Food Chemistry 131 (2012) 754–760

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Food Chemistry

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Antimicrobial, antioxidant and phytochemical investigations of seabuckthorn (Hippophaë rhamnoides L.) leaf, stem, root and seed

Thomas Michel a, Emilie Destandau a,⇑, Gaëtan Le Floch b,c, Marie Elisabeth Lucchesi b,d, Claire Elfakir a

a Université d’Orléans, CNRS UMR 6005, Institut de Chimie Organique et Analytique (ICOA), BP 67059, rue de Chartres, 45067 Orléans Cedex 2, Franceb Université Européenne de Bretagne, Francec Université de Brest, EA 3882 Laboratoire Universitaire de Biodiversité et d’Ecologie Microbienne, IFR148 ScInBios, ESMISAB, Technopôle Brest Iroise, 29280 Plouzané, Franced Université de Brest, EA 3887 Laboratoire d’Ecophysiologie et de Biotechnologie des Halophytes et des Algues Marines, Institut Universitaire Européen de la Mer,Technopôle Brest Iroise, 29280 Plouzané, France

a r t i c l e i n f o

Article history:Received 8 June 2011Received in revised form 22 July 2011Accepted 12 September 2011Available online 24 September 2011

Keywords:Hippophaë rhamnoidesAntimicrobialAntioxidantPhenolic compoundsProanthocyanidinsHPTLC

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.09.029

⇑ Corresponding author. Tel.: +33 238417074; fax:E-mail address: emilie.destandau@univ-orleans.fr

a b s t r a c t

The antimicrobial and antioxidant activities of crude ethanolic extract from Hippophaë rhamnoides L.(Elaeagnaceae) leaf, stem, root and seed, and their respective fractions, obtained by liquid–liquid extrac-tion (LLE) using hexane (HF), ethyl acetate (EAF) and water (WF), were investigated. The crude extractwas obtained by Pressurised Liquid Extraction (PLE), using ethanol at 100 bar and 60 �C. Antimicrobialactivity was tested against food-borne and clinical microorganisms. Antioxidant activity was measuredusing the DPPH-radical scavenging and the ferric reducing antioxidant power (FRAP) assays. The phyto-chemical contents were examined by colorimetric methods. The results showed that crude extracts wereactive against Gram � and + strains, and that seed and root extracts were better radical scavengers thanleaf and stem extracts. For all organs, the two activities tested were found to be higher in WF. Theseactivities were correlated with the presence of phenolic compounds in active fractions. High PerformanceThin Layer Chromatography (HPTLC) fingerprints confirmed presence of phenolic compounds in activeextracts and fractions.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

There is much evidence that consumption of fruits, vegetablesand other derived plant products is beneficial for human health be-cause of the presence of bioactive molecules (Crozier, Jaganath, &Clifford, 2009). These substances are secondary metabolites, bio-synthesised by plants to prevent pathogen attack, UV stress or toattract pollinator insects. The phenolic compounds including flavo-noids, phenolic acid and tannin are a major group of phytochemi-cals which exhibited strong antioxidant (Pietta, 2000) andantibacterial activities (Mayer et al., 2008; Saleem et al., 2010).Furthermore, because of the resistance that microorganisms builtagainst antibiotic and antiseptic agents (Saleem et al., 2010), anddue to the toxicity of food and cosmetic preservatives (i.e. butyl-ated hydroxyanisole and butylated hydroxytoluene, parabens)(Darbre et al., 2002; Moure et al., 2001), there is an increasing de-mand for the search of new bioactive molecules from naturalsources.

Hippophaë rhamnoides L. (Elaeagnaceae), commonly known assea buckthorn (SBT), is an Eurasian nitrogen-fixing actinomycetesplant species, producing yellow-orange berries at the end of

ll rights reserved.

+33 238417281.(E. Destandau).

