives, thirty-one flavonols and nine flavones) in the negative and positive modes. Centaurea aegyptiaca demonstrated outstanding results against Hep-G2, MCF-7, HCT-116 and HELA cell lines with IC50 of 12.1, 30.9, 11.7 and 19.5 μg/mL respectively compared and doxorubicin as a reference drug. Weak antiviral activity was seen against hepatitis A virus (HAV) and no impact against herpes simplex virus type 1 (HSV 1).

Conclusion:

This study provides a better understanding of the chemistry of C. aegyptiaca that announces itself as a promising cytotoxic agent.

Keywords: Centaurea aegyptiaca, Cytotoxicity, MTT assay, flavonoid

Introduction

The Asteraceae (Compositae) is the biggest family of flowering plants with more than 24000 - 30000 species and 1600 -1700 genera, distributed worldwide aside from Antarctica with an observable financial and medicinal contribution (Funk et. al, 2005).

Centaurea, the fourth biggest genus in the family Asteraceae, consists of 600 species around the world, especially in Mediterranean regions and in Western Asia, (Garcia-Jacas et al, 2000). Around 13 species of Centaurea are growing in Egypt. Centaurea species, have been the object of various phytochemical studies, with a diversity of bioactive phytochemicals and prevalence of flavonoids (Flamini et al., 2001; Shoeb et al., 2007; Seghiri et al, 2009) and sesquiterpene lactones (Koukoulitsa et al., 2002; 2005). Centaurea species are claimed for their uses in gastrointestinal and inflammatory disorders, hypotensive and cytotoxic activities, in addition to the anti-bacterial impacts alone or when mixed with different plants (Farrag et al., 1993; Kargıoglu et al, 2010; Köse et al., 2007). Centaurea aegyptiaca (Centaurea cancellata Sieber ex Spreng., Calcitrapa aegyptiaca (L.) Sweet, C. iberica) is growing wild in Sinai (St. Katherine), Egyptian eastern desert, the Red Sea coastal strip, and Gebel Elba. Investigations of C. aegyptiaca revealed the presence of guaianolide (Orabi et al, 2013; Sarg et al, 1987) which showed potential cytotoxic activities against liver and larynx carcinoma cell lines. The studies on this species deal basically with sesquiterpene lactones, while flavonoids are not well investigated.

Due to scarcity of information regarding the chemical composition of C. aegyptiaca, it is valuable to carry out an in-depth phytochemical investigation on this plant to characterize its main active constituents utilizing HPLC-MS-MS analysis and Xcalibur software with the screening of its cytotoxic and antiviral activities.

Plant Material

Aerial parts of Centaurea aegyptiaca L. were gathered during the flowering stage in February 2015 from km 67, Cairo-Suez road, Egypt. The plant was identified by Prof. Dr. Abdel-haleem Abdel-mogaly, Department of Plant Taxonomy, Herbarium of Horticultural Research Institute, Agricultural Research Center, Dokki, Cairo, Egypt. A voucher specimen (RS013) was deposited in Herbarium of Faculty of Pharmacy, October University of Modern Sciences and Arts.

Material and Methods

Preparation of the plant extract

Three hundred grams of plant material were exhaustively extracted with methanol. The combined methanolic extracts (CME) were filtered, concentrated under vacuum at 50°C, dried and left for LC-HRESI-MS-MS analysis, and to evaluate the cytotoxic and antiviral activities.

Cytotoxic activity

The viability of control and treated cells were explored at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University. The CME was tested against the following human tumor cell lines: hepatocellular carcinoma (HepG-2), breast adenocarcinoma (MCF-7), colorectal carcinoma (HCT-116), cervix adenocarcinoma (HELA) utilizing MTT (3-(4, 5-dimethylthiazolyl-2)- 2, 5-diphenyltetrazolium bromide) test in triplicate (Fotakis and Timbrell, 2006). Untreated cells represented the negative control while doxorubicin was the positive control. The absorbance was measured at 570 nm using a microplate ELISA reader (Sun Rise TECAN, Inc, USA). The absorbance of untreated cells was considered as 100%. The results were determined by three independent experiments (Wilson, 2000).

The percentage cell viability was calculated with the Microsoft Excel®. Percentage cell viability was calculated as follows: % Cell viability = (Mean Abs control – Mean Abs test metabolite X 100)/ Mean Abs control Where: Abs: absorbance at 570 nm

The graphic plots were used for estimation of the 50% inhibitory concentration (IC50). STATA statistical analysis package was used for the dose response curve drawing in order to figure IC50.

Antiviral activity

Cytopathic effect inhibition assay was performed following Hu and Hsiung, (1989). The cultures were treated with two fold serial dilutions of C. aegyptiaca extract. Six wells were utilized for every concentration of C. aegyptiaca extract. Untreated vero cells in absence of CME represented the control. Inverted microscope was used for watching the virus in the control wells every 24hr until complete viral-induced cytopathic effects (CPE). Antiviral activity was evaluated by the inhibition of cytopathic effect in comparison with control where scoring of the protection level offered by Centaurea extract was estimated (Vijayan et al., 2004). The experiment was performed thrice. Acyclovir, the treatment clinically used for herpetic viral disease, was utilized as a positive control (Dargan, 1998).

