Food Chemistry 131 (2012) 754760

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Food Chemistry
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Antimicrobial, antioxidant and phytochemical investigations of sea

buckthorn (Hippopha rhamnoides L.) leaf, stem, root and seed
Thomas Michel a, Emilie Destandau a,, Gatan Le Floch b,c, Marie Elisabeth Lucchesi b,d, Claire Elfakir a
a
Universit dOrlans, CNRS UMR 6005, Institut de Chimie Organique et Analytique (ICOA), BP 67059, rue de Chartres, 45067 Orlans Cedex 2, France
b
Universit Europenne de Bretagne, France
c
Universit de Brest, EA 3882 Laboratoire Universitaire de Biodiversit et dEcologie Microbienne, IFR148 ScInBios, ESMISAB, Technople Brest Iroise, 29280 Plouzan, France
d
Universit de Brest, EA 3887 Laboratoire dEcophysiologie et de Biotechnologie des Halophytes et des Algues Marines, Institut Universitaire Europen de la Mer,
Technople Brest Iroise, 29280 Plouzan, France

a r t i c l e i n f o a b s t r a c t

Article history: The antimicrobial and antioxidant activities of crude ethanolic extract from Hippopha rhamnoides L.
Received 8 June 2011 (Elaeagnaceae) leaf, stem, root and seed, and their respective fractions, obtained by liquidliquid extrac-
Received in revised form 22 July 2011 tion (LLE) using hexane (HF), ethyl acetate (EAF) and water (WF), were investigated. The crude extract
Accepted 12 September 2011
was obtained by Pressurised Liquid Extraction (PLE), using ethanol at 100 bar and 60 C. Antimicrobial
Available online 24 September 2011
activity was tested against food-borne and clinical microorganisms. Antioxidant activity was measured
using the DPPH-radical scavenging and the ferric reducing antioxidant power (FRAP) assays. The phyto-
Keywords:
chemical contents were examined by colorimetric methods. The results showed that crude extracts were
Hippopha rhamnoides
Antimicrobial
active against Gram  and + strains, and that seed and root extracts were better radical scavengers than
Antioxidant leaf and stem extracts. For all organs, the two activities tested were found to be higher in WF. These
Phenolic compounds activities were correlated with the presence of phenolic compounds in active fractions. High Performance
Proanthocyanidins Thin Layer Chromatography (HPTLC) ngerprints conrmed presence of phenolic compounds in active
HPTLC extracts and fractions.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
There is much evidence that consumption of fruits, vegetables
and other derived plant products is benecial 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 to
attract pollinator insects. The phenolic compounds including avo-
noids, phenolic acid and tannin are a major group of phytochemi-
cals which exhibited strong antioxidant (Pietta, 2000) and
antibacterial activities (Mayer et al., 2008; Saleem et al., 2010).
Furthermore, because of the resistance that microorganisms built
against antibiotic and antiseptic agents (Saleem et al., 2010), and
due 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 natural
sources.
Hippopha rhamnoides L. (Elaeagnaceae), commonly known as
sea buckthorn (SBT), is an Eurasian nitrogen-xing actinomycetes
plant species, producing yellow-orange berries at the end of
Corresponding author. Tel.: +33 238417074; fax: +33 238417281.
E-mail address: emilie.destandau@univ-orleans.fr (E. Destandau).
summer (Rousi, 1971), from which beverages, jams, candies and cos-
metics are manufactured. SBT has recently gained in interest for its
nutritional and medicinal values (Guliyev, Gul, & Yildirim, 2004;
Zeb, 2004). Literature provided abundant information about health
benets and chemical composition of H. rhamnoides berries and
seeds. For instance, they are well known for their antioxidative prop-
erties, attributed to hydrophilic and lipophilic compounds including
ascorbic acid, avonoids, proanthocyanidins and carotenoids (Fan,
Ding, & Gu, 2007; Gao, Ohlander, Jeppsson, Bjrk, & Trajkovski,
2000; Michel, Destandau, & Elfakir, 2011; Rsch, Bergmann, Knorr,
& Kroh, 2003). Recently the leaves of H. rhamnoides were also consid-
ered for their antioxidant potential correlated to avonoids and phe-
nolic acids derivatives (Kim, Kwon, Sa, & Kim, 2011; Sharma et al.,
2008; Upadhyay, Yogendra Kumar, & Gupta, 2011). Antimicrobial
activities have also been reported for SBT berries (Puupponen-Pimi
et 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 on
the 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 therapeutic
potential and their phytochemical contents.
The objective was (i) to evaluate the antimicrobial capacities
of SBT organs and fractions against food-borne and clinical