summer (Rousi, 1971), from which beverages, jams, candies and cos-metics are manufactured. SBT has recently gained in interest for itsnutritional and medicinal values (Guliyev, Gul, & Yildirim, 2004;Zeb, 2004). Literature provided abundant information about healthbenefits and chemical composition of H. rhamnoides berries andseeds. For instance, they are well known for their antioxidative prop-erties, attributed to hydrophilic and lipophilic compounds includingascorbic acid, flavonoids, proanthocyanidins and carotenoids (Fan,Ding, & Gu, 2007; Gao, Ohlander, Jeppsson, Björk, & Trajkovski,2000; Michel, Destandau, & Elfakir, 2011; Rösch, Bergmann, Knorr,& Kroh, 2003). Recently the leaves of H. rhamnoides were also consid-ered for their antioxidant potential correlated to flavonoids and phe-nolic acids derivatives (Kim, Kwon, Sa, & Kim, 2011; Sharma et al.,2008; Upadhyay, Yogendra Kumar, & Gupta, 2011). Antimicrobialactivities have also been reported for SBT berries (Puupponen-Pimiäet al., 2001), seeds (Chauhan, Negi, & Ramteke, 2007; Negi, Chauhan,Sadia, Rohinishree, & Ramteke, 2005) and leaves (Upadhyay et al.,2011). Despite the well documented SBT berry and seed, data onthe other SBT organs remain disparate and unequal. In this sense,we focused our work on four SBT organs (leaf, stem, root and seed)which were simultaneously investigated for their therapeuticpotential and their phytochemical contents.

The objective was (i) to evaluate the antimicrobial capacitiesof SBT organs and fractions against food-borne and clinical

Table 1Bacterial strains and yeast used in this study.

Organism and grammorphologies

Species ATCC BCC

BacilliGram � Escherichia coli 10536 3.11.006

Pseudomonas 9027 3.11.008

T. Michel et al. / Food Chemistry 131 (2012) 754–760 755

microorganisms, (ii) to assess antioxidant activity of SBT organsand fractions via the commonly 2,20-diphenyl-1-picrylhydrazil(DPPH) free radical scavenging and ferric reducing antioxidantpower (FRAP) methods, and (iii) to investigate the composition ofSBT organs and fractions by High Performance Thin Layer Chroma-tography (HPTLC) and using the total phenolic (TPC) and con-densed tannin (CTC) contents.

aeruginosaGram + Bacillus cereus 6464 3.05.002

CocciGram + Staphylococcus aureus 25923 3.11.005

Enterococcus durans 6056 3.08.023

Yeast Candida albicans 2091 2.11.002

2. Material and method

2.1. Plant material

Sea buckthorn (H. rhamnoides L.) were purchased from the treenursery PLANFOR (Uchacg, France) and were grown outside for1 year before being harvested. The leaves, stems and roots werethen separated from each other, washed and dried at room temper-ature. The seeds, obtained from NatVit (Claret, France), are a by-product of the SBT juice production. All these organs were groundto a powder with a basic electric grinder and stored in the dark atroom temperature before use.

2.2. Chemicals

Methanol, ethanol, acetonitrile (ACN), ethyl acetate, chloroform(CHCl3) and hexane were of analytical grade and provided by SDSCarlo Erba (Val-de-Reuil, France), water (H2O) was purified (resis-tance < 18 MX) from ultra pure water using an Elgastat UHQ IIsystem (Elga, Antony, France). Formic acid (HCOOH), 2,2a-diphe-nyl-1-picrylhydrazil (DPPH) free radical, Folin–Ciocalteu reagent,sodium acetate trihydrate, sodium sulphate (Na2SO4), hydrochloricacid (HCl), b-sitosterol, gallic acid, glucose, quercetin-3-O-galacto-side, saccharose, valine and xylose were purchased from Sigma–Aldrich (Saint Quentin Fallavier, France). Ferric chloride hexahy-drate (FeCl3, 6H2O) and ferrous sulphate heptahydrate (FeSO4,7H2O) were obtained from Acros organics (Geel, Belgium). Isorham-netin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside,kaempferol, oleanolic acid and ursolic acid were supplied by Extra-synthese (Genay, France). The 2,4,6-tripyridyl-S-triazine (TPTZ) wasobtained from JT Baker Chemicals (Deventer, Holland) and sodiumcarbonate (Na2CO3) from Merck (Val de Fontenay, France).

2.3. Extraction and fractionation procedure

The extraction of SBT organs was carried out by Pressurised Li-quid Extraction (PLE), using an Accelerated Solvent Extraction (ASE100) system from Dionex (Voisins le Bretonneux, France), with a34 ml stainless steel vessel. The ground powder of each organ(3 g) was mixed with Na2SO4 (6 g) and extracted with two differentsolvents (i.e. ethanol and ethyl acetate) using five static cycles for5 min each, a flush volume of 70% and a purge with nitrogen gas of100 s at the end of each extraction. Extractions were carried out at60 �C and under a pressure of 100 bar. The liquid extract was thenevaporated at 40 �C using a rotary evaporator (Buchi LabortechnikAG, Switzerland) under vacuum to obtain a dried crude extract,which was then protected from light and stored at 2 �C beforeuse. Each extract was replicate thrice.

