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Anti oxidant activity and nthocyanin content in flower of Mirabilis jalab
L. collected from Yemen.
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Abstract: The
aim of this study was to investigate and compare Mirabilis jalab L. extracts
obtained in
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Ultrasonic
condition with different water/methanol and water/ethanol extraction mixture
acidified with 0.1% HCl. The extracts were analyzed for monomeric
anthocyanins contents and antioxidant activities. The highest anthocyanins
content (3197.8 mg/L) and the best antioxidant
activity were obtained for the Mirabilis jalab extract with 100% ethanol.
Also, there is a good correlations between antioxidant activity (R2=
0.9332 and 0.9712) for water/ethanol and methanol series extracts
respectively.
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Keywords: Mirabilis jalab,
anthocyanins, antioxidant activity, free radical scavenging activity.
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1 Introduction
Mirabilis jalapa
is an erect herb that can grow up to 1 m tall. Leaves are simple, heart-shaped,
3–12 cm long, opposite, tapering to a pointed end. Flowers are bisexual, red,
pink, yellow or white, with perianth distinctly constricted above, and they
bloom late in the afternoon. Fruits are black and globose, 5–8 mm in diameter.
The use of traditional medicine at the primary health care level is widespread
in Yemen; traditional healers use
many local plants for the treatment of microbial disease. Mirabilis jalapa,
a wild grown plant, is used to cure externally infected wounds. In Latin
America, this plant is used in traditional medicine because of the purgative or
emetic cathartic properties contained in its roots (Perrot, 1943). In
Malagassy, it is used to treat intestinal pains. In South Africa, the roots are
used as a purgative drug and it is reputed that the flowers of this plant at
night exhale a strong odour which will stupefy or drive away mosquitoes. The
roots contain oxymethylanthraquinone
(Watt and Breyer-Brandwijk, 1962).Furthermore,
it has been reported that Mirabilis jalaba harbor antiviral activity
against viruses (Kubo et al, 1990), exhibit antifungal activity (De
Bolle et al., 1995; Wang et al., 2001) and showing features as
anticarcinogen chemotherapeutic potential. Moreover, in recent years,
researches determining the anthocyanin content, relation with chlorophyll and
regulation of pigments with genes (Russell., 1997; Kubo et al, 1990;
Vivanco et al., 1999; Dela et al., 2003; Halbwirth et al.,
2003). Anthocyanins are
representative of plant pigments widely distributed in colored fruits and
flowers. Because anthocyanins are widely consumed, finding out additional
biological activities related to these compounds would be of great interest
(Andreia et al., 2007). Anthocyanins are normally obtained
by extraction from plants and the extraction methods currently employed are
with the use methanol, ethanol, acetone, water or mixtures as solvents. In fact,
the color stability of anthocyanins depends on a combination of factors, such
as the structure and concentration of the anthocyanin, pH, temperature and
presence of complex agents such as phenols and metals (Padma
et al.,
2010). The most common solvents used for
anthocyanins extraction are aqueous mixtures of ethanol, methanol or acetone
(Kahkonen et al., 2001). The adverse
effects of oxidative stress on human health have become a serious issue (Duduku
et al., 2011). Under stress, our
bodies produce more reactive oxygen species (ROS) such as; superoxide anion
radicals, hydroxyl radicals and hydrogen peroxide than enzymatic antioxidants such as; superoxide dismutase (Riley, 1994).,
glutathione peroxidase, and catalase and non-enzymatic antioxidants such as;
ascorbic acids, glutathione, carotenoids, and flavonoids. This imbalance leads
to damage of biological structures such as proteins, lipids and DNA and induce
a variety of human diseases (Elahi and Matata, 2006; Thrasivoulou et al., 2006; Aruoma, 1998; Lefer and
Granger, 2000; Smith et al., 2000;
Bhatia et al., 2003; Peuchant et
al., 2004). Antioxidants from fruits and
vegetables, especially with an intense colouration, are considered an important
protection factor against oxidative stress and its deleterious consequences to
human health (Vollmannova et al.,
2009; Pineli et al., 2011).
