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Comparative free radical scavengingpotential and cytotoxicity of differentextracts from Iris pseudopumila Tineoflowers and rhizomesDaniela Rigano a , Filomena Conforti b , Carmen Formisano a ,Francesco Menichini b & Felice Senatore aa Department of Chemistry of Natural Products, University ofNaples “Federico II”, Naples, Italyb Department of Pharmaceutical Sciences, University of Calabria,via Pietro Bucci, Rende (CS), ItalyPublished online: 29 Oct 2009.

To cite this article: Daniela Rigano , Filomena Conforti , Carmen Formisano , Francesco Menichini& Felice Senatore (2009) Comparative free radical scavenging potential and cytotoxicity ofdifferent extracts from Iris pseudopumila Tineo flowers and rhizomes, Natural Product Research:Formerly Natural Product Letters, 23:1, 17-25, DOI: 10.1080/14786410701740237

To link to this article: http://dx.doi.org/10.1080/14786410701740237

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Natural Product ResearchVol. 23, No. 1, 10 January 2009, 17–25

Comparative free radical scavenging potential and cytotoxicity of different

extracts from Iris pseudopumila Tineo flowers and rhizomes

Daniela Riganoa*, Filomena Confortib, Carmen Formisanoa, Francesco Menichinib

and Felice Senatorea

aDepartment of Chemistry of Natural Products, University of Naples ‘‘Federico II’’, Naples, Italy;bDepartment of Pharmaceutical Sciences, University of Calabria, via Pietro Bucci, Rende (CS),Italy

(Received 28 June 2007; final version received 9 October 2007)

Different Iris species are known as medicinal plants. The aim of this study was toevaluate the in vitro antioxidant and cytotoxic activities of different extracts fromIris pseudopumila Tineo flowers and rhizomes. The radical scavenging activity wasassessed by means of DPPH assay. The antioxidant activity was assessed bymeans of two tests: bleaching of �-carotene and lipid peroxidation of liposomes.Methanolic and chloroform extracts from the flowers of I. pseudopumila showed asignificant antiradical effect with IC50 of 101 and 83 mgmL�1, respectively. Asregards to lipid peroxidation, the best activity was showed by methanolic extractof flowers (IC50 of 18 mg mL�1) and chloroformic extract of rhizomes (IC50

33mgmL�1). The cytotoxic activity was carried out using the SRB assay. Thechloroform extract from rhizomes demonstrated a good cytotoxic activity againstamelanotic melanoma cancer cell line (C32) with an IC50 of 57mgmL�1. Theresults obtained support the ethnomedical claims for the plant.

Keywords: Iris pseudopumila; flavonoids; antioxidant; cytotoxicity; lipid perox-idation; radical scavenger

1. Introduction

Iris is the largest genus in the Iridaceae family and comprises about 210 species occurringin Eurasia, North Africa, and North America (Mabberley, 1997). Peeled and driedrhizomes of various Iris species, collectively known as Rhizoma iridis, enjoy popularity intraditional medicine due to their emetic, cathartic, diuretic, stimulant, antispasmodic, andexpectorant properties (Steinegger & Hansel, 1988). In some countries, Iris species are usedin the treatment of cancer, inflammation, bacterial, and viral infections (Han, 1988). Theliterature reports that various Iris sp. possess different activities such as antiulcer,antibacterial, anti-inflammatory, piscicidal, antineoplastic, antioxidant, hypolipidemic,and antitubercolosis (Atta-ur-Rahman et al., 2003; Benoit-Vical, Imbert, Bonfils,& Sauvaire, 2003; Bonfils, Pinguet, Culine, & Sauvaire, 2001; Choudhary, Naheed,Jalil, Alam, & Atta-ur-Rahman, 2005; Han, 1988; Hideyuki, Miyake, & Yoshida, 1995;

*Corresponding author. Email: drigano@unina.it

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2009 Taylor & Francis

DOI: 10.1080/14786410701740237

http://www.informaworld.com

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Orhan et al., 2003; Takahashi, Suzuki, Hano, & Nomura, 2002; Wollenweber et al., 2003;

Wong, Oshima, Pezzuto, Fong, & Farnsworth, 1986; Yong-Qiang, Jun-Jie, Chang-Heng,

Shan-Hao, & Da-Yuan, 2003).Iris pseudopumila Tineo is a dwarf bearded species endemic of Southern Italy, where it

grows as an ornamental plant. It can be yellow or violet, and it grows in shallow stony soils

(Pignatti, 1982). Some of ethnomedical and reported biological activities of Iris sp. may be

due to their antioxidant nature; for this reason, we have recently assayed the methanolic

extract from I. pseudopumila rhizomes and its metabolites using the luminol-dependent

chemiluminescence, finding a significant antioxidant activity (Rigano et al., 2007).

