Int. J. Med. Arom. Plants, ISSN 2249 – 4340RESEARCH ARTICLE

Vol. 2, No. 1, pp. 151-154, March 2012

*Corresponding author: (E-mail) medjroubi<A.T.>yahoo.fr http://www.openaccessscience.com©2012 Open Access Science Research Publisher ijmap@openaccessscience.com

Phytochemical and biological activity of Algerian Centaurea melitensis

Radia AYAD1, Zine El Abidine ABABSA1 , F. Zohra BELFADEL1, Salah AKKAL1, Francisco

LEÓN2 , Ignacio BROUARD2, Kamel MEDJROUBI*1

1Laboratoire de Phytochimie et Analyses Physico-chimiques et Biologiques, Département de Chimie, Faculté

des Sciences Exactes, Université Mentouri de Constantine, Route d’Aïn El Bey 25000 Constantine Algérie.2Instituto de Productos Naturales y Agrobiologia - C.S.I.C.- Instituto Universitario de Bio-Organica ‘‘Anto-

nio Gonzalez’’, Universidad de La Laguna, Av. Astrofısico F. Sanchez 3, 38206 La Laguna, Tenerife, Spain

Article History: Received 5th December 2011, Revised 22nd January 2012, Accepted 22nd January 2012.

Abstract: The aerial parts of Centaurea melitensis (Asteraceae) yielded the germacranolides 8α-O-Isobutyroylsalonitenolide (1), Actiopicrin (2) and Onopordopicrin (6), the elimanolides Dehydromelitensin-β-hydroxyisobutyrate (4) 8α-O-(4-hydroxymethacrylate) melitensine (5), together with two flavonoids Hispidulin (3), Ne-petin (7) and a phenolic acid protocatechuic acid (8), the structures of these compounds were determinated by directcomparison of the spectral data with published data. The antioxidant property of the extract was determinated by usingthe DPPH method.

Keywords: Asteraceae; Centaurea melitensis; Elimanolides; Flavonoids; Germacranolides; Sesquiterpene lactones.

Introduction

The family Asteraceae is one of the largestangiosperm family and comprises about 1300genera and 25000 spices distributed over threesubfamilies and 17 tribes (Bremer 1994). Thelarge genus Centaurea (Asteraceae) comprisesabout 500 spices which are predominately dis-tributed around the Mediterranean area andWestern Asia (Mabberlay 1997). Amongst thebiological effects exerted by Centaurea species,it is noteworthy that several species were partic-ularly recommended against inflammatory con-ditions such as abscesses (Centaurea ibericaTrev. ex Sprengel), asthma (Centaurea ibericaTrev.ex Sprengel), hemorrhoids (Centaureadrahifolia Sm), wound healing (Centaurea ibe-rica Trev.ex Sprengel, Centaurea virgata Lam.,Centaurea pterocaula Trautv.), to reduce fever(Centaurea calcitrapa L. , Centaurea jacea L.,Centaurea iberica Trev.ex Sprengel, Centaureasolstitialis ssp. solstitialis), and headache (Cen-taurea solstialis L. ssp.solstitialis) (Yesilada2002). A wide range of therapeutic effects havealso been attributed to Centaurea species in tra-ditional medicines worldwide including endo-crine diseases (diabetes), inflammatory disord-

ers (rheumatic pain, antipyretic), gastrointestinalsymptoms (diarrhea, indigestions, and stomach-ic), urogenital aliments (diuretic, to induce men-struation), cardiovascular problems (hypoten-sive), parasitic and microbial infections (anti-bacterial, antimalarial…), etc. (Kaij-A-Kamb etal.1992 ; Farrag et al.1993 ; Barrero et al.1997 ;Orallo et al.1998). Centaurea melitensis L. is aperennial plant belonging to the Asteraceae fam-ily that abundantly grows in Algeria (Queseland Santa 1963), this spices is used in folk med-icine for treating hypoglycemia (Kamanzi et al.1983). In the present study, we investigated thephytochemical composition and the antioxidantactivity of C. melitensis.

