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Executive oxidative damage to lipids, proteins, enzymes and

Executive
Summary

Free radical species produced during metabolism result in
oxidative stress, which can cause damage of DNA, proteins, and lipids; and
their occurrence has been associated with some diseases including cancer,
diabetes, and several cardiovascular and neurodegenerative diseases. At a state
of high free radical formation, internal antioxidants become insufficient for
the counteraction of free radicals; therefore, external antioxidants are a
necessity in the prevention of the oxidative damages. In the present study, the
main objective is to evaluate the total content of flavonoids, the antioxidant
capacity and the scavenging activity of methanolic plant extracts of Brassica oleracea var. sabauda, Helianthus
agrestis, and Murraya Koenigi species. A preliminary phytochemical
screening of the extracts will reveal the bioactive constituents of the plants
studied. The total content of
flavonoids for each plant species will be estimated by using the aluminum
chloride colorimetric method. The free radical scavenging capacity of the
extracts will be determined by the 1,1,-diphenyl-2-picryl hydrazyl (DPPH)
method. Finally, the phosphomolybdenum method will be used to evaluate the
total antioxidant capacity of each plant species. Since flavonoids are
prominent constituents of plants that possess an immense spectrum of
biochemical activities necessary to maintain a stable human health, the present
study will lead to the future isolation and characterization of the active
compounds responsible for the pharmacological activity related to these plants.
This further investigation will allow for a greater understanding of the
mechanism of action of the bioactive compounds from the extracts against
oxidative-stress-related diseases in order to develop it as a promising drug
for pharmaceutical and therapeutic distribution.

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Statement of Need

Reactive oxygen species, such as
singlet oxygen, hydroxyl ion, superoxide ion and hydrogen peroxide, are highly
reactive, toxic molecules, which are generated normally in cells during
metabolism. These free radicals cause severe oxidative damage to lipids,
proteins, enzymes and DNA by covalent binding and lipid peroxidation, with
subsequent tissue injury. (Baba, S. A., & Malik, S. A, 2015) In recent
years, natural antioxidant agents have attracted much interest because of their
ability to tremendously diminish the damage due to oxidants by neutralizing the
free reactive species before they begin their onslaught on the cells, and
effectively obstruct damage to lipids, proteins, enzymes and DNA. (Saeed N., Khan
M.R., & Shabbir M., 2012). Free radicals have been implicated in the
development of a number of disorders, including neurodegeneration,
inflammation and cancer, giving rise to studies of antioxidants for the
prevention and treatment of diseases. (Peschel W., Sanchez-Rabaneda F., 
Dieckmann W., Plescher A., & Gartzia I., 2006).

Flavonoids are phenolic substances that act in plants as
antioxidants, antimicrobials, photoreceptors, visual attractors, feeding
repellants, and for light screening. Many studies have suggested that
flavonoids exhibit biological activities, including antiallergenic, antiviral, anti-inflammatory,
and vasodilating actions. However, most interest has been devoted to the
antioxidant activity of flavonoids, which is due to their ability to reduce
free radical formation and to scavenge free radicals (Pietta P., 2000). Indeed,
many recent investigations have reported a significant correlation between the
total content of flavonoids and the antioxidant activity of plant species.

The present study hypothesizes that the species with the
highest content of flavonoids will also show the highest scavenging and
antioxidant activity. The main objective of this investigation is to evaluate
the total content of flavonoids, antioxidant capacity and the scavenging
activity of methanolic plant extracts of Brassica
oleracea var. sabauda, Helianthus agrestis, and Murraya Koenigi species. These
species are commonly used by the world population on a daily basis and can be
easily acquired in the local markets; they are accessible and inexpensive.
Moreover, very little research on the antioxidant potential of these plant species
have been done before, which makes them an excellent source for new discoveries
and innovation. This research has great significance because if the plants studied
are found to be rich in natural antioxidants, they can be further investigated
for their effects in the maintenance of the human health (i.e. as strong anticancerous
and in the protection against heart disease), and in the food industry as
consumers move towards functional foods with specific health effects.

 

Background/Literature
Review

Since
ancient human civilizations, plant-based sources have been invaluable remedy in
the health care system to ease and cure diseases. For thousands of years, the
widespread use of plants for medicinal means have been embedded in our culture
and even showed high potential to be used in modern medicine and in other
future applications (Bhaigyabati, Bag, & Grihanjali Devi, 2014). According to Kamboj
(2000), herbal medicine is still the mainstay of about 75–80% of the world population,
mainly in the developing countries, for primary health care because of better
cultural acceptability, better compatibility with the human body and lesser
side effects. They also offer therapeutics for age-related disorders like
memory loss, osteoporosis, immune disorders, etc. for which no modern medicine
is available (Kamboj, 2000).