LC-HRESI-MS-MS apparatus

The study was performed on a Bruker micro-TOF-Q Daltonics (API) Time-of-Flight mass spectrometer (Bremen, Germany), coupled to 1200 series HPLC system (Agilent Technologies, Waldbronn, Germany), equipped with a high-performance auto-sampler, binary pump, and PDA detector G 1314 C (SL). Chromatographic separation was performed on a Superspher 100 RP-18 (75 × 4mm i.d.; 4 μm) column (Merck, Darmstadt, Germany).

Identification of Phenolic Compounds

Centaurea aegyptiaca CME was investigated following Hassaan et al, (2014). The mobile phase consists of two solvents, (A) 2% acetic acid (pH 2.6) and (B) 80% methanol. Gradient elution was used at a flow rate of 100 ^L/min, from 5% to 50% B at 30°C. Ion spray (pneumatically assisted electrospray) ionization system was utilized. Spectra were recorded in positive and negative ion mode between m/z 120 and 1,500 with capillary voltage, 4000V and heated dry nitrogen gas temperature, 200°C and flow rate 10 L/min. The gas flow to the nebulizer was set at pressure 1.6 bar. For collision-induced dissociation (CID) MS-MS estimations, the voltage over the collision cell ranged between 20 to 70 eV. Argon was utilized as collision gas. Sodium formate was utilized for calibration toward the end of LC-MS run. Interpretation for ESI-MS was performed by Xcalibur 2.1 software from Thermo Scientific (Berlin, Germany).

Results

Phenolics identified

The phytochemical fingerprint of C. aegyptiaca was determined using an LC-HRESI-MS-MS. Analyses were carried out by using a full scan and MS2 data-dependent operative mode.

The combination of ESI positive and negative modes permitted the tentative identification of a total of sixty-one compounds by the interpretation of their fragmentation patterns combined with the available literature information. Twenty-one phenolic acid and their derivatives have been identified in the negative mode only (Table 1), while thirty-one flavonol and nine flavones were tentatively identified based on their MS/MS fragmentation in the negative and positive modes and listed in table 2.

Table 1.

Phenolic acids and their derivatives tentatively identified in Centaurea aegyptiaca

Rt (min.) [M-H]m/z Fragment ions Tentatively identified compound Ref.
1 1.68 341.11 179.08 (-162) 161.17 143.09 119.09 113.04 Caffeoyl hexoside Dartora et al, 2011
2 3.26 191.06 111.01 173.16 127.10 85.05 Quinic acid Dartora et al, 2011
3 3.59 493.16 191.21 330.43 402.4 473.24 Quinic acid derv
4 8.54 315.07 153.07 (-162) 165.14 135.19 109.09 225.2 (-90) 195.05 (-120) Protocatechuic acid hexoside Chen et al, 2012
Vallverdu-Queralt et al., 2015
5 9.27 515.14 179.06 353.23 341.17 191.08 Dicaffeoyl quinic acid Chen et al, 2012
6 12.45 353.09 191.11 179.13 135.15 Chlorogenic acid
Dicaffeoyl quinic acid		 Dartora et al, 2011
7 14.90 285.06 153.10 109.18 Protocatechiuc acid pentoside
8 14.91 447.12 315.21 411.22 271.25 152.18 163.10 179.15 Protocatechiuc acid pentoside hexoside
9 23.52 499.15 191.15 337.26 3 caffeoyl 5 coumaroyl quinic acid Jaiswal et al, 2010
10 15.31 337.09 163.14 3-p-Coumaroyl quinic acid Jaiswal et al, 2010
11 24.30 337.09 173.08 4p-Coumaroyl quinic acid Jaiswal et al, 2010
12 19.64 529.16 173.13 191.11 367.21 355.21 4-Feruloyl 5caffeoylquinic acid isomer Jaiswal et al 2010
13 21.54
21.69		 447.11 152.12 401.19 285.26 241.24 163.16 177.2
401.26 285.18 152.15 163.16		 Protocatechuic acid hexoside pentoside
14 21.96 193.05 134.11 149.07 Ferulic acid Vallverdu-Queralt et al., 2015
15 22.25 417.10 241.18 152.07 285.22 163.15 179.15 Dihydroxybenzoic acid-O-dipentoside Beelders et al, 2014
16 23.62 529.16 173.13 193.08 4-feruloyl-5-Caffeoyl quinic acid Dartora et al, 2011; Jaiswal et al, 2010
17 35.14 367.10 173.16 191.14 4-Feruloyl quinic acid Jaiswal et al, 2010
18 42.74 367.10 191.12 173.13 193.14 5-Feruloyl quinic acid Jaiswal et al, 2010
19 32.15 325.09 163.05 119.09 101.08 113.18 Coumaroyl hexoside Vallverdu-Queralt et al., 2015
20 59.68 543.21 349.21 367.20 193.22 173.13 Diferuloylquinic acid isomer
21 60.69 473.11 311.27 341.20 Dicaffeoyltartaric acids (chicoric acids)
Open in a new tab

Table 2.