Materials and methods

Plant material

Aerial parts of Centaurea melitensis L. werecollected from Boussaâda (Algeria) in June2008, and authenticated by Dr Gérard De Belair(Department of biology, Annaba University,Algeria). A voucher specimen is deposited inthe Herbarium of our laboratory.

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Extraction and isolation and structures elucida-tion

Dried and ground aerial parts (1070g) ofplant were extracted with MeOH (3times) atroom temperature, and the combined extractswere evaporated under reduced pressure. Me-thanolic extract (150ml) was suspended in H2Oand then partitioned with CH2Cl2, Ethyl acetateand n-butanol successively (each300ml×3times). The combined CH2Cl2 andEthyl acetate fractions were separately concen-trated using a rotary evaporator at a maximumtemperature of 45°C, which afforded 11g, 13gextracts, respectively. CH2Cl2 fraction (9g) wassubjected to silica gel column chromatography,starting the elution with CH2Cl2 and graduallyincreasing the polarity of the solvent by the ad-dition of Acetone, and finally with 100% Ace-tone. One hundred and twenty four fractions(124 Frs) were collected and monitored by TLC.Based on TLC profiles, four fractions ; F1 (Frs :7→53, 400mg), F2 (Frs : 57→59 ; 40mg), F3(Frs : 66→76, 75.8 mg), F4 (Frs : 80→83 ;36,7mg) were selected for further purification.F1 was chromatographed over silica gel columnto afford a mixture of sesquiterpene lactones,purification of the intermediate fractions bymeans of preparative TLC (CH2Cl2-Acetone:90:10), gave compounds (1) and (2). F2 waschromatographed over sephadex LH-20 (100%MeOH) to afford compound (3). F3 was chro-matographed on preparative TLC (CH2Cl2: Ace-tone: 80:20) gave tow compounds (4), (5). F4contained considerable amont of compound (6)which precipitated in crystalline form. Ethylacetate fraction (8g) was chromatographed oversilica gel column eluted initially with n-hexaneand then with n-hexane: ethylacetate mixtures(10%→100% EtoAC), EtoAC: MeOH(10%→50% MeOH) and finally with 100%MeOH. Eighty fractions (80 Frs.) were collectedand monitored by TLC. Based on TLC profiles,tow fractions were selected for further purifica-tion. F1 (Frs: 77-79, 44 mg) was subjected topreparative TLC (n-hexane: EtoAC: 50:50) toafford compound (7), F2 was also chromato-graphed on preparative TLC (CH2Cl2: MeOH:80: 20) to provide compound (8). The com-pounds were identified by comparison of theirspectral data with literature values for 8α-O-Isobutyroylsalonitenolide (1) (Miski et al.1967),

Actiopicrin (2) (Barrero et al.1989), Hispidulin(3) (Agrawal et al. 1989 ; Yuldashev et al.1996 ), Dehydromelitensin-β-hydroxyisobutyrate (4) (Gonzalez et al.,1975),8α-O-(4-hydroxymethacrylate) melitensine (5)(Marco et al. 1994), Onopordopicrin (6)(Drożdż et al.1968), Nepetin (7) (Agrawal etal.1989), protocatechuic acid (8) (Collado et al.1985).

Biological material

Antioxidant activity

DPPH: 2, 2-diphenyl-1-picrylhydrazyl (Mo-lecular formula C18H12N5O6) solution in MeOH(100µg/ml) was used in this assay (Takao et al.1994) to assess the free radical scavenging (an-tioxidant) property of the extract. Quercetin, awell known natural oxidant was used as refer-ence.

Determination of Antioxidant activity

Qualitative assay

Test extract of C.melitensis (5mg/ml) andQuercetin were dissolved in MeOH and appliedon a TLC plate (Merck, Germany).after thisplate was sprayed with DPPH solution using anatomizer and allowed to develop for 30 min.The white spots against a pink background indi-cated the antioxidant activity.