Most plant
species are abundant source of potent free radical scavenging molecules
(antioxidants), which are bioactive compounds such as carotenoids, flavonoids,
dietary glutathione, and vitamins (Kalita , Barman Tapan , Pal Tapas , & Kalita , 2013). Antioxidants are powerful preventive agents that
can inhibit or delay reactive oxygen species and other forms of free radicals
in the human body. This characteristic feature
of an antioxidant is due to its redox hydrogen donators and singlet oxygen
quencher (Senguttuvan, J., Paulsamy, S.,
& Karthika, K, 2014).

In
contemporary research, antioxidants have gained popularity. Free radical
species produced during metabolism result in oxidative stress, which can cause
damage of DNA, proteins, lipids, and so on. Some diseases correlated to the
occurrence of free radicals include several cardiovascular and
neurodegenerative diseases, cancer and diabetes (Chun, Frei,
Gardner, Lee Alekel, & Killen Jr., 2013). At a state of high
free radical formation, internal antioxidants become insufficient to counteract
the effects of free radicals, so external antioxidants are needed in order to
prevent the oxidative damages (Kalita et al., 2013).

In the present literature review, different
scientific methods to identify the bioactive phytochemical constituents of
plants, evaluate their total content of flavonoids, antioxidant capacity and scavenging
activity are investigated.  It is
hypothesized that the species with the highest reported content of flavonoids
will also show the highest percent of scavenging and antioxidant activities.
Furthermore, this literature review examines suitable methods for the mentioned
tests, and attempts to demonstrate and support the stated hypothesis.

In an effort to identify the bioactive
constituents present in a variety of Argentine medicinal plants, Coussio, J., Rondina, R., Mendiondo, M., &
Bandoni, A. (1976) conducted a preliminary qualitative phytochemical
analysis of all species studied. To
complete this analysis, Coussio et al., (1976)
performed successive extractions with solvents of increasing polarity,
from non-polar (hexane) to polar solvent (methanol) that drew high range of
plant constituents. Therefore, the methanolic extracts of each species were
used to estimate the total flavonoids content and antioxidant activity. The
test revealed the presence of trepenoids, tannis, phenols, amino acids, terpenoids, anthraquiones, alkaloids, cardiac
glycosides, reducing sugars, saponins, and flavonoids as the plants’ bioactive
constituents. These secondary metabolites have many biological and therapeutic
properties; hence, the species tested were expected to have a great variety of
medicinal uses (Senguttuvan et al., 2014).

Flavonoids are defined as polyphenolic compounds comprising
fifteen carbons with two aromatic rings connected by a three-carbon bridge.
These compounds are the most numerous of the phenolics and are found throughout
the plant kingdom (Cartea, M. E.,
Francisco, M., Soengas, P., & Velasco, P., 2010). Flavonols are the most
widespread of the flavonoids. Quercetin, is the most abundant dietary flavonol,
and it is a potent antioxidant because it has all the right structural features
for free radical scavenging activity (Cartea et. al., 2010). Based on this
fact, quercetin was used as the standard to construct the calibration curve for
total flavonoid content test in Chang, C., Yang,
M., Wen, H., & Chern, J. (2002) experiment. Moreover, Chang et al., (2002)
developed the aluminum chloride colorimetric method to identify the
total content of flavonoids present in propolis, the material used by bees to
protect their hives. In this method, diluted standard solutions of quercetin
were separately mixed with 95% ethanol, 10% aluminum chloride, 1M potassium
acetate and distilled water; incubated at room temperature for 30 min; and
their absorbance measured at 415nm (maximum wavelength of quercetin) with a
Shimadzu UV-160A spectrophotometer. As specified by Bhaigyabati et al., (2014),
the principle involved in aluminum chloride (AlCl3) colorimetric method is that
AlCl3 forms acid stable complexes with the C-4 keto groups and either the C-3
or C-5 hydroxyl group of flavones and flavonols. In addition, Bhaigyabati et
al., (2014) explains that AlCl3 also forms acid labile complexes with the
orthodihydroxyl groups in the A- or B-ring of flavonoids. Likewise, aluminum
chloride colorimetric method was used to determine the total content of
flavonoids in methanolic extract of Biophytum Sensitivum Linn in Kalita et al.,
(2013) research. In this experiment, the final concentration values of plant
extracts were obtained from the quercetin standard curve by interpolating to
the x- axis. The total content of flavonoids was calculated in this experiment
by using the following formula, where “R” represents the results obtained from
the standard quercetin curve, “V” is the volume of stock solution, “D.F” the
dilution factor, and “W” the weight of dry sample plant used in experiment.
(Kalita et al., 2013)