Flavonoids tentatively identified in Centaurea aegyptiaca

No RT [M-H]-m/z Fragment ions [M-H]+ m/z Fragment ions Tentatively identified compound Ref.
1 38.23 563.14 473.26 [M-H-90] 443.31 [M-H-120] 353.22 [M-H-120-90] 383.25 ------ Apigenin 8C-hexoside 6C-pentoside Ibrahim et al.,2015
2 38.83 785.21 315.19 [isorh, M-H-2hexose-rh), 623.26 [M-H-162] 477.23 (M-H-162-146] 300.16 787.24 625.14 [M+H-162] 479.17 [-162-146] 317.13 Isorhamnetin dihexoside rhamnoside
3 40.46 479.08 317.15 [M-H-162] 357.15 481.10 319.08 [M+H-162] Quercetagenin-O-hexoside Pajero et al.,2004
4 40.57 317.16 271.15 287.13 166.03 139.14 299.18 243.14 227.17 179.06 319.12 273.09 301.03 245.07 181.02 153.08 136.37 Quercetagenin
5 41.36 625.14 317.16 [M-H-308] 461.25 341.18 627.16 481.18 [M+H-146] 319.14 [M+H-146-162] 463.23 [M+H+162] Quercetagenin-rhamnoside-hexoside (rutinoside)
6 42.22 641.27 317.15[M-H-2hexose] 243.22 225.30 479.35 [M-H-162] 447.23 ------ Quercetagenin dihexoside
7 44.10 639.16 477.19 [M-H-162] 315.18 [M-H-162-162] 447.22 300.26 271.21 641.17 479.17 [M+H-162] 317.16 [M+H-162-162] Isorhamnetin -O-dihexoside Lin and Harnly, 2010
8 44.36 623.13 285.15 [M-H-162-176] 447.22 [M-H-176] 337.23 357.13 257.18 243.26 625.14 287.12 449.17 [M+H-176] Kaempferol hexoside glucuronide
9 44.55 463.09 301.14 465.10 303.14 Quercetin-O-hexoside Vallverdu-Queralt et al., 2015.
10 45.46 301.18 283.13 271.16 255.17 227.10 179.07 8165.10 151.18 303.12 285.14 273.13 179.14 169.06 137.02 Quercetin Vallverdu-Queralt et al.,2015.
11 48.01 269.14 251.16 209.23 225.26 165.27 189.26 271.21 242.13 253.15 153.10 225.20 211.17 rt48.70 Apigenin
12 48.31 431.10 311.22 [M-H-120] 341.25 [M-H-90] 433.11 415.16 367.16 337.14 313.17 Apigenin C-glucoside (Isovitexin) Irahim et al.,2015.
13 49.05 639.16 331.20 [M-H-162-146] 316.20 - Patuletin hexoside rhamnoside Parejo et al.,2004
14 49.08 493.10 331.16 [M-H-162] Patuletin hexoside Parejo et al.,2004
15 49.10 331.05 331.08 316.17 317.18 [M-H-14] 333.06 318.09 301.12 155.05 169.10 Patuletin Parejo et al.,2004
16 49.37 623.15 315.14 300.22 271.18 625.18 479.17 [M+H-146] 317.07 [M+H-146-162] 463.20 [M+H-162] Isorhamnetin 3-O-hexoside 7-O-rhamnoside Stintzing et al., 2004 Ibrahim et al., 2015
17 49.51 609.18 301.18 [M-H-308] 269.14 199.22 611.19 449.10 [M+H-162] 271.11 303.13 [M+H-146] 465.10 [quercetin+162] 431.15 413.26 Quercetin hexoside rhamnoside
18 49.73 285.19 267.11 253.27 239.12 185.13 137.06 287.13 RT59 269.22 259.14 241.14 213.12 164.89 153.02 Kaempferol Schmeda-Hirschmann Et al., 2015
19 50.52 315.05 300.20 317.07 302.11 Isorhamnetin Pajero et al., 2004
20 53.79 477.10 315.18 [M-H-162] 301.19 357.19 [MH-120] 415.35 479.12 317.13 [M+H-162] 419.94 360.94 Isorhamnetin 3-O-hexoside Pajero et al.,2004.
21 50.54 593.15 285.15 [M-H-146-162] 429.28 531.28 269.17 595.17 287.13 [M+H-162-146] 449.11 [M+H-146] 213.27 Kaempferol O-hexosiderhamnoside Ibrahim et al.,2015.
22 53.13 477.07 301.13 [M-H-176] 415.29 Quercetin glucuronide
23 54.10 431.10 269.18 [M-H-162] 311.16 [M-H-120] 433.11 271.10 [M+H-162] 415.21 Apigenin hexoside
24 54.5 461.11 299.19 [M-H-162] 339.17 --- Hispidulin hexoside Akkal et al.,2003
25 55.91 593.28 269.29 [M-H-162-162] 431.36 [M-H-162] 417.36 285.24 245.27 225.20 595.30 577.22 [M+H-18] 415.16 [M+H-180] 253.21 235.20 325.14 271.27 Apigenin -O- dihexoside
26 56.13 521.09 317.16 [M-H-204] 399.29 429.21 523.11 319.09 [M+H-204] 503.25 455.14 Quercetagenin acetyl hexoside Parejo et al.,2004
27 56.95 505.10 301.16 [M-H-204] 343.21 [M-H-162] 461.30 [M-H-44] 179.09 151.13 507.11 303.12 [M+H-204] 385.22 447.23 [M+H-60] Quercetin acetyl hexoside Oszmiański et al., 2015
28 57.03 491.20 476.28 [M-H-15] 315.23 [M-H-176] 329.22 [M-H-162] 431.32 [M-H-60] 175.16 161.12 Isorhamnetin glucuronide
29 58.59 607.26 269.27 [M-H-176-162] 431.37 [M-H-176] 383.21 353.16 609.28 253.08 235.14 429.03 [M+H-180] 431.28 Apigenin glucuronide hexoside
30 58.70 461.07 285.18 219.23 175.11 339.24 461.09 287.13 [M+H-176] 445.24 [M+H-18] 175.07 Kaempferol gucuronide Schmeda-Hirschmann Et al., 2015
31 59.03 625.12 463.19 [M-H-162] 315.15 301.19 [MH-162-162] Quercetin 3, 7 di-O-hexoside Ibrahim et al.,2015.
32 61.34 447.09 327.14 [M-H-120] 357.06 [M-H-90] 387.31 [M-H-60] 285.29 429.28 241.25 175.16 449.11 287.13 431.19 383.12 329.17 276.17 246.19 Luteolin C-hexoside Brito et al.,2014
33 61.87 491.21 285.16 473.36 431.41 [M-H-60] 315.24 [M-H-176] 329.33 [M-H-162] 175.13 179.18 167.19 493.23 33.13 475.29 [M+H-15] 269 155.05 Kaempferol methyl ether glucuronic acid Schmeda-Hirschmann Et al., 2015
34 62.41 535.11 331.19 [M-H-204] 373.09 [M-H-162] 471.31 [M-H-62] 537.23 478.98 333.18 [M+H-204] 419.88 375.31 [M+H-162] Patuletin acetyl hexoside
35 63.18 519.11 315.23 [M-H-204] 357.36 [M-H-162] 521.13 317.12 [M+H-204] 501.27 359.34 [M+H-162] Isorhamnetin acetyl hexoside
36 63.76 639.17 519.27 [M-H-120] 549.24 [M-H-90] 607.34 [M-H-32] 463.22 [M-H-176] 301.21 [M-H-176-162] 219.17 --- Quercetin C-hexoside glucuronide
37 64.24 521.33 359.45 [M-H-162] 503.39 [M-H-18] 489.26 [M-H-32] 475.22 341.44 315.21 523.35 343.13 [M+H-180] 361.13 [M+H-162] 505.21 [M+H-18] 318.14 325.13 307.23 Centaurein (jaceidin hexoside) Flamini et al.,2001
38 68.03 299.06 284.06 [M-H15-] 301.07 286.09 [M+H129.12 147.11 [15- Hispidulin Flamini et al.,2001
39 69.35 507.35 345.41 [M-H-162] 331.10 299.35 161.19 417.41 509.13 347.17 [M+H-162] 303.12 449.17 [M+H-60] Axillarin hexoside Parejo et al.,2004
40 74.61 609.41 315.27 [M-H-162-132] 293.34 246.23 225.09 447.39 [M-H-162] -- Isorhamnetin hexoside pentoside
Open in a new tab