Furthermore, the same extract showed a good antimicrobial activity against different

Gram positive and Gram negative bacteria (Rigano et al., 2006).In a previous paper (Basile et al., 2006), we showed that the results of

chemiluminescence studies do not necessarily coincide with those of cell free systems.

This may happen because some constituents can modulate leukocyte respiratory burst.

Furthermore, most oxidant agents act directly via a chemical mechanism rather than

through a cellular mediator, such as PMNs. Hence, antioxidant activity tested by cell-free

systems can be closer to antioxidant systems acting in the plant exposed to oxidative stress.

Antioxidant activity by chemiluminescence can show antioxidant and anti-inflammatory

properties, while the cell-free systems can show strict antioxidant properties. For this

reason, in this article, we have evaluated the in vitro antioxidant activity of different

extracts from flowers and rhizomes of I. pseudopumila using different cell-free methods: the

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, bovine brain peroxida-

tion assay and �-carotene bleaching test, which allow different steps of oxidation to be

followed (Gordon, 1990). The detection of the interested antioxidants by chromatographic

techniques are also presented. Furthermore, since the literature shows that different

Iris species exert cytotoxic activity on different cultured human tumour cell lines (Bonfils

et al., 2001; Han, 1988, Hideyuki et al., 1995; Wong et al., 1986; Yong-Qiang et al., 2003),

the aim of this article was also to evaluate the cytotoxic activity of extracts from

I. pseudopumila against three human cancer cell lines, MCF-7, ACHN, and C32, using the

SRB assay.

2. Materials and methods

2.1. General experimental procedures

NMR spectra were acquired on a Bruker DRX-600 (1H at 599.19MHz, 13C at

150.86MHz) spectrometer, � (ppm), J in Hz, spectra referred to CHD2OD as internal

standard. Mass spectra were recorded with a API 2000 (Applied Biosystem). UV spectra

were measured on a UV/VIS Jasco V530 spectrophotometer. Reverse-phase HPLC was

performed with a TSP SpectraSeries P 100 equipped with a rheodyne injector and a

refractive index detector, using a column C18 �-Bondapack (Waters, 500� 10mm, flow

rate 2.5mLmin�1). Thin layer chromatography (TLC) was performed on plates coated

with silica gel 60 F254 Merck, 0.25mm.

2.1.1. Collection of plant material

Iris pseudopumila flowers and rhizomes were collected in May 2003 in the ‘Parco Nazionale

del Cilento’ (Salerno, Southern Italy). A voucher specimen (NAP # 68) is deposited at the

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Herbarium Neapolitanum (NAP), Dipartimento di Biologia Vegetale, Universita degliStudi di Napoli ‘‘Federico II’’, Italy.

2.1.2. Preparation of the extracts

Iris pseudopumila fresh flowers (304 g) were air-dried, cut into small pieces and thensequentially extracted by cold maceration with petroleum ether (40–60�) (3� 2.5 L),CHCl3 (3� 2.5L) and CH3OH (3� 2.5 L). The petroleum ether extract yielded a yellowishsolid (0.8%), the chloroform extract yielded a brown solid residue (1.8%), while themethanol extract yielded blackish brown solid (2.9%).

The methanol and chloroform extracts of I. pseudopumila rhizomes were obtained asdescribed previously (Rigano et al., 2007).

2.1.3. Isolation of compounds from I. pseudopumila flowers methanolic extract

The methanolic solution from I. pseudopumila flowers was concentrated under reducedpressure, and the residue (9.11 g) was chromatographed in 2 g lots on a Sephadex LH-20(Pharmacia) column eluting with CH3OH to afford 12 fractions of 20mL each.The fractions were analyzed by TLC using n-BuOH/CH3COOH/H2O (60: 15: 25, v/v)as eluent and Ce(SO4)2 in H2SO4 as spray reagent. Fractions 13 (188.5mg), 14–17(464.4mg), and 23 (110.7mg) that were shown to be the most active in preliminarybiological assays (data not shown) were further purified by HPLC on a C18 m-Bondapakcolumn:

Fraction 13 was eluted with CH3OH/H2O 50 : 50 to yield pure isovitexin (1; Rt¼ 10min)(11.8mg, 0.13%).

Fractions 14–17 were eluted with CH3OH/H2O 50 : 50 to yield pure isoorientin-6-O00-�-D-glucopyranoside (2; Rt¼ 7.5min) (14.9mg, 0.16%) and isovitexin-6-O00-�-D-glucopyrano-side (3; Rt¼ 10.2min) (17.1mg, 0.19%).