The conventional liquid–liquid extraction (LLE) method wasused to separate crude extracts in three fractions of different polar-ities. Ethanolic crude extract (80 mg for stems and roots, 300 mgfor leaves and seeds) was respectively dissolved in a 20 or100 ml H2O/EtOH (90/10 v:v) solution. Then, this H2O/EtOH (90/10 v:v) solution was partitioned successively with equal volumes(3 � 20 mL or 3 � 100 mL) of hexane (Hex) and ethyl acetate (EA)to constitute HF and EAF. Water fraction (WF) corresponds toaqueous residue obtained at the end of LLE.

2.4. Antimicrobial tests

Antimicrobial activity was tested against a panel of microorgan-isms, including food-borne and clinical microorganisms (Table 1):gram positive bacteria (cocci and bacilli), gram negative bacteriaand one yeast. All these microorganisms were obtained fromAmerican Type Culture Collection (ATCC) or from Brittany CultureCollection (BCC, http://www-tmp.univ-brest.fr/souchotheque).Strains were grown in liquid nutrient broth (Difco, Surrey, Eng-land), at 37 �C, for 24 and 48 h, for bacteria and yeast, respectively,before being used for antimicrobial tests. Methanolic solution ofSBT extract was dropped in sterile 96-well plate (NUNC microplate,Fisher Bioblock) at a final concentration of 100 lg/ml, and thenevaporated by slight heating. Afterwards, a 100 ll of microorgan-ism suspensions (102 cells/ml), obtained by dilution from theculture tube (108 cells/ml), were added in the wells. Cell concen-tration was estimated by haemocytometer. Antibiotic solution(mixture of streptomycin and penicillin G at 5 and 10 mg/ml,respectively) was dropped in the presence of microorganisms forpositive control. The microplate was aseptically sealed and incu-bated at 37 �C for 13, 24 or 48 h for Staphylococcus aureus, otherbacteria strains and yeast, respectively. After agitation, microor-ganism growth was estimated by reading the absorbance at405 nm with a microplate spectrophotometer (Multiskan FC, Ther-mo scientific). The results were expressed as percentage of inhibi-tion according to the formula:

%inhibition ¼ 1� ðAsample � ApcÞðAnc � ApcÞ

� �� 100

where Asample is the absorbance value of the tested solution, Apc isthe absorbance value of the positive control and Anc is the absor-bance value of the negative control. Six replicates were made foreach extract and fractions.

2.5. Antioxidant tests

For all the following tests the samples were diluted in MeOH ata 1 mg/ml concentration and all the tests were miniaturised to berealisable in sterile 96-well plate (NUNC microplate, Fisher Bio-block). All samples were analysed at least four times. The readingswere done with a microplate spectrophotometer (Multiskan FC,Thermo scientific).

2.5.1. DPPH-radical scavenging activityThe radical scavenging activity, using free-radical DPPH assay,

was determined according to the method introduced by Blois(1958). An aliquot of 2 ll of each extract was mixed with a198 ll methanolic solution of DPPH (75 lM). Extract was substi-tuted by methanol blank. Decolourisation of purple free radicalDPPH solution was measured at 517 nm after 30 min incubation

756 T. Michel et al. / Food Chemistry 131 (2012) 754–760

in the dark and at room temperature. A trolox calibration curvewas done between 0.1 and 1 mg/ml. Results were expressed inmg of trolox equivalents/g of dry extract (mg TE/g).

2.5.2. The ferric reducing antioxidant power (FRAP) assayThe FRAP assay was carried out according to the Benzie and

Strain method (1996), with some modifications. The FRAP reagentwas prepared daily from 300 mM acetate buffer (pH 3.6), 20 mMFeCl3–6H2O and 10 mM TPTZ made up in 40 mM HCl. All the abovethree solutions were mixed together in the ratio of 10:1:1 (v/v/v),respectively and then warmed at 37 �C. Extracts, standard (FeSO4–7H2O) or ultra pure water for blank (2 ll) was first mixed with ul-tra pure water (20 ll) and then with FRAP reagent (178 ll). Thechange in absorbance from the red to the blue, was followed at593 nm after 10 min. A trolox calibration curve was done between0.1 and 1 mg/ml. Results were expressed in mg of trolox equiva-lents/g of dry extract (mg TE/g).

