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Mutagenic, antimutagenic andantioxidant activities of a newpolyphenolic and a flavonoidsubstances isolated from AnagallismonelliSamia Ammar a , Mohamed Ali Mahjoub a , Houda Ben Abdelkadera , Kaouther Liouane a , Ines Skandarani b , Leila Chekir-ghedira b

& Zine Mighri aa Faculté des Sciences de Monastir, Laboratoire de Chimie desSubstances Naturelles et de Synthèse Organique 99/UR/12-26,5000 Monastir, Tunisieb Faculté de Pharmacie de Monastir, Unité de Pharmacognosie/Biologie moléculaire 99/UR/07-03, Rue Avicenne 5000 Monastir,TunisiePublished online: 19 May 2008.

To cite this article: Samia Ammar , Mohamed Ali Mahjoub , Houda Ben Abdelkader , KaoutherLiouane , Ines Skandarani , Leila Chekir-ghedira & Zine Mighri (2008) Mutagenic, antimutagenic andantioxidant activities of a new polyphenolic and a flavonoid substances isolated from Anagallismonelli , Natural Product Research: Formerly Natural Product Letters, 22:8, 658-665, DOI:10.1080/14786410701669287

To link to this article: http://dx.doi.org/10.1080/14786410701669287

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Natural Product Research, Vol. 22, No. 8, 20 May 2008, 658–665

Mutagenic, antimutagenic and antioxidant activities

of a new polyphenolic and a flavonoid substances

isolated from Anagallis monelli

SAMIA AMMARy, MOHAMED ALI MAHJOUBy, HOUDABEN ABDELKADERy, KAOUTHER LIOUANEy, INES SKANDARANIz,

LEILA CHEKIR-GHEDIRAz and ZINE MIGHRI*y

yFaculte des Sciences de Monastir, Laboratoire de Chimie des SubstancesNaturelles et de Synthese Organique 99/UR/12-26, 5000 Monastir, Tunisie

zFaculte de Pharmacie de Monastir, Unite de Pharmacognosie/Biologie moleculaire99/UR/07-03, Rue Avicenne 5000 Monastir, Tunisie

(Received 13 April 2007; in final form 6 September 2007)

A new polyphenolic natural substance: 2,5-dihydroxy-benzoic acid 30-formyl-50-hydroxy-phenyl ester (1), Anamighrinal and 3-(O-�-L-rhamnosyl) quercetin (2) have been isolatedfrom the methanolic aerial part extract of the plant Anagallis monelli by chromatographicseparation. Their structures have been deduced essentially by one and two NMR spectroscopicprocedures and mass spectrometry. Antioxidant, mutagenic, antimutagenic activities, of thenatural products were realised and positive results were recorded.

Keywords: Anagallis monelli; NMR; New substance; Antioxidant; Mutagenic; Antimutagenic

1. Introduction

As a part of our investigation on some medicinal plants growing in Tunisia [1,4] andin order to continue our search on potential biological natural compounds, we reportbelow the isolation, the structure elucidation and biological activities of a newpolyphenolic compound, and an usual heterosid, from the methanolic extract of thewhole Anagallis monelli var. linifolia (L.) (Primulaceae). We note that the Tunisian floracounts six species of Anagallis, the most preponderant one is A. arvensis [5]. Accordingto our bibliographic studies, not much is known on the chemistry of A. monelli. Wenote that in folk medicine the decoction of some Anagallis species has fungistaticproprieties [6]. Occurrence of saponins in A. arvensis species [7,8] explains its traditionaluse as antifungic and antiviral [9]. With an aim to focus in the biological interest ofthe species monelli, mutagenic and antimutagenic activities were realised based on the

*Corresponding author. Fax: þ21673 500278. Email: zine_mighri2002@yahoo.fr

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 online � 2008 Taylor & Francis

http://www.tandf.co.uk/journals

DOI: 10.1080/14786410701669287

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SOS Chromotest and antioxidant activities were realised based on the 2,20-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) tests. The structures of the natural products were establishedby the spectroscopic means.

