Variation in chemical composition and biological activities of two species of Opuntia flowers at four stages of flowering

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Industrial Crops and Products 37 (2012) 34– 40

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Industrial Crops and Products

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ariation in chemical composition and biological activities of two species ofpuntia flowers at four stages of flowering

mène Ammara, Monia Ennouria,b,∗, Bassem Khemakhemc, Thabèt Yanguid, Hamadi Attiaa

Alimentary Analysis Unit, National Engineering School of Sfax, BPW 3038, Sfax, TunisiaHigher Institute of Applied Sciences & Technology of Mahdia, Sidi Messaoud 5111 Mahdia, TunisiaLaboratory of Plant Biotechnology, Department of Biology, Faculty of Sciences of Sfax, BP 1171, 3000 Sfax, TunisiaLaboratory of Environmental Bioprocess, Center of Biotechnology of Sfax, BP1177, 3018 Sfax, Tunisia

r t i c l e i n f o

rticle history:eceived 6 May 2011eceived in revised form3 September 2011ccepted 23 November 2011vailable online 7 January 2012

eywords:

a b s t r a c t

The chemical composition of hexane extracts from flowers belonging to two species of pricklypear, Opuntia ficus-indica (L.) Miller and Opuntia stricta (Haw.) Haworth has been studied by gaschromatography–mass spectrometry in four developmental stages of flower: vegetative, initial flower-ing, full flowering, and post-flowering stages. Remarkable differences were noted between the flowers’compositions. The main compounds were carboxylic acid (28–97%), terpenes (0.2–57%), esters (0.2–27%),and alcohols (<1.8%).

The study of the biological activities showed that extracts were active in vitro towards four bacteria

puntia ficus indicapuntia strictalowerhemical compositionlowering stage

and two fungal strains. It exhibited remarkable activity against Pseudomonas aeruginosa, Staphylococcusaureus, and Escherichia coli.

Antioxidant activity of the flowers extracts was evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH)radical method.

Our findings demonstrate the interest of Opuntia flowers extract as a source of bioactive substancesand its potential preservative use in food.

. Introduction

Opuntia ficus-indica (L.) Miller and Opuntia stricta (Haw.)aworth belongs to the Cactaceae family and the order Centrosper-ae. These plants grow wild in arid and semi-arid regions, where

he production of more succulent food plants is severely limited.. ficus-indica species has gradually attained economic importancef the international scientific community, through FAO, also con-ributes to the diffusion of this cultivation (Galati et al., 2002).

Many uses of cactus pear fruit and cladodes are reportedHoffmann, 1980). Cladodes have been investigated as a possi-le treatment for gastritis, hyperglycemia, ateriosclerosis, diabetes,nd prostatic hypertrophy (Frati-Munari et al., 1990; Hegwood,990; Palevitch et al., 1993). Cladodes are also highly used for theood industry; they have been studied for their gelling propertiesSepúlveda et al., 2007) and they are also considered as a source of

ber and color in the formulation of cake (Ayadi et al., 2009).

The fruits are used for the manufacture of food products suchs juices (Espinosa et al., 1973; Ennouri et al., 2006), alcoholic

∗ Corresponding author at: Alimentary Analysis Unit, National Engineering Schoolf Sfax, BPW 3038, Sfax, Tunisia. Tel.: +216 98 278 684; fax: +216 74 221 160.

E-mail address: [email protected] (M. Ennouri).

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.11.027

© 2011 Elsevier B.V. All rights reserved.

beverage (Bustos, 1981), jam (Sawaya et al., 1983), natural liquidsweetener (Saenz et al., 1996).The flower was little studied becauseof their limited bloom duration and in addition to that the diffi-culty to get them. They generally appear on one-year-old cladodes.The color of the Opuntia flower is generally yellow, but there arealso orange, pink, purple, red, white or mottled flowers (Anderson,2001). Floral development from bud to anthesis requires between21 and 75 days (Grant and Grant, 1979; Barbera et al., 1992; Niedduand Spano, 1992).Opuntia flower is useful in numerous fields; suchas traditional medicines thanks to its effect which are defined asdepurative and in particularly diuretic and relaxant of renal excre-tory tract.