Cytotoxic activity

Centaurea aegyptiaca CME was investigated against four human cell carcinoma (HepG-2, MCF-7, HCT-116, and HELA) using MTT assay. The higher potency was observed with HCT-116 followed by HepG-2 then HELA cells (Table 3) with IC50 less than 20 μg/mL compared with doxorubicin as a standard cytotoxic agent with a range of 0.46-0.23 μg/mL.

Table 3.

Cytotoxic activity of Centaurea aegyptiaca extract

Cell line HepG-2 MCF-7 HCT-116 HELA
CME Dox CME Dox CME Dox CME Dox
IC50 (μg/mL) 12.1 ± 3.89 0.467 ± 0.2 30.9 ± 2.62 0.426 ± 0.35 11.7 ± 1.44 0.23 ± 0.17 19.5± 2.73 0.456± 0.35
Open in a new tab

(CME) and Doxorubicin standard (Dox) against hepatocellular carcinoma cells (Hep-G2), Human breast adenocarcinoma cells (MCF-7), colon carcinoma cells (HCT-116) and cervical cell carcinoma (HELA)

Antiviral activity

Centaurea aegyptiaca CME exhibited weak antiviral activity against hepatitis A virus (HAV), and no effect was observed with herpes simplex virus type 1(HSV 1).

Discussion

Liquid chromatography/tandem mass spectrometry (LC/MS/MS), represents the most popular technique used to unravel the complexity of phenolic compounds. Those non-volatile, polar and thermally labile compounds are found as complex mixtures in plants [Parejo et al., 2004]. Centaurea aegyptiaca has not been studied for its phenolic content before.

