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ORIGINAL ARTICLE/ARTICLE ORIGINAL Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds Activite ´ antibacte ´ rienne et antifongique des fruits et des graines immatures de sept populations tunisiennes de Citrullus colocynthis Schrad B. Marzouk a,1, * , Z. Marzouk b,1 , R. Décor c , L. Mhadhebi d , N. Fenina b , M. Aouni a a Laboratoire des maladies transmissibles et des substances biologiquement actives, faculte ´ de pharmacie de Monastir, rue Avicenne, 5000 Monastir, Tunisia b Unite ´ de recherche de pharmaco-e ´conomie et de ´veloppement des me ´dicaments, laboratoires de biologie ve ´ge ´tale et de pharmacologie, faculte ´ de pharmacie de Monastir, Monastir, Tunisia c BCT Consulting, rue du 13-Aout-1956, 5000 Monastir, Tunisia d Unite ´ de recherche de substances actives marines, laboratoire de pharmacologie, faculte ´ de pharmacie, Monastir, Tunisia Received 13 March 2010; received in revised form 21 May 2010; accepted 24 May 2010 Available online 24 July 2010 KEYWORDS Citrullus colocynthis Schrad.; Geographical distributions; Immature fruit; Immature seed; Flavonoids; Alkaloids; Antibacterial activity; Anticandidal activity Summary Objective. Skin infections and gynaecological and pulmonary problems produced by micro- organisms are widespread in the entire globe. The treatment of these infections is mainly based on the use of synthetic drugs, many of which have lost their effectiveness, in recent years, due to the development of resistant strains, and to the rise of opportunistic fungal infections. The aim of this study was to assess in vitro antibacterial and anticandidal activity of aqueous immature fruit and seed extracts, screened for the alkaloid and flavonoid levels, of seven populations of Citrullus colocynthis Schrad. Material and method. C. colocynthis Schrad. samples were harvested and their identification verified. Aqueous extracts (from immature fruits and seeds) were screened for activity against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Enterococcus faecalis and Staphylococcus aureus) bacteria and four Candida spp. (Candida glabrata, Candida albicans, Candida parapsilosis and Candida krusei). Microdilution method was used to investigate the antimicrobial activity. Journal de Mycologie Médicale (2010) 20, 179184 * Corresponding author. E-mail addresses: [email protected] (B. Marzouk), [email protected] (R. Décor). 1 Same contributions. 1156-5233/$ see front matter # 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2010.05.006

Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

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Page 1: Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

ORIGINAL ARTICLE/ARTICLE ORIGINAL

Antibacterial and antifungal activities of severalpopulations of Tunisian Citrullus colocynthis Schrad.immature fruits and seedsActivite antibacterienne et antifongique des fruits et des graines immatures desept populations tunisiennes de Citrullus colocynthis Schrad

B. Marzouk a,1,*, Z. Marzouk b,1, R. Décor c, L. Mhadhebi d,N. Fenina b, M. Aouni a

a Laboratoire des maladies transmissibles et des substances biologiquement actives, faculte de pharmacie de Monastir,rue Avicenne, 5000 Monastir, TunisiabUnite de recherche de pharmaco-economie et developpement des medicaments, laboratoires de biologie vegetale et depharmacologie, faculte de pharmacie de Monastir, Monastir, Tunisiac BCT Consulting, rue du 13-Aout-1956, 5000 Monastir, TunisiadUnite de recherche de substances actives marines, laboratoire de pharmacologie, faculte de pharmacie, Monastir, Tunisia

Received 13 March 2010; received in revised form 21 May 2010; accepted 24 May 2010Available online 24 July 2010

KEYWORDSCitrullus colocynthisSchrad.;Geographicaldistributions;Immature fruit;Immature seed;Flavonoids;Alkaloids;Antibacterial activity;Anticandidal activity

SummaryObjective. — Skin infections and gynaecological and pulmonary problems produced by micro-organisms are widespread in the entire globe. The treatment of these infections is mainly basedon the use of synthetic drugs, many of which have lost their effectiveness, in recent years, due tothe development of resistant strains, and to the rise of opportunistic fungal infections. The aimof this study was to assess in vitro antibacterial and anticandidal activity of aqueous immaturefruit and seed extracts, screened for the alkaloid and flavonoid levels, of seven populations ofCitrullus colocynthis Schrad.Material and method. — C. colocynthis Schrad. samples were harvested and their identificationverified. Aqueous extracts (from immature fruits and seeds) were screened for activity againstGram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Enterococcusfaecalis and Staphylococcus aureus) bacteria — and four Candida spp. (Candida glabrata,Candida albicans, Candida parapsilosis and Candida krusei). Microdilution method was usedto investigate the antimicrobial activity.