The valorization of natural resources such as plant extracts andproducts of plant secondary metabolism, particularly used by thepopular tradition is for a great interest. In fact, plant extracts haveattracted a great deal of scientific interest due to their poten-tial as a source of natural antioxidants and biologically activecompounds, such as antibacterial, antifungal and insecticidal sub-stances (Celiktas et al., 2007).

The nutritional and pharmacological benefits of the different

parts of the prickly pear, in addition to its increasing importanceat industrial level motivated our investigation on the chemicalcontent of the flowers which are less known. In the literaturefew reports are focused on Opuntia flowers. Bergaoui et al. (2007)

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I. Ammar et al. / Industrial C

ave reported the chemical composition of volatiles fractions fromqueous distillate obtained from three Opuntia species (Opun-ia lindheimeri, Opuntia macrohiza and Opuntia microdasys). Theecent study of De Léo et al. (2010) describes the chemical contentf methanol extract O. ficus-indica flowers. However, there is noeport concerning the chemical analyses of Opuntia hexane extract.herefore, to provide major information about the chemical char-cterization of O. ficus-indica and O. stricta flowers, we performedhe analysis of hexane extract of plant material from Tunisia.

The aim of the present work was to study the chemical com-osition by GC–MS, the antioxidant, and antimicrobial activities ofhe hexane extracts isolated from Opuntia flowers.

. Materials and methods

.1. Plant material

Two species were tested in our study: O. ficus-indica (L.) Mill.nd O. stricta (Haw.) Haworth. Samples of Opuntia flowers growingild in the region of Sfax, Tunisia, were collected during vegetative

A), initial flowering (B), full flowering (C) and post-flowering (D)tages in May–June 2009.

The Opuntia flowers were harvested at four stages. In vegetativetage, we have green closed petal flowers. In initial flowering stage,tamens are grouped together around the style in the beginning,nd become separated in the full flowering stage. The flower is inull and starts nectar production. In the post flowering stage, theower becomes closed and dry.

Harvesting of O. ficus-indica for vegetative and initial floweringtage is taking place in the period ranging from 20 to 25 May. Theowers at full flowering stage were harvested in the beginning of

une. For O. stricta the harvesting period starts in the middle of Juneor the three first stages. The post flowering stage flowers for thewo species were collected in the last week of June.

Harvested flowers for every growth stage and for the two speciesere divided into two batches: the first one (Table 1) was used for

he hexane extraction; the second (50 g) was dried and used for thextraction of polyphenols.

.2. Isolation of hexane extract

For every flowering stage, appropriate amounts of Opuntia flow-rs (Table 1) were cut into small parts and transferred into glassasks of 1 l volume, filled with hexane then the mixture was kept

n the dark for a week in order to have a better maceration. Thextracts were filtered using Whatman filter paper (No. 1) and thenoncentrated under reduced pressure at 40 ◦C using a rotary evap-rator. The yield extracts was shown in Table 1.

Extracts were stored in a freezer at 4 ◦C for further tests.

.3. Hexane extracts analysis

Hexane extracts were analyzed by using an Agilent-echnologies 6890 Network GC system equipped with a flameonization detector and HP-MS capillary column (30 m × 0.25 mm,lm thickness 0.25 �m, Agilent-Technologies, Little Falls, CA, USA).or MS detection, the electron ionization mode with ionizationnergy of 70 eV was used, with a mass range at m/z 50–550. Thenjector and detector temperatures were set at 250 ◦C and 280 ◦C,espectively. The column temperature was programmed from5 ◦C to 250 ◦C at a rate of 5 ◦C/min, with the lower and upperemperatures being held for 1 and 6 min, respectively. The flow

ate of the carrier gaz (helium) was 1.0 ml/min. A sample of 1 �las injected, using split less mode. All the quantifications were

arried out using a built-in data-handling program provided byhe manufacturer of the gas chromatograph. The composition

nd Products 37 (2012) 34– 40 35

was reported as a relative percentage of the total peak area. Theidentification of the hexane extract compounds was based ona comparison of their retention times to n-alkanes, comparedto published data and spectra of authentic compounds. TheseCompounds were further identified and authenticated using theirmass spectra compared to the Wiley version 7.1 library.

