Indigofera suffruticosa: An Alternative Anticancer Therapy
Indigofera suffruticosa: An Alternative Anticancer Therapy
Jeymesson Raphael Cardoso Vieira 2
Ivone Antoˆ nia de Souza 1
Silene Carneiro do Nascimento 1
Soˆ nia Pereira Leite 0
0 Universidade Federal de Pernambuco/Departamento de Histologia e Embriologia do Centro de Cieˆ ncias Biolo ́ gicas , Recife-PE , Brazil
1 Universidade Federal de Pernambuco/Departamento de Antibio ́ ticos do Centro de Cieˆ ncias Biolo ́ gicas , Recife-PE
2 Universidade Federal de Pernambuco/Departamento de Patologia do Centro de Cieˆ ncias da Sau ́ de Recife-PE
Indigofera suffruticosa Mill (Fabeceae) occurs in the Northeast countryside and has intensive popular use in the treatment of infectious, inflammatory and other processes. The main aim of the present work was to investigate the cytotoxic and antitumor effects of aqueous extracts of leaves of I. suffruticosa obtained by infusion and maceration as well as to evaluate the toxicological properties. Aqueous extracts did not exhibit cytotoxicity against HEp-2 (human epidermoid cancer cell) cell lines by MTT method. From the aqueous extract by infusion, the toxicological assay showed low order of toxicity. The antitumor effect of aqueous extracts by infusion (64.53%) and maceration (62.62%) against sarcoma 180 in mice at a dose of 50 mg kg 1 (intraperitoneally), based on low order of toxicity was comparable to the control group, which showed 100% development. Considering the low order of toxicity and that it is highly effective in inhibiting growth of solid tumors, the aqueous extracts of leaves of I. suffruticosa may be used as an alternative anticancer agent.
antitumor - aqueous extract - cytotoxicity - Indigofera suffruticosa - toxicity
Introduction
Cancer is the leading cause of mortality worldwide and the
failure of conventional chemotherapy to effect major reduction
in the mortality indicates that new approaches are critically
needed (
1
). An extremely promising strategy for cancer
prevention today is chemoprevention, which is defined as the
use of synthetic or natural agents to block the development of
cancer in humans (
2
). A variety of bioactive compounds and
their derivatives have been shown to inhibit carcinogenesis in a
number of experimental systems involving initiation,
promotion and progression (
3,4
). Plants, vegetables and herbs used
as folk and traditional medicine have been accepted currently
as one of the main sources of cancer chemoprevention drug
discovery and development (5). In Brazil traditional system of
medicine supports a growing interest in the pharmacological
evaluation of various plants.
Species belonging to the Fabaceae, the Indigofera
suffruticosa Mill (Fig. 1) have been used as an infusion or decoct
(flavor extract by boiling 1 liter of hot water per 5 g of leaves)
(
6
). This plant is found in tropical and subtropical areas and
well adapted to growth in semi-arid regions and soil of low
fertility (
7–9
). It occurs in Northeast Brazil and has intensive
popular use in the treatment of inflammation (
10,11
) and other
diseases such as epilepsy in human (12) and in animal models
(
13–15
). Recently, embryotoxic effects and antimicrobial
activity have been reported (
16,17
). A chemical investigation
of extract of leaves of I. suffruticosa in Natural Products Alert
(18) and Chemical Abstracts databases have revealed the
presence of alkaloids, flavanoids, steroids, proteins,
carbohydrates and indigo. Antitumor activities have been reported
in several plant species (
2,19,20–24
), however, up to now,
few researches have been done to investigate this
traditionally used plant in the recognition of their mechanism,
guaranteeing in the future its scientific and therapeutic use.
For allopathic drug development, even when traditional
formulations are taken into consideration, traditional medical
systems are very rarely regarded in the same way (
25
). The aim
of the present study was to carry out a brief basic toxicological
analysis and establish the safety of aqueous extract of leaves of
I. suffruticosa focusing on its cytotoxic and antitumor
activities.
Methods
Plant Material
The leaves of I. suffruticosa were collected in June 2000 in
Sa˜o Caetano, State of Pernambuco/Brazil and authenticated by
the Biologist Marlene Barbosa from the Botany Department,
Federal University of Pernambuco (UFPE), Brazil. A voucher
specimen number 32 859 has been deposited at the Herbarium
of the Botany department.
