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PHYTOCHEMICAL, ANTIOXIDANT AND CYTOTOXICITY OF
HYDROETHANOLIC EXTRACTS OF CROTALARIA RETUSA L.
Mathias Tawiah Anim1, Christopher Larbie1*, Regina Appiah-Opong2, Isaac Tuffour2,
Kofi Baffour-Awuah Owusu2 and Abigail Aning2
1Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science
and Technology, Kumasi-Ghana.
2Noguchi Memorial Institute of Medical Research, University of Ghana, Legon-Ghana.
ABSTRACT
For centuries now, plants have served as a great source of compounds
with pharmacological properties. Some plants like weeds, however,
have not been fully exploited for their medicinal value. In this
research, the phytochemical, total phenolic content, antioxidant and
antiproliferative effect of Crotalaria retusa, a weed reported to have
potential to fight tumours was assessed. The study focused on the leaf,
stem, seed, pod and flower of this plant as well as fractions of its stem
was assessed. Standard methods such as the MTT, the DPPH and the
Folin-Ciocalteau assays were used. The phytochemicals present
included saponins, tannins, alkaloids and sterols. The leaf was found to
have the highest concentration of phenols (67.35±1.153 mg GAE/g of
extract) and the best free radical scavenging activity with an EC50
value of 0.222±0.004 mg/mL. All the extracts induced cytotoxicity in a
dose dependent manner. The study revealed that the stem of C. retusa exhibited the highest
cytotoxicity against the selected cancer cells. However, extracts of the plant parts were
observed to be non-selective towards cancer cells since they were equally toxic to the normal
human liver (WRL 68) cells. All the fractions tested against the cancer cells did not exhibit
any significant increase in cytotoxicity. The study reveals the antiproliferative nature of C.
retusa, with Jurkat being the most sensitive cell line. The antioxidant potential of this plant
could be due in part, to its total phenol content. Further study is however needed to determine
the active principle of C. retusa.
World Journal of Pharmaceutical Research
SJIF Impact Factor 5.990
Volume 5, Issue 2, 162-179. Research Article ISSN 2277– 7105
Article Received on
04 Dec 2015,
Revised on 24 Dec 2015,
Accepted on 14 Jan 2016
*Correspondence for
Author
Dr. Christopher Larbie
Department of
Biochemistry and
Biotechnology Kwame
Nkrumah University of
Science and Technology
Kumasi-Ghana.
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KEYWORDS: Crotalaria retusa, Phytochemical, Antioxidant Cytotoxicity, Jurkat.
INTRODUCTION
Plants are nature’s gift to mankind in terms of providing us with food, oxygen, as well as
shelter. Since time immemorial, they have served as the first line of defence used by our
forefathers to fight diseases. Most orthodox drugs administered today were derived from
plants (Ncube et al., 2008).
The plant Euphobia peplus contains a compound called ingenol mebutate (Picato) which is
used to treat skin cancer (Zarchi and Jemec, 2015). The common drug quinine was also
derived from the bark of Cinchona officinalis, and this drug is widely prescribed for the
treatment of malaria in countries that cannot afford the more expensive anti-malaria drugs
(Reyburn et al., 2009). Some types of plants have been under-utilized and are as such
destroyed when they appear above the ground. Typical examples are weeds and ornamentals.
Native to Africa, the weed Crotalaria retusa L., commonly called the devil bean or rattle
box, is one of the numerous weeds in the Fabaceae family. It is a legume that has the
capability of accumulating monocrotaline, an important toxicant with a wide degree of
toxicity in animals (World Health Organization, 1988). This compound has been suggested to
have a potential for killing tumours since it is capable of killing lung cells (Schoental and
Head, 1955). Very little is however known of its antiproliferative activity on cancerous cells.
Cancer incidence and mortality has quickly increased over the past decade and is gradually
becoming a menace especially in less developed countries. Most patients in less developed
countries are unable to treat the disease mostly due to the high cost involved.Assessing the
anticancer potential of these locally available plants brings us one step closer to discovering
an alternative source of chemotherapy against cancer. This will possibly be less expensive
and affordable since these plants are readily available for preparation and administration.
The objective of this research was to assess the antiproliferative activity of 50%
hydroethanolic extracts of C. retusa parts. The phytochemical, antioxidant and total phenolic
content (TPC) of its leaf, stem, pod, seed and flower were analyzed.
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MATERIALS AND METHODS
Cell lines and reagents
The cell lines used (Jurkat, MCF 7, PC 3, WRL 68, HepG2) were obtained from RIKEN
BioResource Centre Cell Bank (Japan). Culture media (RPMI and α-MEM), 96 well plates,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye, isopropanol, HCl,
trypan blue solution, absolute ethanol, foetal bovine serum (FBS), antibiotics (penicillin and
streptomycin), 2, 2-diphenyl-1-picryl hydrazyl (DPPH) and phosphate buffer saline were
obtained from Sigma-Aldrich Company (St. Louis, MO, USA).