2.6. Determination of total phenolic content (TPC)

TPC was determined by a miniaturisation of method developedby Singleton and Rossi (1965). For a 200 ll final volume, 2 ll of ex-tract (1 mg/ml) or 2 ll of aqueous standard solution (gallic acid) or2 ll of ultra pure water for blank were first mixed with 10 ll of Fo-lin–Ciocalteu reagent. 30 ll of a Na2CO3 aqueous solution (20%)was then added, between 1 and 8 min later, completed with ultrapure water. Afterwards, the blue mixture was incubated at roomtemperature for 2 h and its absorbance was read at 760 nm witha microplate spectrophotometer (Multiskan FC, Thermo scientific).Results were expressed in mg of gallic acid equivalents/g of dry ex-tract (mg GAE/g) using a linear equation based on the calibrationcurve of gallic acid from 0.1 to 0.5 mg/ml. All samples were ana-lysed at least four times.

2.7. Determination of condensed tannin content (CTC)

CTC was determined by a miniaturised procedure developed bySun, Ricardo-da-Silva, and Spranger (1998). Firstly, a 10 ll metha-nolic solution of samples (1 mg/ml), catechin or blank were intro-duced in wells. Afterwards a 4% vanillin methanolic solution(120 ll) was added, followed by addition of concentrated HCl(60 ll). The red mixture was allowed to stand for 15 min and theabsorbance was measured at 500 nm using a microplate spectro-photometer (Multiskan FC, Thermo scientific). Results were ex-pressed in mg of catechin equivalents/g of dry extract (mg CE/g)using a linear equation based on the calibration curve of catechinfrom 0.05 to 1 mg/ml. All samples were analysed at least fourtimes.

2.8. HPTLC analyses

2.8.1. Instrumentation and operating conditionsA Camag (Muttenz, Switzerland) HPTLC system equipped with

an automatic TLC sampler (Linomat 4) and a horizontal developingchamber were used for the phytochemical analyses. Before the de-posit step, plates were washed with methanol and dried. Sample(10 ll at 10 mg/ml) and standard (5 ll at 1 mg/ml) solutions werelaid on using an automated TLC sampler in 7 mm bands, at 10 mmfrom the bottom, 5 mm from the sides and with 3.5 mm space be-tween the two bands. The HPTLC plate and solvent system werechosen according to phytochemicals studied. After development,the plate was removed, dried and spots were visualised under vis-ible and UV (254 and 366 nm) light. The plates were then sprayedwith a specific reagent.

2.8.2. Amino acids and sugarsDevelopment was done on HPTLC silica gel 60 F254 10 � 20 cm

plates (Merck, Germany) in ACN:H2O (75:25 v/v). Two specific re-agents were used to detect, respectively, amino acid and sugar.Treatment of plates with ninhydrin (0.1% in EtOH) leads to aminoacid characteristic spots after heating at 120 �C for 5 min. Sugarswere then detected using Molich indicator as follows. Solution A(2 g of a-napthol in 100 ml of (v/v) EtOH) was first applied, andafter a drying step solution B (H2SO4 at 5% (v/v) in EtOH) wassprayed. The plate was then put into an oven at 120 �C, for approx-imately 5 min, to reveal sugar spots with a purple colour. Valine,xylose, glucose and saccharose were used as amino acid and sugarstandards.

2.8.3. Terpenoids and phytosterolsTerpenoids and phytosterols were separated on HPTLC silica gel

60 F254 10 � 20 cm plates (Merck, Germany) in CHCl3:MeOH:H2O(90:20:1.5 v/v), afterwards detection was done by application ofthe Liebermann–Buchard (LB) reagent. LB was prepared carefullyby adding 0.5 ml of anhydride CH3COOH and 0.5 ml of concen-trated H2SO4 in 50 ml of EtOH cooled in ice. The sprayed platewas warmed at 120 �C for 5–10 min and then looked at under vis-ible and UV (k = 366 nm) light. Oleanolic acid, b-sitosterol andursolic acid were used as terpene standards.

2.8.4. PolyphenolsAfter developing the HPTLC Lichrospher RP18 WF254s plates

(Merck, Germany) in ACN: H2O:HCOOH (50:50:5 v/v), fluorescenceof phenolic compounds was observed after application of NEU re-agent followed by PEG reagent. NEU reagent was obtained by mix-ing 1 g of diphenyl boric acid ethylamino ester in 100 ml of MeOHand PEG reagent was a solution of polyethylene glycol (PEG) 4000at 5% in EtOH. The sprayed plate was then studied under UV light(k = 366 nm). Isorhamnetin, isorhamnetin-3-O-glucoside, isorham-netin-3-O-rutinoside, quercetin-3-O-galactoside, kaempferol andgallic acid were used as polyphenolic standards.