Journal de Mycologie Médicale (2010) 20, 179—184

* Corresponding author.E-mail addresses: [email protected] (B. Marzouk), [email protected] (R. Décor).

1 Same contributions.

1156-5233/$ — see front matter # 2010 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.mycmed.2010.05.006
Page 2: Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

Results. — Quantitative screening proved that Medenine Citrullus population was the richest inalkaloids while Hammamet Citrullus population presented the highest flavonoid level. AllC. colocynthis extracts showed antibacterial and anticandidal properties against the testedstrains. However, samples from southern populations were the most active.Conclusion. — This study provides scientific insight into the ancient practice of usingC. colocynthis Schrad. immature fruits and seeds as an antimicrobial agent and demonstratethe geographical distribution influence on the chemical composition and therefore, on theantimicrobial efficacy.# 2010 Elsevier Masson SAS. All rights reserved.

MOTS CLÉSCitrullus colocynthisSchrad. ;Distributionsgéographiques ;Fruit immature ;Graine immature ;Flavonoïdes ;Alcaloïdes ;Activitéantibactérienne ;Activité anti-Candida

Resume

Objectif. — Les infections cutanées, gynécologiques et pulmonaires causées par les micro-organismes existent dans le monde entier. Le traitement de ces infections est principalementbasé sur l’utilisation des médicaments synthétiques qui deviennent, ces dernières années, peuefficaces, à cause de la résistance des souches bactériennes et la multiplication des champignonsopportunistes. Le but de ce travail est de tester les activités antibactérienne et anti-Candida, invitro, des extraits aqueux des fruits et des graines immatures de sept populations de Citrulluscolocynthis Schrad. dont on a déterminé les teneurs en alcaloïdes et en flavonoïdes.Materiel et methodes. — Ces pouvoirs antimicrobiens sont évalués selon la méthode de micro-dilution vis-à-vis de bactéries Gram-négatives (Escherichia coli et Pseudomonas aeruginosa) etGram-positives (Enterococcus faecalis et Staphylococcus aureus) et vis-à-vis de diverses espècesde Candida (Candida glabrata, Candida albicans, Candida parapsilosis et Candida krusei).Resultats. — Les résultats montrent que c’est la population de Médenine qui est la plus riche enalcaloïdes tandis que la population de Hammamet renferme la teneur la plus élevée enflavonoïdes. Tous les extraits testés de C. colocynthis montrent une activité antimicrobiennevis-à-vis de toutes les souches expérimentées. Les populations du Sud tunisien sont les plusactives.Conclusion. — Cette évaluation confirme que la composition chimique et le pouvoir antimicro-bien de la coloquinte sont influencés par la distribution géographique de cette espèce.# 2010 Elsevier Masson SAS. Tous droits réservés.

180 B. Marzouk et al.

Introduction

Many plants used in Tunisian traditional medicine have thepotential to provide pharmacologically active natural pro-ducts. Interest in ethnopharmacy as a source of these com-pounds has increased worldwide, particularly in the searchfor drugs to counter multi-resistant microorganisms. Plantswith antimicrobial activities have become more interestingbecause many people are aware of problems associated withthe over-prescription and misuse of traditional antibiotics.Nevertheless, only approximately 20% of the plants found inthe world have been submitted to pharmacological or bio-logical testing [10].

Citrullus colocynthis Schrad., belonging to the family ofCucurbitaceae, is endemic in the south of Tunisia [13]. Thismedicinal plant popularly known as Handhal, Hdaj or DellaaEl-Wad, is widely used in Tunisian folk medicine for treatingmany diseases such as rheumatism [8,4], hypertension [4]and various contagious diseases, including dermatologicalproblems and gynaecological, urinary and pulmonary infec-tions [8,4,9].

In Tunisia, as in the rest of the Mediterranean region [2],the parts of plants most often used for medicinal purposesare fruits and/or seeds. In a previously published paper,immature fruits and seeds are demonstrated the mostefficient of all [9].