Plant Preparation
C. retusa samples were handpicked from the Tech Credit Union Building environs
(6°40'13.7"N 1°34'28.2"W), on the KNUST campus in April, 2014 before 9.00 am. Specimen
of the plant was sent to the Department of Herbal Medicine, Faculty of Pharmacy and
Pharmaceutical Sciences, KNUST, Kumasi for authentication by a taxonomist and a voucher
specimen was deposited at the Herbarium for reference purpose (KNUST/HMI/2014/L092).
The plant was sorted into leaf, pod, seed, flower and stem. The stem component was chopped
into pieces and all the parts were washed separately with water three times and air dried at
room temperature for three weeks. The dried samples were separately pulverized and
packaged in zip-locks for further use. Preparation of 50% hydroethanolic extracts of the
various parts was carried out separately, by suspending 50 g of the powder of each part in 500
mL of 50% ethanol (50:50 v/v). The extraction was done by cold maceration for 24 hrs at
room temperature on a shaker. The extracts were filtered through cotton wool, concentrated
using a rotary evaporator and freeze-dried to obtain the C. retusa hydroethanolic leaf, pod,
seed, flower and stem crude extracts.
Fractionation of hydroethanolic stem extract
Fractionation of the hydroethanolic stem extract of C. retusa was carried out in a separating
funnel using solvents of increasing polarity: petroleum ether, chloroform and ethyl acetate. A
mass of 2.5 g of crude hydroethanolic stem extract of C. retusa was dissolved in 25 mL of
50% ethanolic solution and was successively partitioned with petroleum ether, then with
chloroform and finally with ethyl acetate, each having a volume of 50 mL, to obtain
petroleum ether, chloroform and ethyl acetate fractions. This was done for two to three times
as polarity increased. The remaining portion was designated as hydroethanolic fraction.
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Phytochemical Screening
The presence of general glycosides, anthracene glycosides, saponins, tannins, alkaloids,
flavonoids, sterols and triterpenoids was analyzed using standard methods (Trease and Evans,
1989; Sofowora, 1993; Harborne, 1998).
Determination of total phenols
Total phenolic content (TPC) was determined using the Folin–Ciocalteau assay with slight
modification (Marinova et al., 2005). To a volume of 10 μL of sample, 790 μL of distilled
water was added. The concentration of the crude extracts tested was 5 mg/mL. A volume of
50 μL of Folin–Ciocalteau reagent was added to the diluted samples and thoroughly mixed.
The mixtures were incubated in the dark for 8 mins. Subsequently, 150 μL of 7% Na2CO3
was added before incubation of the mixture for 2 hrs in the dark at room temperature.
Triplicate experiments were performed. The absorbance was read at a wavelength of 750 nm
using a microplate reader (Tecan Infinite M200, Austria). Gallic acid (GA) was used as the
standard phenolic compound. A GA calibration curve was plotted and used to determine the
total phenolic content. The results were expressed in milligrams of GA equivalents per gram
dry mass (mg GAE/g DM).
Antioxidant assay
The antioxidant activity of C. retusa leaf, pod, seed, flower and stem extracts was determined
using the free radical scavenging activity by DPPH method with some modification (Blois,
1958). Methanolic solution of DPPH (0.5 mM) was added to equal volumes of various
concentrations of each extract (concentration range 0-5 mg/mL). After 20 mins incubation at
room temperature, the absorbance was read at a wavelength of 517 nm (Tecan Infinite M200
Pro plate reader, Austria). The inhibition concentration at 50% (IC50) value of each extract
was calculated from the following formula.
% Antioxidant activity = [(A0−A1)/A0 × 100]
where A0 is the absorbance of negative control (methanol) and A1 is the absorbance of test
sample with DPPH. Butylated hydroxytoluene (BHT) was used as standard control. Triplicate
experiments were performed. The EC50 value, which is the concentration of the extracts that
can cause 50% free radical scavenging activity, was determined.
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MTT assay
L-RPMI and α-MEM culture media respectively, supplemented with 10% foetal bovine
serum (FBS) containing penicillin, streptomycin and L-glutamine were maintained in culture
at 37°C in a humidified 5% CO2 atmosphere. The tetrazolium-based colorimetric assay
(MTT) was used to determine the cytotoxicity of C. retusa on the cancer and normal cell
lines (Ayisi et al., 2011). Cells were seeded into the 96-well plates at the concentration of
1×104 cells/well, treated with varying concentrations of the plant extracts (0-1000 μg/mL)
and incubated as indicated above for 72 hrs. A color control plate was also setup for each
extract including the positive control, curcumin. MTT solution (0.5 mg/mL) was added to
each well on the plate, and incubation continued for further 4 hrs. The reaction was stopped
with acidified isopropanol solution, and the plate incubated in the darkness overnight at room
temperature before reading the absorbance at 570 nm using a microplate reader (Tecan
Infinite M200 Pro, Austria). The percentage cell viability was determined as follows.
