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Antioxidant activity and chemicalcomposition of three Tunisian Cistus:Cistus monspeliensisCistus villosus andCistus libanotis☆

Marcello Nicolettia, Chiara Tonioloa, Alessandro Vendittiab,Maurizio Brunoc & Mariem Ben Jemiad

a Dipartimento di Biologia Ambientale, Università Sapienza diRoma, Rome, Italyb Dipartimento di Chimica, Università Sapienza di Roma, Rome,Italyc STEBICEF, Università degli Studi di Palermo, Viale delle Scienze,Parco d'Orleans II, I-90128 Palermo, Italyd Laboratoire des Plantes Extremophiles, Biotechnologic CenterBorj-Cedria Technopark, B.P. 901, 2050 Hammam-Lif, TunisiaPublished online: 12 Aug 2014.

To cite this article: Marcello Nicoletti, Chiara Toniolo, Alessandro Venditti, Maurizio Bruno &Mariem Ben Jemia (2014): Antioxidant activity and chemical composition of three Tunisian Cistus:Cistus monspeliensisCistus villosus and Cistus libanotis☆, Natural Product Research: FormerlyNatural Product Letters, DOI: 10.1080/14786419.2014.947486

To link to this article: http://dx.doi.org/10.1080/14786419.2014.947486

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Antioxidant activity and chemical composition of three Tunisian Cistus:Cistus monspeliensis, Cistus villosus and Cistus libanotis q

Marcello Nicolettia, Chiara Tonioloa, Alessandro Vendittiab*, Maurizio Brunoc and

Mariem Ben Jemiad

aDipartimento di Biologia Ambientale, Universita Sapienza di Roma, Rome, Italy; bDipartimento diChimica, Universita Sapienza di Roma, Rome, Italy; cSTEBICEF, Universita degli Studi di Palermo, Vialedelle Scienze, Parco d’Orleans II, I-90128 Palermo, Italy; dLaboratoire des Plantes Extremophiles,Biotechnologic Center Borj-Cedria Technopark, B.P. 901, 2050 Hammam-Lif, Tunisia

(Received 8 May 2014; final version received 17 July 2014)

The chemical composition of three rockrose Cistus species, Cistus monspeliensis,Cistus libanotis and Cistus villosus, collected in Tunisia, was studied by HPTLC,focusing on the terpenes and phenols constituents. Diterpenes of Cistus are important asthe main constituents of the leaf sticky aromatic resin, known as labdanum, which arehighly appreciated in perfumery. Polyphenols in the methanolic extracts of eachspecies were identified, quantified as total and as flavonoids and tannins, and tested forantioxidant activity. Diterpenes were evident in C. libanotis and C. monspeliensis,whereas they were practically absent in C. villosus; C. libanotis had higher phenolicamount, whereas antioxidant activities were important, but different according to thefollowing tests: DPPH radical scavenging, conversion of the Fe3þ/ferricyanidecomplex and inhibition of b-carotene bleaching. The reported data confirm the validityof utilisation of Cistus sp. in marketed herbal products, as well as the relevant presenceof diterpenes in species actually not used for labdanum production.

Keywords: Cistus; HPTLC; antioxidant activity; terpenes; phenols

1. Introduction

The rockrose Cistus is a genus of Cistaceae family containing about 20 species in form of

perennial shrubs present on dry or rocky soils in Europe (Tabacik & Bard 1971; Ellul et al.

2002). A total of 17 species are typical examples of the Mediterranean Flora and widely spread

mainly near the coasts, forming dense populations.

Since ancient times the importance of Cistus species was known. The leaves of several

species, i.e. Cistus ladanifer and Cistus creticus, are coated with an abundant highly sticky

brown aromatic resin, also known as labdanum or labdan (Oyaizu 1986). The Egyptians, at the

times of pharaohs, used the plant as a medicine and the resin for embalming and for aphrodisiac

purposes (Lucas 1926; Newberry 1929). Later in Greek times, as early as Herodotus and

Theophrastus, this plant has been prized as the source of the resin. Nowadays the use of

labdanum in perfumery is really prevalent and appreciated, because of its resemblance to

ambergris (Brady et al. 2002; Clarke 2006). Ambergris became very difficult to find, when whale

fishing was banned by many countries. Therefore, labdanum is the main ingredient used when

making the scent of ambergris in perfumery. Ambergris and labdanum contain similar diterpenic

constituents. On exposure to air and sunlight, the main compound of ambergris, the triterpene

q 2014 Taylor & Francis

qThis manuscript was submitted to SIF 2013, XIII National Congress of Societa Italiana di Fitochimica(Italian Phytochemical Society), Gargnano (Italy), 19–21 September 2013.