2. Materials and methods

2.1. General experimental procedures

1H, 13C NMR and two-dimensional NMR spectra were obtained with Bruker WP 300spectrometer at 300MHz for 1H NMR and 75MHz for 13C NMR. Measurements weremade in Pyridine-d5 and Acetone-d6 at 25

�C, and the resonance of residual solvent wereused as internal references. Hxcoqf and inv4lplrndqf hetero nuclear pulse programswere used, respectively, for CHCorr and HMBC experiments; 2D homonuclearcorrelation pulse programs, Cosyqf45 and Cosyqfrl were used for COSY experiments.ESMS spectrum was recorded in negative and positive switching mode, using a HP 1100MSD apparatus. FTIR spectrum was measured on a Perkin–Elmer 157G infraredspectro-photometer. Optical density values were mesured on Hitachi Fluorescence(model 4010) and Shimadzu UV-2101 PC Spectrophotometers, respectively, forgenotoxicity and antioxidant activities.

2.2. Plant material

Aerial parts of the plant A. monelli (1200 g) were collected at the beginning of May 2002in Sousse (centre of Tunisia). A voucher specimen (A.M.C02-01) was deposited at theherbarium in the Faculty of Sciences, University of Monastir, Tunisia.

2.3. Extraction and isolation

Dried and finely powdered aerial parts (1200 g) were immersed in MeOH (4� 2L) atroom temperature for a week. Methanolic extract was filtered then evaporated todryness. The MeOH extract (360 g) was extracted with AcOEt (1L). Removal of thesolvent from the AcOEt solution gave a methanolic residue noted E1 (147 g) and an oilysubstance (212 g). Extraction of the oily substance with petroleum ether yield anethyl acetate residue noted E2 (127 g) and a petroleum ether extract (85 g). The ethylacetate residue E2 was fractioned by silica gel column chromatography (CC) elutedwith a step-gradient of AcOEt in petroleum ether. Fifteen fractions (F1–F15) werecollected. Repeated CC separations of the E6 fraction (206mg) and E10 fraction(1600mg) afforded in pure form the natural compounds 2,5-dihydroxy-benzoic acid30-formyl-50-hydroxy-phenyl ester (48mg), and 3-(O-�-L-rhamnosyl) quercetin (96mg).

2.4. Chemicals

The mutagen Nifuroxazide, the O-nitrophenyl-�-D-galactopyranoside and n-nitrophe-nyl-phosphate, for colorimetric evaluation of �-galactosidase and alkaline

New polyphenolic and flavonoid substances from A. monelli 659

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phosphatase, were purchased from Sigma. (ABTS), (DPPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were also obtained from Sigma.All other chemicals were of analytical grade purity.

2.5. Escherichia coli mutagenicity assay

Genotoxicity assay of 2,5-dihydroxy-benzoic acid 30-formyl-50-hydroxy-phenyl esterand 3-(O-�-L-rhamnosyl) quercetin was carried out using SOS chromotest. This test isbased on using the genetically modified E. coli PQ37 strain, in which the lacZ is underthe control of the sfiA geneb [10]. In short, genotoxicity samples was detectedby measuring the activation of SOS response of tested organism by evaluating�-galactosidase induction and alkaline phosphatase expression. The �-galactosidaseand alkaline phosphatase activities were calculated according to Quillardet andHofnung [11], Units¼U(�,p)¼A420� 103 t�1 (A420¼optical density at 420 nm forthe tested molecule at a determined concentration; t: substrate conversion time in min).The induction factor (IF) was calculated as the ratio R/R0 with R¼U�/Up(U�¼ �-galactosidase activity and Up¼ alkaline phosphatase activity at compoundsand crude extracts determined concentration; R0¼ �-galactosidase activity/alkalinephosphatase activity at test compound concentration zero). A positive controlNifuroxazide was used as reference genotoxic compound.

2.6. Antimutagenic activity

The antimutagenic activity was carried out with the same method as that describedfor the study of the mutagenic activity, except the incubation of the mutagen agentat the same time as the tested substance [12]. The same concentrations of molecules usedfor the study of the mutagenic activity are used for the study of the antimutagenicactivity. All experiments were performed in triplicate.

2.7. Radical cation ABTS1.

scavenging activity

The standard method described by Miller et al. [13] has been adopted with minormodifications. This assay assesses the total radical scavenging capacity based on theability of a compound to scavenge the stable ABTS radical (ABTSþ

.

). The blue greenABTS radical form was produced through the reaction between ABTS and potassiumpersulfate in water. A concentrated ABTSþ

.

stock solution was diluted with phosphatebuffered saline (PBS), pH 7.4 to a final absorbance of 0.70� 0.02 with a wavelength of734 nm and at a temperature of 37�C. Solutions with different diluted concentrations ofour two natural products were prepared in ethanol. Ten microliters of an antioxidantcontaining studied solution were added to 990mL ABTSþ

.

solution and the absorbanceat 734 nm was measured. Sample absorbance was compared to a blank where 10 mLof the solvent were added to 990mL of the ABTSþ

.

solution. Absorbance was measuredat 5, 10, 15 and 20min after addition of the antioxidant. Results were expressed ininhibition percentage versus samples concentrations (mmol) at different times.