2.9. Statistical analysis

Data were expressed as the mean ± standard deviation of atleast three measurements. One-way analysis of variance (ANOVA),correspondence analysis and determination of the Pearson correla-tion coefficient (q) was used during this work to evaluate and cor-relate results between them. Differences with p-value superior to0.05 were not considered significant. The data were statisticallyanalysed using Microsoft XLSTAT software (Microsoft Corporation,Redmond, USA).

3. Results and discussion

H. rhamnoides is a good source of bioactive compounds due toits content of various phytochemicals, however most of the litera-ture report activities from SBT berries (Gao et al., 2000; Guliyevet al., 2004). For this reason our study was focused on originalSBT organs: leaf, stem, root and seed. Furthermore, to the best ofour knowledge SBT stem and root extracts have been never inves-tigated for antimicrobial and antioxidant activities.

3.1. Extraction and fractionation of SBT crude extracts

The PLE extraction procedure, which used organic solvent athigh pressure and temperature, was chosen because it is a wellestablished technique in natural product extractions and it offersadvantages like rapidity, efficiency, weak solvent consumptionand clean-up extracts (Kaufmann & Christen, 2002). The SBT

0

5

10

15

20

25

Leaf Stem Root Seed

Extra

ctio

n yi

eld

%

EthanolEthyl acetate

Fig. 1. Extraction yield obtained by PLE of different SBT organs using two differentorganic solvents. Results are expressed as the mean ± standard deviation (n = 3).⁄⁄⁄, ⁄⁄ and ⁄ denotes significantly different at 0.1%, 1% and 5% level, respectively. a:significantly different against ethyl acetate (p < 0.05).

Table 2Mass yields of fractions obtained from the ethanolic extract of SBT leaf, stem, root andseed.

Organs Mass yield

Water (%) Ethyl acetate (%) Hexane (%)

Leaf 40.60 20.26 37.60Stem 49.75 22.75 30.75Root 29.75 22.75 25.00Seed 34.16 18.75 27.60

T. Michel et al. / Food Chemistry 131 (2012) 754–760 757

organs were extracted using two solvents ethanol and ethyl ace-tate. Fig. 1 showed that the yield of ethanol extracts were signifi-cantly higher than the yield of ethyl acetate extracts. Thisdifference could be attributed to the selectivity of extraction. Eth-anol is a polar solvent known to extract a wide range of moleculesincluding sugar, glycoside and weakly polar compounds. Whereas,ethyl acetate is an intermediary apolar solvent that extracts prefer-entially more hydrophobic compounds like aglycone and long car-bon chain ones. Such better extraction, using an alcohol solvent,has already been reported for extraction of SBT seeds (Negi et al.,2005). Furthermore, ethanol is considered as a low toxic solvent,

Table 3Antimicrobial activity of SBT ethanolic extracts (leaf, stem, root, seed) and fractions tested

Sample Bacillus cereus Pseudomonas aeruginosa Escherichia

Leaf CEE 32 ± 6 24 ± 3 42 ± 1WF 71 ± 12 24 ± 16 31 ± 8EAF 9 ± 3 34 ± 1 24 ± 11HF / / /

Stem CEE 41 ± 9 22 ± 1 39 ± 1WF 71 ± 4 45 ± 4 33 ± 17EAF 35 ± 10 36 ± 4 /HF / / /

Root CEE 45 ± 6 16 ± 3 40 ± 1WF 67 ± 6 43 ± 5 37 ± 4EAF 15.9 ± 6.6 26 ± 2 23 ± 8HF / / /

Seed CEE 64 ± 4 28 ± 9 38 ± 11WF 89 ± 5 36 ± 2 45 ± 3EAF 12 ± 9 24 ± 2 23 ± 3HF / / /

CEE: crude ethanolic extract; WF: water fraction; EAF: ethyl acetate fraction; HF: hexan

thus its use is preferable to minimise impact on the environment.In this sense, ethanol was chosen for the extraction of bioactivecompounds from SBT organs. Fig. 1 showed also that extractionwas more efficient for leaves and seeds with a yield tenfold higherthan for stems and roots. A brief temperature optimisation usingleaf sample, showed that use of increasing temperature improvedextraction yield. Temperature set at 40, 50 and 60 �C induced yieldvarying from 24% to 30% (±0.03%), respectively. The temperaturewas not set at values higher than 60 �C in order to avoid degrada-tion of thermolabile compounds. The extraction procedure wasconsequently achieved using ethanol at 100 bar and 60 �C.

