Fitoterapia 77 (2006) 94–99

www.elsevier.com/locate/fitote

Medicinal potential from in vivo and acclimatized plants of

Cleome rosea

Claudia Simoes a,*, Jose Carlos P. De Mattos b, Katia C.C. Sabino c,

Adriano Caldeira-de-Araujo b, Marsen G.P. Coelho c,

Norma Albarello a, Solange F.L. Figueiredo a

a Departamento de Biologia Vegetal/Laboratorio de Biotecnologia de Plantas (LABPLAN), Instituto de Biologia Roberto Alcantara Gomes,

Universidade do Estado do Rio de Janeiro (UERJ), Rio de Janeiro, Brazilb Departamento de Biofısica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro (UERJ),

Rio de Janeiro, Brazilc Departamento de Bioquımica , Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro (UERJ),

Rio de Janeiro, Brazil

Received 1 December 2004; accepted 17 November 2005

Available online 10 January 2006

Abstract

Methanolic extracts obtained from different organs of Cleome rosea, collected from its natural habitat and from in vitro-

propagated plants, were submitted to in vitro biological assays. Inhibition of nitric oxide (NO) production by J774 macrophages

and antioxidant effects by protecting the plasmid DNA from the SnCl2-induced damage were evaluated. Extracts from the stem of

both origins and leaf of natural plants inhibited NO production. The plasmid DNA strand breaks induced by SnCl2 were reduced by

extracts from either leaf or stem of both sources. On the other hand, root extracts did not show any kind of effects on plasmid DNA,

and presented significant toxic effects to J774 cells. The results showed that C. rosea presents medicinal potential and that the

acclimatization process reduces the plant toxicity both to plasmid DNA and to J774 cells, suggesting the use of biotechnology tools

to obtain elite plants as source of botanical material for pharmacological and phytochemical studies.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Medicinal plant; In vitro propagation; NO inhibition; DNA protection

1. Introduction

Cleome rosea is an annual herbaceous plant, native to Brazil. It is frequently found near the coast, in areas of

restinga, an ecosystem submitted to an intense degradation process. Several species of Cleome have been target of

phytochemical studies. Compounds such as triterpenes and flavonoids have already been isolated [1–4].

Moreover, several pharmacological activities such as anti-inflammatory [5,6], antioxidant [7], antineoplasic [8,9],

antimicrobial [10] and analgesic effects [11] have already been demonstrated for crude extracts or compounds

obtained from different Cleome spp.; however, the C. rosea species has not yet been studied.

0367-326X/$ - s

doi:10.1016/j.fit

* Correspondin

E-mail addre

ee front matter D 2005 Elsevier B.V. All rights reserved.

ote.2005.11.001

g author. Tel.: +55 21 25877361; fax: +55 21 25877655.

ss: csimoes04@yahoo.com.br (C. Simoes).

C. Simoes et al. / Fitoterapia 77 (2006) 94–99 95

Studies about the medicinal potentiality of a species demand the continuous supply of botanical material. The use

of in vitro-propagated plants as source of raw material for phytochemical and pharmacological studies has increased

[12]. This technique presents advantages, when compared to the conventional propagation methods, including the

production of a great number of plants in a short period of time and in reduced space, besides permitting continuous

production during the year round and without seasonal changes. Thus, these plants are renewable resources allowing

supply without reaccessing the source material in its natural habitat.

Among the pharmacological activities detected in plant extracts, one of the most frequent is anti-inflammatory

action. As nitric oxide (NO) is a well-known mediator and the reactive oxygen species (ROS) are substances released

by the inflammatory processes, the search for plant extracts or their isolated compounds with antioxidant and/or NO

production inhibitory effects has been largely stimulated as potential anti-inflammatory therapeutics. The presence of

substances with antioxidant activity has widely been studied in plants also on account of the narrow relationship

between free radicals and the aging process [13,14] or neoplasic process [15,16].

This work aimed at evaluating the medicinal potential of C. rosea methanolic extracts obtained from different plant

organs, collected from their natural habitat and from acclimatized plants obtained by in vitro propagation. The

potentiality of the extracts to inhibit NO production by activated macrophages and its antioxidant action by inhibiting

the DNA damage induced by SnCl2 was evaluated.