However, plants in the environment are exposed to arange of abiotic stresses like osmotic, salinity, temperatureand heavy metal toxicity which affects their growth and

other metabolism process such as alkaloid and flavonoidproductions. These secondary metabolites are synthesizedby plants to defend themselves against the harmful action ofexternal agents [7,15]. Thus alkaloid and flavonoid contentsdepend on the geographical distributions.

The current study quantified the alkaloid and flavonoidcontents and measured the in vitro antibacterial and antic-andidal activities of seven populations of C. colocynthisSchrad. using the broth serial dilution (microdilutionmethod). Preparation and testing were carried out on thereconstituted lyophilized aqueous extracts.

Material and methods

Plant material

C. colocynthis Schrad. plants were collected in August 2007from seven stations (Table 1 and Fig. 1). The identificationwas performed according to the flora of Tunisia [13] andvoucher specimens deposited in the biological laboratory ofthe Faculty of Pharmacy of Monastir.

Extraction protocol

The extraction was performed on 100 g of fresh immaturefruits and seeds separately. For preparing the aqueousextract, organs were ground with a mixer and added to500 ml of distilled water. The mixture was allowed to refluxfor 30 min, after which the solution was allowed to cool (4 h

Page 3: Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

[(Figure_1)TD$FIG]

Figure 1 Geographical position of the Citrullus colocynthisSchrad populations.Position geographique des prelevements de Citrullus colo-cynthis Schrad.

Table 1 Sites and voucher specimens of Citrullus colo-cynthis Schrad. populations.Origine geographique des echantillons de Citrullus colocynthisSchrad.

Population Site Voucherspecimens

Hammamet Hammamet Sud(36825 N, 10834 W)

C.C-07.07

Mahdia Chorban (35820 N, 10832 W) C.C-06.07Kasserine Mozgam (35811 N, 8846 W) C.C-05.07Sbeitla Sbitla (35814 N, 9807 W) C.C-04.07Sidi-Bouzid Jelma (35816 N, 9825 W) C.C-03.07Sfax Skhira (34810 N, 10804 W) C.C-02.07Medenine Sidi Makhlouf

(33833 N, 10827 W)C.C-01.07

N: north; W: west.

Antimicrobial activity of several populations of Citrullus colocynthis Schrad 181

at 4 8C). The mixture was then filtered using filter paper(Whatman No. 1) under the vacuum of a water pump. Thefiltrate obtained was lyophilized, yielding the lyophilizedaqueous extract. Yields are given in Table 2.

Quantitative alkaloid screening

Alkaloids were quantified according to the volumetric pro-cedure of the Official Method of European Pharmacopoeia.Briefly, on 3 g of each dried organ, ammonium hydroxide(5 ml), ethanol (10 ml) and diethyl ether (30 ml) were suc-cessively added. After 4 h of maceration the mixture waslixiviated by 150 ml of ether and 50 ml of chloroform. Theobtained solution was reduced to 50 ml by a rotavapor(Heidolph) and then treated by sulphuric acid (0.5 N,3 � 20 ml). Bases were removed from the extract throughprecipitation with an excess of ammonium hydroxide (3 ml).The supernatant was then extracted with chloroform(3 � 10 ml). The precipitate containing alkaloids was sepa-rated by means of filtration and treated again by chloroformwhich was evaporated under low pressure. The presence ofalkaloids was verified by Dragendorff’s reagent confirmedwith Bouchardat’s and Meyer’s reagents. On the residue,20 ml of sulphuric acid were added. The excess of sulphuricacid was titrate by sodium hydroxide (0.02 N) using thephenolphthalein indicator. The percentage of total alka-loids was then calculated according to the followingformula: alkaloid le ð%Þ ¼ VH2SO4 � VH2SO4ð Þ � 5:788 : Wð Þ;

Table 2 Extraction yields (w/w %) of alkaloid and flavonoid of CRendements (w/w %) d’extraction des alcaloıdes et des flavonoıd

Population of Citrullus colocynthis Schrad.

Hammamet Mahdia Kasserine

Seeds Fruits Seeds Fruits Seeds Fruits

Extraction yields 1.68 0.93 1.90 1.70 2.88 2.94Alkaloid levels 2.01 1.57 3.10 2.08 2.74 1.61Flavonoid levels 0.24 — 0.18 — 0.18 —

Extraction yields: the lyophilized aqueous extract weights are determinacalculated according to formulas cited in the text.

with VH2SO4 = 20 ml, VH2SO4 ex = volume of sulphuric acidexcess, W = 3 g and 1 ml of sulphuric acid correspond to5.788 mg alkaloids.

