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Since most of the standard anticancer treatments are
not selective and affect both tumor and normal cells, thereby causing systemic
toxicity or increased risk of other cancers. Thus, there is a need for the
development of safer alternatives for the treatment of cancer which are
affordable, accessible, having less toxicity and minimum side effects. This
paper is carried out to investigate the anticancer MCF7 (breast cancer), HeLa
(cervical cancer) cell line, antioxidant (iron chelating properties and
chemiluminescence assay), cytotoxicity activities and phytochemical
investigation Sudanese medicinal plant A.
arabica leaves and gum.
Plant parts were extracted using 80% methanol. The
anticancer activity was examined by using MTT assay. And determine their
antioxidant activities, screened for their cytotoxicity using - (4,5-Dimethyl
thiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), filter and kept in dark,
prepared freshly.
The extract A.
arabica gum is showed high activity against chemiluminescence assay IC50
values 27.3 μg/ml and no activity in leaves >100 μg/ml, antioxidant (Iron
chelating properties) no activity levels of inhibition % in A. arabica gum 11.92 μg/ml. The methanol
extracts of the plant showed considerable activity against MCF7 (breast cancer)
according to the inhibition percentage A.
arabica gum. Also A. arabica
leaves and gum against HeLa (cervical cancer) cell line no activity IC50
values>100 μg/ml.
Phytochemical screening of the extracts A. arabica leaves indicated presence of
alkaloids, cumarin, triterpenes, saponins in trace amount and sterols in
moderate amount and tannins, flavonoids in high amount and anthraqunones were
not detected. A. arabica gum
indicated presence of alkaloids, cumarin in trace amount and triterpenes in
moderate amount and tannins, flavonoids, sterols, saponins in high amount and
anthraqunones were not detected.
Keywords:
Anticancer, Chemiluminescence, Phytochemical, HeLa
INTRODUCTION
Two classes
of use of medicinal preparations are commonly recognized, often under the
titles herbal and pharmaceutical. Pharmaceuticals, discussed below, are refined
or synthesized drugs. The World Health Organization has defined medicinal
herbals as follows [1]. Finished, labeled medicinal products that contain as
active ingredients aerial or underground parts of plants or other plant
material or combinations thereof, whether in the crude state or as plant
preparations. Plant material includes juices, gums, fatty oils, essential oils
and any other substances of this nature. Herbal medicines may contain
excipients [inert additives such as starch used to improve adhesive quality in
order to prepare pills or tablets in addition to the active ingredients. Medicines
containing plant material combined with chemically defined active substances,
including chemically defined, isolated constituents of plants, are not
considered to be herbal medicines. Exceptionally, in some countries herbal
medicines may also contain, by tradition, natural organic or inorganic active
ingredients which are not of plant origin [1].
Botanical description
First
described in Africa by the Swedish botanist Carl Linnaeus in 1773. They are
pod-bearing, with sap and leaves typically bearing large amounts of tannins and
condensed tannins that historically found use as pharmaceuticals and
preservatives. The genus Acacia previously contained roughly 1300 species,
about 960 of them native to Australia, with the remainder spread around the
tropical to warm-temperate regions of both hemispheres, including Europe,
Africa, southern Asia and the Americas. However, in 2005 the genus was divided
into five separate genera under the tribe “Acacieae”. The genus Acacia was
retained for the majority of the Australian species and a few in tropical Asia,
Madagascar and Pacific islands. Most of the species outside Australia and a
small number of Australian species were reclassified into vachellia and
senegalia. The two final genera, Acaciella and Mariosousa, each contain about a
dozen species from the America.
Biological activity
Acacia species
have possible uses in folk medicine. A 19th century Ethiopian
medical text describes a potion made from an Ethiopian mixed with the roots of
the tacha, then boiled, as a cure for rabies [2]. An astringent medicine high
in tannins, called catechuor cutch, is procured from several species, but more
especially from Acacia catechu.by boiling down the wood and evaporating the
solution so as to get an extract [3]. The catechu extract from Acacia catechu figures in the history of
chemistry in giving its name to the catechin, catechol and catecholamine
chemical families ultimately derived from it [2].