Mouse Experiments
Male Swiss albino mice weighing 20–25 g were purchased
from the animal house of Centro de Pesquisas Aggeu
Magalha˜es–Pernambuco, Brazil. They were housed in
standard environmental conditions of temperature, humidity and
under clear and dark cycles of 12 h. The mice were fed on diet
of the biotery (LABINA Purina Brazil) and water ad libitum.
All procedures described were reviewed and approved by the
University Animals Ethical Committee.
Cell Lines
Sarcoma 180, solid tumor and HEp-2 (human epidermoid
cancer cell) cell lines were obtained from Department of
Antibiotics/UFPE, Brazil. The solid tumor was maintained in
Swiss albino mice and the HEp-2 cells in minimum essential
medium, DMEM (Dulbeccos’ Modified Eagle medium).
Preparation of Extracts
Two extracts were prepared by infusion and maceration from
150 g of leaves of I. suffruticosa. The leaves were weighed;
chopped and extracted with solvents and water. The infusion
was prepared with 75 g of fresh leaves in 2 · 200 ml of
increasing polarity solvents (hexane, ethyl acetate and
methanol) at 40 C for 10 min and removing solid matter by
filtration. After this preliminary step, the same plant material
was extracted in boiling distilled water using the same
conditions, and the maceration obtained following the
abovementioned process at room temperature (28 C) overnight. The
solvents were removed by rotary evaporation. The yields (w/
w) of the infusion and the maceration were hexane (0.67 and
0.74%), ethyl acetate (0.39 and 0.34%) methanol (3.9 and
1.88%) in terms of newly collected plant material. After
lyophilization, the aqueous extracts yielded 4.20 and 1.75%
and the dried material was stored at 20 C (
26,27
). The
aqueous extracts by infusion and maceration were used for
cytotoxic and antitumor activities.
In vitro Cytotoxicity
The HEp-2 cells (human epidermoid cancer cells) were
investigated by the MTT method [3-(4,5-dimetyl
(thiazol-2yl)-2,5 diphenyltetrazolium bromide)]. Extracts with
concentrations more than 30 mg ml 1 are considered citotoxic
(
28
). They were trypsinized, counted and prepared in a
suspension with 105 cells per ml of DMEM and distributed
in a plate with 96-wells, which was incubated at 37 C in a
humidified atmosphere for 24 h. The aqueous extracts of
leaves of I. suffruticosa obtained by infusion and maceration
were dissolved in DMSO (dimethylsulfoxide) in
concentrations of 6.25, 12.5, 25 and 50 mg ml 1 and put in the wells
with the HEp-2 cells. Each concentration was tested in
quadruplicate. As the control, DMEM with DMSO was
used. After 72 h, 25 ml of MTT and 5 mg ml 1 of PBS
was added to the wells and the plate was incubated for 2 h.
The optical density was measured at 550 nm with ELX 800
reader.
Acute Toxicity Study in Mice
Thirty-six healthy Swiss albino mice male, weighing 20–25 g,
were divided in groups of six. The animals were on fasting
for 18 h before being submitted to the experiment. The
aqueous extract of I. suffruticosa by infusion was dissolved/
suspended in distilled water and administered by the
intraperitoneal (i.p.) route in doses of 50, 150, 300, 600,
1200 and 2400 mg kg 1. The general behavior of mice was
observed continuously for 1 h after the treatment and then
intermittently for 4 h, and thereafter over a period of 24 h
(
29
). The mice were further observed for up to 14 days
following treatment for any signs of toxicity and deaths. The
LD50 value was determined according to the method of
Litchfield and Wilcoxon (
30
).
Antitumor Assays
Male albino Swiss mice were divided into six groups of five
animals. In all groups 0.3 ml of Sarcoma 180 cells from a solid
tumor (around 3 · 106 cells) i.p were injected. After 48 h of the
implant, 0.2 ml kg 1 i.p. of saline solution was administered in
control group (groups 1 and 2). The chemotherapy was
initiated making use of the aqueous extract of leaves of
I. suffruticosa by infusion (groups 3 and 4) and by maceration
(groups 5 and 6) in daily concentration of 50 mg kg 1 i.p. for
seven consecutive days. The dose of the extract was based on
the LD50. On the 8th day, the mice were sacrificed for analysis
of tumor development.