2. Experimental

2.1. Plant material

The plants (9 months old) were collected after fructification on the Itaipuacu restinga (Rio de Janeiro, Brazil) and

acclimatized plants (6 months old) originating from in vitro cultures propagated on MS medium [17] supplemented

with the plant growth regulator 6-benzyladenine [18]. Dr. Rosa Fuks, from the Rio de Janeiro Botanical Garden,

authenticated the taxonomic identity of C. rosea Vahl ex DC. (Capparaceae). A voucher (HRJ 7185) has been

deposited in the Herbarium of the Rio de Janeiro State University.

2.2. Preparation of extracts

Leaf (90 g), stem (140 g) and root (40 g) samples, from both origins, were fragmented, dried at 45 8C for 48 h

(leaf—14 g, stem—25 g, root—6 g) and extracted with MeOH for 14 days at environmental conditions. The ex-

tracts were filtered and evaporated to dryness in vacuo, yielding 270 mg (leaf), 195 mg (stem) and 60 mg (root) for

both origins.

2.3. NO production assay

The macrophages-cell-line J774 was distributed in 96-well tissue culture plates at 5�105 cell ml�1 of RPMI

medium supplemented with 10% fetal calf serum, 10 Ag ml�1 penicillin G and 10 UI ml�1 streptomycin. The cells

were activated by the addition of LPS (5 Ag ml�1) and IFN-g (10 UI ml�1). The plant extracts were first diluted with

dimethyl sulfoxide (DMSO) and afterwards with supplemented RPMI medium until the final concentrations of 100,

200 and 500 Ag ml�1 in the culture. The maximal final concentration of DMSO in culture was 0.1%. After incubation

for 24 h at 37 8C, 5% CO2 and humidified atmosphere, samples of culture supernatants (100 Al) were collected for

nitrite determination. The control culture consisted of cells incubated with DMSO 0.1% (final concentration), LPS

and IFN-g and the supernatant nitrite concentration considered as 100% of NO production. The cells were also

cultured with LPS and IFN-g, without DMSO.

2.4. Nitrite determination

Nitrite, an indicator of NO synthesis, was measured in the activated J774 cell supernatants as described by Green

et al. [19]. Samples of culture supernatant (100 Al) were mixed to 100 Al of Griess reagent (1% sulfanilamide/0.1%

naphthylethylenediamine dihydrochloride/5% H3PO4) and the mixture maintained for 10 min at r.t. prior to determine

the absorbance at 550 nm, using a microplate reader (AQuant). To avoid interference on nitrite determination, blanks

C. Simoes et al. / Fitoterapia 77 (2006) 94–9996

were performed by incubating only the culture medium with the different amounts of extracts. The nitrite

concentrations in the culture supernatants were calculated from a standard curve of sodium nitrite. Inhibition

index of NO production was calculated as: Inhibition index=100� [(NS�100)/NC], where NS represents the nitrite

concentration of the sample and the NC the nitrite concentration of the control culture. Experiments were carried out

in triplicate.

2.5. Cytotoxicity assay

The extracts cytotoxicities were determinated by cell viability, assessed by the mitochondrial-dependent reduction

of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan crystals [20]. Briefly, J774 cells

(5�105 cells ml�1 of supplemented RPMI medium) were incubated for 24 h (37 8C, 5% CO2, humidified

atmosphere) in 96-well tissue culture plates (final volume of 200 Al) with different final concentrations of C.

rosea extracts (100, 200 and 500 Ag ml�1), prepared as described previously for NO production, with DMSO 0.1% as

maximal final concentration. Then, 100 Al of culture supernatants was removed by aspiration, 10 Al MTT solution (5

mg ml�1 in phosphate buffer 0.01 M pH 7.2 with NaCl 0.15 M) were added to cells and the plates incubated for 30

min at 37 8C and 5% of CO2 atmosphere. Finally, the formazan crystals were dissolved with 100 Al of sodium dodecyl

sulfate solution (10% in 0.01 N HCl) and the absorbance read at 570 nm using a micro-plate reader. The cells

incubated with supplemented RPMI medium with DMSO 0.1% (final concentration) was considered as control

culture and its absorbance as 100% of viability. The cells were also cultured without DMSO. Experiments were

carried out in triplicate. Cytotoxicity was determined as: Cytotoxicity=100� [(AbsS�100)/AbsC], in which AbsScorresponds to the absorbance of the sample and AbsC the absorbance of control culture.