The IC50 values were determined from the plot of percent cell viability on the y-axis against
extract concentrations on the x-axis.
Statistical analysis
Data were analyzed by one-way analysis of variance and the means assessed by Tukey’s test
at 5% level of significance (p < 0.05) using Graph pad Prism version 5.0.
RESULTS
Phytochemical screening
Table 1 shows the presence and levels (marked by intensity of colour or froth) of general
glycosides, anthracene glycosides, saponins, tannins, alkaloids, flavonoids, sterols and
triterpenoids. In all the samples tested for, only the stem showed the presence of general
glycosides. None of the samples, however was observed to possess anthracene glycosides.
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Table 1: Phytochemical constituents present in 50% hydroethanolic crude extracts of C.
retusa parts.
Phytochemical
Seed
Pod
Flower
Stem
Leaf
General glycoside
++
++
+++
++
++
Anthracene glycoside
-
-
-
-
-
Saponins
+++
+++
+++
+++
+++
Tannins
+++
+++
+++
+++
+++
Alkaloids
+++
++
++
++
++
Flavonoids
+++
+++
+++
+++
-
Sterols
+
+++
+++
++
++
Triterpenoids
-
+++
-
-
-
The data show the intensities of observed colours or froths as compared to standard. +++
present at high concentration; ++ present in moderate concentration; + present in low
concentration; - absent.
Total Phenol Content
From the standard linear plot constructed, the total phenolic content of all the plant extracts
were extrapolated. The leaf of the C. retusa plant recorded the highest concentration of
phenols (67.35±1.153 mg Gallic acid equivalent/g) while the seed was least in total phenolic
content. The total phenol content of the leaf, stem, seed, pod and flower is shown in Table 2.
Figure 1: Standard calibration plot obtained from the various gallic acid
concentrations.
Table 2: Total phenolic content of hydroethanolic extracts of C. retusa.
Plant
Part
Total phenolic content
(mg GAE/g)
P-value
C. retusa
Seed
34.70±0.573
< 0.0001
Pod
61.22±0.0
Flower
37.15±0.870
Stem
50.62±0.290
Leaf
67.35±1.153
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Antioxidant activity
The ability of the extracts to scavenge DPPH free radical was used to assess it antioxidant
activity. The leaf of C. retusa exhibited the strongest antioxidant activity with an EC50 value
of 0.222 mg/mL. The antioxidant activity of the standard (BHT), stem, leaf, flower, seed and
pod of C. retusa is shown in figure 2.
Antiproliferative activity of extracts and curcumin
All the extracts exhibited cytotoxicity towards Jurkat, MCF 7, PC 3 and WRL 68 in a dose
dependent manner. Only the stem extract was active against all the tested cell lines with its
strongest inhibition against Jurkat (IC50 value = 221.97µg/mL). Curcumin inhibited the
growth of the normal cell (WRL 68), likewise the pod, flower, stem and leaf. Figures 3, 4, 5
and 6 show the antiproliferative activity of the parts of C. retusa on the cells.
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Figure 2: Antioxidant activity of (A) C. retusa stem, (B) C. retusa leaf, (C) C. retusa seed,
(D) C. retusa pod, (E) C. retusa flower and (F) BHT. Each point represents a mean of
three determinations.
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Figure 3: Antiproliferative activity of (A) C. retusa stem, (B) C. retusa leaf, (C) C. retusa
seed, (D) C. retusa pod, (E) C. retusa flower and (F) Curcumin on Jurkat cells. Each
point represents a mean of three determinations.
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Figure 4: Antiproliferative activity of (A) C. retusa stem, (B) C. retusa leaf, (C) C. retusa
seed, (D) C. retusa pod, (E) C. retusa flower and (F) Curcumin on MCF 7 cells. Each
point represents a mean of three determinations.
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Figure 5: Antiproliferative activity of (A) C. retusa stem, (B) C. retusa leaf, (C) C. retusa
seed, (D) C. retusa pod, (E) C. retusa flower and (F) Curcumin on PC 3 cells. Each point
represents a mean of three determinations.
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Figure 6: Antiproliferative activity of (A) C. retusa stem, (B) C. retusa leaf, (C) C. retusa
seed, (D) C. retusa pod, (E) C. retusa flower and (F) Curcumin on WRL 68 cells. Each
point represents a mean of three determinations.
Antiproliferative activity of C. retusa stem fractions and curcumin
Petroleum ether, chloroform, ethyl acetate and hydroethanolic fractions of the most active
part of the plant, its stem, were also assessed for their level of cytotoxicity. Figures 7, 8 and 9
show the antiproliferative activity of these fractions on Jurkat, MCF 7 and HepG2 cells
respectively.