*Corresponding author. Email: alessandro.venditti@uniroma1.it

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alcohol (2 )-ambrein, decomposes into labdanes, ambrox, amberlyn and ambroxan, which are

responsible for the fragrance properties (De Pascual Teresa et al. 1985; Hashimoto et al. 1998;

Castro et al. 2002). Labdanum can be considered a valid substitute of ambergris for the presence

of similar labdane constituents (Ben Jemia et al. 2013).

Besides its importance in perfumery, labdanum has been used for centuries as herbal popular

medicine to treat colds, coughs, menstrual problems, rheumatism, diarrhoea as well as an

incense (Abrahams 1979). Actually, it is often used in making cosmetic creams, because of its

anti-wrinkle properties and shampoos, as it strengthens the hair follicle. Nowadays there is an

interest on the traditional uses, due to the benefits of Cistus sp. essential oil and tea, owing to a

significant market in herbal products, like botanical food supplements. However, more scientific

pieces of evidence and additional information about the chemical constituents of each species

are needed (Tabacik & Bard 1971; De Pascual Teresa et al. 1981; Weyerstahl et al. 1998).

Several chemical studies on Cistus monspeliensis leaves have been reported, including catechin-

related compounds in aqueous extracts (Berti et al. 1967; Angelopoulou et al. 2001;

Kalpoutzakis et al. 2003; Pomponio et al. 2003) and reports on biological properties (Chinou

et al. 1994; de las Heras et al. 1994; De Andres et al. 1999; Kupeli & Yesilada 2007; Barrajon-

Catalan et al. 2010; Barrajon-Catalan et al. 2011). The composition of essential oil and aqueous

extracts from Cistus libanotis has also been reported (Zidane et al. 2013). No previous reports on

the composition of Cistus villosus are available. In this paper, in continuation of our research on

Cistus sp., we focused on three species collected in Tunisia: C. monspeliensis L., C. libanotis L.

and C. villosus L., as a contribution in order to evaluate their potentiality in health products

market, after a preliminary study on antiproliferative activity on the hexane extracts (Ben Jemia

et al. 2013).

2. Results and discussion

Research on the three Tunisian Cistus sp. is depicted in three steps: (a) determination, as

complete as possible, of the chemical composition; (b) focus on terpenes and phenols

constituents, using selected markers and quantification analyses; (c) test for antioxidant activity.

Interest on diterpenes is related to labdanum, whereas polyphenols are considered responsible

for a reported efficacy on respiratory diseases, including influenza, by CYSTU052, a rich-

polyphenolic extract of Cistus incanus (Droebner et al. 2007; Kalus et al. 2009; Kalus et al.

2010).

HPTLC fingerprint analysis was selected for the first step, in consideration of its high

sensitivity and the possibility of evidencing ‘as many small molecules as possible’ (Reich &

Schibli 2007; Nicoletti 2011; Gallo et al. 2012). The analysis was supported by the availability of

standards obtained in previous researches on Cistus sp. (Venditti et al. 2014) and by comparison

with a sample of C. monspeliensis, collected in Italy. In fact, C. monspeliensis is widely

distributed in the Mediterranean basin, whereas the other two species are less common.

The results of HPTLC analysis are partially reported in Figures 1 and 2, whereas other

results are reported in the additional data section (Figures S1–S4). The sequence of the spots

in the tracks can be used for interpretation of the metabolic production of each species.

By comparison of fingerprints and standards, we can obtain the following information: (a) each

species presents an individual fingerprint, which allows its identification. The two samples of

C. monspeliensis are in good accordance, considering that the strong red spots present in track

5 at the bottom of the plate (Figure S1, Additional data) must be assigned to waxes and

chlorophylls constituents, dependent on the freshness of the Italian sample; (b) selected

standards allow to identify the Rf sectors pertaining to the different classes of metabolites.