Fraction 23 was eluted with CH3OH/H2O 50 : 50 to yield pure isoorientin(4; Rt¼ 7.5min) (4.2mg, 0.04%).

The structures of these compounds were determined by comparison of their spectroscopicdata (NMR and MS) with literature values (Agrawal, 1989).

2.1.4. 2,2-Diphenyl-1-picryl-hydrazil (DPPH) free radical-scavenging activity

Free radical scavenging activity was measured using DPPH assay, which was adaptedfrom Wang et al. (1998) modified as reported by Conforti et al. (2006). The samples weretested at different concentrations. The absorbance was measured using a Perkin ElmerLambda 40 UV/VIS spectrophotometer at 517 nm. Acid ascorbic was used as positivecontrol.

2.1.5. Bovine brain peroxidation assay

The in vitro antioxidant activity tests were carried out using the TBA test (Fernandez,Perez-Alvarez, & Fernandez-Lopez, 1997) modified as reported by Conforti et al. (2006).The TBA reaction is based on the fact that peroxidation of most membrane systems leadsto formation of small amounts of free malonaldehyde (MDA). The incorporation of any

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antioxidant compound in the bovine brain peroxidation assay reaction mixture will lead toa reduction of the extent of peroxidation. The extracts were tested for their antioxidantactivity against liposomes, which were prepared from bovine brain extract in phosphatebuffered saline (5mgmL�1). Propyl gallate was used as a positive control (Jacobi,Hinrichsen, Web, & Witte, 1999).

2.1.6. �-Carotene bleaching test

Antioxidant activity was determined using �-carotene bleaching test (Amin, Zamaliah,& Chin, 2004) modified as reported by Conforti et al. (2006). The absorbance of thesamples, standard and control was measured at 470 nm using a Perkin Elmer Lambda 40UV/VIS spectrophotometer. The measurement was carried out at initial time (t¼ 0) andsuccessively at 30 and 60min. The antioxidant activity (AA) was measured in terms ofsuccessful bleaching �-carotene.

2.1.7. Cytotoxicity assay

The protein-staining sulforodamine B (SRB) assay, developed by the National CancerInstitute for in vitro antitumor screening (Rubinstein et al., 1990), was used in this study toestimate cell number indirectly by providing a sensitive index of total cellular proteincontent, which is linear to cell density. SRB is an anionic protein stain containing twosulphonic groups that bind electrostatically to basic aminoacid residues of cellular proteinunder mildly acid conditions.

The human breast adenocarcinoma MCF-7 (ATCC No.: HTB-22), amelanoticmelanoma C32 (ATCC No.: CRL-1585), and renal cell adenocarcinoma ACHN (ATCCNo.: CRL-1611) cell lines were used in this experiment. The MCF-7 cell line were culturedin DMEM medium. The C32 and the ACHN cell lines was cultured in RPMI 1640medium. All were supplemented with 10% foetal bovine serum, 1% L-glutamine, 1%penicillin/streptomycin.

The following protocol is based on that originally described by Skehan et al. (1990)with modifications (Conforti, Marrelli, Statti, & Menichini, 2006). The absorbance of eachwell was read on a Spectra Max plate reader at 492 nm. Cell survival was measured as thepercentage absorbance compared to the control (non-treated cells).

3. Statistical analysis

Data were expressed as means�SD. Statistical analysis was performed by using Student’st-test. Differences were considered significant at p� 0.05. The inhibitory concentration50% (IC50) was calculated from the Prism dose–response curve (statistical programme)obtained by plotting the percentage of inhibition versus the concentrations.

4. Results and discussion

Iris species are known traditionally as medicinal plants; they are used as cough remedies,to treat wounds, as laxatives, to treat migraines and against cancer, inflammation,bacterial and viral infections. Plants of this genus were reported to have differentbiological properties, including anti-inflammatory and anticancer. To validate some of theethnopharmacological uses of the plant, the dried flowers and rhizomes of I. pseudopumila

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were defatted with petroleum ether and then extracted with chloroform and MeOH.