Quantitative assay

Stock solutions of the test extract of C. meli-tensis were prepared to obtain different concen-trations (0.0012, 0.0025, 0.005, 0.01, 0.02, 0.03,0.05, 0.1, 0.2, 0.4, 0.5, 0.7 mg/ml). Prepared so-lutions (15µl each) were mixed with DPPH(1.5ml) and allowed to stand 15min for anyreaction to occurs. The UV absorbance of thesesolutions was recorded at 517nm. The experi-ment was performed in triplicate and the aver-age absorption was noted for each concentra-tion. The same procedure was followed for theQuercetin (used as reference). The percentage (I%) of inhibition of DPPH by the methanolic ex-tract of C. melitensis was calculated from theequation :

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I% = [AC– AE] / AC× 100.

AC: Absorption in the absence of the inhibitedcontrol (negative control).

AE: Absorption in the presence of the inhibitedsample (extract).

Results and discussion

The methanolic extract of C. melitensis ex-hibited a very important antioxidant in theDPPH assay with a maximal inhibition activityof 89.02% at the high concentration of0.7mg/ml.

Table 1: Antioxidant activity of the methanolicextract of C. melitensis at different concentra-tions (mg/ml).

Concentrationsof extract(C.melitensis) /Quercetin

I% of DPPHradical byextract(C.melitensis)

I% ofDPPHradical byQuercetin

0.0012 01.95% 46.45±1.78%0.0025 05.32% 94.72±0.30%0.005 11.41% 95.81±0.13%0.01 13.04% 95.91±0.00%0.02 17.71% 96.06±0.04%0.03 22.82% ND0.05 34.13% ND0.10 55.10% ND0.20 87.71% ND0.40 88.80% ND0.50 88.91% ND0.7 89.02% ND

ND: not done; Concentrations values are inmg/ml; %: Percentage of inhibition

The results summarized in Table 1 showedthat the percentage of inhibition of DPPH radi-cal increase proportionally with the increase ofextract concentration, the obtained inhibition (I%) varied from 1.95% to 89.02%; these resultswere compared with those of the Quercetin.

It is generally assumed that the radical sca-venging or antioxidant activity of plants is aconsequence of the presence of the natural phe-nolic antioxidants, like: lignans, flavonoids andphenolic acids. The antioxidant activity of phe-nolic natural products is predominantly due totheir redox properties, i.e. the ability to act asreducing agents, hydrogen donors and singletoxygen quenchers, and to some extent, could

also be due to their metal chelation potential(Shoeb et al. 2006).

Relationship between antioxidant activity andconcentration of the extract of C. melitensis

The correlation between antioxidant activityand concentrations of the extract of C. meliten-sis is shown (Figure 1), The R2 value was 0.74.The present study shows that highly positiverelationship exists between the antioxidant ac-tivity and the increasing in concentrations of theextract. Previous work (Cai et al. 2004; Shan etal. 2005) showed that there were highly positivelinear correlation between antioxidant capacityand total phenolic content of a large number ofspices and herbs.

Figure 1: Relationship between concentrationsof the extract (mg/ml) and inhibition of DPPH(I%).

Conclusion

In conclusion, the present study addressedthe phytochemical and biological investigationof C. melitensis leading to the identification ofeight (8) different compounds (sesquiterpenelactones, flavonoids and a phenolic acid). Theimportant antioxidant activity of the extract,suggests the potential presence of phenoliccompounds in C. melitensis.

References

Agrawal, P.K., Thakur, R.S., Bansal, M.C.1989. Flavonoids. In: Agrawal, P.K. (Eds.),Carbon-13 NMR of Flavonoids. Elsevier,Amsterdam.

Barrero, A. F., Sanchez, J. F., Rodriguez, I. andSoria Sanz, C. 1989. Germacranolides from

154Int. J. Med. Arom. Plants Centaurea melitensis as a source of secondary metabolites

Ayad et al. http://www.openaccessscience.comijmap@openaccessscience.com

Centaurea melitensis. Phytochemistry, 28:1975-1976.

Barrero, A.F., Herrador, M., Arteaga, M., Ca-brera, P., Rodriguez-Garcia, E., Garcia-Moreno, I.M., Gravalos, D.G. 1997. Cyto-toxic activity of flavonoids from Cartha-mus arborescens. Ononis natrix ssp. ramo-sissima and Centaurea malacitana. Fitote-rapia, 68:281-283.