2.4. Study of the antioxidant power of Opuntia flowers extracts

2.4.1. Extraction of the antioxidants50 g of powdered dried flowers was mixed with 150 ml ethanol

(95%). The mixture was homogenized 24 h at 30 ◦C. After being cen-trifugated at 1000 × g for 30 min, the supernatant was collected. Theprocess of extraction is repeated twice. Supernatants were evapo-rated to dryness and residues were suspended in 500 �l of ethanol(Maisuthisakul et al., 2007). This solution was used for estimationof the antioxidant power of extracts and its content in polyphenols.

2.4.2. Determination of total phenolics contentsTotal phenolics contents of samples obtained from flowers

ethanol extracts was determined using the Folin–Ciocalteu reagentaccording to the modified method of Singleton and Rossi (1965)with gallic acid as standard. The amount of total phenolics com-pounds was calculated as mg of gallic acid equivalents (GAE) andexpressed as mg gallic acid/g dry weight (DW) of the plant material.

The calibration equation for gallic acid was y = 0.0053x − 0.0030(R2 = 0.999) where y is the absorbance and x is the concentration ofgallic acid in mg/ml.

2.4.3. Antioxidant activity evaluation: DPPH assayThe antioxidant capacity was tested by DPPH method which was

the most used for the evaluation of the antioxidant properties andantiradical activity of natural products. It is strongly correlated tothe phenolic compounds (Katalinic et al., 2006; Miliauskas et al.,2004; Maisuthisakul et al., 2007).

The DPPH radical-scavenging activity of flowers hexane extractswas determined by the method of Kirby and Schmidt (1997) withsome modifications. A volume of 500 �l of the extract at differ-ent concentrations (1.0–1000 �g/ml) was added to 375 �l of 99%methanol and 125 �l of DPPH solution (0.2 mM in methanol) asfree radical source. The mixtures were incubated for 60 min in thedark. Scavenging capacity was measured spectrophotometricallyby monitoring the decrease in absorbance at 517 nm. In its radicalform, DPPH has an absorption band at 517 nm which disappearsupon reduction by an antiradical compound. Lower absorbanceof the reaction mixture indicated higher free radical-scavengingactivity. BHA (butylated hydroxyanisole), BHT (butylated hydrox-ytoluene), and ascorbic acid were used as positive control. DPPHradical-scavenging activity was calculated as:

DPPH radical-scavenging activity = A control − A sampleA control

× 100

where “A control” is the absorbance of the control reaction (con-taining all reagents except the sample) and “A sample” is theabsorbance of the hexane extracts of Opuntia flowers. Tests werecarried out in triplicate.

2.5. Antibacterial activity determination

2.5.1. Microbial strainsAntibacterial activity of Opuntia flowers extract was tested

against four strains of bacteria and fungi kindly provided byPr. Abdelhafidh Dhouib from the Tunisian microbial collection(CTM) of the Center of Biotechnology, Sfax-Tunisia: Staphylococcusaureus (CTM 50 041), Escherichia coli (CTM 50 227), Pseudomonas

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36 I. Ammar et al. / Industrial Crops and Products 37 (2012) 34– 40

Table 1Hexane extracts obtained yields from Opuntia ficus indica and Opuntia stricta at four flowering stage.

Opuntia ficus indica Opuntia stricta

(A) (B) (C) (D) (A) (B) (C) (D)

Flowers (g) 360 587 502 147 233 653 509 30143

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Extract (g) 1.5 1.45 0.85

Yield (% DM) 2.25 1.56 1.09

eruginosa (CTM 50 238) and Bacillus subtilis (CTM 50 077). Anti-ungal activity was tested using Aspergillus niger (CTM 10 099) andandida lipolytica (CTM 30 027).