Chloroformic and methanolic extracts were then evaluated for their antioxidant and

cytotoxic properties.The antioxidant potential of extracts of I. pseudopumila was determined by two

complementary methods (�-carotene bleaching test and bovine brain peroxidation assay),

while radical scavenging activity was carried out using DPPH test. The reduction of DPPHabsorption is in fact indicative of the capacity of the extracts to scavenge free radicals,

independently of any enzymatic activity. The scavenging effects of plant extracts

and fractions on DPPH were examined at different concentrations(25, 50, 100, 250, 500, 1000 mgmL�1). The absorbance decreases as a result of a colour

change from purple to yellow as the radical is scavenged by antioxidants. All samples were

able to reduce the stable free radical DPPH to the yellow-coloured 1,1-diphenyl-2-picrylhydrazyl (Table 1). Methanolic and chloroform extracts from the flowers showed the

highest antiradical effect, with IC50 of 101 and 83 mgmL�1, respectively (Figure 1a), while

the methanol and chloroform extracts from rhizomes showed similar lower activity (IC50

of 279 and 249 mgmL�1, respectively) (Figure 2a). The samples exhibited a significant

antioxidant capacity also in the �-carotene-linoleic acid test system (Figures 1cA; B and

2cA; B). Inhibition of the breakdown of lipid hydroperoxides to unwanted volatileproducts allowed us to determine secondary antioxidants in related mechanisms. In the

absence of antioxidants, oxidation products (lipid hydroperoxides, conjugated dienes, andvolatile byproducts) of linoleic acid simultaneously attack the �-carotene, resulting in

bleaching of its characteristic yellow colour in ethanolic solution. In the presence of the

total extract, oxidation products were scavenged and bleaching was prevented. A higherlevel of antioxidant activity was observed for methanolic extract from flowers after 30min

of incubation with IC50 of 2 mgmL�1 (Figures 1cA;B). As a third test method employed,

inhibition bovine brain peroxidation was also used to measure antioxidant capacityexpressed as protective action to MDA formation. Potent antioxidant activity, using

liposomes prepared from bovine brain extract, was showed by methanolic extract of

flowers with an IC50 of 18 mgmL�1 (Figure 1b) but chloroform extract from rhizomes alsoshowed a good activity (IC50 of 57 mgmL�1) (Figure 2b). These results agree with those

Table 1. IC50 values of radical scavenging and antioxidant activities of I. pseudopumila extracts(n¼ 3).

IC50 (mgmL�1)

�-Carotene bleaching test

Sample DPPH Lipid peroxidation30min ofincubation

60min ofincubation

MeOH extract of flowers 101� 0.231 18� 0.071 2� 0.032 8� 0.055Chloroform extract of flowers 83� 0.195 61� 0.131 5� 0.045 6� 0.062MeOH extract of rhizomes 274� 0.934 108� 0.251 26� 0.081 41� 0.085Chloroform extract of rhizomes 249� 0.831 57� 0.121 4� 0.035 6� 0.056aPropyl gallate – 7� 0.072 1� 0.041 1� 0.034aAscorbic acid 2� 0.031 – – –

Note: aPropyl gallate and ascorbic acid were used as positive control.

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previously obtained using the chemiluminescence method (Rigano et al., 2007), confirmingthe antioxidant properties of the plant.

The cytotoxic activity of samples was carried out using the SRB assay. Three humancancer cell lines, MCF-7, ACHN, and C32, were used. All three cell lines used in this assayare capable of attachment to form a homogeneous monolayer on plastic substratum of theculture wells, which is ideal for the SRB assay. For each cell line there was a linear

0.0 0.2 0.4 0.6 0.8 1.00

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100 Methanol extract

Ascorbic acid

Chloroform extract

Concentration (mgml−1)0.00 0.25 0.50 0.75 1.00

020

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120 Methanol extractPropyl gallateChloroform extract

Concentration (mgml−1)

0.000 0.005 0.010 0.015 0.020 0.0250

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120 Methanol extractAscorbic acidChloroform extract

Concentration (mgml−1)

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0.000 0.005 0.010 0.015 0.020 0.0250

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Ascorbic acid

Chloroform extract

Concentration (mgml−1)

(a)

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Figure 1. Antioxidant activity of Iris pseudopumila flowers on: (a) DPPH free radical; (b) Lipidperoxidation of liposomes assay; (c) �-carotene bleaching test: (A) after 30min of incubation and (B)after 60min of incubation. All samples were assayed in triplicate and averaged.

0.000 0.005 0.010 0.015 0.020 0.0250

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Ascorbic acid

Chloroformextract

Concentration (mg ml−1)

0.0 0.2 0.4 0.6 0.8 1.00

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Ascorbic acid

Chloroformextract

Concentration (mg ml−1)0.00 0.25 0.50 0.75 1.00

020

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Concentration (mg ml−1)

0.000 0.005 0.010 0.015 0.020 0.0250

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Figure 2. Antioxidant activity of Iris pseudopumila rhizomes on: (a) DPPH free radical; (b) Lipidperoxidation of liposomes assay; (c) �-carotene bleaching test: (A) after 30min of incubation and (B)after 60min of incubation. All samples were assayed in triplicate and averaged.