Quantitative oligo-flavonoid screening

In order to obtain total oligomer flavonoids (TOF), thepowdered organs (100 g) were macerated separately for

itrullus colocynthis Schrad.es de Citrullus colocynthis Schrad.

Sbeitla Sidi-Bouzid Sfax Medenine

Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits

1.93 2.68 1.76 2.44 2.15 2.81 2.94 2.762.48 1.55 2.90 1.71 3.90 2.86 3.87 2.900.20 — 0.17 — 0.13 — 0.10 —

ted and than yields are calculated; alkaloid and flavonoid levels are

Page 4: Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

182 B. Marzouk et al.

24 h in a mixture of acetone/water (2:1). Each extract wasthen filtered and the acetone was evaporated under lowpressure. Tannins were removed from the aqueous phasethrough precipitation with NaCl for 24 h at 5 8C and thesupernatant was then extracted with ethyl acetate, concen-trated, and precipitated with an excess of chloroform. Theprecipitate containing TOF was separated by means of filtra-tion [12]. The presence of flavonoids was verified by thereaction of ‘cyanidine’ in the presence of hydrochloric acidand of magnesium [14]. After release of hydrogen, a colora-tion of orange with the red purple indicates the presence ofthe flavonoids. The developing of the spots corresponding tothe flavonoids on the plates of thin layer chromatography(TLC) is done by pulverization using a solution of AlCl3 inmethanol 2%. The plates of TLC are then observed under UVwith 366 nm: the spots corresponding to the flavonoids havea yellow fluorescence at an intense yellow fluorescence [12].The percentage of TOF was then calculated according to thefollowing formula: total oligomer flavonoids level(%) = (weight of total oligomer flavonoids/powdered organweight) � 100.

Antibacterial and anticandidal activities

MicroorganismsFour reference strains were chosen for antibacterial inves-tigation: two Gram-positive cocci (Enterococcus faecalis,ATCC 29212 and Staphylococcus aureus, ATCC 25923) andtwo Gram-negative bacilli (E. coli, ATCC 25922 and Pseudo-monas aeruginosa, ATCC 27853).

Table 3 Antibacterial minimal inhibitory concentration (MIC) (mglyophilized aqueous extracts.CMI (mg/mL) et CMB (mg/mL) antibacterienne des extraits aque

Gram-negative bacilli

E. coli P. aeru

ATCC 25922 ATCC

MIC MBC MIC

Hammamet Seeds 1.63 3.25 0.20Fruits 1.63 3.25 3.25

Mahdia Seeds 3.25 6.50 1.36Fruits 1.63 3.25 3.25

Kasserine Seeds 1.63 6.50 3.25Fruits 1.63 3.25 1.63

Sbeitla Seeds 1.63 3.25 1.63Fruits 0.81 1.63 1.63

Sidi-Bouzid Seeds 1.63 3.25 0.81Fruits 0.81 1.63 1.63

Sfax Seeds 0.41 0.81 0.41Fruits 0.20 0.41 0.20

Medenine Seeds 0.41 0.81 0.81Fruits 0.20 0.41 0.20

E. coli: Echerichia coli; P. aeruginosa: Pseudomonas aeruginosa; S. aurepositive control: levofloxacin (E. coli: 0.61 mg/ml; P. aeruginosa: 0.3 m

The antifungal effect of the various C. colocynthis Schrad.extracts was also tested against four pathogenic referenceyeasts (Candida albicans ATCC 90028, Candida glabrata ATCC90030, Candida krusei ATCC 6258 and Candida parapsilosisATCC 22019).

Minimal inhibitory concentration (MIC) and minimalbactericidal concentration (MBC)/minimal fungicidalconcentration (MFC) determinationsThe use of antifungal susceptibility testing against fungi hasincreased during the last few years. The reference methoddeveloped by the Clinical and Laboratory Standards Institute(CLSI), is time consuming and needs many reagents, whichmay pose a problem for the laboratories of institutions with alarge number of samples to process. This is the main reasonfor exploring other methodologically easier methods such asthe E test, the sensitive yeast test and the microdillutionmethod using 96-well microplates.