.5.2. Evaluation of microbial and antifungal activity of hexanextracts isolated from flowers: agar diffusion method

Antimicrobial activity essays were performed according to theisc diffusion method. Hexane extract (100 �l) was dissolved

n 100% dimethylsulfoxide (DMSO) (900 �l). Culture suspen-ion (200 �l) of tested microorganisms (106 colony-forming unitscfu/ml) of bacteria cells (estimated by absorbance at 600 nm) and08 spores/ml of fungal strains (measured by Malassez blade) waspread on PCA medium and PDA medium, respectively. Then, wells7 mm diameter) were made using a sterile borer and were loadedith 100 �l of Opuntia flowers extract at 100 mg/ml. A well with

nly 100 �l of DMSO (without extract) was used as a negative con-rol. Gentamycin and Neomycin were used as positive referencesor bacteria. The Petri dishes were kept, first for 1 h at 4 ◦C (Hajjit al., 2010), and then incubated for 24 h at 37 ◦C for bacteria and2 h at 30 ◦C for fungal strains. Antimicrobial activity was evaluatedy measuring the diameter of the zones inhibition in millimetersincluding well diameter of 7 mm). The measurements of zonesnhibition were carried out for three sample replications and valuesre the averages of three replicates.

. Results and discussion

.1. Chemical composition of the hexane extracts

The obtained extracts have a yellow color and a viscous form,ith a perfumery odor. In our samples, the extracts yields wereifferent at four flowering stages (Table 1). The highest yield wasoticed in the full flowering stage for O. ficus-indica (3.76% DM)hile for O. stricta the extracts yield in generally, is lower than O.

cus-indica, specially in the stage of full flowering (0.67% DM). Thextract’s yields are highly variable and depend on the species andhe flowering stage.

The hexane extracts from Opuntia flowers were analyzed byC–MS and the individual identified components, with their rel-tive percentages, are summarized in Table 2.

Fifty different components were detected. The identified com-ounds constituted about 85–99.7% of total integrated peak areaf each sample. In the view of the results, similarity of the chemi-al composition profile of all samples was shown. Qualitative anduantitative differences in the isolated composition from flowersollected in different stages were observed.

As can be seen from Table 2, the results revealed the pres-nce of secondary metabolites belonging essentially to carboxyliccids, terpenes, esters, and alcohols classes in the Opuntia flowersxtracts.

All the analyzed extracts were dominated by carboxylic acidraction (28–97%). The 9,12-octadecadienoic acid (linoleic acid)nd hexadecanoic acid (palmitic acid) are the most representa-

ive compounds (6–79%) and (5–43%) respectively. Several typesf carboxylic acids (acid octanoic, butanedioic and pentanedioic)re present only in the post flowering stage for O. ficus-indica. Hex-decanoïc acid reaches his highest concentration at full flowering

.44 0.26 1.09 0.75 1.01

.76 0.68 1.18 1.26 0.67

stage 29.9% for O. ficus-indica and 42.9% for O. stricta, this can beexplained by the development of this acid during flowering. Indeed,during the full flowering stage, all the indispensable elements tohis smell are gathered. The mentioned fatty acids, being a part ofcarboxylic acids, are often found in plants extracts. They serve asenergy sources and sometimes as defensive compounds of the plant(Alipieva et al., 2003).

The analyzed fractions showed a relatively low content of aro-matic compounds (<9.3%) which is brought with the extract. Itis essentially phenolic compounds (2-methoxy-4-vinylphenol andphenol). A low content in monoterpenes was registered, rep-resenting from 0.1 to 0.8% which are d1-limonene and linalool(oxygenated monoterpenes). The analyzed extracts are also low insesquiterpenes represented by germacrene D and aromadendrene.These last two compounds were identified by De Léo et al. (2010),emitted in vivo by dried O. ficus-indica flowers, containing 12.6% ofgermacrene D and 4% of aromadendrene.

During the vegetative stage and the initial flowering stage of O.ficus-indica specie, as well as for post flowering stage of O. strictaspecie, flowers showed the presence of squalene, in the proportionsof 23.6%, 6.8% and 3.7% respectively. Beside its role in the biosyn-thesis, squalene possesses a considerable biological role, indeed itis endowed with bactericidal and anti-tumoral activity (Harborneand Baxter, 1993).