Diterpenes are evident in C. libanotis and C. monspeliensis, whereas they are practically

absent in C. villosus; this is interesting since the two first species are not preferred in labdanum

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Figure 1. HPTLC analysis of Cistus species from Tunisia and Italy and selected standards. Mobile phase:AcOEt–CH2Cl2–CH3COOH–HCOOH–H2O 100:25:8:8:8 (v/v/v/v/v). Visualisation: UV 254 nm. Tracks:1, C. villosus (Tunisia); 2. C. libanotis (Tunisia); 3, C. monspeliensis (Tunisia); 4, C. monspeliensis (Italy);5, clerodane (15-acetoxy-cis-clerodan-3-ene-18-oic acid); 6, labdane (8-hydroxylabdan-15-oic acid); 7,flavonol (myricetin 3,7,40,50-tetramethyl ether).

Figure 2. The same plate as Figure 1. Visualisation: UV 254 nm. Derivatisation: NPR þ anisaldehyde.

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production, C. creticus and C. incanus being traditionally used; (c) polyphenols are present in

different concentrations, as evident in the fluorescent spots present at different Rf values;

(d) flavonoids are present in accordance with the following series (in order of decreasing

concentration): C. libanotis . C. monspeliensis . .C. villosus, albeit the differences in

class should be considered, because for tannins the situation appears different, as

C. villosus . C. libanotis . C. monspeliensis. The analysis of buds of C. monspeliensis was

also included, considering the use of this raw material as food supplements in bud therapy. The

track of bud extract evidences a clear similarity with the track of leaves extract, but with some

differences. Therefore, HPTLC analysis is able to evidence the presence of secondary

metabolites in food supplements used in bud therapy and that these products are not totally

identical with those used in other herbal medicines, Figure 3.

2.1. Total phenolic, flavonoid and tannin contents

Former data now can be completed by those regarding the quantity of polyphenols. Based on the

absorbance values of extract solutions reacted with the Folin–Ciocalteu reagent and compared

with the standard solutions of gallic acid equivalents, total phenolic contents are given in

Table 1. C. libanotis had a higher phenolic content (40.51mg GAE g21 DW) than C. villosus and

C. monspeliensis (32.51mg GAE g21 DW and 33.16mg GAEg21 DW, respectively).

These data were in agreement with other studies (Zidane et al. 2013) that mentioned the

presence of phenols content of C. libanotis (40.02 FA mg/g DW), although the variation in total

phenol content could be due to various factors. One of such factors may be the genetic potential

of individual species for polyphenol biosynthesis. Apart from the genetic background,

maturation stage may also be critical in this respect. According to Table 1, we noted that

flavonoid contents varied with species. C. villosus leaves had the highest amount of flavonoids

Figure 3. Densitometric analysis of tracks of plate in Figures 1 and 2. Tracks 1–4 extracts of Cistus sp. anda–c selected standards, as already reported.

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(9.44mg EC g21 DW) followed by C. libanotis (8.49mg EC g21 DW) and C. monspeliensis

(5.59mg EC g21 DW) extracts. These findings were consistent with HPTLC analysis and

previous work (Zidane et al. 2013), which noted that C. libanotis is rich in total flavonoids (24.96

QE mg/g DW).

Concerning proanthocyanidins, Table 1 shows that condensed tannin contents in C. villosus

(7.36mg EC g21 DW) were higher than those in C. libanotis (6.87mg EC g21 DW) and

C. monspeliensis (3.52mg EC g21 DW). These data were in agreement with other studies

(Petereit et al. 1991; Pomponio et al. 2003) which exhibited the richness of Cistus sp. in tannins.

2.2. Antioxidant capacity

The synthetic DPPH radical is widely used to evaluate the free radical scavenging activity in

many plant extracts (Brand-Williams et al. 1995). The assessment of antioxidant activity showed

that the examined extracts were able to scavenge this radical (Table 2). C. monspeliensis

displayed higher activity than C. villosus and C. libanotis (IC50 values were 3.0, 22 and

28mgmL21, respectively). In addition, methanol extracts of C. monspeliensis exhibited higher

DPPH radical scavenging activity than that of the control (BHT: 12mg/mL). In the same context,

the results of DPPH tests of methanolic extracts of C. incanus and Cistus parviflorus from Libya

showed that IC50 values obtained for the samples subjected to DPPH assay were in the range

from 4.75 to 17.75mg/mL.

