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In vitro antioxidant, total phenolic, total flavonoid content and biosynthesis

of silver nanoparticles using Cassia auriculata leaves extracts

Article · May 2021

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 International Journal of Botany Studies
www.botanyjournals.com
ISSN: 2455-541X
Received: 30-04-2021, Accepted: 14-05-2021, Published: 31-05-2021
Volume 6, Issue 3, 2021, Page No. 476-480

In vitro antioxidant, total phenolic, total flavonoid content and biosynthesis of silver nanoparticles
using Cassia auriculata leaves extracts

Thirumal Sivakumar
Department of Botany, Annamalai University, Annamalai Nagar, Tamil Nadu, India

Abstract
Biosynthesis of nanoparticles is an emerging field of nanotechnology, select over physical, chemical and biological synthesis
due to their protection, cost-effectiveness nature, bio-compatibility, environmentally and scalable properties. In the present
study was carry out biosynthesis of silver nanoparticles using leaves extract of Cassia auriculata and also antioxidant activity,
total phenolic, total flavonoids content were analyzed. AGNP and leaf extracts prepared individually in different solvents such
as ethanol, ethyl acetate, hexane and aqueous were evaluated for their bio-efficacies. The free radical scavenging activity, total
antioxidant, total phenols and flavonoids in AgNPs containing leaves extract were 82.96 (Scavenging activity %), 240.68(µg
AA/mL), 250.14 (µg GAE/g extract), and 890.56 (µg QE/g extract) respectively. Biosynthesized AgNPs showed a more
activity for increased concentration of leaves extract. It was concluded that C. auriculata leaves extracts can be extensively
used in the production of capacity antioxidant activity.

Keywords: C. auriculata, AgNPs, total phenolic, total flavonoids, antioxidant

Introduction functions than chemically synthesized nanoparticles

Nanotechnology is one of the most important and attracting (Choudhury et al., 2016) [7].
in this era, growing day by day, influencing various aspects Previous studies have suggested the use of various plant
of human life (Roco and Bainbridge, 2005) [1]. Past few parts such as leaf, stem, bark, fruit, bud, wood, and root, for
decades, nano scale particles have been attracting more and the synthesis of silver nanoparticles. Plant-mediated
more attention owing to their compatibility in the field of synthesis of silver nanoparticles is preferred over microbe-
pharmaceuticals, biology, materials science and electronics mediated synthesis. The latter is not feasible and requires
at nanoscale level (Yokoyama & Welchons, 2007) [2]. Even more aseptic conditions, a time-consuming process and a
though accessible in many ways, the synthesis of longer incubation period. Furthermore, the reduction
nanoparticles is important with a biological approach properties of secondary metabolites of plants are attributed
(Kaushik et al., 2010) [3]. In the recent situation, the to the more ability of plant extracts to synthesize
biosynthesis of nanoparticles mediated plant extracts is nanoparticles with enhanced properties (Kharissova et al.,
much simpler than the chemical synthesis method and can 2013) [8].
be used for many ameliorate treatments (Mittal et al., 2012) Free radical plays an important role in oxygen based
[4]
. Moreover, the synthesis of nanoparticles using plant (aerobic) living systems. They are part of cell respiration
extract reduction and covering agents is more beneficial and other key cellular processes, but are also involved in
than the microbial synthesis. Recently, the collection of NPs aging and disease development (Harrman, 1992; Bagchi and
using living agents (fungicides, bacteria and plant extracts) Puris, 1994) [9-10]. Free radicals are unstable molecules with
is preferred over conventional methods because of their free unconnected electrons. They are very reactive because
simpler process, stability, rapidness, high drug loading free electrons constantly bind to other electrons to form
capacity, non-pathogenic, low cost and eco-friendliness covalent pairs. In this activity, free radicals eliminate the
(Boomi et al., 2019) [5]. electrons from other molecules. Beyond affecting the
Presently, the plant-mediated green synthesis of silver regulation of cells, it can also injury molecules known as
nanoparticles has grown into a new and key branch of carbohydrates, fats, proteins and nucleic acids (Young and
nanotechnology. Since it is environmentally friendly and Woodside, 2001; Valko et al., 2007) [11-12]. There are various
cost effective, it is less toxic when associated with chemical sources of free radicals within the cell and its environment.
hazards, so it has emerged and gained prominence. In the In aerobic organisms, free radicals are developed during
physical and chemical nanoparticles synthesis, the green normal metabolic processes (Mohammed and Ibrahim,
synthesis has many benefits (Chanel et al., 2017) [6]. In the 2004) [13]. Key sources include electron transfer in the
physical and chemical nanoparticles synthesis, the green plasma membrane and cell respiration in the mitochondrial
synthesis has many benefits. For example (i) low toxic and membrane. Mitochondria are the main source of antioxidant
hazardous substances and environmentally friendly solvents, damage because free radicals such as superoxide can run
(ii) simple, fast and inexpensive, (iii) uses low energy and away from the electron transport chain (Shigenaga et al.,
operates under moderate operating conditions, (iv) combines 1994; Liu et al., 1999) [14-15]. To exempt intracellular
the energy of silver nanoparticles and plant active damage by free radicals, cells have formed an endogenous
ingredients. In this respect, plant-mediated silver antioxidant system. This system converts free electrons into
nanoparticles are reported to contain higher biological non-reactive form by proteins.