In order to get information about molecular family responsibleof activity, ethanolic crude extracts of each organ were separatedin three fractions by classic LLE using hexane (HF), ethyl acetate(EAF) and water (WF). The mass yields are resumed in Table 2.For all organs, the aqueous WF was always the largest, with a massyield higher than the other fractions.

3.2. Antimicrobial activity

Table 3 showed inhibition percentage of SBT ethanolic extractsand fractions against several bacteria and one yeast, at a concentra-tion of 100 lg/ml which was the best compromise between activ-ity and concentration found. We did not use the conventionalMinimum Inhibitory Concentration (MIC) value to determine anti-microbial activity because an inhibition of 100% was never ob-tained whatever concentration (50, 100, 150, 200, 250 and300 lg/ml) and extract studied. The SBT crude extracts all depictedan antimicrobial activity with some differences between organs.The maximum inhibition (%) was varied according to organs andstrains: S. aureus (72 ± 3%) for leaf extract, Bacillus cereus(64 ± 4%) for seed extract, Enterococcus durans (63 ± 6% and68 ± 13%) for root and seed extracts, respectively. The minimuminhibition was always obtained for Pseudomonas aeruginosa, andthe less effective organ was stem. P. aeruginosa is known to easilyacquire resistance, via mutations, to a diverse class of antibiotics(Livermore, 2002). This phenomenon could explain the loweractivity of SBT ethanolic extracts observed against this strain bydevelopment of a more complex resistance mechanism. Antimicro-bial activity of SBT extracts was found against both Gram � and +strains which was already found by Negi et al. (2005) when workedon H. rhamnoides seed.

The WF was the most efficient fraction, particularly for B. cereusand S. aureus. For P. aeruginosa and Escherichia coli the activity wasfound in both WF and EAF, but the activity of WF was always high-

at 100 lg/ml, and expressed as inhibition percentage (%).

coli Staphylococcus aureus Enterecoccus durans Candida albicans

72 ± 3 40 ± 6 67 ± 785 ± 12 / /26 ± 9 / // / /

36 ± 8 34 ± 4 53 ± 1148 ± 15 / /22 ± 6 / // / /

25 ± 10 63 ± 6 55 ± 321 ± 2 / // / // / /

41 ± 12 68 ± 13 68 ± 142 ± 10 / // / // / /

e fraction.

758 T. Michel et al. / Food Chemistry 131 (2012) 754–760

er than that of EAF, except for leaf fraction against P. aeruginosa.The HF was not active against all microorganisms tested. SBT frac-tions did not affect E. durans and Candida albicans.

3.3. Antioxidant activity

To investigate the antioxidant activities of SBT organs we usedtwo different in vitro assays, the DPPH radical scavenging andthe FRAP assays. Results are resumed in Table 4.

The DPPH scavenging activity of extracts ranged from 174.8 and528.6 mg TE/g Root (500 ± 64 mg TE/g) and seed (529 ± 79 mg TE/g) extracts had significantly higher antioxidant properties than leaf(175 ± 57 mg TE/g) and stem (210 ± 42 mg TE/g) extracts(p < 0.0001). This result was correlated with FRAP assay where root(311 ± 31 mg TE/g) and seed (454 ± 82 mg TE/g) extracts were alsofound significantly more active than leaf (171 ± 19 mg TE/g) andstem (137 ± 9 mg TE/g) extracts (p < 0.0001). Significantly higheractivities were seen between seed and root (p < 0.0001) and be-tween leaf and stem (p = 0.044) with the FRAP assay. Upon organstested, antioxidant potential were determined to be in the follow-ing range seed > root > leaf > stem extracts. This result is in correla-tion with data found by Sharma et al. (2008), who revealed thatseeds have more antioxidant capacity than leaves. It can be alsonoticed that as in antimicrobial results stem extract were seen asthe least effective.

All fractions depicted antioxidant activity with the both assays.However, activity was mainly found in WF and EAF, and especiallyin WF in case of roots and seeds. For all organs, HF was regarded asthe least antioxidant, which can be explained by the presence ofno-scavenger molecules like pigments (chlorophyll) or wax. TheDPPH scavenging activity of fractions showed that WF was signif-icantly more antioxidant than EAF (p < 0.0001). On the contrary, re-sults from FRAP describe that only root and seed present a WFsignificantly more active than the EAF (p < 0.0001). No significantdifference can be seen in case of leaves and stems.