All the studied samples contained significant amounts of hydro-carbons which are mainly saturated and in linear chains; theyvary from 14 to 22 atoms of carbons. They are present in variedproportions for the various flowering stages. The highest content(3.5%) is observed in the vegetative stage of O. ficus-indica. Twotypes of hydrocarbons which are nonadecane and heptadecadi-ene, obtained only in O. stricta flowers. Hexacosane, tetradecane,and docosane are present specifically in the vegetative stage of O.ficus-indica. Hydrocarbons found by De Léo et al. (2010) (tetrade-cane, pentadecane, hexacosane) are similar to those of our study.As for Benzoic acid, it is present only in O. ficus-indica extracts.We noticed that ethyl linoleate is the dominating ester in theflowers extracts in full and post flowering stages of O. ficus-indica (23.7% and 26.9%, respectively). Similar results were reportedby Bergaoui et al. (2007) for volatile flowers extract of O. lind-heimeri and O. microdasys, mainly constituted by esters (34.8% and34.1%, respectively) and carboxylic acids (20.7% and 17.7%, respec-tively).

Aldehydes are present as tracks in most of the studied samples.Tetradecanal in the buds extracts of the vegetative stages seems tobe replaced by the decanal in the post flowering stage (Table 2).Besides, they often possess allelochemical functions even in lowconcentrations: aldehydes can serve as attractive substances, insec-ticides, pheromones, etc., (Wang et al., 1999; Alipieva et al., 2003).The study of De Léo et al. (2010) concerning the dry Opuntia flow-ers shows more important amounts of aldehydes which are about10.7% classified in nonanal (2.5%) and decanal (8.2%).

As for alcohols, they are present in low concentrations; thephenylethyl alcohol detected fraction is known for its antimicro-

bial activity (Harborne and Baxter, 1993). In the present study thiscompound is only reported for O. stricta.

The two species extracts show some interesting features. Infact, the main compound is hexadecanoic acid in all cases, and the

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I. Ammar et al. / Industrial Crops and Products 37 (2012) 34– 40 37

Table 2Chemical Composition of Opuntia flowers extracted by hexane at four flowering stage.

Compounda RT (min)b cContent (%)

Opuntia ficus indica Opuntia stricta

(A) (B) (C) (D) (A) (B) (C) (D)

Aromatic compoundsFurane 10.75 – 0.16 – – – – – –Benzofurane 18.01 – – 0.26 – – – – –2-Methoxy-4-vinylphenol 20.28 0.13 0.30 0.83 1.05 1.23 8.92 0.3Phenol 13.79 – – 0.12 – – – 0.36 –

Monoterpenesdl-Limonene 11.87 0.10 0.54 – – – – – –Linalool L. 14.09 0.25 0.27 0.08 – – – – 0.22TerpenoideCamphor 15.52 32.4 9.03 5.74 1.14 0.1 5.01 0.21 0.45

SesquiterpenesAromadendrene 24.06 0.08 – 2.15 0.23 – – – –Germacrene D 24.57 0.76 0.28 0.16 – 0.09 0.05 0.05 –

TriterpeneSqualene 39.80 23.6 6.8 – – – – – 3.71

Carboxylic acidsButanedioic acid 16.50 – – – 1.28 – – – –Octanoic acid 16.91 – – – 0.44 – – 0.18 –Pentanedioic acid 19.30 – – – 0.63 – – – –Benzoic acid 25.49 0.35 1.38 – – – – – –Nonanoic acid 25.10 – – – 2.46 – – 0.10 0.2Tetradecanoic acid 31.54 – – 2.15 20.8 1.84 0.74 4.3 –Dodecanoic acid 33.70 – – – 1.65 0.13 0.08 0.48 5.01Hexadecanoique acid 35.45 12.4 11.8 29.9 15.6 4.6 6.43 42.9 35.6Octadecanoic acid 35.56 5.31 14.2 – 5.46 – 3.90 – –9,12,15-Octadecatrienoic acid 38.93 – – – – 15.1 – – –9,12-Octadecadienoic acid 39.20 10.1 34.5 24.7 5.73 74.9 79.4 33.44 33.45