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Antioxidants are very important for the protection of cells min at room temperature. Absorption was then recorded
and biological macromolecules from degenerative reactions using a spectrophotometer at 517nm and taken as standard
produced by free radicals and reactive oxygen species compared to vitamin C (ascorbic acid). The experiments
(Hermans et al., 2007) [16]. Antioxidant properties of were repeated three times. The antioxidant contents of
different plant products, i.e. polyphenolic substances (e.g. leaves extracts prepared in various solvents and AgNPs was
flavonoids and tannins) derived from various plant and calculated as the percentage reduction of DPPH
herbal extracts (Agati et al., 2012; Dajas, 2012) [17-18] and discoloration using the following equation:
Elumbotti, Sweet Potato, Datura, Sesuvium portulacastrum, Percent Inhibition: = 100X1− (Asample /Ablank),
Green tea, Tuber extract (sweet potato), (Sivakumar and where, Ablank is absorbance of the solution when nothing
Gajalakshmi, 2013; Sivakumar et al., 2010; Sivakumar and was added; Asample is absorbance of the solution when
Panneerselvam, 2011; Anburaj et al., 2012; Senthilkumar extract or AgNPs was added
and Sivakumar, 2014; Sivakumar et al., 2015) [19-24].
Cassia auriculata comes under the family Leguminosae and Total antioxidant activity
subfamily of Fabaceae and is broadly used as conventional Total antioxidant capacity (TAC), phosphomolybdenum
medicine in India and Southeast Asia. It is an vital herb that was analyzed according to the technique followed by
is widely distributed in India, Vietnam, Sri Lanka, Eddine et al., (2016) [28]. One mg of various solvents was
Indonesia, Malaysia, Australia, and Japan tropical Africa dissolved in leaves extracts AgNPs in 10μL DMSO
and the southwestern parts of the People's Republic of (dimethyl sulfoxide) and volume was made up to 1mL using
China. C. auriculata is a key medicinal plant and is used in methanol. 1mg/mL ascorbic acid solution was used as
traditional medicine, Ayurvedic, homeopathic remedies and positive control. A stock solution of ascorbic acid (1mg/mL)
also used in the cure of various diseases such as eye was prepared using methanol. In the reaction mixture,
complaints, stomach problems, heart disease, respiratory 100μL test samples was added to the test tube containing
disorders, constipation, dyspepsia, hemorrhoids. C. 1mL of freshly prepared Phosphomolybdate reagent
auriculata plant extract has been reported to have solution (28mM sodium dihydrogen orthophosphate, 0.6mM
antibacterial and antidiabetic activity (Parkas 2006; sulphuric acid, and 4mM ammonium molybdate
Thirumal Sivakumar, 2021) [25-26]. In the present research tetrahydrate) and shaken vigorously and test tubes were
work, an attempt has been made to synthesize silver covered with an aluminum foil and incubated in a water
nanoparticles and characterization of silver nanoparticles bath at 90°C for 2h. After incubation, the test tubes were
using the leaf extract of Cassia ariculata. The synthesized allowed to cool to room temperature and reading was
silver nanoparticles were analyzed for their in vitro measured at 695nm using a UV-Vis spectrophotometer and
antioxidant, total phenolic, total flavonoids content of leaves methanol was taken as a negative control.
extracts of Cassia ariculata (L).
Total phenolic content determination
Materials and Methods Total phenolic content (TPC), the Folin-Ciocalteu
Collection and synthesis of silver nanoparticles evaluation was followed by slight modification protocol as
Cassia auriculata (L.). leaves were collected from suggested by Slinkard and Singleton, (1977) [29]. Test
Elusempon, Villupuram District, Tamil Nadu, India. The samples of 100μL aliquots were taken and total volumes
collected leaves were separated and thoroughly washed with were made up to 3mL using distilled water. Under dark
sterile double distilled water and dried under room conditions, 0.5 mL of Folin Ciocalteu reagent was mixed to
temperature then grind with the help of mortar and pestle. the reactive sample, mixed well and allowed to stand for 3
The leaves were grind until it reaches to a paste form and min. 2mL of 20% (w/v) Na2CO3 (anhydrous) solution was
then the leaves extracts was filtered with Whatmann no.1 added and well mixed. The reaction sample was later
filter paper and filtrate saved. The silver nanoparticles were incubated in the dark for 60min. For the blank, the reaction
synthesized by adding 90ml of 0.1mM aqueous AgNO3 compound was taken with methanol instead of the test
silver nitrate solution and mixed with 10ml of Cassia samples. Absorbance was recorded at 650nm using a UV-
auriculata leaves extracts. The synthesis was carried out in Vis spectrophotometer and total phenolic content was
a dark condition to minimize the photo activation of silver expressed as μg of gallic acid (GA)/mg of dry extract
nitrate. The development of silver nanoparticles was (μgGA/mg).
indicated by the color change. After the synthesis, the
reaction mixture was shaken thoroughly and kept into the Total flavonoid content determination
oven for one week. The pellets containing silver Total flavonoid content determination (TFC) of each leaves
nanoparticles was collected and dried at room temperature extract prepared separately in different solvents (ethanol,
and also dried powder was used for further experiments. ethyl acetate, hexane and methanol) compared to AgNPs
was assessed using aluminum chloride using colorimetric
Antioxidant assay method and Quercetin was used as standard solution. In a
DPPH radical scavenging assay reaction mixture, 125μL of each test sample was added to a
The antioxidant activity, the free radical scavenging of test tube containing 37.5μL of NaNO2 (5% w / v) solution.
DPPH (2,2-diphenyl 1-picrylhydrazyl) was analyzed using a The mixture was then stirred well and incubated at room
modified method followed by MacDonald-Wicks et al., temperature for 7 min. After incubation, 75μL of AlCl3(10%
(2006) [27]. The reaction mixture was prepared by adding w/v) mixture was added, followed by 250μL of NaOH(1N).
50μL of various concentrations of leaves solvents extract, The total volume of the reaction mixture was added to 2.5
combining AgNPs with 75μL of methanol and then adding mL of distilled water. Absorption of the reaction compound
100μL of 0.1mm concentration to the DPPH solution. The was recorded at 510nm using a UV-Vis spectrophotometer
mixture was stirred for 10 min and then incubated for 25