In the current study, the MIC preventing visible bacterialor fungal growth was measured by the broth dilution method[3,6]. All lyophilized aqueous extracts stock solution wereprepared by dissolution in 10% dimetyl sulfoxyde (DMSO). Theplant extracts concentrations tested ranged from 0.10 to6.50 mg/ml. The MIC of each extract was defined as thelowest concentration which inhibited either bacterial orCandida growth, after incubation at 37 8C. between 18 to24 h. The MBC and the MFC were determined by subcultureon blood agar at 37 8C between 18 and 24 h. Levofloxacin wasused as antibacterial positive control, and amphotericin B forthe anticandidal one.

/ml) and minimal bactericidal concentration (MBC) (mg/ml) of

ux lyophilises.

Gram-positive cocci

ginosa S. aureus E. faecalis

27853 ATCC 25923 ATCC 29212

MBC MIC MBC MIC MBC

0.41 0.41 0.81 1.63 3.256.50 3.25 6.50 3.25 6.50

3.25 3.25 6.50 1.63 3.256.50 3.25 6.50 3.25 6.50

6.50 0.81 1.63 1.63 3.253.25 1.63 3.25 3.25 6.50

3.25 0.81 1.63 0.81 1.633.25 1.63 3.25 0.81 1.63

1.63 0.41 0.81 0.81 1.633.25 1.63 3.25 1.63 3.25

0.81 0.81 1.63 0.81 1.630.41 0.81 1.63 0.81 1.63

1.63 0.81 1.63 0.81 1.630.41 0.81 1.63 0.41 0.81

us: Staphylococcus aureus; E. faecalis: Enterococcus faecalis. MICg/ml; S. aureus: 0.3 mg/ml; E. faecalis: 1.22 mg/ml).

Page 5: Antibacterial and antifungal activities of several populations of Tunisian Citrullus colocynthis Schrad. immature fruits and seeds

Antimicrobial activity of several populations of Citrullus colocynthis Schrad 183

Results

Extraction yields and quantitative alkaloid andoligo-flavonoid screening

Seeds and fruits from Kasserine, Sfax and Medenine pre-sented significantly much higher extraction yields than theother populations. Hammamet populations show the low-est yields (Table 2). Alkaloids presence was reported in allC. colocynthis organs except the roots [9] namely imma-ture seeds and fruits whose alkaloids are quantified in thisstudy. Results (reported in Table 2) were dependent uponthe population. Sfax (3.90 and 2.86%, respectively forseeds and fruits) and Medenine (3.87 and 2.90% corre-spondingly for seeds and fruits) were the richest in alka-loids contents, but Hammamet organs presented thelowest amounts of all. Differences were also notedbetween plant organs: seeds showed the better percen-tages (from 2.01 to 3.90%) than fruits (from 1.55 to 2.90%).Flavonoids which are only detected in seeds [9] werepresent just at low levels (from 0.10 to 0.24%, Table 2).Hammamet was the population richer in these compo-nents.

Antibacterial activity

All the extracts tested from Tunisian C. colocynthis Schrad.showed antibacterial activity against all tested strains. MICand MBC were tested for concentrations ranging from 0.10 to6.50 mg/ml (see Table 3). The strongest inhibitions wereobtained with Sfax and Medenine. The lowest activity was

Table 4 Antifungal minimal inhibitory concentration (MIC) (mglyophilized aqueous extracts.CMI (mg/mL) et CMB (mg/mL) anti-Candida des extraits aqueux l

C. albicans C. glab

ATCC 90028 ATCC

MIC MBC MIC

Hammamet Seeds 0.41 0.81 0.81Fruits 0.81 1.63 0.41

Mahdia Seeds 0.81 0.81 0.20Fruits 0.81 1.63 0.41

Kasserine Seeds 0.41 0.81 0.81Fruits 0.20 0.41 0.20

Sbeitla Seeds 3.25 6.50 1.63Fruits 0.20 0.41 0.20

Sidi-Bouzid Seeds 0.20 0.41 0.20Fruits 0.20 0.41 0.81

Sfax Seeds 0.20 0.41 0.20Fruits 0.41 0.81 0.41

Medenine Seeds 0.20 0.41 0.20Fruits 0.41 0.81 0.41

C. albicans: Candida albicans; C. glabrata: Candida glabrata; C. kruscontrol with amphotericin B (MFC, 0.5 mg/ml).

observed for seeds and fruits of Mahdia and Hammamet fruits(MIC ranging from 1.63 to 3.25 mg/ml). The plant organs withthe highest antibacterial properties were immature fruits forSfax and Medenine and immature seeds for Hammamet,Kasserine and Sidi-Bouzid. Seeds and fruits of Sbeitla andMahdia presented close activities. The most active organs ofall were Medenine immature fruits. The strongest inhibitionsfor the most active populations (Sfax and Medenine) wereobtained against E. coli and P. aeruginosa with immaturefruits. For both strains, the MIC was of 0.20 mg/ml and theMBC of 0.41 mg/ml.