2.6. Antioxidant assay

The antioxidant potential was evaluated according to Caldeira-De-Araujo et al. [21]. This assay is based on the

inhibition of the powerful reduced agent stannous chloride (SnCl2) to induce strand breaks in DNA molecule

mediated by the production of ROS [22]. The SnCl2-induced lesions are responsible for plasmid DNA conformational

changes, leading to modification in migration pattern, during agarose gel electrophoresis. Briefly, Plasmid DNA (pUC

9.1) was obtained through alkaline lysis as described by Sambrook et al. [23]. Electrophoresis assay was carried out to

evaluate a possible oxidant or antioxidant effect of the crude MeOH extracts. Three different extract concentrations (5,

25 and 50 Ag ml�1) were incubated with 200 ng of plasmid DNA, in the presence, or not, of SnCl2 (200 Ag ml�1). All

dilutions were made with ultrapure water (Milli-Q system) and the reaction mixtures were incubated at r.t. for 40 min.

Then, aliquots of each sample (10 Al) were mixed with 2 Al of loading buffer (0.25%; xylene cyanol; 0.25%

bromophenol blue; 30% glycerol in water), applied to a horizontal gel plate (0.8% agarose) in TAE 1� buffer and

submitted to electrophoresis at 7 V/cm for 30 min. Gel electrophoresis was performed in order to separate different

conformations of plasmid DNA: native conformation (supercoiled) and resulting from strand breaks (open circle).

Afterwards, this gel was stained with ethidium bromide (0.5 mg ml�1) and the DNA bands were visualized by

fluorescence in an ultraviolet transilluminator system (UVP transilluminator). The gel bands were then digitalized and

the results obtained provide only a qualitative analysis.

2.7. Statistical analysis

The variance analysis of data of NO production and cytotoxicity were determined by one-way ANOVA and the

significant differences by Dunnet’s comparison test.

3. Results and discussion

3.1. Effects on NO production and cytotoxicity

All methanolic extracts at 100 Ag ml�1 did not inhibit NO production or exhibited cytotoxicity (Table 1). Extracts

from leaf (200 Ag ml�1) and stem (500 Ag ml�1) of plants collected on the restinga, inhibited the NO production in

45F19% and 36F13%, respectively, and did not show cytotoxicity at these concentrations, differently from the root

Table 1

Effects of C. rosea leaf, stem, root methanolic extracts on inhibition of NO production by J774 macrophages and cytotoxicity

Parts Extract (Ag ml�1) Plants from restinga Acclimatized plants

NO production inhibition (%) Cytotoxicity (%) NO production inhibition (%) Cytotoxicity (%)

Leaf 100 22F3 9F2 12F6 3F2

200 45F19* 5F3 18F7 2F1

500 78F22** 73F18** 18F13 5F3

Stem 100 12F7 3F1 11F9 2F1

200 18F11 4F3 11F8 7F2

500 36F13* 6F2 55F14** 6F3

Root 100 15F9 13F9 15F10 12F6

200 35F10* 40F6** 31F12* 9F9

500 84F12** 79F12** 78F6** 74F8**

Data represent meansFS.D. of three independent assays with triplicates.

* P b0.05.

** P b0.01 in relation to control culture, by Dunnet’s comparison test.

C. Simoes et al. / Fitoterapia 77 (2006) 94–99 97

of the same origin. Similar results of NO synthesis inhibition by macrophages were demonstrated by Fushiya et al. [6]

with MeOH extract obtained from the aerial parts of C. droserifolia and with substances isolated from other plants as

Lycium chinense [24] andMagnolia sieboldii [25]. The inhibition of NO production by C. rosea extracts (Table 1) did

not seem to result from DMSO effects, since the nitrite concentration in control culture (4.32F0.07 AM, with LPS,

IFN-g and DMSO 0.1%) was not different from that obtained by activated cell culture without DMSO (4.38F0.01

AM) as well as the cell viability in both conditions by the MTT assay (Abs 570 nm of 0.399F0.078 and

0.413F0.107, respectively). Absence of effect in cell culture by this DMSO concentration has already been

demonstrated by Hall et al. [26]. Root (200 Ag ml�1) from both origins inhibited the NO production but only root

from the restinga presented cytotoxic effects at this concentration. Moreover, the leaf lost the cytotoxicity after the

acclimatization process. Extracts from stem of acclimatized plants (500 Ag ml�1) remained with their inhibitory effect

on NO production without cytotoxicity.