Phenolic acids and their derivatives

Chlorogenic acids (CGAs) are a group of esters formed between a quinic acid and certain trans-cinnamic acids, most commonly caffeic, p-coumaric, and ferulic acid (Clifford et al, 2006).

The neutral loss of 162, 132, 146, 176 mass units revealed the presence of compounds with a hexose (galactose or glucose), pentose (arabinose or xylose), deoxyhexose (rhamnose) and glucuronic acid unit, respectively (Cuyckens and Claeys 2004).

Compound 1 was identified as caffeoyl hexoside with base peak 179, while compound 2 was tentatively identified as quinic acid (MS2 at m/z 111, 173). Many derivatives were assigned as dicaffeoyl quinic acid (compound 5; MS2 at m/z 191, 353), chlorogenic acid (compound 6; MS2 at m/z 191, 179), caffeoyl coumaroyl quinic acid (compound 9; MS2 173, 191), 4 and 5 p-coumaroyl quinic acid (compounds 10 and 11; 163, 191 respectively) in addition to several isomers of Feruloyl caffeoyl quinic acid with various fragmentation pattern (compound 12 and 16). Phenolic acid glycosides, such as protocatechuic acid hexoside (compound 4), protocatechuic acid pentoside (compound 7) and protocatechuic acid pentoside hexoside (compound 8), giving the corresponding fragments for the loss of a hexose [M-H-162], a pentose [M-H-132] and the consecutive loss of both. In addition, coumaric acid hexosides (compound 19) was identified based on the fragments at m/z 163 (loss of a hexose moiety) and the typical fragment of coumaric acid at m/z 119 (Fischer et al., 2011). Compound 15 was assigned as dihydroxybenzoic pattern previously reported with the same fragmentation pattern and attempted to be protocatechuic acid dipentoside. Many isomers of feruloyl quinic acid have been identified with different fragmentation pattern (Compound 17 and 18; MS2 173 and 191 respectively) (Table 1).

Flavonoid glycosides

Based on previous information concerning Centaurea genus, methoxylated derivatives of flavone and flavonol glycosides were expected. Through this study, C. aegyptiaca was claimed as a rich source of flavonoids. In the MS identification of C-glycosides, the key fragmentations utilized were [M-H-60]-, [M-H-90]- and [M-H-120]-.

Apigenin and hispidulin that were previously identified in Centaurea furfuracea (Akkal et al., 2003) represented the principle identified flavones. Compounds 1, 11, 12, 23, 25 and 29 were tentatively identified as apigenin derivatives with [A-H]- at m/z 269.14 and characteristic peaks at m/z 225 and 209. Compounds 12 and 23 were tentatively identified as Apigenin 8C-glycoside and 6C-glycoside, while compounds 24 and 37 were identified as hispidulin hexoside, and hispidulin, respectively.

Methoxylated flavonol represented the majority of identified flavonols including isorhamnetin and patuletin derivatives in addition to jaceidin, axillarin. Compound 2, with a [M-H]- at m/z 785 and MS2 ions at m/z 315 (M-324, - dihexose moiety, -rhamnose), 623 [M-H-162]- and 477 [M-H-162-146]- was tentatively identified as Isorhamnetin dihexoside rhamnoside. Quercetagenin (compound 4), with its derivatives quercetagenin O-hexoside, rutinoside, and dihexoside with loss of 162, 308 and 324 daltons respectively were also tentatively identified (Compounds 3, 5 and 6), in addition to the acetylated derivative (compound 26), with [M-H] at m/z 521 and MS2 at 317 and the characteristic loss of 204 denoting acetylated hexoside and tentatively identified as quercetagenin acetyl hexoside. Kaempferol, (compound 18), was also identified with its characteristic peak in the positive mode at m/z 213. Compound 8 was tentatively identified as kaempferol hexoside glucuronide with the characteristic loss of 176 in the positive and negative mode. Kaempferol hexoside rhamnoside was also identified with [M+H] at m/z 595 and characteristic fragments at m/z 287, 449 [M+H-146] and 213 (compound 21). Quercetin (Compound 10), was observed with six derivatives tentatively identified as quercetin-O-hexoside (compound 9), quercetin rutinoside (compound 17) with the characteristic loss of 308 daltons, quercetin glucuronide (compound 22), quercetin dihexoside (compound 31), quercetin hexoside glucuronide (compound 36) and the acetylated derivative of quercetin hexoside (compound 27) with MS2 at m/z 301 [M-H-204], 343 [M-H-162] and 461 [M-H-44]. Patuletin (compound 15) and its derivatives, patuletin hexoside (compound 14) and hexoside rhamnoside (compound 13) have been also identified with [A-H] 331. Acetylated hexoside derivatives (compound 34), has been also identified with [M-H] at m/z 535 and base peak 331 [M-H-204] and characteristic fragment at m/z 373 [M-H-162]. Centaurein (compound 37), was tentatively identified with [M-H] at m/z 521 and characteristic fragments at 359 [M-H-162] indicating its aglycone jaceidin. Patuletin and Centaurein were previously identified in Centaurea species (Akkal et al., 2003; Flamini et al., 2001).