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and the results were expressed as quercetin equivalent (QE) depicted to the dose-dependent higher in the total
/mg fresh extract (μgQE / mg). antioxidant activity of the exposed samples. Related to our
results, Nakala and Sadras (2016) [37], exhibited higher
Statistical analysis antioxidant activity of AGNPs synthesized using P. longum
Statistical analysis was performed using SPSS/20 software. fruit extract. Present reports indicate that the combination of
The experimental data were expressed as mean±standard phenols, alkaloids, and flavonoid compounds may have
deviation and were obtained from biological and technical enhanced antioxidant activity of AgNPs. Present reports
three replicates of each experiment. show that phenols, alkaloids and fl avonoid
phytocompounds may have increased antioxidant activity of
Results and Discussion AgNPs due to the diffusion of plant extracts into the metal
Antioxidant potential (Phull et al., 2016) [38]. In this regard, the total phenolic and
Present research DPPH and phosphomolybdenum studies flavonoid content of AGNPs and plant extracts equipped
were carried out to study the antioxidant capacity of AGNPs into different solvents were evaluated.
compared to crude extract prepared separately in various
solvents. In DPPH methods, the nitrogen atom of DPPH
contains the odd electron, which is reduced by accepting
hydrogen ion and free electrons from the antioxidant
compounds before converting the purple colored DPPH into
the yellow colored hydrazine molecule. The DPPH free
radical scavenging activities relative to the size of the test
samples expressed in percentage were recorded as depict in
(Figure. 1). The results exhibited a higher in scavenging
activity by increasing the concentration of the samples. In
various treatments, AGNPs exhibited the increased
antioxidant activity (82.96%), followed by aqueous, ethanol,
ethyl acetate and hexane extract 71.29, 68.45, 62.49 and Fig 2: Total antioxidant capacity of various C. auriculata leaves
53.86% scavenging activity at 1mg/mL, dose of test extracts and AgNPs according to phosphomolybdate assay,
samples, respectively. Present results combined the intense expressed as μg/mL of ascorbic acid (AA).
scavenging activity of AGNPs synthesized using C.
auriculata leaves extract. The powerful active scavenging The TPC (250.14μg GA/mg fresh extract) of the synthesized
activity of AGNPs may be due to the presence of significant AgNPs was found to be the increased, followed by aqueous
amounts of phenols, flavonoids and alkaloids in the leaves extract, ethanol, ethyl acetate and hexane extract 221.03,
extract, which may play an vital role as capping and 180.36, 135.45 and 102.59μg GA/mg respectively. The total
stabilizing agents. The same results, (Abel-Aziz et al.,2014; phenolic content of AgNPs and the leaves extract
Senthilkumar et al., 2017; Jothi et al., 2019; Sivakumar, formulated in different solvents are shown in Figure 3.
2019; Angelin et al., 2020; Thiyagarajan and Sivakumar,
2020; Sivakumar, 2021) [30-36], showed higher antioxidant
activity of leaves extract containing AgNPs.