The loss of NO production inhibitory effect and the reduction of cytotoxicity found on some extracts from

acclimatized plants could be related to a possible alteration in the chemical composition of the plant induced by the

acclimatization process. Moreover, plants extracts can enclose substances with opposite effects. Such fact was verified

by Fushiya et al. [27] when methanolic extract from aerial parts of Acer nikoense did not show inhibition of NO

production by mouse peritoneal macrophages. However, when this extract was fractioned into butanol and ethyl

acetate sub-fractions, a significant inhibition level (30%) was verified in the last one.

3.2. Antioxidant potential

The electrophoretic mobility of untreated plasmid DNA is shown in Fig. 1. (I and II , lane 1), with a predominance

of the supercoiled form, while the efficient cleavage of the plasmid DNA induced by SnCl2-generated ROS is shown

in Fig. 1 (I and II, lane 2), evidenced by the formation of open-circle form.

Leaf and stem extracts obtained from plants collected in the restinga (Fig. 1I.A and I.B) and from acclimatized

plants (Fig. 1II.A and II.B), provided protection against the DNA strand breaks induced by SnCl2. This protecting

action of the extracts could be due to the presence of metabolites which could react with the stannous ion, preventing

its oxidation and consequently blocking ROS production, which would be the main factor responsible for the breaks

found in the plasmid DNA. The decrease in genotoxic potential of SnCl2 by increasing stem extract concentrations

(Fig. 1I.B and II.B—lanes 6, 7, 8) suggests a C. rosea antioxidant property. In the Cleome spp., the presence of

compounds with antioxidant potential has been already reported in C. arabica [7]. A dose-dependent protection

against the genotoxic SnCl2-induced damage was also reported by Reiniger et al. [28] in the same experimental

model, using different concentrations of the alkaloid boldine, obtained from Peumus boldus.

The incubation of plasmid DNA in the presence of the highest concentrations of leaf and stem extracts of plants

collected in the restinga (Fig. 1I.A and I.B—lane 5) induced a low level of strand breaks. This deleterious effect

should be due to the concentration used. In the same way, boldine possesses an antioxidant action in low doses and

induce DNA strand breaks in high concentrations [28]. Nevertheless, this boldine toxic effect had already been

DNA / SnCl2 / Extracts (µg.mL-1)

5 25 50

DNA / Extracts (µg.mL-1)

5 25 50 DNA DNA / SnCl2

Fig. 1. Antioxidant properties of C. rosea leaf (A), stem (B), root (C) methanolic extracts (I: plants collected in restinga; II: acclimatized plants).

C. Simoes et al. / Fitoterapia 77 (2006) 94–9998

reported elsewhere [29]. This lesive effect was not verified in the C. rosea stem and leaf extracts from acclimatized

plants (Fig. 1II.A and II.B—lanes 3, 4, 5). Also the root only presented toxic effects to DNA plasmid when the

extracts were prepared from plants of the restinga (Fig. 1I.C and II.C—lanes 1, 3, 4, and 5). As mentioned already,

this differentiated response may be associated to the variation of levels of specific metabolites in crude extracts. The

in vitro culture conditions can be interfering in the production of chemical substances. They usually demand the

manipulation of the culture media ensuring the increase or even the production of specific metabolites. The chemical

diversity of medicinal plant species under different in vitro conditions has been studied [30,31].

The root extracts from both origins (Fig. 1I.C and II.C) did not show protective effect (lanes 2, 6, 7 and 8) against

the genotoxic SnCl2-induced damage. This inefficiency could be due to the absence of metabolites with the capacity

to block the formation of ROS or even scavenge them. It could also be related to the concentrations used, perhaps

insufficient to express antioxidant action. The same result was verified for flavonoid rutin, isolated from Ruta

C. Simoes et al. / Fitoterapia 77 (2006) 94–99 99

graveolens, which in the concentrations tested by Bernardo et al. [32], was not able to protect the plasmid DNA

against the oxidizer action of SnCl2, even though rutin is known as a powerful antioxidant [33–35].

The results suggest that plants collected in the restinga as well as acclimatized plants of C. rosea present medicinal

potential as NO production inhibition and plasmid DNA protection against the strand breaks induced by SnCl2. It also

indicated that the acclimatization process reduces plant toxic effects to both plasmid DNA or J774 cells. Finally, the

present work demonstrates the viability to use plants obtained from biotechnological techniques as raw material

source for pharmacological and phytochemical studies, in addition to contributing to the preservation of the restinga

ecosystem, avoiding plant retreat in their natural habitat.

Acknowledgements

The authors acknowledge the financial support given by the National Research Council (CNPq) and the Rio de

Janeiro State University (UERJ).

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