PHYTOCHEMICAL PROPERTIES
Acacias contain
a number of organic compounds that defend them from pests and grazing animals [4].
Many of these compounds are psychoactive in humans. The alkaloids found in
Acacias include dimethyltryptamine (DMT), 5-methoxy-dimethyltryptamine
(5-MeO-DMT) and N-methyltryptamine (NMT). The plant leaves, stems and/or roots
are sometimes made into a brew together with some MAOI-containing plant and
consumed orally for healing, ceremonial or religious uses. Nineteen different
species of Acacia in the Americas contain cynogenic glycosides, which, if
exposed to an enzyme which specifically splits glycosides, can release hydrogen
cyanid (
MATERIALS AND METHODS
Collection of tested
plant parts
Tested plant parts of the Acacia arabica leaves and gum were
collected from Elsunut Forest, Khartoum State, collected of during the period
June and July 2010 and identified of taxonomist team of MABRI (Medicinal and
Aromatic Plants Research Institute, National Center of Research) Khartoum,
Sudan. And herbarium voucher deposit at herbarium medicinal plants in the
MAPRI.
Preparation of crude
plant extract
100 g of each plant sample was art coarsely
powdered using Mortar and pistil and extracted with 80% methanol extracted for
18 h with by using shaker (Stuart scientific, flash shaker, S F 1, U K). The
extract was filter and evaporated using rotary evaporator at 40°C (Buchi, 461,
Switzerland).
Culture media and human tumor cell lines human cell lines
MCF7 (breast cancer), HeLa (cervical cancer)
cell line were obtained frozen in liquid nitrogen (-180°C), the tumor cell
lines were maintained in the Institute of ICCB, University of Karachi Pakistan.
Culture media
RPMI-1640 medium was used for culturing and
maintenance of the human tumor cell lines. The medium was supplied in a soluble
form. Before using the medium it was warm at 37°C in a water bath and
supplemented with penicillin/streptomycin and Fetal bovine serum (FBS) with 10% concentration.
The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 and were sub cultured twice a week.
Procedure
Maintenance of the human cancer cell lines in the
laboratory: A cry
tube containing frozen cells was taken out of the liquid nitrogen container and
then thawed in a water bath at 37°C. The cry tube was opened under strict
aseptic conditions and its content were supplied by 5 ml complete media
(RPMI-1640 in 10% fetal bovine serum) drop by drop in a 50 ml disposable sterile falcon tubes and were centrifuged
at 1200 rpm for 10 min to discard the preserving solution. The supernatant was
discarded and the cell pellet was seeded in 5 ml complete media in T25 Nunclon
sterile tissue culture flasks. The cell suspension was incubated at 37°C in a
humidified atmosphere with 5% CO2 and followed up daily with changing the supplemented
medium every 2-3 days. Incubation was continued until a confluent growth was
achieved and the cells were freshly sub cultured before each experiment.
Collection of cells
by trypsinization: The
media was discarded. The cell monolayer was washed twice with 5 ml phosphate
buffered saline and all the adherent cells were dispersed from their monolayer
by the addition of 1 ml trypsin solution (0.025 % trypsin w/v) for 2 min. The
flask was left in the incubator till complete detachment of all the cells and
checked with the inverted microscope (Olympus). Trypsin was inactivated by the
addition of 5 ml of the complete media. The trypsin content was discarded by
centrifugation at 1200 rpm for 10 min. The supernatant was discarded and the
cells were separated into single cell suspension by gentle dispersion several
times, then suspended and seeded in 5 ml complete media in T25 Nunclon sterile
tissue culture flasks.
Determination and
counting of viable cells: 50 μl of fresh culture media was added to 50 μl of the single cell
suspension. The cells were examined under the inverted microscope using the hemocytometer.