EstersLinalyl propionate 16.78 0.53 – – – – – – –Ethyl tridecanoate 34.82 – – – 0.22 0.18 – – –Ethyl linoléate 39.08 5.68 6.63 23.7 26.9 – – – –

AldehydesNonanal 14.13 – 0.35 – 0.91 – 0.21 0.53 1.551-Borneol 15.56 0.09 – – – – –Decanal 17.13 – – – 0.07 – – – 0.14Tetradecanal 32.08 0.92 0.51 0.25 – 0.06 – –

ketonesCyclohexanone 14.13 – – 0.12 – – – – –Ethanone 24.72 0.38 0.43 – – – – 0.08 –2-Pentadecanone 32.57 – – – – 0.58 – 1.09

AlcoholsPhenylethyl alcool 14.52 – – – – – – 0.23 –1-Hydoxylinalool 21.58 – 0.48 – – – – – –8-Hydroxylinalool 21.51 0.19 0.8 – – – – –1-Heptadecanol 33.32 0.66 – – – – – – 0.611-Hexadecanol 33.31 – 0.55 – – – – – –

HydrocarbonesTetradecane 22.39 0.35 0.18 0.12 – 0.08 0.13 – 0.50Pentadecane 24.89 0.26 – 0.18 0.12 0.13 –Hexacosane 31.93 0.81 – – – – – – –Nonadecane 33.59 – – – – 0.18 0.13 0.32 0.09Heptadecadiene 28.80 – – – – – 1.6 – 0.29Docosane 39.29 2.29 – – – – – – –

Others4-Piperidinone 14.54 0.26 0.15 – – – – – –6,10,14-Trimethyl-2-pentadecanone 32.57 – – – – – – – 1.28

–: not detected; (A) the vegetative phase, (B) the initial flowering phase, (C) the full flowering phase, (D) the post-flowering phases.

electi

hsbscofa

of flowering phases and to localize the best biological activities and

a Identification of components based on GC–MS Wiely 7.1 version library.b Retention time.c Percentages were obtained from electronic integration measurements using a s

ydrocarbon class is represented in similar way. Although the twopecies contain good amounts of oxygenated terpenoids, the distri-ution is different. Even if certain compounds are present in the twopecies as the camphor and the germacrene D, the others are spe-ific for the species and are identified only in an extract or the other

ne, for example, the limonene, which is present in small quantitiesor O. ficus-indica, it is absent in the stricta specie, and esters whichre present in important quantities for O. ficus-indica, they are

ve mass detector.

absent in O. stricta. During the Opuntia flowers evolution, it is possi-ble that a number of metabolic reactions occurs coming along withmore or less important variations. Stage of development wouldprobably be an important factor to identify variations with the level

compounds aiming at greater functional properties. Our findingscan not allow us to conclude to a direct relation between maturitystage and chemical composition.

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38 I. Ammar et al. / Industrial Crops a

Table 3Content in polyphenols of ethanol extracts flowers during four stage of flowering.

Species Flowering stage Polyphenols content(mg of gallic acid/g ofextract)

Opuntia ficus indica (A) 16.30 ± 0.90(B) 13.40 ± 0.70(C) 13.30 ± 0.90(D) 49.00 ± 0.80

Opuntia stricta (A) 16.70 ± 0.40(B) 15.30 ± 0.97(C) 13.10 ± 0.43(D) 80.90 ± 1.30

Values represent averages ± standard deviations for triplicate experiments.

Table 4Reductive potential ability of Opuntia ficus indica and Opuntia stricta flower extractsat four stage of flowering and from BHA, BHT and ascorbic acid.

Species Flowering stage Antiradical activityIC50 (�g/ml)

Opuntia ficus indica (A) 205(B) 240.5(C) 100(D) 60

Opuntia stricta (A) 147(B) 62(C) 110.2(D) 50.1

Synthetic antioxidantsBHA 12.9

3

teosTA2stctfihsflerg

Table 5.