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.09.029
 T. Michel et al. / Food Chemistry 131 (2012) 754760 755

microorganisms, (ii) to assess antioxidant activity of SBT organs Table 1

and fractions via the commonly 2,20 -diphenyl-1-picrylhydrazil Bacterial strains and yeast used in this study.

(DPPH) free radical scavenging and ferric reducing antioxidant Organism and gram Species ATCC BCC
power (FRAP) methods, and (iii) to investigate the composition of morphologies
SBT organs and fractions by High Performance Thin Layer Chroma- Bacilli
tography (HPTLC) and using the total phenolic (TPC) and con- Gram  Escherichia coli 10536 3.11.006
densed tannin (CTC) contents. Pseudomonas 9027 3.11.008
aeruginosa
Gram + Bacillus cereus 6464 3.05.002
2. Material and method Cocci
Gram + Staphylococcus aureus 25923 3.11.005
2.1. Plant material Enterococcus durans 6056 3.08.023
Yeast Candida albicans 2091 2.11.002
Sea buckthorn (H. rhamnoides L.) were purchased from the tree
nursery PLANFOR (Uchacg, France) and were grown outside for
1 year before being harvested. The leaves, stems and roots were
then separated from each other, washed and dried at room temper- 2.4. Antimicrobial tests
ature. The seeds, obtained from NatVit (Claret, France), are a by-
product of the SBT juice production. All these organs were ground Antimicrobial activity was tested against a panel of microorgan-
to a powder with a basic electric grinder and stored in the dark at isms, including food-borne and clinical microorganisms (Table 1):
room temperature before use. gram positive bacteria (cocci and bacilli), gram negative bacteria
and one yeast. All these microorganisms were obtained from
American Type Culture Collection (ATCC) or from Brittany Culture
2.2. Chemicals
Collection (BCC, http://www-tmp.univ-brest.fr/souchotheque).
Strains were grown in liquid nutrient broth (Difco, Surrey, Eng-
Methanol, ethanol, acetonitrile (ACN), ethyl acetate, chloroform
land), at 37 C, for 24 and 48 h, for bacteria and yeast, respectively,
(CHCl3) and hexane were of analytical grade and provided by SDS
before being used for antimicrobial tests. Methanolic solution of
Carlo Erba (Val-de-Reuil, France), water (H2O) was puried (resis-
SBT extract was dropped in sterile 96-well plate (NUNC microplate,
tance < 18 MX) from ultra pure water using an Elgastat UHQ II
Fisher Bioblock) at a nal concentration of 100 lg/ml, and then
system (Elga, Antony, France). Formic acid (HCOOH), 2,2a-diphe-
evaporated by slight heating. Afterwards, a 100 ll of microorgan-
nyl-1-picrylhydrazil (DPPH) free radical, FolinCiocalteu reagent,
ism suspensions (102 cells/ml), obtained by dilution from the
sodium acetate trihydrate, sodium sulphate (Na2SO4), hydrochloric
culture tube (108 cells/ml), were added in the wells. Cell concen-
acid (HCl), b-sitosterol, gallic acid, glucose, quercetin-3-O-galacto-
tration was estimated by haemocytometer. Antibiotic solution
side, saccharose, valine and xylose were purchased from Sigma
(mixture of streptomycin and penicillin G at 5 and 10 mg/ml,
Aldrich (Saint Quentin Fallavier, France). Ferric chloride hexahy-
respectively) was dropped in the presence of microorganisms for
drate (FeCl3, 6H2O) and ferrous sulphate heptahydrate (FeSO4,
positive control. The microplate was aseptically sealed and incu-
7H2O) were obtained from Acros organics (Geel, Belgium). Isorham-
bated at 37 C for 13, 24 or 48 h for Staphylococcus aureus, other
netin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside,
bacteria strains and yeast, respectively. After agitation, microor-
kaempferol, oleanolic acid and ursolic acid were supplied by Extra-
ganism growth was estimated by reading the absorbance at
synthese (Genay, France). The 2,4,6-tripyridyl-S-triazine (TPTZ) was
405 nm with a microplate spectrophotometer (Multiskan FC, Ther-
obtained from JT Baker Chemicals (Deventer, Holland) and sodium
mo scientic). The results were expressed as percentage of inhibi-
carbonate (Na2CO3) from Merck (Val de Fontenay, France).
tion according to the formula:
 