Viable cells were counted and the following equation was used to calculate the
cell count/ml of cell suspension.
Viable cells /ml= number of cells
in 4 quarters × 2 (dilution factor) × 104 / 4
The cells were then diluted to give the
concentration of single cell suspension required for each experiment. The cell
count was adjusted to 1 × 104-105 cells/ml using medium
containing 10% fetal bovine serum.
Cryopreservation of cells:
To avoid the loss of the cell line, excess cells were preserved in liquid
nitrogen as follows: Equal parts of the cell suspension and freezing medium
(10% DMSO in complete media) were dispersed to cry tubes. The cry tubes were
racked in appropriately labeled polystyrene boxes gradually cooled till
reaching -80°C. Then the cry tubes were transferred to a
liquid nitrogen (-196°C).
Micro culture
tetrazolium (MTT) assay
MTT assay: In order to evaluate the
cytotoxicity effect of the extracts and compounds, the following procedure of
the MTT was used.
MTT procedure: Serial dilutions of extract were
prepared in a 96 well flat bottomed plate (Nalge Nunc, Inter.). The outer walls
of the plate were filled with 250 µl of incomplete culture medium except the
last row 6 middle wells (B-G), which were used for the negative control
receiving 50 µl of culture medium and 2 µl of sterile 0.5% Triton X.
To the rest of the plate, 50 µl/wells (CCM)
were added and 30 µl more were added to second column wells (B-G) that were
used as first extract dilution wells. To the first dilution wells in the row,
500 µg of c suspension extract were added to the 80 µl. extract were then
serially diluted by two-fold dilution from well B3 till B11 by transferring 250
µl to the next well after proper mixing. From the last dilution wells (B-11),
50 µl were discarded. Each compound was tested in triplicate. Cell suspension
in a complete culture medium containing 2.5 × 105/ml was properly
mixed and 150 µl of it were transferred into each well of the plate. The plate
was covered and
placed in 5% CO2 incubator at 37°C for
three-five days (72-120 h). On the third/fifth day, the supernatant was
removed from each well without detaching cells. MTT stock (5 mg/ml) was
prepared earlier in 100 ml PBS. MTT suspension was vortexed and kept on
magnetic stirrer until all MTT dissolved. The clear suspension was filter
sterilized with 0.2 µ Millipore filter and stored at 4°C or –20 until use. MTT was
diluted (1:3.5) in a culture medium and brought to room temperature. To each
well of the 96 well plates, 50 µl of diluted MTT were added. The plate was incubated
further at 37°C for 2-3 h in CO2 incubator. MTT was removed carefully without
detaching cells and 200 µl of DMSO were added to each well. The plate was
agitated at room temperature for 15 min then read at 540 nm using micro plate
reader.
% Inhibition = [(AControl
– ASample) / AControl] × 100
Where, AControl is the absorbance
of the negative control and ASample the absorbance of tested samples
or standard. All data are an average of triplicate analyses.
Antioxidant assay
Metal chelating
activity assay: The
iron chelating ability was determined according to the modified method of Dinis
et al. [5]. The Fe+2 were monitored by measuring the formation of
ferrous ion-ferrozine complex. The experiment was carried out in 96 micro titer
plates. The plant extracts were mixed with FeSO4. The reaction was
initiated by adding 5 mM ferrozine. The mixture was shaken and left at room
temperature for 10 min. The absorbance was measured at 562 nm. EDTA was used as
standard and DMSO as control. All tests and analysis were run in triplicate.
Chemiluminescence assay: Luminol or lucigenin-enhanced
chemiluminescence assay was performed. Briefly, 25 μL diluted whole blood (1:50
dilution in sterile HBSS++ (Hanks Balance Salt Solution with Ca and Mg) or 25
µL of PMNCs (polymorph nuclear cells) (1 × 106) or MNCs (mono
nuclear cells) (5 × 106) cells were incubated with 25 µL of serially
diluted plant extract at concentration ranges between 6.25 and 100 μg/mL.