TE

Si

Ascorbic acid 15BHT 145.8

.2. Total phenolic content

It is well known that phenolic compounds contribute directlyo the antioxidant activity and therefore, it is quite important tovaluate the total phenolic content in tested extracts. The amountf total phenolics in ethanol extracts from different developmentaltages varies from 13 to 80.9 (mg of gallic acid/g of extract) (Table 3).his is in accordance with the result obtained from the flowers oflpinia zerumbet (57 mg of gallic acid/g of extract) (Elzaawely et al.,007). By comparing these contents with regard to the studiedpecies, we notice that O. stricta contains more important quan-ities in polyphenols than O. ficus-indica specie. This differencean be explained by the variation in chemical composition forhe two species. The amounts of polyphenol were similar in therst three flowering stages. The vegetative stage of both speciesas similar contents (≈16 mg of gallic acid/g of extract). Duringtages of initial and full flowering, this content decreases. For fullowering, this content reach 13.3 and 13.1 mg of gallic acid/g of

xtract of O. ficus-indica and O. stricta respectively. Same results areeported by Sellami et al. (2009) concerning sweet marjoram (Ori-anium marjorana L.). In the post-flowering stage, the polyphenols

able 5valuation of the antibacterial and antifungal activities of Opuntia flowers extracts: inhib

Species of Opuntia Opuntia ficus indica

Flowering stage (C) (D)

Pseudomonas aeruginosa 0 17.6 ±Escherichia coli 0 19.7 ±Staphylococcus aureus 14.8 ± 1.6 12.0 ±Bacillus subtilis 0 0

Aspergillus niger 0 0

Candida lipolitica 0 0

tandard+: T+: (for Bacillus): T+: Neomycine 30 �g/disque: 21 mm. (For Pseudomonas): Tnhibition.

nd Products 37 (2012) 34– 40

content significantly increased to reach the highest content (49 and81 mg of gallic/g acid of extract for O. ficus-indica and O. strictarespectively). That is can be explained by the rearrangements ofcompounds occurring during the flowering metabolism leading toa decrease or change of the phenolic compounds structures. Theseresults suggest that the post flowering stage can be characterizedby the maximum growth. In fact, it could be postulated that duringthe post flowering stage, flowers accumulate phenolics to prepareitself to the lignification process. Indeed, many studies reportedthat lignin is a polymer synthesized from phenolic compounds ofphenylpropane type and occurring under well-defined molecularassociations (Boudet, 1998; Brunow, 1998).

3.3. DPPH free radical-scavenging activity

The antiradical activity is under the influence of extract’s phe-nolic composition (Cheung et al., 2003). So, antioxidant activityof flowers extracts can be partially awarded to their contentsin phenolic compounds. Indeed, O. stricta extracts in the post-flowering stage, contain the highest content in polyphenols andpossesses the best antioxidant activity (Tables 3 and 4). How-ever, a high content of phenolic compounds does not correspondautomatically to a high antioxidant activity. Despite the initialflowering’s extract of O. stricta contains a low amount of total phe-nolics (15.3 mg of gallic acid/g of extract), the radical-scavengingactivity was important (62 �g/ml). This activity can be due, par-tially, to components others than polyphenols (Elzaawely et al.,2007). Among these components there are the flower pigmentswhich belongs to the class of flavonoïdes for example quercetinand isorhamnetin 3-glucosides and 3-rutinosides, isorhamnetin3-rhamnosylgalactoside, and kaempferol 3-galactoside (Clark andParfeitt, 1980). Besides, the antioxidant potential of the pheno-lic compounds depends on the number and the arrangements ofhydroxyls groups as well of the presence of constituents’ donors ofelectrons (Lapornik et al., 2005).

As a general rule, lower is the inhibitive concentration IC50,higher is the antioxidant power of the extract, so, post-flowersextracts of O. ficus-indica and O. stricta have the most importantactivities (IC50 = 60 and 50.1 �g/ml, respectively) (Table 4).