Acetylated derivatives have been reported before as, quercetin 3-0-[(2,3,4-triacetyl-a-rhamnopyranosyl)1“’→6”] 3-acetyl-β-galactopyranoside and quercetin 3-0-[(2,3,4-triacetyl-a-rhamnopyranosyl)1“’→6”]-4-acetyl-β-galactopyranoside from Gentiana spicata, a descendant from Centaurea family (Handoussa et al., 2009).

Cytotoxic activity

When the IC50 value in carcinoma cells, after incubation between 48 and 72 hrs, is under 20 μg/mL, the cytotoxic activity was commonly considered (Boik, 2001). C. aegyptiaca CME demonstrated significant activity against colorectal and hepatocellular carcinoma. This promising cytotoxic action was related to the increased number of OH groups and presence of 3’- OMe as reported by Jeong et al. (2007). Cytotoxic activity was previously reported in various Centaurea species because of phenolic, flavonoid and sesquiterpene contents (Erol-Dayi et al., 2011, Medjroubi et al., 2005, Shoeb et al., 2007, Saroglou et al., 2005 and Ahmed et al, 2014).

Conclusion

Centaurea aegyptiaca is a rich source of phenolic and flavonoid glycosides contributing to a powerful cytotoxic activity that may represent a good candidate for further studies and reflects its traditional uses.

Footnotes

Conflict of interest The authors announce that there are no conflicts of interest.