660 S. Ammar et al.

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2.8. DPPH . scavenging capacity

The DPPH radical scavenging capacity of our two natural products was measured

from the bleaching of purple coloured ethanol solution of DPPH. Method described by

Hatano et al. [14] has been used. Total of 0.5mL of each sample concentration was

mixed with a same volume of DPPH ethanolic solution. After incubation of 30min in

darkness at room temperature, absorbance was read at 520 nm wavelength. A mixture

of 0.5mL of DPPH solution and 0.5mL of ethanol was taken as a blank. Decrease in

absorption induced by the tested compounds was compared to that of the positive

control Trolox. IC50 values calculated denote the concentration (microgram of natural

substance per milliliter of ethanol) required to scavenge 50% of DPPH radicals.

All measurements were performed in triplicate. Results were expressed in inhibition

percentage versus samples concentrations (mgmL�1) at 30min.

3. Results and discussion

3.1. Structure elucidation

NMR spectra were recorded at 300MHz or 75MHz in pyridine-d5. The polyphenolic

substance was isolated as amorphous solid. Positive and negative ion electrospray

ionization mass spectrum of this substance shows the pseudomolecular picks [MþH]þ

and [M�H]� at m/z 275 and 273, respectively, to confirm the molecular formula

C14H10O6.The analysis of 13C NMR spectrum shows the presence of 14 carbon atoms, the

observation of six aromatic CH groups respectively at 96.7; 101.8; 105.9; 116.5; 118.7

and 121.5 ppm. An aldehydic and an ester groups were also observed respectively

at 184.7 and 167.6 ppm. These observations were supported by the DEPT 135

experiment, which shows the disappearance of eight peaks that belong to quaternary

carbons.The analysis of 1H spectrum shows a singlet at 6.81 ppm integrated for two protons

(H-20 and H-40), two singlets respectively at 7.01 and 7.99 ppm integrated for one proton

corresponding to H-60 and H-6, two large signals at 9.67 and 13.8 ppm integrated,

respectively, two and one proton and relative to three phenolic groups (table 1).Aldehydic group was confirmed by the IR spectrum, which revealed a characteristic

band at 2853 cm�1. Based on the same spectrum the strong band at 1706 cm�1 also

confirms the presence of an ester group.Scalar correlations revealed from the total COSY spectrum were in agreement

with the hydrocarbon skeleton of the polyphenolic substance, particularly H-20/H-40,

H-40/H-60, H-4/H-3, H-6/H-3 and H-6/H-4. Correlations H-40/C-60, H-60/C-40, H-40/

C-30, H-60/C-50, H-40/C-70, H-4/C-6 and H-4/C-5 revealed from the HMBC spectrum

prove the total structure of the natural product Anamighrinal (figure 1).Compound 2 was obtained as amorphous solid. Positive ion electrospray ionization

mass spectrum of this substance shows two pseudomolecular peaks [MþNa]þ and

[MþK]þ at m/z 471 and 487, respectively, and confirms the molecular formula

C21H20O11. All spectroscopic data of this substance named 3-(O-�-L-rhamnosyl)

quercetin were consistent with those reported in the literature [15].

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Our bibliographical study shows that the compound 1 is new. Its systematical name is2,5-dihydroxy-benzoic acid 30-formyl-50-hydroxy-phenyl ester, while its trivial nameis Anamighrinal.

The water solubility of compounds 1 and 2 encouraged us to focus on its biologicalactivity and especially on its prevention of mutagenicity and radical toxicity.

3.2. E. coli mutagenicity assay

The genotoxicity of the new substance (1) and the O-heterosid compound 2 was studiedusing the SOS chromotest (table 2). At different concentrations and even with highconcentration (50mgmL�1). Substance 1 seems cause no mutagenic effects, neverlessthe O-heterosid causes important mutagenic effects. Compared with the positivecontrol (IF¼ 23.64 at a concentration of 1mgmL�1) the new substance inductionfactor was constantly feeble (IF� 1) contrary compound 2 induction factor wasinteressant. According to Mesch–Sundermann, a compound with an IF lower then 2.00was classified as non-genotoxic and a compound with an IF higher then 2.00 wasclassified as very genotoxic [16]. Therefore, we can prove that the new substance isa non-genotoxic compound and the O-heterosid compound is genotoxic.