Statistical Analyses
The experimental results of antitumor assays were expressed
as Median (min–max). Data were assessed by ANOVA
followed by Kruskal–Wallis. P < 0.001 was considered as
statistically significant.
Results
Cytotoxic Effect of Aqueous Extracts of I. suffruticosa
Aqueous extracts of leaves of I. suffruticosa by infusion and
maceration in concentrations of 6.25–50 mg ml 1 were tested
on HEp-2 cell lines by MTT method. The aqueous extracts
did not produce any cytotoxic effect to HEp-2 cell lines
(>30 mg ml 1) when compared with control DMEM and
DMSO (Table 1).
Acute Toxicity in Mice
There were no deaths but some low signs of toxicity were
observed after i.p. injection of aqueous extracts of leaves of
I. suffruticosa by infusion at any dose level up to the highest
dose tested (2400 mg kg 1) whose effects were more
pronounced (Table 2). Some adverse effects, such as agitation,
piloerection, exhaustion and sleepiness, were seen
immediately after the i.p. injection while others (irritability,
exhaustion, agitation and spasm) were observed later, and they were
more pronounced at the higher dose. The acute toxicity (LD50)
of aqueous extract of leaves of I. suffruticosa by infusion at
different doses in mice did not show rates of mortality during
72 h of observation in the preliminary assay.
Antitumor Effects of Aqueous Extracts on Sarcoma 180
The effects of the aqueous extracts of leaves of I. suffruticosa
by infusion and maceration on Sarcoma 180 is shown in Fig. 2.
The extract by infusion reduced significantly the mean volume
of Sarcoma 180 via i.p. administration in dose of 50 mg kg 1
64.53% [0.64 mg (0.01–22.10)] and the maceration extract
reduced 62.62% [(0.64 mg (0.01–2.10)] at the same dose when
compared to the mean volume of the tumor of control group
treated with saline solution which showed a tumor
development of 100% [1.97 (1.27–2.87)]. As shown, the inhibition was
dose-dependent and similar to that promoted by the maceration
and infusion extracts at the same dose.
Discussion and Conclusions
The treatment of cancer may benefit from the introduction
of novel therapies derived from natural products. Natural
products have served to provide a basis for many of the
pharmaceutical agents in current use in cancer therapy (
31
).
The use of chemotherapeutic drugs in cancer involves the risk
of life threatening host toxicity. The search, therefore, goes on
to develop the drugs, which selectively act on tumor cells. The
plants belonging to family Fabacea have medicinal properties,
especially the plant I. suffruticosa (
11,16,17
). The present
investigation shows that aqueous extracts revealed absence of
D/T ¼ Dead/treated mice: 0/6 ¼ toxic symptoms during the observation period
after the injection. Mice in each dose group (n ¼ 6) were carefully examined
for any signs of toxicity (behavioral changes) for 14 days.
*The lyophilized aqueous extract of leaves of I. suffruticosa was dissolved in
distilled water and administered as single i.p. dose to group of mice.
cytotoxic effects, low toxicity dose-dependent and antitumor
effect. Our results concerning the aqueous extracts of
I. suffruticosa leaves obtained by infusion and maceration
on HEp-2 cell lines did not reveal cytotoxic potential. Reports
showed that cytotoxicity with extracts of leaves of
I. suffruticosa were not encountered in the literature and it
confuses the comparison of these results with others using the
same conditions. Extracts of Umbelliferous plants retarded the
development of solid and ascites tumors and increased the life
span of these tumor-bearing mice. Tritiated thymidine uridine
and leucine incorporation assay suggested that fraction acts
directly on DNA synthesis (
20
). Extract of Emilia sonchifolia
was cytotoxic on tumor cells with reduction in tritiated
thymidine but not to normal human lymphocytes (
32
). Many
pharmacological effects observed in animals can extend result
in high value of application for the human species. Based on
cytotoxicity bioassays, over 400 compounds have been
isolated from plants, marine organisms and microorganisms
between 1996 and 2000 (
33
). As a result, this is the main
reason for large use of toxicological test for determination of
toxicity and safety when using drugs (
34
). The aqueous extract
of I. suffruticosa leaves obtained by infusion showed low
order of toxicity in mice at least up to the maximum dose of
2400 g kg 1 and no death but some low signs of toxicity.