2.3. Extraction and fractionation procedure Asample  Apc
%inhibition 1   100
Anc  Apc
The extraction of SBT organs was carried out by Pressurised Li-
where Asample is the absorbance value of the tested solution, Apc is
quid Extraction (PLE), using an Accelerated Solvent Extraction (ASE
the absorbance value of the positive control and Anc is the absor-
100) system from Dionex (Voisins le Bretonneux, France), with a
bance value of the negative control. Six replicates were made for
34 ml stainless steel vessel. The ground powder of each organ
each extract and fractions.
(3 g) was mixed with Na2SO4 (6 g) and extracted with two different
solvents (i.e. ethanol and ethyl acetate) using ve static cycles for
5 min each, a ush volume of 70% and a purge with nitrogen gas of 2.5. Antioxidant tests
100 s at the end of each extraction. Extractions were carried out at
60 C and under a pressure of 100 bar. The liquid extract was then For all the following tests the samples were diluted in MeOH at
evaporated at 40 C using a rotary evaporator (Buchi Labortechnik a 1 mg/ml concentration and all the tests were miniaturised to be
AG, Switzerland) under vacuum to obtain a dried crude extract, realisable in sterile 96-well plate (NUNC microplate, Fisher Bio-
which was then protected from light and stored at 2 C before block). All samples were analysed at least four times. The readings
use. Each extract was replicate thrice. were done with a microplate spectrophotometer (Multiskan FC,
The conventional liquidliquid extraction (LLE) method was Thermo scientic).
used to separate crude extracts in three fractions of different polar-
ities. Ethanolic crude extract (80 mg for stems and roots, 300 mg 2.5.1. DPPH-radical scavenging activity
for leaves and seeds) was respectively dissolved in a 20 or The radical scavenging activity, using free-radical DPPH assay,
100 ml H2O/EtOH (90/10 v:v) solution. Then, this H2O/EtOH (90/ was determined according to the method introduced by Blois
10 v:v) solution was partitioned successively with equal volumes (1958). An aliquot of 2 ll of each extract was mixed with a
(3  20 mL or 3  100 mL) of hexane (Hex) and ethyl acetate (EA) 198 ll methanolic solution of DPPH (75 lM). Extract was substi-
to constitute HF and EAF. Water fraction (WF) corresponds to tuted by methanol blank. Decolourisation of purple free radical
aqueous residue obtained at the end of LLE. DPPH solution was measured at 517 nm after 30 min incubation
756 T. Michel et al. / Food Chemistry 131 (2012) 754760
in the dark and at room temperature. A trolox calibration curve
was done between 0.1 and 1 mg/ml. Results were expressed in
mg of trolox equivalents/g of dry extract (mg TE/g).
2.5.2. The ferric reducing antioxidant power (FRAP) assay
The FRAP assay was carried out according to the Benzie and
Strain method (1996), with some modications. The FRAP reagent
was prepared daily from 300 mM acetate buffer (pH 3.6), 20 mM
FeCl36H2O and 10 mM TPTZ made up in 40 mM HCl. All the above
three 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 rst mixed with ul-
tra pure water (20 ll) and then with FRAP reagent (178 ll). The
change in absorbance from the red to the blue, was followed at
593 nm after 10 min. A trolox calibration curve was done between
0.1 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 developed
by Singleton and Rossi (1965). For a 200 ll nal volume, 2 ll of ex-
tract (1 mg/ml) or 2 ll of aqueous standard solution (gallic acid) or
2 ll of ultra pure water for blank were rst mixed with 10 ll of Fo-
linCiocalteu reagent. 30 ll of a Na2CO3 aqueous solution (20%)
was then added, between 1 and 8 min later, completed with ultra
pure water. Afterwards, the blue mixture was incubated at room