Control wells received HBSS++ and cells but no extract. Tests were performed in
white 96 wells plates, which were incubated at 37°C for 30 min in the
thermostated chamber of the luminometer. Opsonized zymosan-A or PMA 25 μL,
followed by 25 µL luminol (7 × 105 M) or lucigenin (0.5 mM) along
with HBSS++ were added to each well to obtain a 200 µL volume/well. The
luminometer results were monitored as chemiluminescence RLU (reading
luminometer unit) with peak and total integral values set with repeated scans
at 30 s intervals and one second points measuring time.
Phytochemical
screening
General phytochemical screening to detect the
chemical groups was carried out for all extracts using the methods described by
various researchers [6-9] with few modifications.
Identification of
tannins
0.2 g of each extract was dissolved in 10 ml
of hot saline solution and divided in two test tubes. In the first tube 2-3
drops of ferric chloride were added and 2-3 drops of gelatin salt reagent were
added to the other tube. The occurrence of a blackish blue color in the first
test tube and turbidity in the second one indicates the presence of tannins.
Test of sterols and triterpenes
0.2 g of each
extract was dissolved in 10 ml of chloroform, to 50 ml of
the solution 0.5 ml acetic anhydride was added and then 3 drops of conc.
Sulphuric acid at the bottom of the test tube. At the contact
zone of the two liquids a gradual appearance of green, blue pink to purple
color is indicates the presence of sterols (green to blue) and or triterpenes
(pink to purple) in the sample.
Test for alkaloids
0.5 g of each extract was dissolved in 2 ml
of 2 N HCl in water bath and stirred while heating for 10 min, then cooled
filtered and divided into two test tubes. To the first test tube a few drops of
Mayer’s reagent were added while to the other tube a few drops of Valser’s
reagent were added. A slight turbidity or heavy precipitate in either of the
two test tubes indicates the presence of alkaloids.
Tests for flavonoids
0.5 g of each
extract was dissolved in 30 ml of 80% ethanol and filtered. The filtrate was
used for following tests:
(i) To 3
ml of the filtrate in a test tube 1 ml of 1% aluminum chloride solution was in
methanol was added. Formation of a yellow color indicated the presence of
flavonoids. Flavones and or chalcone.
(ii) To 3
ml of the filtrate in a test tube 1 ml of 1% potassium hydroxide solution was
added. A dark yellow color indicated the presence of flavonoids compounds
(flavones or flavonones) chalcone and/or flavonols.
(iii) To 2
ml of the filtrate 0.5 ml of magnesium turnings were added. Production of a
faint pink or red color was taken as presumptive evidence that flavonones are
present in the plant sample.
Test for saponins
0.3 g of each extract was placed in a clean
test tube. 10 ml of distilled water were added, the tube stoppered and
vigorously shaken for about 30 s. The tube was then allowed to stand and
observed for the formation of foam, which persisted for at least 1 h, was taken
as evidence for presence of saponins.
Test for cumarins
0.2 g of each extract dissolved in 10 ml
distilled water in test tube and filter paper attached to the test tube to be
saturated with the vapor after a spot of 0.5 N KOH put on it. Then the filter
paper was inspected under UV light, the presence of coumarins was indicated if
the spot was found to have adsorbed the UV light.
Test for anthraquinone
glycosides
0.2 g of each extract was boiled with 10 ml
of 0.5 N KOH containing 1 ml of 3% hydrogen peroxide solution. The mixture was
extracted by shaking with 10 ml of benzene. 5 ml of the benzene solution was
shaken with 3 ml of 10% ammonium hydroxide solution and the two layers were
allowed to separate. The presence of anthraquinones was indicated if the
alkaline layer was found to have assumed pink or red color.