3.4. Total phenolic content (TPC) and condensed tannin content (CTC)

The TPC of SBT ethanolic extracts ranged from 65 ± 14 to139 ± 22 mg GAE/g. The TPC of root and seed extracts weresignificantly higher than leaf and stem extracts (p < 0.0001). Nosignificant differences were seen between root and seed, or be-tween leaf and stem. With regard to the TPC of fractions, the trendwas almost similar to antioxidant tests with a higher significant

Table 4Free radical scavenging activity (DPPH), ferric reducing antioxidant power (FRAP), totalextracts (leaf, stem, root, seed) and fractions.

Sample DPPH (mg troloxequivalent/g dry extract)

FRAP (mg troloequivalent/g dr

Leaf CEE 175 ± 57 171 ± 20WF 275 ± 57 148 ± 16EAF 161 ± 17 106 ± 17HF 87 ± 8 16 ± 3

Stem CEE 210 ± 42 137 ± 9WF 263 ± 19 118 ± 6EAF 172 ± 25 69 ± 10HF 41 ± 9 5 ± 1

Root CEE 500 ± 64 311 ± 31WF 304 ± 13 367 ± 32EAF 63 ± 9 5 ± 3HF 31 ± 6 2 ± 0.2

Seed CEE 529 ± 79 454 ± 82WF 368 ± 15 324 ± 41EAF 81 ± 5 28 ± 3HF 19 ± 6 7 ± 1

CEE: crude ethanolic extract; WF: water fraction; EAF: ethyl acetate fraction; HF: hexan

phenolic content for the WF (p < 0.01). Determination of Pearsoncorrelation coefficients between TPC and DPPH (q = 0.811,p < 0.0001) and between TPC and FRAP (q = 0.688, p < 0.0001) indi-cated that antioxidant activity and TPC could be correlated. Thus,bioactivities found in crude extract and WF could be attributedto phenolic compounds. This result is in correlation with previouswork on SBT leaf and seed extracts which found that antimicrobialand antioxidant potential were correlated to the presence of phe-nolic compounds (Kim et al., 2011; Negi et al., 2005; Upadhyayet al., 2011). Furthermore, from TPC data it can be observed thatdifferences between fractions were less important in contrast toantioxidant tests. For instance, no significant difference betweenEAF and HF were recorded for all organs (p > 0.01). This less consid-erable differentiation between fractions can be attributed to TPCassay, which has been described to react toward various compoundclasses including phenols, vitamins and unsaturated fatty acid(Everette et al., 2010).

The CTC assay evaluates the content in condensed tannin orproanthocyanidin (polymeric flavan-3-ol derivative) of a plant ex-tract. The CTC values of SBT ethanolic extracts varied from 13 ± 6 to176 ± 27 mg CE/g. The seed crude extract contained significantlymore condensed tannins than the other organs (p < 0.0001). Thelowest content was found in leaf extract, whereas no significantdifference was seen between stem and root (p > 0.05). All fractionsshowed a basic response to CTC assay, however only WF of seedexhibited a high content in condensed tannin that is significantagainst the other fractions (p < 0.0001). This result suggests thatantimicrobial and antioxidant activities obtained from seed crudeextract and WF could be attributed to condensed tannin presentin higher proportion in this part of the plant. A previous study(Fan et al., 2007) has also shown that proanthocyanidins werethe main antioxidant molecules of SBT seed. In addition, proanth-ocyanidins have been well recognised for their antimicrobial activ-ity (Mayer et al., 2008).

3.5. Phytochemical screening

HPTLC, combined with chemical detection, is an effective tech-nique for the phytochemical screening of plant extracts. Detectionof three molecule families was attempted using different station-ary-mobile phases and using specific derivation system. Presenceand/or absence of specific molecules were identified by matchingthe colours of spots after development and after derivatisation.The HPTLC fingerprint of SBT ethanolic extracts and fractions are

phenolic content (TPC) and condensed tannin content (CTC) of SBT crude ethanolic

xy extract)

TPC (mg gallic acidequivalent/g dry extract)

CTC (mg catechinequivalent/g dry extract

65 ± 14 13 ± 692 ± 23 5 ± 1253 ± 3 20 ± 1564 ± 2 36 ± 12

84 ± 29 26 ± 1395 ± 9 14 ± 1252 ± 17 34 ± 1664 ± 23 33 ± 16

139 ± 22 42 ± 12106 ± 14 16 ± 1336 ± 10 29 ± 1542 ± 5 20 ± 14

120 ± 14 176 ± 27138 ± 28 161 ± 1349 ± 9 39 ± 1458 ± 19 57 ± 12

e fraction.