The second method investigated in this paper, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free
radical method, aims to determine the scavenging activity of plant species. The DPPH is a stable radical with a maximum absorption
at 517 nm that can readily undergo scavenging by antioxidant (Lu and Yeap
Foo, 2001). It has been widely used to test the ability of compounds as free-radical
scavengers or hydrogen donors, and to evaluate the antioxidative activity of
plant extracts (Porto et al., 2000). In the DPPH assay
conducted in Sowndhararajan, K., & Kang, S. C. (2013) research, the absorbance at 517 nm decreased as
the concentration of the extracts increased, thus increasing the concentration
of the antioxidants. This reduction of absorbance is due to the odd electron of
DPPH that generally yields a strong absorbance at 517 nm while being quenched
by the introduced extract. Lastly, the percent scavenging radical activity was
calculated for each extract sample using the formula in Zheleva-Dimitrova, D., Nedialkov, P., & Kitanov, G. (2010)
publication:

           A
correlation between the increase in DPPH
scavenging activity and the increase in flavonoid content is evident in
all Zheleva-Dimitrova et al., (2010),
Sowndhararajan, et al., (2013), and Bhaigyabati et al., (2014) experiments, which support the initial hypothesis.  

       
The last method discussed in this paper involves the
phosphomolybdenum complex, and aims to
determine the total antioxidant activity of plant extracts. In Waheed,
I., Ahmad, M., Syed, N., & Ashraf, R. (2014) research article, the total
antioxidant potential of methanol extracts and fractions of Ballota limbata
(Lamiaceae) was measured by employing phosphomolybdenum complex formation
method. In this assay, the conversion of Mo (V) from Mo (VI) by reduction
occurred by various fractions of plants which were detected at an absorbance of
695 nm by the spectrophotometer. This method was also used by Miladi S, Damak
M., (2008) research team, who described the method to be simple and independent
after using it to successfully quantify vitamin E in seeds. More proof for the
efficiency of the method is obtained in Esmaeili A.
K., Taha R. M., Mohajer S., & Banisalam B., (2015) experiment, where the results
reported show a correlation between phytochemical contents and the values
obtained for both radical scavenging activity and antioxidant ability of the
extract of red clover and its various soluble fractions. In this experiment,
flavonoid contents exhibited excellent association with DPPH, superoxide, ABTS
radical scavenging activities, and reducing power. In the same way, results
reported by Senguttuvan et al., (2014) showed a
strong correlation between the contents of total flavonoids and radical
scavenging activity, which indicates that flavonoids are major contributors to
the antioxidant potential of plant species. (Senguttuvan et al., 2014)

Taken together, the present literature review
confirms that a preliminary phytochemical study provides invaluable information
of the different phytochemicals present in herbal extracts consumed on a daily
basis by the population. This preliminary analysis is a necessary step that
predicts whether or not the plant species studied is expected to contain high
content of flavonoids and other bioactive constituents. Additionally, aluminum
chloride colorimetric method, DPPH radical scavenging assay and phosphomolybdenum
method were found to be suitable techniques to assess the total flavonoid
content, free radical scavenging capacity and overall total antioxidant
activity of methanolic extracts of plant
species respectively. All findings in the present study support the view
that many medicinal plants are promising sources of potential antioxidant and
may be efficient as preventive medication sources in many diseases. Most
importantly, the research articles analyzed support the initial hypothesis by explaining
in multiple instances the direct relationship between the total content of
flavonoids in plant extracts and their antioxidant and scavenging
activity.  

 

Project
Description

Collection of Sample

Fresh samples of B. oleracea
var. sabauda, H. agrestis, and M. koenigi were already collected from a
farmer’s market at Homestead, Florida. The handpicked species were washed well
using tap water and once again using distilled water. They were cut into small
pieces and dried in shade for a period of ten days at room temperature of 22oC.
The dried samples were grinded using a mortar and pestle. They were further
stored in sterile flasks and refrigerated.