Test for cyanogenic
glycosides
0.2 g of extract fraction was placed in
Erlenmeyer flask and sufficient amount of water was added to moisten the
sample, followed by 1 ml of chloroform (to enhance every activity). A piece of
freshly prepared sodium picrate paper was carefully inserted between a split
cork which was used to stopper the flask, a change in color of the sodium
picrate paper from yellow to various shades of red was taken as an indication
of the presence of cyanogenic glycoside.
STATISTICAL ANALYSIS
All data are presented as mean ± standard
deviation of the mean – statistical analysis for all the assays result were
done using students t-test significance was tribute to probability values
P<0.05 or P>0.01 in some cases.
RESULTS AND
DISCUSSION
The importance of medicinal plants, and the
contribution of phytomedicine to the well-being of a significant number of the
world's population, has attracted interest from a variety of disciplines. The
isolated polyphenols from strawberry including kaempferol, quercetin,
anthocyanins, coumaric acid and ellagic acid were shown to inhibit the growth of
human cancer cell lines originated from breast (MCF-7), oral (KB, CAL-27),
colon (HT-29, HCT-116) and prostata (LNCaP, DU-145) [10].
The antioxidants may prevent and cure cancer
and other diseases by protecting the cells from damages caused by ‘free radicals’
the highly reactive oxygen compounds. Many naturally occurring substances
present in the human diet have been identified as potential chemo-preventive
agents; and consuming relatively large amounts of vegetables and fruits can
prevent the development of cancer. Many plant-derived products have been
reported to exhibit potent anti-tumors activity against several rodent and
human cancer cell lines [11].
So the main objective of this paper was to
screen Sudanese medicinal plant parts A.
arabica for their anticancer and antioxidant activity to find medicinal
plants having potent anticancer activity.
In the present work the first experiment was determined the anticancer
activity against MCF7
(Breast cancer) and HeLa (cervical cancer) used traditionally to treat various
diseases followed by two methods determine the antioxidant activity of the
crude extracts which were Iron chelating properties and chemiluminescence assay.
The most frequent types of cancer differ between
men and women, about 30% of cancer deaths are due to the five leading
behavioral and dietary risks: high body mass index, low fruit and vegetable
intake, lack of physical activity, tobacco use and alcohol use, tobacco use is
the most important risk factor for cancer causing 22% of global cancer deaths
and 71% of global lung cancer deaths, cancer causing viral infections such as
HBV/HCV and HPV are responsible for up to 20% of cancer deaths in low- and
middle-income countries, about 70% of all cancer deaths in 2008 occurred in
low- and middle-income countries, deaths from cancer worldwide are projected to
continue rising, with an estimated 13.1 million deaths in 2030 [12]. The
antioxidants may prevent and cure cancer and other diseases by protecting the cells
from damages caused by ‘free radicals’ the highly reactive oxygen compounds.
Many naturally occurring substances present in the human diet have been
identified as potential chemo-preventive agents; and consuming relatively large
amounts of vegetables and fruits can prevent the development of cancer. Many
plant-derived products have been reported to exhibit potent anti-tumors
activity against several rodent and human cancer cell lines [11].
Very high activity against PC3 cell lines (IC50 39.4 µg/ml) the antioxidant activity using the DPPH assay
of the gum was very high (IC50 1.009 µg/ml) whereas the leaves showed high activity with
(IC50 12.8 µg/ml) [13].