Fig 3: Total phenolic content of C. auriculata leaves extract

prepared in various solvents and AgNPs expressed as μg of gallic
acid equivalent (GA)/mg of fresh extract.

Fig 1: Free radical Scavenging capacity of different C. auriculata The total flavonoid content (TFC) of AGNPs 890.56
leaves extracts and AgNPs according to phosphomolybdate assay, μgQE/mg of fresh extract was compared with the leaves
expressed as μg/mL of ascorbic acid (AA).
extracts prepared separately in aqueous, ethanol extract,
ethyl acetate and hexane extract containing 808.21, 725.89,
Furthermore, AgNPs and plant extracts produced in various
568.75 and 389.54μg QE/mg of leaves extract, respectively,
solvents were assessed for total antioxidant capacity (TAC)
as showed in Figure.4. Same to our results (Pull et al., 2016)
using a phosphomolybdate evaluation. In this evaluation, [38]
showed increased TPC and TFC values of AgNPs
molybdenum (VI) is decrease to molybdenum (V) when the
synthesized using Bergenia ciliata compared to crude
incident of antioxidants (reducing agents). The results
extracts. From the above results, a strong correlation was
revealed improved antioxidant capacity of AgNPs
found between TFC, TPC, TAC and DPPH values, resulting
(240.68μg / mL) compared to plant crude extracts, followed
in higher phenolics and flavonoid content observed for their
by aqueous extract, ethanol, ethyl acetate and hexane extract
concluded that the presence of synthesized AgNPs was
of 222.32, 204.12, 190.53 and 170.59 μg/mL, of AA at
responsible for their notable antioxidant activity.
1mg/mL concentrations of samples, respectively. Figure. 2.

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