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relationship between cell number and adsorbance measured at 492 nm in both control anddrug-treated wells. After 48 h of treatment the cytotoxicity of the extracts and fractionswas determined. The chloroform extract from rhizomes demonstrated the better cytotoxicactivity against amelanotic melanoma cancer cell line (C32) with an IC50 of 57 mgmL�1

(Table 2). The samples did not show significant cytotoxic activity against all other tumourcell lines (IC504 100 mgmL�1).

With the aim to characterise the phytochemical profile of I. pseudopumila and toidentify the compounds responsible for the activity, the methanolic extract from theflowers, proved to be the most active in all the antioxidant bioassays, was subjected toa chromatographic separation that led to the isolation of isovitexin (1), isoorientin-6-O00-�-D-glucopyranoside (2), isovitexin-6-O00-�-D-glucopyranoside (3) and isoorientin (4) as maincomponents, never found previously in I. pseudopumila flowers. Being the major phenoliccompounds in I. pseudopumila, the compounds 1–4 are likely to be responsible for theobserved antioxidant activity of the crude methanol extract. In effect, previous papersclearly show that these flavonoids possess antioxidant and radical scavenging properties(Tunalier, Kosar, Kuepeli, Calis, & Baser, 2007). Flavonoid antioxidants may act in avariety of ways, including direct quenching of reactive oxygen species, inhibition ofenzymes involved in the production of the reactive oxygen species, chelation of low-valentmetal ions such as Fe2þ or Cu2þ, and regeneration of membrane bound antioxidants suchas �-tocopherol (Pietta, 2000). The phenolic compounds 1–4 may chelate metal ions(Fernandez, Mira, Florencio, & Jennings, 2002), rendering them inactive to participate infree radical generating reactions. As regards the methanolic extract from rhizomes, whichalso showed a good antioxidant activity although lower than methanolic extract fromflowers in a previous paper (Rigano et al., 2007), we have reported the isolation from it of10 isoflavonoids and 5 flavonoids, showing that some of them, when evaluated for theiractivity by luminol-dependent chemiluminescence assay, possessed interesting antioxidantproperties. The antioxidant activity of natural flavonoids is governed by their conjugatedrings and by number and location of their aromatic hydroxyl groups (Chen, Chan, Ho,Fung, & Wang, 1996). The antioxidant activity may increase with the number of totalhydroxyl groups probably due to the vulnerable loss of a proton and stability of the radicalintermediate because of resonance delocalisation. In the case of flavonoids from flowers,the positive factor was the four hydroxyl groups, which increased the antioxidativeeffectiveness. Flavonoids from rhizomes showed in their structures methoxyl groups thatmay account for the attenuation of lipid peroxidation as well as the major glycosylationreduced the antioxidant activity.

Table 2. Cytotoxic activity of I. pseudopumila extracts.

IC50 (mgmL�1)

Cell line MeOH flowers CHCl3 flowers MeOH rhizomes CHCl3 rhizomes

MCF-7 4100 4100 4100 96� 1.79C32 4100 4100 4100 57� 1.04ACHN 4100 4100 4100 99� 1.95

Notes: Exposure time 48 h� S.E.M. (n¼ 3). MCF-7: human breast cancer cells; ACHN: renal celladenocarcinoma; C32: amelanotic melanoma cells. Vinblastine (2 mgmL�1) was used as positivecontrol.

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Since chloroformic extract from the flowers of I. pseudopumila showed a goodantiradical activity, while chloroformic extract from the rhizomes showed both

antioxidant and cytotoxic activities, further studies are in progress in order to identifythe active compounds from both of these extracts. Previous papers report that cytotoxic

activity in Iris genus is often due to iridals, particular triterpenoids isolated from differentspecies (Bonfils et al., 2001; Han, 1988; Hideyuki et al., 1995; Wong et al., 1986;Yong-Qiang et al., 2003). Work is in progress to confirm this hypothesis.

In conclusion, the present article shows for the first time the free radical scavengingand the antioxidant activities of some extracts from I. pseudopumila rhizomes and flowers.

Furthermore, chloroform fraction from the rhizomes showed high-cytotoxic activity onamelanotic melanoma cell line (C32). Hence, the present investigation provides a proof for

the ethnomedical use of Iris and indicates that the antioxidant and cytotoxic nature of theplant may be responsible for the ethnomedical and reported biological activities.

Acknowledgements

The authors thank Dr Diego Di Novella for kindly providing the plant. Thanks are also due to theC.S.I.A.S of University ‘Federico II’ of Naples for technical assistance.

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