Moreover both tested parts (leaves and gum) were not toxic on normal
cell lines (IC50>100 µg/ml) [13]. Phytochemical screening of the
extracts A. arabica leaves indicated
presence of alkaloids, coumarin, triterpenes, saponins
in trace amount
and sterols in moderate amount
and tannins, flavonoids in
high amount and anthraquenones were not detected. Phytochemical, screening of
the extracts A. arabica gum indicated
presence of alkaloids, coumarin in trace amount and triterpenes in moderate
amount and tannins, flavonoids, sterols, saponins in high amount and
anthraqunones were not detected. Gum of Acacia for their oxidative burst high
activity against whole
blood (IC50 27.3 µg/ml). In
previous study Acacia species have possible uses in folk medicine. A 19th
century Ethiopian medical text describes a potion made from the gum and leaves
and mixed with the roots of the tacha, then boiled and used as a cure for
rabies [2]. An astringent medicine high in tannins, called catechuor cutch, is
procured from several species, but more especially from Acacia catechu by boiling down the wood and evaporating the
solution so as to get an extract [3]. The catechu extract from Acacia catechu figures in the history of
chemistry in giving its name to the catechin, catechol and catecholamine
chemical families ultimately derived from it [2]. Acacia contain a number of
organic compounds that defend them from pests and grazing animals [4]. Many of
these compounds are psychoactive in humans. The alkaloids found in Acacia
include dimethyltryptamine (DMT), 5-methoxy-dimethyltryptamine (5-MeO-DMT) and
N-methyltryptamine (NMT). The plant leaves, stems and/or roots are sometimes
made into a brew together with some MAOI-containing plant and consumed orally
for healing, ceremonial or religious uses. Nineteen different species of Acacia
in the Americas contain cynogenic glycosides, which, if exposed to an enzyme
which specifically splits glycosides, can release hydrogen cyanid (HCN) in the
Acacia “leaves”. If fresh plant material spontaneously produces 200 ppm or more
HCN, then it is potentially toxic. This corresponds to about 7.5 μmol HCN/g of
fresh plant material. It turns out that, if Acacia “leaves” lack the specific
glycoside-splitting enzyme, then they may be less toxic than otherwise, even
those containing significant quantities of cyanic glycosides [4].
1.
WHO (1996) World Health Organization Expert Committee on
Specifications for Pharmaceutical Preparations. WHO (Geneva) Thirty-fourth
report, 1996 (WHO Technical Report Series No. 863), pp: 178-184.
2.
Remington JP, Wood HC (1918) The US Dispensatory. The
Dispensatory of the United States of America, 1918.
3.
FAO (2011) Cutch and catechu plant origin from the Food
and Agriculture Department of the United Nations Annual report.
4.
Meehan C, Olson E, Curry Robert L (2008) Exploitation of
the Pseudomyrmex - Acacia mutualism by a predominantly vegetarian jumping
spider (Bagheera kiplingi) The 93rd
ESA Annual Meeting USA.
5.
Dinis T, Madeira V, Almeida M (1994) Action of phenolic derivates
(acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid
peroxidation and as peroxyl radical scavengers. Arch Biochem Biophys 315:
161-169.
6.
Sofowora A (1993) Medicinal plants and traditional
medicines in Africa. Chichester John, Willey & Sons: New York, USA, p: 256.
7.
Harborne J (1984) Phytochemical methods. 2nd Edn.
Chapman and Hall USA.
8.
Martinez A, Valencia G, Fitoquimica M (2003) In Manual de
prácticas de Farmacognosia y Fitoquímica. 1st Edn. Medellin:
Universidad de Antioquia, Phytochemical screening methods, pp: 59-65.
9.
Wall M, Eddy C, Mc Clenna M, Klump M (1952) Detection and
estimation of steroid and sapogenins in plant tissue. Anal Chem 24: 1337-1342.
10.
Zhang X, Wang Y, Ge H (2008) Association of CDH1 single
nucleotide polymorphisms with susceptibility to esophageal squamous cell
carcinomas and gastric cardiac carcinomas. Hebei Cancer Institute and the
Fourth Affiliated Hospital, Hebei Medical University, Shijiazhuang Hebei
Province, China.
11.
Madhuri S, Govind P (2009) Some anticancer medicinal
plants of foreign origin. J Curr Sci 96: 779-783.
12.
WHO (2012) Fact sheet No 297. February WHO Media centre.
13.
Elnour MA, Elegamy AA, Koko WS, Khalid A, Fadul E (2013)
Antioxidant activity and cytotoxicity of some Sudanese medicinal plants. Int J
Adv Industrial Eng 1: 20-23.
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