References

  • 1.Ahmed S.A, Kamel E.M. Cytotoxic activities of flavonoids from Centaurea scoparia Scientific World Journal 2014, Article ID 274207. 2014:7. doi: 10.1155/2014/274207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Akkal S, Benayache F, Medjroubi K, Tillequin F, Seguin E. Flavonoids from Centaurea furfuracea (Asteraceae) Biochem. Syst. Ecol. 2003;31:641–643. [Google Scholar]
  • 3.Beelders T, de Beer D, Stander M.A, Joubert E. Comprehensive Phenolic Profiling of Cyclopia genistoides (L.) Vent. by LC-DAD-MS and -MS/MS Reveals Novel Xanthone and Benzophenone Constituents. Molecules. 2014;19(8):11760–11790. doi: 10.3390/molecules190811760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Boik J. Natural Compounds in Cancer Therapy. Minnesota, USA: Oregon Medical Press; 2001. [Google Scholar]
  • 5.Brito A, Ramirez J.E, Areche C, Sepúlveda B, Simirgiotis M.J. HPLC-UV-MS Profiles of Phenolic Compounds and Antioxidant Activity of Fruits from Three Citrus Species Consumed in Northern Chile. Molecules. 2014;19:17400–17421. doi: 10.3390/molecules191117400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chen H.J, Inbaraj B.S, Chen B.H. Determination of Phenolic Acids and Flavonoids in Taraxacum formosanum Kitam by Liquid Chromatography-Tandem Mass Spectrometry Coupled with a Post-Column Derivatization Technique. Int. J. Mol. Sci. 2012;13:260–285. doi: 10.3390/ijms13010260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Clifford M.N, Zheng W, Kuhnert N. Profiling the chlorogenic acids of aster by HPLC-MSn. Phytochem. Anal. 2006;17(6):384–393. doi: 10.1002/pca.935. [DOI] [PubMed] [Google Scholar]
  • 8.Cuyckens F, Claeys M. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 2004;39:1–15. doi: 10.1002/jms.585. [DOI] [PubMed] [Google Scholar]
  • 9.Dargan D.J. Investigation of the anti-HSV activity of candidate antiviral agents. In: Brown SM, MacLean AR, editors. Methods in Molecular Medicine, Vol. 10: Herpes Simplex Virus Protocols. Totowa NJ: Humana Press Inc; 1998. pp. 387–405. [DOI] [PubMed] [Google Scholar]
  • 10.Dartora N, de Souza L.M, Santana-Filho A.P, Iacomini M, Valduga A.T, Gorin P.A.J, Sassaki G.L. UPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilex paraguariensis with different growth conditions, treatments and ageing. Food Chem. 2011;129(4):1453–1461. [Google Scholar]
  • 11.Erol-Dayi Ö, Pekmez M, Bona M, Aras-Perk A, Arda N. Total Phenolic contents, Antioxidant Activities and cytotoxicity of three Centaurea Species: calcitrapa subsp. calcitrapa, C. ptosimopappa and C. spicata. Free Radicals Antioxid. 2011;1(2):31–36. [Google Scholar]
  • 12.Farrag N.M, Abd El Aziz E.M, El-Domiaty M.M, El Shafea A.M. Phytochemical investigation of Centaurea araneosa growing in Egypt. Zagazig J. Pharm. Sci. 1993;2(1):29–45. [Google Scholar]
  • 13.Flamini G, Antognol E, Morelli I. Two flavonoids and other compounds from the aerial parts of Centaurea bracteata from Italy. Phytochemistry. 2001;57:559–564. doi: 10.1016/s0031-9422(01)00066-8. [DOI] [PubMed] [Google Scholar]
  • 14.Fotakis G, Timbrell J.A. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett. 2006;160(2):171–177. doi: 10.1016/j.toxlet.2005.07.001. [DOI] [PubMed] [Google Scholar]
  • 15.Funk V.A, Bayer R.J, Keeley S, Chan R, Watson L, Gemeinholzer B, Schilling E, Panero J.L, Baldwin B.G, Garcia- Jacas N, Susanna A, Jansen R.K. Everywhere but Antarctica: using a super tree to understand the diversity and distribution of the Compositae. Biol. Skr. 2005;55:343–374. [Google Scholar]
  • 16.Garcia-Jacas A, Susanna V, Mozaffarian R. Ilarslan, The natural delimitation of Centaurea (Asteraceae: Cardueae): ITS sequence analysis of the Centaurea jacea group. Plant Syst. Evol. 2000;223:185–199. [Google Scholar]
  • 17.Handoussa H, Osmanova N, Ayoub N, Mahran L. Spicatic acid: A 4-carboxygentisic acid from Gentiana spicata extract with potential hepatoprotective activity. Drug Discov Ther. 2009;3(6):278–286. [PubMed] [Google Scholar]
  • 18.Hassaan Y, Handoussa H, El-Khatib A.H, Linscheid M.W, El Sayed N, Ayoub N. Evaluation of Plant Phenolic Metabolites as a Source of Alzheimer’s Drug Leads. BioMed Research International 2014, Article ID 843263. 2014:10. doi: 10.1155/2014/843263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Hu J.M, Hsiung G.D. Evaluation of new antiviral agents I In vitro prospectives. Antiviral Res. 1989;11:217–232. doi: 10.1016/0166-3542(89)90032-6. [DOI] [PubMed] [Google Scholar]
  • 20.Ibrahim R.M, El-Halawany A.M, Saleh D.O, El Naggar E, El-Shabrawya A.O, El-Hawarya S.S. HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities. Rev. Bras. Farmacogn. 2015;25(2):134–141. [Google Scholar]
  • 21.Jaiswal R, Sovdat T, Vivan F, Kuhnert N. Profiling and characterization by LC-MSn of the chlorogenic acids and hydroxycinnamoyl shikimate esters in mate (Ilex paraguariensis) J. Agric. Food Chem. 2010;58:5471–5484. doi: 10.1021/jf904537z. [DOI] [PubMed] [Google Scholar]
  • 22.Jeong J.M, Kang S.K, Lee I.H, Lee J.Y, Jung H. Antioxidant and Chemosensitizing Effects of Flavonoids with Hydroxy and/or Methoxy Groups and Structure-Activity Relationship. J. Pharm. Pharmaceut. Sci. 2007;10(4):537–546. doi: 10.18433/j3kw2z. 2007. [DOI] [PubMed] [Google Scholar]
  • 23.Koukoulitsa E, Skaltsa H, Karioti A, Demetzos C, Dimas K. Bioactive sesquiterpene lactones from Centaurea species and their cytotoxic/cytostatic activity against human cell lines. Cin vitro Planta Med. 2002;68:649–652. doi: 10.1055/s-2002-32893. [DOI] [PubMed] [Google Scholar]
  • 24.Kargıoglu M, Cenkci S, Serteser A, Konuk M, Vural G. Traditional uses of wild plants in the middle Aegean region of Turkey. Human Ecol. 2010;38:429–450. [Google Scholar]