temperature for 2 h and its absorbance was read at 760 nm with
a microplate spectrophotometer (Multiskan FC, Thermo scientic).
Results were expressed in mg of gallic acid equivalents/g of dry ex-
tract (mg GAE/g) using a linear equation based on the calibration
curve 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 by
Sun, 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 the
absorbance was measured at 500 nm using a microplate spectro-
photometer (Multiskan FC, Thermo scientic). 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 catechin
from 0.05 to 1 mg/ml. All samples were analysed at least four
times.
2.8. HPTLC analyses
2.8.1. Instrumentation and operating conditions
A Camag (Muttenz, Switzerland) HPTLC system equipped with
an automatic TLC sampler (Linomat 4) and a horizontal developing
chamber 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 were
laid on using an automated TLC sampler in 7 mm bands, at 10 mm
from the bottom, 5 mm from the sides and with 3.5 mm space be-
tween the two bands. The HPTLC plate and solvent system were
chosen 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 sprayed
with a specic reagent.
2.8.2. Amino acids and sugars
Development was done on HPTLC silica gel 60 F254 10  20 cm
plates (Merck, Germany) in ACN:H2O (75:25 v/v). Two specic re-
agents were used to detect, respectively, amino acid and sugar.
Treatment of plates with ninhydrin (0.1% in EtOH) leads to amino
acid characteristic spots after heating at 120 C for 5 min. Sugars
were then detected using Molich indicator as follows. Solution A
(2 g of a-napthol in 100 ml of (v/v) EtOH) was rst applied, and
after a drying step solution B (H2SO4 at 5% (v/v) in EtOH) was
sprayed. 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 sugar
standards.
2.8.3. Terpenoids and phytosterols
Terpenoids 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 of
the LiebermannBuchard (LB) reagent. LB was prepared carefully
by 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 plate
was warmed at 120 C for 510 min and then looked at under vis-
ible and UV (k = 366 nm) light. Oleanolic acid, b-sitosterol and
ursolic acid were used as terpene standards.
2.8.4. Polyphenols
After developing the HPTLC Lichrospher RP18 WF254s plates
(Merck, Germany) in ACN: H2O:HCOOH (50:50:5 v/v), uorescence
of 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 MeOH
and PEG reagent was a solution of polyethylene glycol (PEG) 4000
at 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 and
gallic acid were used as polyphenolic standards.
2.9. Statistical analysis
Data were expressed as the mean standard deviation of at
least three measurements. One-way analysis of variance (ANOVA),
correspondence analysis and determination of the Pearson correla-
tion coefcient (q) was used during this work to evaluate and cor-
relate results between them. Differences with p-value superior to
0.05 were not considered signicant. The data were statistically
analysed using Microsoft XLSTAT software (Microsoft Corporation,
Redmond, USA).
3. Results and discussion
H. rhamnoides is a good source of bioactive compounds due to
its content of various phytochemicals, however most of the litera-
ture report activities from SBT berries (Gao et al., 2000; Guliyev
et al., 2004). For this reason our study was focused on original
SBT organs: leaf, stem, root and seed. Furthermore, to the best of
our 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 at
high pressure and temperature, was chosen because it is a well
established technique in natural product extractions and it offers
advantages like rapidity, efciency, weak solvent consumption
and clean-up extracts (Kaufmann & Christen, 2002). The SBT
 T. Michel et al. / Food Chemistry 131 (2012) 754760 757