Table 1. 1H- and 13C-NMR chemical shifts of compound 1 in pyridine-d5 and13C, 1H correlations (HMBC).

N� �13C (ppm) � 1H (ppm); J(Hz) HMBC

1 106.9 – –2 160.4 – H-3/C-23 118.7 7.37 (1H, d); J3�4¼ 8.1 H-2/C-34 121.5 7.64 (1H, d); J4�3¼ 8.1 –5 149.6 – H-4/C-56 116.5 7.99 (1H, s) H-4/C-67 167.6 – –10 153.5 – –20 96.7 6.81 (1H, s) H-40/C-20

30 124.8 – H-20/C-30

40 101.8 6.81 (1H, s) H-20/C-40

50 166.7 – H-20/C-50

60 105.9 7.01 (1H, s) –70 184.7 13.8 (1H, s) H-20/C-70

OH

H

H

OH

H

O

O

H

H

O

H

OH

H

1

23

4

56

1′

2′3′

4′

5′6′

7′

7

Figure 1. 2,5-Dihydroxy-benzoic acid 30-formyl-50-hydroxy-phenyl ester (1).

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3.3. Antimutagenic activity

This test was based on the inhibition of the SOS inducing factor (IF), afterco-incubation of the positive mutagenic compound nifuroxazide and the testedsubstances 1 and 2. Results were expressed as percentage of genotoxicity inhibitionrelative to nifuroxazide (table 2). Application of substances with exponential valueconcentrations shows genotoxicity inhibition rising from 56.09 to 83.83% forcompound 1 and from 40.28 to 74.02% for compound 2. As shown in table 2unexpected results were recorded, the new substance was more active with feebleconcentrations, it shows maximum genotoxicity inhibition of 83.83% at a concentrationof 2mgmL�1. Complementary experiments are necessary to explain the concentrationrelated inhibitory effects and the chemical structure-biological activity relationship ofthis natural product, perhaps in the presence of another positive chemical mutagen.

3.4. Radical cation ABTS1.

scavenging activity

Results of our investigation on ABTSþ.

cation radicals scavenging activity, representedby table 3, show maximal inhibition percentage with 90.57% of compound 1 and72.51% with compound 2 at a concentration of 1mgmL�1. At feeble concentration(0.03mgmL�1) and immediately after 5min of incubation the new natural product 1display a value of 40.23% of ABTSþ

.

inhibition. Compared to Trolox (97.9%inhibition at 1mgmL�1 and after 5min of incubation), compound 1 is a powerfulantioxidant substance against ABTSþ

.

cation radicals at a concentration of 1mgmL�1.

3.5. DPPH. scavenging capacity

The result of antioxidant activity of our natural substances 1 and 2, against free radicalDPPH and after 30min of incubation, was represented by table 3. Compared to Trolox

Table 2. Mutagenicity and antimutagenicity results of compound 1 and 2 to Escherichia coli PQ37 againstthe mutagen nifuroxazide.

Concentration(mgmL�1) Uß UP R IF Results

Nifuroxazide 0 3.31� 0.06 12.95� 0.008 0.25 – –þ 36.80� 0.03 7.22� 0.05 5.91 23.64 –

Mutagenic activityCompound 1 50 2.38� 0.02 8.53� 0.03 0.27 1.08 –

10 2.56� 0.03 8.87� 0.03 0.28 1.12 –2 3.04� 0.03 9.27� 0.03 0.32 1.28 –

Compound 2 50 24.18� 0.04 9.90� 0.03 2.44 9.76 –10 12.50� 0.03 8.33� 0.03 1.5 6 –2 17.92� 0.03 9.80� 0.04 1.82 7.28 –

Antimutagenic activity Genotoxcicityinhibition (%)

Compound 1 50 26.40� 0.03 9.66� 0.02 2.73 10.94 56.0910 18.60� 0.03 10.10� 0.03 1.84 7.38 71.812 12.55� 0.03 10.82� 0.02 1.16 4.66 83.83

Compound 2 50 18.23� 0.03 10.59� 0.04 1.72 6.88 74.0210 42.80� 0.02 11.79� 0.04 3.63 14.52 40.282 38.20� 0.01 11.57� 0.03 3.30 13.2 46.11

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as a standard reference product (99.3% inhibition at 1mgmL�1 and after 30min

of incubation), we note that our natural substances also show powerful inhibitionactivity. With tested concentrations the inhibition percentage has a feeble variationfrom 90.22 to 93.44% of compound 1 and from 78.02 to 88.64% of compound 2.