Compared with the synthetic reference antioxidants (Table 4),Opuntia flowers extracts have an IC50 less interesting than the BHA(12.9 �g/ml) and the ascorbic acid (15 �g/ml), while they have anactivity better than the BHT (145.8 �g/ml).

3.4. Antibacterial and antifungal activity of the extracts ofOpuntia flowers

The diameters of inhibition zones (including the diameter of thewell) of various strains with the studied extracts are presented in

The obtained results show various actions and degrees of germssensibility towards the tested extracts. The analysis of these results(Table 5) allows us to conclude that the flowers extracts present an

ition diameter in mm.

Opuntia stricta

(C) (D)

0.6 15.0 ± 0.7 14.4 ± 1.8 1.5 16.4 ± 0.9 10.5 ± 0.3

0.9 11.4 ± 1.2 00 00 11.8 ± 1.70 8 ± 0.2

+: Neomycine30 �g/disque: 25 mm. Standard−: T−: (DMSO) dimethylsulfoxide: no

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ntibacterial activity more marked towards P. aeruginosa for theost flowering stage of O. ficus-indica and for both full and postowering stages of O. stricta. Extracts present a clear antibacte-ial activity face to P. aeruginosa. On the other hand, this inhibitionemains lower than that of positive control (25 mm for Neomycine30 �g/disk)).

S. aureus is sensitive to the majority of the studied extracts,xcept O. stricta extract in post-flowering stage which has no activ-ty towards this strain. The best activity is detected for O. ficus-indicaxtract in full flowering stage. According to the study of Souissi1989), concerning the antibacterial effect of essential oil from postowering stage of O. ficus-indica towards S. aureus and P. aeruginosa,he obtained results are comparable to ours (12 mm and 16 mm ofone inhibition respectively for the two strains). We observed that,he inhibitive power face to the tested microorganisms does notepend on the gram and the morphology. Indeed we showed anntibacterial activity against S. aureus (Gram+ Coccids) as well as. coli (Gram−, Bacilli).

Extract obtained from the post flowering stage present a markedctivity for E. coli and P. aeruginosa with O. ficus-indica. So, theresent results show that the Opuntia flowers extracts are active

n diverse degrees and demonstrate an antibacterial activity bynhibiting the growth in vitro of the bacterial germs. Besides, theesistance is total for B. subtilis which is insensible for all thearious Opuntia flowers extracts while the inhibition diameter ofeomycine towards this strain is of 21 mm.

The most effective extract towards the studied microorganismseems to be the same extract which possesses the best antioxidantctivity for O. ficus-indica and O. stricta, which allows us to concludehat the concordance of these two biological properties is the resultf the richness extract in bioactive compounds.

Concerning the antifungal activity the post flowering stagextract of O. stricta, we obtained an inhibition diameter of 11.8 mmowards A. niger. Only the post flowering stage extract of O. strictaresented a low activity (8 mm) towards C. lipolytica, while for thether studied extracts there is no inhibition. So the antibacterialctivity for Opuntia extracts is more marked than the antifungalctivity.

. Conclusions

The results present in this study are the first information on thehemical composition, antioxidant and antimicrobial activities ofpuntia flowers.

As for the chemical composition, amounts and nature of com-ounds vary with flowering stages and species, suggesting changes

n secondary metabolism of flowers during flowering. Phenolicontent shows marked variation with flowering stage and the max-mum phenolic amounts is detected during post flowering stage forhe two species, this stage is also characterized by the maximum ofntioxidant activity.

In this study the hexane extracts of post and full flowering stagef Opuntia flowers show not only antifungal activity but also sig-ificant antibacterial activity against P. aeruginosa, S. aureus, and E.oli.

In conclusion, the results show that post flowering stage cor-espond to the maximum accumulation of polyphenol, antioxidantnd antibacterial activities. The richness of Opuntia flowers in inter-sting compounds can support the utilization of these flowers inarious fields of application including agro-alimentary, cosmeticnd pharmaceutical.

cknowledgement

We would like to thank M. Hela Abid teacher of English at Sec-ndary School for her proofreading assistance.

nd Products 37 (2012) 34– 40 39

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