 

Preparation of extracts and Phytochemical Screening

The phytochemicals of B.
oleracea var. sabauda, H. agrestis,
and M. koenigi will be determined by
successive extractions with solvents of different polarities (i.e hexane,
methanol, chloroform, and water) according to the procedure described by
Rondina and J. D. Coussio, (1976). Phytochemical screening of all species’
extracts will be carried out to detect the presence of alkaloids, saponins,
tannis and phenols, amino acids, terpenoids, anthraquinones, alkaloids, cardiac
glycosides, reducing sugar and flavonoids. The extracts with the strongest
intensity of flavonoids reported will be chosen, and evaporated in a vacuum
rotary evaporator until dryness. The chosen extracts will be used to study the
total content of flavonoids, antioxidant activity and scavenging capacity of
the selected plants.

 

 

 

Evaluation of Total Flavonoid Content

The total content of flavonoids for each plant extract will
be estimated by using the aluminum chloride (AlCl3) colorimetric
method. Quercetin
will be used as a standard to construct a calibration curve.  Stock solutions of plant extracts will be
prepared by dissolving the extract of each species in methanol. Test solutions
will be prepared by adding 10% aluminum chloride, 1 M sodium acetate and
distilled water to the methanolic extracts. The blank sample will be prepared
in a similar way with the exception that it would not contain 10% aluminum
chloride. The absorbance of all test solutions will be measured against the
blank at 415 nm. (Bag G.C., Grihanjali Devi P., Bhaigabati. Th., 2014).

Determination of Free Radical Scavenging Activity

The free radical scavenging capacity of each species’
extract will be determined by the 1,1,-diphenyl-2-picryl hydrazyl (DPPH)
method. (Kalita , Barman Tapan , Pal Tapas
, & Kalita , 2013) Ascorbic acid will be used as a
reference to construct a standard calibration curve. Various concentrations of
the methanolic extract of B. oleracea
var. sabauda, H. agrestis, and M. koenigi will be prepared with
Tris-HCl ph7 buffer and 0.1 mM DPPH solution. The negative control will be prepared
with methanol, tris-HCl ph7 buffer and DPPH solution. All extracts will be
incubated for 30 minutes at room temperature and their absorbance measured at
517 nm.

 

 

Total Antioxidant Capacity

The phosphomolybdenum method will be used to evaluate the
total antioxidant activity of the extracts of B. oleracea var. sabauda,
H. agrestis, and M. koenigi. (Bhaigyabati, Bag, & Grihanjali Devi, 2014) Ascorbic acid
will be used as a standard to plot a calibration curve. Test solutions will be
prepared by adding ammonium molybdate reagent solution to each species’ methanolic
extract.  All solutions will be incubated
at 95oC for one hour and then cooled down to room temperature. Their
absorbance will be measured at 695 nm using the ammonium molybdate reagent
solution as the control sample.

 

Budget
Analysis

The project will be funded by the ARCOS Undergraduate
Research and Project Opportunity (URPO) Program at Miami Dade College. All
resources will be purchased by Miami Dade College and will be available to use
in January 2018.

 

Table 1. Project Budget

Item Description

Supplier

Catalog #

Price ($)

Quantity

Total ($)

Mercuric chloride (II) 

Sigma Aldrich

215465-5G

24.10

1

24.10

Potassium Iodide

Sigma Aldrich

793582-100G

51.70

1

51.70

Methanol anhydrous, 99.8%

Sigma Aldrich

322415-1L

60.30

2

120.60

Chloroform

Sigma Aldrich

C2432-1L

73.50

1

73.50

Quercetin hydrate

Sigma Aldrich

337951-25G

42.00

1

42.00

L-Ascorbic acid

Sigma Aldrich

A7506-100G

40.30

1

40.30

2,2-Diphenyl-1picrylhydrazyl

Sigma Aldrich

D9132-1G

97.30

1

97.30

Aluminum chloride

Sigma Aldrich

294713-5G

46.00

1

46.00

Sodium Phosphate

Sigma Aldrich

342483-25G

29.20

1

29.20

Ammonium molybdate

Sigma Aldrich

277908-5G

78.10

1

78.10

 

 

 

 

 

$602.80

 

 

Conclusion

This preliminary study will provide invaluable information
of the different phytochemicals contained in herbal extracts available in the
farmer’s market and consumed regularly by the population. The results are
expected to show an equivalent relation between the total flavonoid content,
the scavenging activity and the antioxidant capacity.

Since flavonoids and other of the metabolites studied in
this research are prominent constituents of plants that have a large range of
biochemical activity necessary to maintain a healthy human being, further
investigation will allow for the isolation and characterization of the active
compounds responsible for pharmacological activity related to these plants. This
further examination will lead to a greater understanding of the mechanism of
action of the bioactive compounds from the extracts against oxidative-stress-related
diseases in order to develop it as a promising drug for pharmaceutical and
therapeutic distribution.

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