Bremer, K. 1994. Asteraceae: Cladistics andClassification. Timber Press, Portland.

Cai, Y.Z., Luo, Q., Sun, M., Corke, H. 2004.Antioxidant activity and phenolic com-pounds of 112 traditional Chinese medicin-al parts associated with anticancer. LifeScience, 74: 2157-2184.

Collado, I.G., Macias, F.A., Massanet, G.M.,Luis, F.R. 1985. Flavonoids from Centau-rea clementei. Journal of Natural Products,48: 819-822.

Drożdż, B., Holub, M., Samek, Z., Herout, V.,Sˇorm, F. 1968. The constitution and abso-lute configuration of onopordopicrine, asesquiterpenic lactone from Onopordonacanthium L. Collection of CzechoslovakChemical Communications, 33: 1730–1737.

Farrag, N.M., Abd El Aziz, E.M., El-Domiaty,M.M., El Shafea, A.M. 1993. Phytochemi-cal investigation of Centaurea araneosagrowing in Egypt. Zagrep Journal ofPharmaceutical Sciences, 2: 29–45.

Gonzalez, A. G., Arteaga, J. M. and Brenton, J.L. 1975. Elimanolides from Centaurea me-litensis. Phytochemistry, 14: 2039.

Kaij-A-Kamb, M., Amoros, M., Girre, L. 1992.Chemical and biological activity of the ge-nus Centaurea. Pharmaceutica Acta Helve-tica, 67: 178–188.

Kamanzi, K., Raynaud, J., Voirin, B. 1983. TheC-glycosyl flavonoids from flowers ofCentaurea melitensis. Plantés Medicinal etPhytotherapié, 17: 47–51.

Mabberlay, D.J. 1997. The Plant Book. Secondedition. Cambridge University Press, Cam-bridge.

Marco, J.A., Sanz-Cervera, J.F., Garcia-Lliso,V., Susanna, A. and Garcia-Jacas, N. 1994.Sesquiterpene lactones, Lignans and Aro-matic esters from Cheirolophus species.Phytochemistry, 37: 1101-1107.

Miski, M., Ali, H. M., Mabry, T, J. 1988. Ses-quiterpene lactones of Onopordon Tauri-cum. Phytochemistry, 27(5): 1417-1420.

Orallo, F., Lamela, M., Camina, M., Uriatre, E.,Calleja, M. 1998. Preliminary study of thepotential vasodilator effects on rat aorta ofcentaurein and centaureidin, two flavonoidsfrom Centaurea corcubionensis. PlantaMedica, 64: 116–119.

Quezel, P., Santa, S. 1963. Nouvelle flore del’Algérie et des régions désertiques etméridionale, Tome II, C.N.R.S, Paris,France.

Shan, B., Cai, Y.Z., Sun, M., Corke, H. 2005.Antioxidant capacity of 26 spice extractsand characterization of their phenolic con-stituents. Journal of Agricultural and FoodChemistry, 53: 7749-7759.

Shoeb, M., Macmanus, S.M., Kong-Thoo-Lin,P., Celik, S., Jaspars, M., Nahar, L., Sarker,S. D. 2007. Bioactivity of the extracts andisolation of lignans and a sesquiterpenefrom the aerial parts of Centaurea pamphy-lica (Asteraceae). DARU Journal, 15(3):118-122.

Takao, T., Watanabe, N., Yagi, I., Sakata, K.1994. A simple screening method for anti-oxidants and isolation of several antioxi-dants produced by marine bacteria fromfish and shellfish, Bioscience. Biotechnolo-gy and Biochemistry, 58: 1780-1783.

Yesilada, E., 2002. Biodiversity in Turkish FolkMedicine. In: S¸ ener, B. (Eds.), Biodiver-sity: Biomolecular Aspects of Biodiversityand Innovative Utilization. Kluwer Aca-demic/Plenum Publishers, London, UK, pp.119–135.

Yuldashev, M.P., Batirov, E.K., Malikov, V.M.1996. Flavonoids of the roots of Scutellariacomosa. Chemistry of Natural Compounds,32: 592-593.