25 thus its use is preferable to minimise impact on the environment.

Ethanol
In this sense, ethanol was chosen for the extraction of bioactive
Ethyl acetate
compounds from SBT organs. Fig. 1 showed also that extraction
20
was more efcient for leaves and seeds with a yield tenfold higher
than for stems and roots. A brief temperature optimisation using
leaf sample, showed that use of increasing temperature improved
extraction yield. Temperature set at 40, 50 and 60 C induced yield
Extraction yield %

15 varying from 24% to 30% (0.03%), respectively. The temperature

was not set at values higher than 60 C in order to avoid degrada-
tion of thermolabile compounds. The extraction procedure was
10 consequently achieved using ethanol at 100 bar and 60 C.
In order to get information about molecular family responsible
of activity, ethanolic crude extracts of each organ were separated
in three fractions by classic LLE using hexane (HF), ethyl acetate
5
(EAF) and water (WF). The mass yields are resumed in Table 2.
For all organs, the aqueous WF was always the largest, with a mass
yield higher than the other fractions.
0
Leaf Stem Root Seed

Fig. 1. Extraction yield obtained by PLE of different SBT organs using two different
3.2. Antimicrobial activity
organic solvents. Results are expressed as the mean standard deviation (n = 3).
, and denotes signicantly different at 0.1%, 1% and 5% level, respectively. a: Table 3 showed inhibition percentage of SBT ethanolic extracts
signicantly different against ethyl acetate (p < 0.05). and 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 conventional
Table 2
Minimum Inhibitory Concentration (MIC) value to determine anti-
Mass yields of fractions obtained from the ethanolic extract of SBT leaf, stem, root and
seed. microbial activity because an inhibition of 100% was never ob-
tained whatever concentration (50, 100, 150, 200, 250 and
Organs Mass yield
300 lg/ml) and extract studied. The SBT crude extracts all depicted
Water (%) Ethyl acetate (%) Hexane (%) an antimicrobial activity with some differences between organs.
Leaf 40.60 20.26 37.60 The maximum inhibition (%) was varied according to organs and
Stem 49.75 22.75 30.75 strains: S. aureus (72 3%) for leaf extract, Bacillus cereus
Root 29.75 22.75 25.00 (64 4%) for seed extract, Enterococcus durans (63 6% and
Seed 34.16 18.75 27.60
68 13%) for root and seed extracts, respectively. The minimum
inhibition was always obtained for Pseudomonas aeruginosa, and
organs were extracted using two solvents ethanol and ethyl ace- the less effective organ was stem. P. aeruginosa is known to easily
tate. Fig. 1 showed that the yield of ethanol extracts were signi- acquire resistance, via mutations, to a diverse class of antibiotics
cantly higher than the yield of ethyl acetate extracts. This (Livermore, 2002). This phenomenon could explain the lower
difference could be attributed to the selectivity of extraction. Eth- activity of SBT ethanolic extracts observed against this strain by
anol is a polar solvent known to extract a wide range of molecules development of a more complex resistance mechanism. Antimicro-
including sugar, glycoside and weakly polar compounds. Whereas, bial activity of SBT extracts was found against both Gram  and +
ethyl acetate is an intermediary apolar solvent that extracts prefer- strains which was already found by Negi et al. (2005) when worked
entially more hydrophobic compounds like aglycone and long car- on H. rhamnoides seed.
bon chain ones. Such better extraction, using an alcohol solvent, The WF was the most efcient fraction, particularly for B. cereus
has already been reported for extraction of SBT seeds (Negi et al., and S. aureus. For P. aeruginosa and Escherichia coli the activity was
2005). Furthermore, ethanol is considered as a low toxic solvent, found in both WF and EAF, but the activity of WF was always high-

Table 3
Antimicrobial activity of SBT ethanolic extracts (leaf, stem, root, seed) and fractions tested at 100 lg/ml, and expressed as inhibition percentage (%).