The in vitro test of compound 1 against toxic radical entities ABTSþ.

and DPPHproves the efficiency of this natural substance as an antioxidant agent. Indeed theremarkable activity of the new compound is awaited. Based on the structure-activityrelationship of polyphenols compounds [17], oxidation of hydroquinone skeleton in thenew substance structure (1) results with the formation of the quinone stable form.

4. Conclusion

During this study, we are focusing on the interest of a new natural substance and anusual flavonoid isolated from the plant A. monelli, as agents against the genotoxicityand the oxidative effects caused by free radicals. The safety of compound 1 and thegenotoxicity–anticancer activity relationship [18] were two reasonable reasons tocontinue our research and to focus on the anticancer effects of our natural compound.

Compound 1 (Anamighrinal): Amorphous solid, m.p.: 125–127�C. IR (KBr)�max: 3420, 2853, 1653, 1267 cm�1. 1H and 13C NMR data, see table 1. ES-MS[M�H]� at m/z 273, [MþH]þ at m/z 275 (C14H10O6, 274).

Compound 2 (3-(O-�-L-rhamnosyl) quercetin): Amorphous solid. IR (KBr) �max: 3249,2932, 1656, 1098 cm�1. 1H NMR: �(Acetone d-6) Aglycone moiety; 6.23 (1H, d,J¼ 2.1Hz, H-6), 6.47 (1H, d, J¼ 2.1Hz, H-8), 7.01 (1H, d, J¼ 8.4Hz, H-50), 7.41 (1H,dd, J¼ 8.4 and 2.1Hz, H-60), 7.50 (1H, d, J¼ 2.1Hz, H-20); 13C NMR: �(Acetone d-6)glucose moiety; 71.5, 72.6, 73.6, 74.2, 75.3, 103.2. Aglycone moiety; 95.0 (C-8), 99.2(C-6), 106.7 (4a), 117.3 (C-50), 118.6 (C-20), 123.3 (C-60), 123.3 (C-10), 137.7 (C-3), 147.9

Table 3. ABTSþ. and DPPH. inhibition percentage in the presence of different concentrations ofcompounds 1 and 2.

ABTS percentage inhibition DPPH

Time (min)

Concentration(mgmL�1) 5 10 15 20 30

1 64.74� 1.68 75.79� 1.8 80.95� 1.84 90.57� 1.98 93.33� 0.790.5 55.61� 1.38 66.13� 2.21 73.90� 1.44 80.50� 0.73 93.22� 0.15

Compound 1 0.25 55.12� 1.09 65.49� 2.07 71.45� 1.58 77.22� 1.54 93.44� 0.630.125 44.00� 0.52 50.30� 2.22 57.16� 1.36 64.40� 1.61 93.38� 0.070.06 41.32� 1.53 50.11� 0.19 55.54� 2.11 62.82� 1.77 90.90� 0.230.03 40.23� 1.98 47.73� 2.09 50.60� 1.93 56.74� 1.90 90.22� 0.39

1 60.30� 1.84 67.79� 1.42 72.51� 1.88 77.90� 1.32 88.64� 1.030.5 43.47� 1.61 53.43� 1.18 61.87� 1.90 68.06� 1.76 87.96� 0.23

Compound 2 0.25 39.25� 1.47 48.37� 1.13 56.22� 1.03 61.53� 1.69 87.79� 0.630.125 37.36� 2.19 44.79� 1.71 53.77� 1.53 57.95� 2.26 86.66� 0.630.06 28.99� 1.24 37.85� 0.96 44.57� 1.13 50.79� 1.64 84.97� 1.110.03 17.72� 1.43 19.72� 1.49 27.48� 1.17 44.19� 1.81 78.02� 1.03

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(C-30), 149.6 (C-40), 158.6 (C-8a), 159.3 (C-2), 163.7 (C-5), 165.4 (C-7), 179.9 (C-4).

ES-MS [MþNa]þ at m/z 471, [MþK]þ at m/z 487 (C21H20O11, 448).

Acknowledgements

The authors wish to thank Dr F. Harzallah-Skhiri, laboratoire de Biologie vegetale,

Institut Superieur de Biotechnologie, Monastir, Tunisia, for botanical classification of

the plant material.

References

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