Fig. 2. HPTLC chromatograms of SBT leaf, stem, root and seed crude extracts, its main fractions obtained after LLE, and standard compounds. Plate (a) corresponds to the sugarplates developed onto silica gel 60 F254 plates in ACN:H2O (75:25 v/v) after derivatisation with Molich reagent. Plates (b) and (c) correspond to the non-polar molecule platesdeveloped onto silica gel 60 F254 plates in CHCl3:MeOH:H2O (90:20:1.5 v/v) after derivatisation with LB reagent in visible (b) and at 366 nm (c). Plates (d) and (e) correspondto the polyphenol plates developed onto Lichrospher RP18 WF254s plates in ACN:H2O:HCOOH (50:50:5 v/v) before derivatisation at 366 nm (d) and after derivatisation withNEU + PEG reagents at 366 nm (e). Tracks: 1, valine (v); 10 , oleanolic acid (oa), isorhamnetin (i); 100 , isorhamnetin, isorhamnetin-3-O-glucoside (i3g), isorhamnetin-3-O-rutinoside (i3r); 2, leaf crude extract; 3, leaf WF; 4, leaf EAF; 5, leaf HF; 6, stem crude extract; 7, stem WF; 8, stem EAF; 9, stem HF; 10, root crude extract; 11, root WF; 12, rootEAF; 13, root HF; 14, seed crude extract; 15, seed WF; 16, seed EAF; 17, seed HF; 18, xylose (x), glucose (g), saccharose (s); 180 , b-sitosterol (bs), ursolic acid (ua); 1800 , gallicacid (ga), quercetin-3-O-galactoside (q3g), kaempferol (k). WF, EAF and HF correspond respectively to water, ethyl acetate and hexane fractions.

T. Michel et al. / Food Chemistry 131 (2012) 754–760 759

760 T. Michel et al. / Food Chemistry 131 (2012) 754–760

shown in Fig. 2. HPTLC analyses of crude extract showed that theywere constituted of sugars, terpenoids and phenolic compounds,and that their phenolic fingerprints were different from each othernotably between leaf and other organ extracts.

With regard to fractions, HPTLC analyses provided informationon their phytochemical constituents. Sugars, especially mono-and disaccharides, were found in WF. Terpenoids and sterols weredetected after derivatisation in EAF and HF. Other non-polar com-pounds were present in crude extract and fractions, like chloro-phyll, exhibiting characteristic red-pink spots, in leaf and stemfingerprints. In addition, it seems that extracts and fractions weremostly constituted of phenolic compounds, which are partitionedin all fractions. Furthermore, HPTLC comparisons showed clearlythat the major phenolic compounds of crude extract were foundin WF, confirming that phenolic derivatives could be responsibleof the activities of SBT organs.

4. Conclusion

Present work described the evaluation of antimicrobial andantioxidant activities of SBT leaf, stem, root and seed and theirrespective fractions, as well as their phytochemical investigations.To the best of our knowledge it was the first time that SBT organswere investigated simultaneously in such a manner. It has beendemonstrated that SBT leaf, stem, root and seed present good anti-microbial and antioxidant activities. Seed and root extracts wereseen as the most antioxidant organs and stem as the least antimi-crobial and antioxidant one. In order to elucidate bioactive mole-cules crude extracts were partitioned by LLE. When fractionswere analysed the bioactivity was mainly found in the aqueousfraction WF. The good correlation found between activity and phy-tochemical contents indicates that effects observed could be attrib-uted to phenolic compounds. Furthermore, in the case of seedthese effects could be due to proanthocyanidins. HPTLC finger-prints confirmed that WF was mainly constituted of phenolic com-pounds and that it was constituted of the same major moleculescompared to crude extracts. Nevertheless, further works areneeded to identify bioactive molecules, especially in root, that pre-sents a strong activity and that was never investigated before. Be-sides, its well known fruits and the other organs of H. rhamnoideswere also seen to constitute potent bioactive compounds. The re-sults indicate that SBT leaf, stem, root and seed can be regardedas a natural source of antimicrobials and antioxidants and maybe considered in future to replace synthetic preservatives in food,cosmetic and pharmaceutic products.

Acknowledgements

The authors wish to thank gratefully the society NatVit (Claret,France) for providing by-product materials obtained from its SBTjuice production.

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