Sample Bacillus cereus Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Enterecoccus durans Candida albicans
Leaf CEE 32 6 24 3 42 1 72 3 40 6 67 7
WF 71 12 24 16 31 8 85 12 / /
EAF 93 34 1 24 11 26 9 / /
HF / / / / / /
Stem CEE 41 9 22 1 39 1 36 8 34 4 53 11
WF 71 4 45 4 33 17 48 15 / /
EAF 35 10 36 4 / 22 6 / /
HF / / / / / /
Root CEE 45 6 16 3 40 1 25 10 63 6 55 3
WF 67 6 43 5 37 4 21 2 / /
EAF 15.9 6.6 26 2 23 8 / / /
HF / / / / / /
Seed CEE 64 4 28 9 38 11 41 12 68 13 68 1
WF 89 5 36 2 45 3 42 10 / /
EAF 12 9 24 2 23 3 / / /
HF / / / / / /

CEE: crude ethanolic extract; WF: water fraction; EAF: ethyl acetate fraction; HF: hexane fraction.
758 T. Michel et al. / Food Chemistry 131 (2012) 754760

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 used
two different in vitro assays, the DPPH radical scavenging and
the FRAP assays. Results are resumed in Table 4.
The DPPH scavenging activity of extracts ranged from 174.8 and
528.6 mg TE/g Root (500 64 mg TE/g) and seed (529 79 mg TE/
g) extracts had signicantly 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 also
found signicantly more active than leaf (171 19 mg TE/g) and
stem (137 9 mg TE/g) extracts (p < 0.0001). Signicantly higher
activities were seen between seed and root (p < 0.0001) and be-
tween leaf and stem (p = 0.044) with the FRAP assay. Upon organs
tested, 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 that
seeds have more antioxidant capacity than leaves. It can be also
noticed that as in antimicrobial results stem extract were seen as
the least effective.
All fractions depicted antioxidant activity with the both assays.
However, activity was mainly found in WF and EAF, and especially
in WF in case of roots and seeds. For all organs, HF was regarded as
the least antioxidant, which can be explained by the presence of
no-scavenger molecules like pigments (chlorophyll) or wax. The
DPPH 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 WF
signicantly more active than the EAF (p < 0.0001). No signicant
difference 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 to
139 22 mg GAE/g. The TPC of root and seed extracts were
signicantly higher than leaf and stem extracts (p < 0.0001). No
signicant differences were seen between root and seed, or be-
tween leaf and stem. With regard to the TPC of fractions, the trend
was almost similar to antioxidant tests with a higher signicant
phenolic content for the WF (p < 0.01). Determination of Pearson
correlation coefcients 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 attributed
to phenolic compounds. This result is in correlation with previous
work on SBT leaf and seed extracts which found that antimicrobial
and antioxidant potential were correlated to the presence of phe-
nolic compounds (Kim et al., 2011; Negi et al., 2005; Upadhyay
et al., 2011). Furthermore, from TPC data it can be observed that
differences between fractions were less important in contrast to
antioxidant tests. For instance, no signicant difference between
EAF and HF were recorded for all organs (p > 0.01). This less consid-
erable differentiation between fractions can be attributed to TPC
assay, which has been described to react toward various compound
classes including phenols, vitamins and unsaturated fatty acid
(Everette et al., 2010).
The CTC assay evaluates the content in condensed tannin or
proanthocyanidin (polymeric avan-3-ol derivative) of a plant ex-
tract. The CTC values of SBT ethanolic extracts varied from 13 6 to
176 27 mg CE/g. The seed crude extract contained signicantly
more condensed tannins than the other organs (p < 0.0001). The
lowest content was found in leaf extract, whereas no signicant
difference was seen between stem and root (p > 0.05). All fractions
showed a basic response to CTC assay, however only WF of seed
exhibited a high content in condensed tannin that is signicant
against the other fractions (p < 0.0001). This result suggests that
antimicrobial and antioxidant activities obtained from seed crude
extract and WF could be attributed to condensed tannin present
in higher proportion in this part of the plant. A previous study
(Fan et al., 2007) has also shown that proanthocyanidins were
the 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. Detection
of three molecule families was attempted using different station-
ary-mobile phases and using specic derivation system. Presence
and/or absence of specic molecules were identied by matching
the colours of spots after development and after derivatisation.
The HPTLC ngerprint of SBT ethanolic extracts and fractions are