  • 25.Köse YB, Iscan G, Demirci B, Baser K.H.C, Celik S. Antimicrobial activity of the essential oil of Centaurea aladagensis. Fitoterapia. 2007;78:253–254. doi: 10.1016/j.fitote.2006.12.002. [DOI] [PubMed] [Google Scholar]
  • 26.Koukoulitsa C, Geromichalos G.D, Skaltsa H. VolSurf analysis of pharmacokinetic properties for several antifungal sesquiterpene lactones isolated from Greek Centaurea sp. J. Comput. Aided Mol. Des. 2005;19:617–623. doi: 10.1007/s10822-005-9018-y. [DOI] [PubMed] [Google Scholar]
  • 27.Lin L.Z, Harnly J.M. Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat) Food Chem. 2010;120:319–326. [Google Scholar]
  • 28.Medjroubi K, Benayache F, Bermejo J. Sesquiterpene lactones from Centaurea musimomum Antiplasmodial and cytotoxic activities. Fitoterapia. 2005;76(7-8):744–746. doi: 10.1016/j.fitote.2005.08.005. [DOI] [PubMed] [Google Scholar]
  • 29.Orabi K.Y, Sary H.G, Ayoub N.A, Singab A.N.B. Isolation of a Bioactive Guaianolide from Centaurea aegyptiaca Ethanol Extract. Planta Med. 2013;79:PN62. [Google Scholar]
  • 30.Oszmiański J, Wojdyło A, Nowicka P, Teleszko M, Cebulak T, Wolanin M. Determination of Phenolic Compounds and Antioxidant Activity in Leaves from Wild Rubus L. Species. Molecules. 2015;20:4951–4966. doi: 10.3390/molecules20034951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Parejo I, Jáuregui O, Viladomat F, Bastida J, Codina C. Characterization of acylated flavonoid-O-glycosides and methoxylated flavonoids from Tagetes maxima by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2004;18:2801–2810. doi: 10.1002/rcm.1697. [DOI] [PubMed] [Google Scholar]
  • 32.Sarg T.M, El-Domiaty M, El-Dahmy S. Further guaianolides from Centaurea aegyptiaca. Sci. Pharm. 1987;55:107–110. [Google Scholar]
  • 33.Saroglou V, Karioti A, Demetzos C, Dimas K, Skaltsa H. Sesquiterpene lactones from Centaurea spinosa and their antibacterial and cytotoxic activities. J. Nat. Prod. 2005;68:1404–1407. doi: 10.1021/np058042u. [DOI] [PubMed] [Google Scholar]
  • 34.Schmeda-Hirschmann G, Quispe C, González B. Phenolic Profiling of the South American “Baylahuen” Tea (Haplopappus spp, Asteraceae) by HPLC-DAD-ESI-MS. Molecules. 2015;20:913–928. doi: 10.3390/molecules20010913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Seghiri R, Boumaza O, Mekkiou R, Benayache S, Mosset P, Quintana J, Estévez F, Leo’n F, Bermejo J, Benayache F. A flavonoid with cytotoxic activity and other constituents from Centaurea Africana. Phytochemistry Letters. 2009;2:114–118. [Google Scholar]
  • 36.Shoeb M, Jaspars M, MacManus S.M, Celik S, Nahar L, Kong-Thoo-Lin P, Sarker S.D. Anti-colon cancer potential of phenolic compounds from the aerial parts of Centaurea gigantea (Asteraceae) J. Nat. Med. 2007;61:164–169. [Google Scholar]
  • 37.Koukoulitsa E, Skaltsa H, Karioti A, Demetzos C, Dimas K. Bioactive sesquiterpene lactones from Centaurea species and their cytotoxic/cytostatic activity against human cell lines in vitro. Planta Med. 2002;68:649–652. doi: 10.1055/s-2002-32893. [DOI] [PubMed] [Google Scholar]
  • 38.Koukoulitsa C, Geromichalos G.D, Skaltsa H. VolSurf analysis of pharmacokinetic properties for several antifungal sesquiterpene lactones isolated from Greek Centaurea sp. J. Comput. Aided Mol. Des. 2005;19:617–623. doi: 10.1007/s10822-005-9018-y. [DOI] [PubMed] [Google Scholar]
  • 39.Orabi K.Y, Sary H.G, Ayoub N.A, Singab A.N.B. Isolation of a Bioactive Guaianolide from Centaurea aegyptiaca Ethanol Extract. Planta Med. 2013;79:PN62. [Google Scholar]
  • 40.Parejo I, Jáuregui O, Viladomat F, Bastida J, Codina C. Characterization of acylated flavonoid-O-glycosides and methoxylated flavonoids from Tagetes maxima by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2004;18:2801–2810. doi: 10.1002/rcm.1697. [DOI] [PubMed] [Google Scholar]
  • 41.Sarg T.M, El-Domiaty M, El-Dahmy S. Further guaianolides from Centaurea aegyptiaca. Sci. Pharm. 1987;55:107–110. [Google Scholar]
  • 42.Saroglou V, Karioti A, Demetzos C, Dimas K, Skaltsa H. Sesquiterpene lactones from Centaurea spinosa and their antibacterial and cytotoxic activities. J. Nat. Prod. 2005;68:1404–1407. doi: 10.1021/np058042u. [DOI] [PubMed] [Google Scholar]
  • 43.Schmeda-Hirschmann G, Quispe C, González B. Phenolic Profiling of the South American “Baylahuen” Tea (Haplopappus spp, Asteraceae) by HPLC-DAD-ESI-MS. Molecules. 2015;20:913–928. doi: 10.3390/molecules20010913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Stintzing F.C, Kammerer D, Schieber A, Adama H, Nacoulma O.G, Carle R. Betacyanins and phenolic compounds from Amaranthus spinosus L. and Boerhavia erecta L. Z Naturforsch C. 2004;59(1-2):1–8. doi: 10.1515/znc-2004-1-201. [DOI] [PubMed] [Google Scholar]
  • 45.Vallverdú-Queralt A, Boix N, Piqué E, Gómez-Catalan J, Medina-Remon A, Sasot G, Mercader-Martí M, Llobet J.M, Lamuela-Raventos R.M. Identification of phenolic compounds in red wine extract samples and zebrafish embryos by HPLC-ESI-LTQ-Orbitrap-MS. Food Chem. 2015;181:146–151. doi: 10.1016/j.foodchem.2015.02.098. [DOI] [PubMed] [Google Scholar]
  • 46.Vijayan P, Raghu C, Ashok G, Dhanaraj SA, Suresh B. Antiviral activity of medicinal plants of Nilgiris. Indian J. Med. Res. 2004;120:24–29. [PubMed] [Google Scholar]
  • 47.Wilson A.P. In: Cytotoxicity and viability assays in animal cell culture: A Practical Approach. 3rd ed. Masters J. R. W, editor. Oxford University Press; 2000. [Google Scholar]

Articles from African Journal of Traditional, Complementary, and Alternative Medicines are provided here courtesy of African Traditional Herbal Medicine Supporters Initiative

RESOURCES