The lowest IC50 values found were for C. villosus and C. libanotis, which were richer in

phenols than in C. monspeliensis,which had lower phenols content and a high DPPH scavenging

activity. This finding might be ascribed to certain constituents that are particularly responsible

for strong antioxidant effect (Guendez et al. 2005). The synergic effect of the antioxidants in the

extracts should also be considered (Sun & Ho 2005).

The reducing capacity of extracts may serve as indicator of its potential antioxidant activity.

The presence of reducers (i.e. antioxidants) causes the conversion of the Fe3þ/ferricyanidecomplex to the ferrous form. The EC50 value (Table 2) of reducing power ability of ascorbic acid

Table 2. Antioxidant capacity of C. monspeliensis, C. villosus and C. libanotis.

DPPH IC50

(mg/mL)

Reducingpower EC50

(mg/mL)

Metal chelatingactivity

assay (IP) %

b-Carotene–linoleic acid assayIC50 (mg/mL)

C. monspeliensis 3^ 0.36 142^ 12.46 3.33^ 2.01 20.2^ 5.54C. villosus 22^ 0.07 80^ 0.57 16.32^ 1.77 214^ 2.61C. libanotis 28^ 0.01 28^ 4.71 15^ 2.32 72^ 7.53BHT 12^ 0.13 – – 85^ 0.22Ascorbic acid – 40^ 1.31 – –EDTA – – 97.8^ 0.11 –

Table 1. Total phenolic, flavonoid and tannin contents in Cistus.

Total polyphenol content(mgGAE/gDW)

Total flavonoidcontent

(mgEC/gDW)Tannins content(mgEC/gDW)

C. monspeliensis 33.16^ 0.01 5.59^ 0.01 3.52^ 0.01C. villosus 32.51^ 0.03 9.44^ 0.03 7.36^ 0.02C. libanotis 40.51^ 0.03 8.49^ 0.00 6.87^ 0.02

Notes: Total phenolic contents were expressed as mg GAE g21 DW. Total flavonoid and total tannin contents wereexpressed as mg CE g21 DW.

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(40mg mL21) was lower than that of methanol extract of C. libanotis (EC50 ¼ 28mgmL21).

C. libanotis showed higher reducing ability than C. monspeliensis and C. villosus. These results

suggest that the extract of C. libanotis was electron donors reacting with free radicals to convert

them into more stable products and to terminate radical chain reactions as described by Shimada

et al. (1992).

Concerning the test of b-carotene, it undergoes rapid discolouration in the absence of an

antioxidant. The presence of antioxidant compounds such as phenolics can hinder the extent of

b-carotene destruction by ‘neutralising’ the linoleate-free radical and any other free radicals

formed within the system (Kamath & Rajini 2007). Table 2 depicts the inhibition of b-carotenebleaching by the leaves extracts of Cistus, and by the positive control BHT. In terms of

b-carotene bleaching effect, those samples exhibited the following order: C. monspeliensis .C. libanotis . BHT . C. villosus. C. monspeliensis extract exhibited a marked antioxidant

activity (IC50 ¼ 20.2mg/mL) compared with that of BHT (IC50 ¼ 85mg/mL), while C. villosus

extract was less active, with IC50 ¼ 214mg/mL.

The ability to chelate transition metals can be considered as an important antioxidant mode

of action. In fact, the chelation and deactivation of transition metals prevent these species from

participating in metal-catalysed initiation and hydroperoxide decomposition reactions

(Dastmalchi et al. 2007). Numerous studies indicated that plant extracts enriched in phenolic

compounds are capable of complexing with and stabilising transition metal ions, rendering them

unable to participate in metal-catalysed initiation and hydroperoxide decomposition reactions

(Bourgou et al. 2008). As can be seen in Table 2, none of the three extracts of Cistus was as

effective as the positive control EDTA (IP ¼ 97.8%).

3. Experimental

See supplementary materials.

4. Conclusions

The HPTLC analysis clearly confirmed the separation of Cistus sp. in two groups, between

species containing high concentrations of diterpenes, useful for labdanum production, and others

devoid of diterpenes. Also polyphenols content varies according to the species. Use of Cistus

sp. in botanical food supplements was also confirmed by the antioxidant tests, although different

biological activities should be considered. Further chemical analyses and pharmacological

experiments are required to confirm the above data and to complete the information about this

important genus of Mediterranean flora.

Supplementary material

Experimental details relating to this paper are available online, alongside Figures S1–S5.

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