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Figure 7: Antiproliferative activity of C. retusa stem petroleum ether (A), chloroform
(B), ethyl acetate (C), hydroethanolic (D) fractions and curcumin (E) on Jurkat cells.
Each point represents a mean of three determinations.
Figure 8: Antiproliferative activity of C. retusa stem petroleum ether (A), chloroform
(B), ethyl acetate (C), hydroethanolic (D) fractions and curcumin (E) on MCF 7 cells.
Each point represents a mean of three determinations.
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Figure 9: Antiproliferative activity of C. retusa stem petroleum ether (A), chloroform
(B), ethyl acetate (C), hydroethanolic (D) fractions and curcumin (E) on HepG2 cells.
Each point represents a mean of three determinations.
DISCUSSION
All the crude extracts of the various parts of the plant considered expressed a rather high level
of saponins and tannins, with alkaloids and sterols varying in concentration.Earlier studies
have shown the presence of alkaloids and sterols in the leaf of C. retusa (Dhole et al., 2012).
The presence of alkaloids and sterols in the leaf of this plant was confirmed in this research.
Flavonoids and triterpenoids were absent from the leaves of this plant. Only the stem of C.
retusa was observed to contain general glycosides.
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Phytochemicals such as phenols and polyphenolic compounds like flavonoids are generally
present in medicinal plants and these compounds have been shown to possess good
antioxidant activities (Van Acker et al., 1996). An earlier study revealed that C. retusa
possessed the strongest antioxidant activity compared with other Crotalaria species
(Devendra et al., 2012). Results from this research showed that C. retusa indeed has great
antioxidant activity, predominantly found in its leaf. There was significant difference
between the observed total phenolic contents of the various parts analyzed (p-value <
0.0001).
Devendra et al. (2012) indicated compounds isolated from ethanolic extracts of Crotalaria
species, possess pharmacological properties and potential to develop natural compound-based
pharmaceutical products. The findings of this study confirm the presence of potential
compounds with the ability to scavenge free radical and potential anti-carcinogenic and anti-
inflammatory agents because of the observed high antioxidant activity (Stavric, 1993;
Elangovan et al., 1994; Marrin et al., 2002).
The MTT cell viability assay showed that only the stem of this plant had antiproliferative
activity against all the three cancer cells (Jurkat, MCF 7 and PC 3). Jurkat was most sensitive
to all the C. retusa extracts used in this research compared to the other cells.
The seed of C. retusa has been reported by Maia et al. (2013), to have high levels of the
hepatotoxic alkaloid, monocrotaline, which requires bioactivation to become toxic to
hepatocytes (John et al., 2005). Thus, the flower (other than the seed) of C. retusa was the
most hepatotoxic part of this plant (toxic to WRL 68 cells), eventhough the seed has the
highest levels of monocrotaline. These findings suggest that indeed, monocrotaline, though
hepatotoxic would require bioactivation (John et al., 2005), hence its reduced toxicity in
vitro. The flower could possibly contain certain compounds that would not require
bioactivation consequently, its observed toxicity in vitro.
All the extracts analyzed were cytotoxic towards the normal human liver cell (WRL 68), thus
exhibiting a rather poor selectivity. This suggests some degree of toxicity when any part of
this plant is ingested for the purpose of alleviating a disease condition. However, further in
vivo studies will be required to confirm this.
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All the fractions obtained from the stem of this plant were relatively less toxic against the
cancer cells as compared to the crude extract. This could be attributed to the fact that the
active molecules in the extracts worked in a synergistic manner (or the activity of the active
compound was complemented by another compound) and individually was not that effective.
On the other hand, it is possible that the solvent used for fractionation was unsuitable for the
purpose. Hence the fractionation procedure did not yield any significant increase in
cytotoxicity.
CONCLUSION
Data from this study suggests C. retusa leaf is a good source of phenolic compounds with
good antioxidant properties. The stem of C. retusa, is effective in inhibiting the growth of
Jurkat, MCF 7 and PC 3 cancer cells. However, its cytoxicity towards normal cell (WRL 68)
renders it a less desirable chemotherapeutic alternative.
Purification of C. retusa by Srinivas et al. (2014) yielded monocrotaline, which was much
more toxic towards VERO (kidney) and HeLa (cervical) cancer cells. Though fractions from
the stem had a lower cytotoxicity effect on cancer cells relative to the crude extract, it is
possible that further purification and isolation of a pure compound from the stem of C. retusa
is likely to improve the observed cytotoxicity on the cancer cell lines and possibly reduce its
toxicity towards normal cell. This could serve as a lead to identifying and developing a
potential chemotherapeutic agent against cancer.
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