The antioxidative capacity, which is defined as the capacity to inhibit or
delay the oxidation of other molecules, anthocyanins and their aglycones
(anthocyanidins) and their free radical scavenging activity have been revealed
(Wang et al., 1999; Nakajima et al., 2004; Orak, 2006). The
antioxidant activity of berries is directly proportional to the anthocyanins
content (Adina et al., 2010). In
response to the increased popularity and greater demand for medicinal plants, a
number of conservation groups are recommending that wild medicinal plants be
brought into cultivation. Various herbs and spices have been reported to
exhibit anthocyanin content in their flowers including Mirabilis jalaba (Padma
and Dhara, 2010) and it is reported
that,various herbs and species have been reported to exhibit antioxidant
activity, Ocimum sanctum, Piper cubeba, Allium sativum, Terminalia
bellerica, Camellia sinensis and Zingiber officinale (Nooman et al ., 2008).Various analytical
methods have been used to evaluate the antioxidant properties of phenolic
compounds: the 1, 1-diphenyl- 2-picrylhydrazyl (DPPH) assay proves the capacity
of the antioxidants to quench the PPH radical, whereas the ORAC method is based
on the loss of fluorescence of the -phycoerythrin protein or of fluorescein
upon oxidation (Cao, et al., 1993;
Ninfali, et al., 2005). Reactive
oxygen species (ROS), including super oxide anion (O2-),
hydroxyl radical (OH) and hydrogen peroxide (H2O2), exist
in living organisms (Riley, 1994). The red colour of the wild Yemeni Mirabilis
jalaba is a consequence of its anthocyanin contents that was not well
scientifically investigated in extracts in Yemen. Therefore in the present
study, Mirabilis jalaba flower plant was collected from Ibb region in
Yemen to study its anthocyanin content and antioxidant properties.
2 Material and methods
2.1 Material
Fresh petals of the (Mirabilis jalaba)
were collected randomly from the Ibb region during June 2013
2.2 Extraction of
anthocyanins
The anthocyanins were
extracted according to the methodology of Lees and Francis (1972). Solvents
such as methanol and ethanol were used at concentrations of 100, 75, 50, 25 and
0.0% in water, acidified with 0.1% hydrochloric acid (HCl) (Synth 37%) Twenty
five g of fresh petals of Mirabilis jalab were treated with 100 ml of
different water/alcohol solutions acidified with 0.1% HCl (Merck, 37%) as
extracting material (solid to solvent ratio 1:4 w/v). Each solution was transferred to a 500 ml
beaker, covered with parafilm and stored overnight at 4 °C. The mixture was
then filtered under vacuum using nº 1 Whatman paper and a Buchner funnel.
Filtrate solution was taken and then 200 ml of solvent was added to complete
the mixture. This mixture was later filtered and the residue washed with
solvent until obtained a total of 500 ml solution. A 5 ml aliquot was removed
from each extract, placed in a 50 ml volumetric flask, the volume completed
with two buffer solutions: potassium chloride buffer 0.025 M (pH 1.0) and
sodium acetate buffer 0.4 M (pH 4.5) and then the absorbance was measured
simultaneously at 516 nm and 700 nm after 15 minutes of incubation at room
temperature. Absorbance readings were made at room temperature against
distilled water as blank was used for measurements.
2.3 Quantitative
determination of the anthocyanins
Determination of total
monomeric anthocyanins content was quantified using a pH differential method
described by Giusti and Wrolstad (2001). The absorbance was measured
simultaneously at 516 nm and 700 nm after 15 minutes of incubation at room
temperature. Absorbance readings were made at room temperature against
distilled water as blank. A Jasco V 530 UV-Vis spectrophotometer was used for
measurements. The monomeric anthocyanin pigment concentration was calculated
according to the following equation:
Monomeric anthocyanin
pigment (mg/L) = (A x MW x DF x 1000)/ (εx1) Where A=(A510–A700)pH 1.0–(A516–A700)pH
4.5, MW is the molecular weight (449.2) and ε is the molar absorptivity,
(26,900) and DF is the dilution factor.
2.4 Determination of
antioxidant activity by the DPPH method
The 1,
1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging assay was first
described by Blois in 1958 and was later modified slightly by numerous
researchers. It is one of the most extensively used antioxidant assays for
plant samples. DPPH is a stable free radical that reacts with compounds that
can donate a hydrogen atom. This method is based on the scavenging of DPPH
through the addition of a radical species or an antioxidant that decolourizes
the DPPH solution. The antioxidant activity is then measured by the decrease in
absorption at 515 nm. In this method, a 0.1mM solution of DPPH in methanol is
prepared (4 mg DPPH /100 ml methanol), and then stored at -20 and 2 ml of this
solution are added to 0.5 ml of the sample solution in methanol. The mixture
was left to stand at room temperature for 30 min in the dark before Absorbance
measurement at 517 nm to assess the stability of the coloured reactive action,,
A large decrease in the absorbance of the reaction mixture indicates
significant free radical scavenging activity of the compound. The antioxidant
activity of the extracts was estimated by the ability to scavenging the DPPH
radical. The DPPH concentration in the reaction medium was calculated from the
calibration curve (Figure 1) with the following equation determined by linear
regression (R2 = 0.988).