Table 4
Free radical scavenging activity (DPPH), ferric reducing antioxidant power (FRAP), total phenolic content (TPC) and condensed tannin content (CTC) of SBT crude ethanolic
extracts (leaf, stem, root, seed) and fractions.

Sample DPPH (mg trolox FRAP (mg trolox TPC (mg gallic acid CTC (mg catechin
equivalent/g dry extract) equivalent/g dry extract) equivalent/g dry extract) equivalent/g dry extract
Leaf CEE 175 57 171 20 65 14 13 6
WF 275 57 148 16 92 23 5 12
EAF 161 17 106 17 53 3 20 15
HF 87 8 16 3 64 2 36 12
Stem CEE 210 42 137 9 84 29 26 13
WF 263 19 118 6 95 9 14 12
EAF 172 25 69 10 52 17 34 16
HF 41 9 51 64 23 33 16
Root CEE 500 64 311 31 139 22 42 12
WF 304 13 367 32 106 14 16 13
EAF 63 9 53 36 10 29 15
HF 31 6 2 0.2 42 5 20 14
Seed CEE 529 79 454 82 120 14 176 27
WF 368 15 324 41 138 28 161 13
EAF 81 5 28 3 49 9 39 14
HF 19 6 71 58 19 57 12

CEE: crude ethanolic extract; WF: water fraction; EAF: ethyl acetate fraction; HF: hexane fraction.
 T. Michel et al. / Food Chemistry 131 (2012) 754760 759

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 sugar
plates 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 plates
developed 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) correspond
to 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 with
NEU + 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, root
EAF; 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 , gallic
acid (ga), quercetin-3-O-galactoside (q3g), kaempferol (k). WF, EAF and HF correspond respectively to water, ethyl acetate and hexane fractions.
760 T. Michel et al. / Food Chemistry 131 (2012) 754760
shown in Fig. 2. HPTLC analyses of crude extract showed that they
were constituted of sugars, terpenoids and phenolic compounds,
and that their phenolic ngerprints were different from each other
notably between leaf and other organ extracts.
With regard to fractions, HPTLC analyses provided information
on their phytochemical constituents. Sugars, especially mono-
and disaccharides, were found in WF. Terpenoids and sterols were
detected 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 stem
ngerprints. In addition, it seems that extracts and fractions were
mostly constituted of phenolic compounds, which are partitioned
in all fractions. Furthermore, HPTLC comparisons showed clearly
that the major phenolic compounds of crude extract were found
in WF, conrming that phenolic derivatives could be responsible
of the activities of SBT organs.
4. Conclusion
Present work described the evaluation of antimicrobial and
antioxidant activities of SBT leaf, stem, root and seed and their
respective fractions, as well as their phytochemical investigations.
To the best of our knowledge it was the rst time that SBT organs
were investigated simultaneously in such a manner. It has been
demonstrated that SBT leaf, stem, root and seed present good anti-
microbial and antioxidant activities. Seed and root extracts were
seen 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 fractions
were analysed the bioactivity was mainly found in the aqueous
fraction 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 seed
these effects could be due to proanthocyanidins. HPTLC nger-
prints conrmed that WF was mainly constituted of phenolic com-
pounds and that it was constituted of the same major molecules
compared to crude extracts. Nevertheless, further works are
needed 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. rhamnoides
were also seen to constitute potent bioactive compounds. The re-
sults indicate that SBT leaf, stem, root and seed can be regarded
as a natural source of antimicrobials and antioxidants and may
be 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 SBT
juice production.
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