A number of species of Indigofera contain an amino acid
indospicine (
35,36
), and nitropropanoyl esters of glucose
(
37,38
) as some natural toxic product. Phytochemical analysis
suggests that the presence of biologically activity compounds
(alkaloids, steroids, flavanoids, proteins—lectin,
carbohydrates, indigo, etc.) in the aqueous extracts of leaves of
I. suffruticosa could be correlated to anti-inflammatory and
antimicrobial activities (
11,21
). Biological activities of the
compounds detected in the aqueous extracts of leaves of
I. suffruticosa by infusion and maceration could be linked to
antitumor activity.
Aqueous extracts of leaves of I. suffruticosa by infusion
and maceration were used on Sarcoma 180 in mice at dose of
50 mg kg 1 i.p. The aqueous extracts showed a tumor
reducing activity. Our results concerning the aqueous extracts
of leaves of I. suffruticosa corroborate with those found in
aqueous extract of Emblica officinalis (Eupherbiacase),
which showed a reduction of solid tumor in mice. The extract
inhibits cell cycle regulating enzymes cdc 25 phosphatase
(
16,21
).
Actual mechanism by which aqueous extracts of leaves of
I. suffruticosa by infusion and maceration showed antitumor
activity is not known. The results of the present study indicate
that antitumor activity of aqueous extracts of I. suffruticosa
may be due to its interference with cell development. This will
be the object of future researches as modulating lipid
peroxidation and augmenting antioxidant defense system
(superoxide dismutase and catalase) (
2,19,24
), DNA synthesis
(
20
), interaction with cell cycle regulation (
21
) and
nonsteroidal anti-inflammatory activity (
1
). More recently,
molecular biomarkers have been used for oncological
management as prohibitin, mortalin and HSP 60/HSP 10 (
39
);
hyaluronic acid (HA) (
40
); epidermal growth factor receptor
(EGFR) and cytokeratins (
41
); research of tumor angiogenesis
with CD105 antigen (
42
); alpha-fetoprotein (AFP) (
43
);
cyclooxygenase-2 (COX-2) (
44
) and immunohistochemical
markers, such as CD34 and CD117 (
45
). Besides assessment
of prognostic factors as clinical (tumor growth rate and
inflammatory signs), to also analyze parameters like
hemoglobin content and red blood cell count, and histological (tumor
stage, grading, tumor necrosis, lymph nodes status and
margins status).
Same mechanisms of aqueous extract of leaves of
I. suffruticosa by infusion may be involved in the embryo
development in mice (
16
). The first cleavages in mammalian
embryogenesis are symmetrical mitotic divisions that increase
the number of blastomeres by partitioning the oocyte without a
net change in embryo size. Through the 8-cell stage the
blastomeres of murine embryos are totipotent (
46–48
). The
cell cycle of the two-cell embryos in the mouse has duration of
20–26 h with G1 and S phases that last 1–2 and 6–7 h; these
phases are followed by an extended G2 phase (12–15 h) and M
(mitosis) phase of 1–2 h (
49
). Many plant extracts have been
used as a source of medicinal agents to cure urinary tract
infections, cervicitis vaginitis, gastrointestinal disorders,
respiratory diseases, cutaneous problems, helmintic infections,
parasitic protozoan diseases, and antitumor and antimicrobial
(
17,50–53
). The results of this investigation may improve our
understanding in usage of this plant as an alternative
anticancer therapy. Identification of the active principles and
their mechanisms of action remain to be studied. This is a
promising plant for further studies toward drug development.
Acknowledgment
We wish to express our appreciation to CAPES (Coordenac¸a˜o
de Aperfeic¸oamento de Pessoal de N´ıvel Superior) for its
financial support.
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Received July 5 , 2006 ; accepted October 30, 2006 ology