A515=11.368x
-0.0437.
Figure1. Concentration
response curve for DPPH at 515 nm
3. Results and
Discussions
The paper describes the
extraction method of anthocyanins and antioxidant activity of Mirabilis jalab selected
from Yemen. The changes in total anthocyanins content depending on the
water/alcohol ratio are showed in Fig. 1. The monomeric anthocyanins content
increases with increasing the percentage of methanol in the extraction system.
This tendency also observed for water/ethanol extraction, where high values
were obtained for 100 % ethanol extracting system. The amount of monomeric
anthocyanins in Mirabilis
jalab extracts ranged from 3197.8 mg/L to 55.7 mg/L for water/ethanol
extraction, and from 946 mg/L to 278.3 mg/L for water/ethanol extraction.
Fig.1. Comparison of anthocyanins
content from extracts obtained in water/methanol and water/ethanol systems.
Figure 2 shows the
percentage of anti oxidant activity after 2 hours of reaction between the
extracts and DPPH radical for the two studied cases. The lower this value, the
higher is antiradical efficiency activity of Mirabilis jalab extracts increases
with increasing the percentage of methanol and ethanol in the extraction
system. Comparing antioxidant activities of the Mirabilis jalaba
extracts in the two cases, it is observed similar antioxidant activities in the
ethanol and methanol series.
Fig.2. Comparison of remaining DPPH of
extracts obtained in water/methanol and water/ethanol systems.
The correlations
between antioxidant activity and monomeric anthocyanins content for the two
extraction systems are showed in Fig. 3. The following equations determined by
linear regression regarding the relationship between antioxidant activity and
anthocyanins content was obtained, Eq. (1) for ethanol series and Eq. (2) for
methanol series. There is good correlation between antioxidant activity and
anthocyanins content for the Mirabilis jalab extracts from methanol and
ethanol series.
y
= -0.0077x + 39.478 (R² = 0.9591) (1)
y = -0.0682x + 79.824 (R² = 0.839) (2)
Fig.3. Correlation between anthocyanin
contents and anti oxidant activity.
Also, notice a good
correlation between anti oxidant activity and extraction systems content for ethanol
and methanol series extracts. The values of the determination coefficients are
acceptable, indicating a high correlation by linear regressions between
antioxidant activities and extraction systems Eq. (3) for ethanol series and
Eq. (4) for methanol series
y
= -24.938x + 43.818 R² = 0.9332 (3)
y
= -51.636x + 68.801 (R² = 0.9712) (4)
Fig.4. Correlation between alcohol
concentrations and anti oxidant activity.
The anti oxidant activity
by the studied extracts show similar pattern curves of anti oxidant activity
versus time. The reaction occurs rapidly in the first minutes and then slowed.
The lower step can be due to the antioxidant properties of the slow reacting
components originally present in the sample and/or due to the reaction products
formed during rapid phase (Tsimogiannis
and Oreopoulou, 2006). The percentage of free radical scavenging activity
against reaction time is exemplified in Fig. 5and 6 for the extracts obtained
in water/methanol system and water/ethanol system respectively.
Fig.5. DPPH scavenging kinetic curves
for Mirabilis jalaba extracts obtained in water/ethanol system
Fig.6 scavenging kinetic curves for Mirabilis
jalaba extracts obtained in water/methanol system
4. Conclusions
The performed studies
indicate that Mirabilis jalab extracts
are a rich source of anthocyanins and possess a significant anti oxidant
activity. The best results regarding monomeric anthocyanins content and
antioxidant activity were obtained at extraction with 100% methanol and
ethanol. However, for food industry, the extractions with ethanolic solution
are more convenient. The correlations between anthocyanins content, and
antioxidant activity depend on the extraction solvent, the best determination
coefficients was found for Mirabilis
jalab extracts obtained in water/ethanol
systems Fig 2.
5-
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