Antifungal activity

Anticandidal property is reported as MIC and MFC (Table 4).All extracts showed significant antifungal activity against alltested yeasts. Overall, the best populations with antifungalactivities were Sidi-Bouzid and Sfax (for both immature seedsand fruits). For all extracts, MIC are ranged from 0.20 to1.63 mg/ml. Immature seeds were as efficient as immaturefruits for Mahdia and Medenine. Immature fruits were moreanticandidal than immature seeds for Kasserine and Sbeitla.A close property was noted with seeds and fruits for Ham-mamet, Sidi-Bouzid and Sfax.

Discussion and conclusion

Tunisia recently increased research in Traditional HerbalMedicines following scientific confirmation of their effec-tiveness in treating conditions for which they were tradi-tionally prescribed. The present investigation has explored

/ml) and minimal fungicidal concentration (MFC) (mg/ml) of

yophilises.

rata C. krusei C. parapsilosis

90030 ATCC 6258 ATCC 22019

MBC MIC MBC MIC MBC

1.63 0.81 1.63 0.41 0.810.81 0.41 0.81 0.81 1.63

0.41 0.41 0.81 0.20 0.410.81 0.41 0.81 0.41 0.81

1.63 0.41 0.81 0.81 1.630.41 0.20 0.41 0.41 0.81

3.25 1.63 3.25 3.25 3.250.41 0.81 1.63 1.63 3.25

0.41 1.63 3.25 0.81 1.630.81 0.81 1.63 1.63 3.25

0.41 0.20 0.41 0.41 0.810.81 0.20 0.41 0.20 0.20

0.81 0.81 1.63 0.20 0.410.81 0.41 0.41 0.41 0.81

ei: Candida krusei; C. parapsilosis: Candida parapsilosis. Positive

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184 B. Marzouk et al.

the use of one such plant, C. colocynthis Schrad., endemic inTunisia, for treating infectious diseases.

The antimicrobial and anticandidal screening showedinteresting activities at low concentrations. The study clar-ified complexes of ethnopharmacology and biodiversity pic-tures in terms of plant organs and plant geographicaldistribution. Results previously obtained [9] showed theimportance of organs: immature seeds and fruits, the com-positions of fruits and seeds differed, as made obvious bytheir extraction yields and antimicrobial and anticandidalactivities. In addition, the present investigation showed, fora given organ, that the antimicrobial effect can change froma population to another one.

For the antibacterial activity, the plant extracts wereactive against both Gram-positive and Gram-negative bac-teria, though more against the latest. Activity dependedfrom the bacterial strain, the plant organ and the population.Concerning the anticandidal property, the present studyshowed MICs and MFCs significant for crude plant extractsagainst all strains and for all populations with some differ-ences.

These antimicrobial effects may be attributed, possibly incombination, to various phytochemicals detected during theextracts chemical screening and which are known to causedamage to cell membranes, causing leakage of cellularmaterials and ultimately the microorganism death [11,1].The trend over all the strains to have higher activity forimmature fruit aqueous extracts and immature seeds onespoints towards various active, potentiate or antagonisticcompounds present in various concentrations according tothe plant population, especially alkaloids [16] and flavonoids[5] which possess antimicrobial properties. These two phy-tochemical key families are quantified and results indicatedthat the geographical distribution of C. colocynthis Schrad.influenced secondary metabolite levels and subsequentlytheir biological activities.

With all these wide spectrum antibacterial and antic-andidal properties, C. colocynthis Schrad. can be consideredan effective antimicrobial agent to treat infectious diseases.Differences in the composition levels and in the activitiesmay be attributed to climatic conditions. Despite the com-plexity of the chemistry of the inorganic and organic nature,it is possible to find some general trends like genetics andbreeders which are more and more interested to the linkbetween genetics factors and phenotypic variation of qua-litative and quantitative composition.

Conflict of interest statement

The authors have not declared any conflict of interest.

Acknowledgements

We would like to thank Pr. Abderrahman Bouraoui andPr. Maha Mastouri for their precious help to realize this work.

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