Comparative Phytochemical Investigation and Antioxidant Activity in Different Parts of Acacia nilotica Seed


*Corresponding Author:

Manjoosha Srivastava

Phytochemistry Division,

Council of Scientific and Industrial Research-National Botanical Research Institute,

Lucknow,

Uttar
Pradesh 226001

E-mail: [email protected]







Date of Received 24 December 2020
Date of Revision 26 October 2021
Date of Acceptance 05 May 2022
Indian J Pharm Sci 2022;84(3):552-559  

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Abstract

Acacia nilotica, indigenously known as ‘Babool’ of family Leguminosae has many traditional and ethnobotanical uses. All the parts of tree are important and seeds are most abundantly available which are rich in different phytochemicals. Thus, the aim of this study was qualitative and quantitative analysis of phytochemicals and evaluation of the antioxidant activity in Acacia nilotica seed along with their parts viz. cotyledon and endosperm as a potential source in nutritional and medicinal applications. Phytochemicals as carbohydrate, protein, alkaloid, flavonoids, phenolics, saponin and tannin were present in seed, cotyledon and endosperm. Cotyledon was rich in sugar, carbohydrate, protein and oil, i.e. 4.73 %, 17.83 %, 30.45 % and 11.78 % respectively whereas endosperm was rich in phenolics and flavonoids i.e. 2.66 mg/g gallic acid equivalent and 7.66 mg/g quercetin equivalent respectively. Antioxidant activity studies showed that endosperm exhibited maximum percentage inhibition i.e. 74.07 % compared to the standard butylated hydroxyanisole having 82.88 % inhibition of 2,2-diphenyl-1-picrylhydrazyl radicals. The study revealed that the specific utilization of Acacia nilotica seed have scope in nutritional supplements and as useful bioactive extracts for sustainable health benefits.

Keywords

Acacia nilotica, 2,2-diphenyl-1-picrylhydrazyl, gallic acid equivalent, quercetin equivalent,
nutritional, phytochemicalsuse

Plants and their parts are good source of effective
medicines and nutritional supplements for human health
and well-being. Nearly three quarter population of the
world depends upon the plants and their products for
healthcare management and about 30 % plant species
are in medicinal use. In Indian medicine system such as
Ayurveda and Unani, drugs of herbal origin have been
used traditionally and ethnbotanically since ancient
times [1].

Acacia nilotica (A. nilotica), indigenously known as
‘Babool’, belonging to subfamily Mimosoideae [2,3] and
family Leguminosae [4,5], is an important and evergreen
tree of Indian dried regions. Tree is generally 10 m in
height, bark is longitudinally fissured with dark brown
or black color and young plant exhibit terete, pubescent
and slender branchlets [6]. A. nilotica recognized as a
pioneer species for high bioactive compounds and
economically important source of gum, tannins, fodder,
fuel and timber from long time [7,8]. The species are
widespread throughout Asia, Africa and America [9],
and naturally found in Botswana, Kenya, Egypt, Zimbabwe, India, Saudi Arabia, Burma, Tanzania,
Nepal, Pakistan, Nigeria, Sri Lanka, Indonesia, Sudan,
Iraq, Iran, Namibia, Yemen and Ethiopia [10]. The plant A.
nilotica
has been used in traditional medicines and their
all parts viz. gum, seeds, pods, roots, leaves, flowers
and bark are medicinally important [11-13]. The plant is
rich in different phytochemicals viz. carbohydrates,
alkaloids, proteins, fatty acids, phenolics, flavonoids,
tannins, saponins, cyanogenic glycosides, diterpenes,
phytosterols, triterpenegenins, amino acids viz. cystine,
methionine, threonine, lysine, tryptophan and macro
elements such as potassium, phosphorus, magnesium,
iron and manganese [14,15]. A. nilotica possesses
antioxidant, antimicrobial, antifungal, antibacterial,
antipyretic, antiinflammatory, antidiarrhoeal,
antiulcer, antihelmintic, wound healing, diuretic, antispasmodial, antihypertensive, antitumor and
anticancer properties [16-19] and effective in coughs, cold,
congestion, nerve stimulation, leucorrhea, dysentery,
ophthalmia, hemorrhages and sclerosis relief [20].
Antioxidant property plays a vital role for the treatment
of inflammation and cancer which are caused by free
radicals [21] and presence of tannin which is beneficial for
activation of glucose transfer and prevents lipolysis [22].
Phenolic compounds in A. nilotica makes the species
potential for antioxidant property due to free radical
scavenger activity [23,24].

A. nilotica seed exhibit edible characteristics and
has nutritive and medicinal values due to various
phytochemicals. Although, some studies are
available on seed [25], but part specific study of seed
on phytochemicals and bioactivity has not been
reported yet. Thus, A. nilotica seed was exploited for
phytochemical investigation and antioxidant property.

Materials and Methods

Plant material:

Seeds were collected from Kalli Pashchim region of
Lucknow district (Uttar Pradesh), India. The material
was authenticated (LWG No.102994) and specimen
was deposited in the herbarium at Council of Scientific
and Industrial Research (CSIR)-National Botanical
Research Institute (Uttar Pradesh), India. Different parts
of the seed viz. cotyledon and endosperm (seed gum)
were separated mechanically using grinder (Philips
grinder) and sieved by 40 mesh sieve to obtain 40 mesh
powder of each part for study.

Chemicals viz. Gallic acid, Quercetin, 2,2-Diphenyl-1-
Picrylhydrazyl (DPPH) and Butylated Hydroxyanisole
(BHA) of Merck, solvents viz. n-hexane, chloroform,
acetone, ethanol and methanol of Qualigens and
glasswares made of Borosil were used.

Preliminary study:

Powdered materials were examined for organoleptic
characteristics viz. colour, odour, taste and texture.
The solubility was studied in solvents viz. hexane,
chloroform, acetone, ethanol, distilled water and
boiling water [26].

Extraction and phytochemical screening:

Materials were extracted under cold and hot conditions
using different polarity gradient solvents viz. n-hexane,
chloroform, acetone, alcohol and water. Cold extraction
was carried out using percolation method by exhausting 50 g powder materials each in 250 ml solvents for 24
h at room temperature (25°-30°). Extracts were then
filtered using Whatman filter paper (No. 42). Hot
extraction was carried out through successive soxhlet
extraction method by taking 50 g powder materials and
250 ml different solvents in soxhlet apparatus on water
bath. The extracts were concentrated through Rotary
Evaporator (IKA-RV 10 digital) at 40°-55° and dried
at low temperature (-80°) and pressure (0.200 millibar
(mBar)) using lyophilizer (Labconco- FreeZone Plus
4.5). Dried extracts were used in qualitative tests
for the identification of various phytochemicals viz. carbohydrates, sugar, starch, glycosides, alkaloids,
phenolics, flavonoids, saponins, tannins and sterols
which were estimated quantitatively [27,28].

Phytochemical estimation:

Phytochemicals identified in different polarity gradient
solvent extracts, viz. n-hexane, chloroform, acetone,
ethanol and water were estimated quantitatively
according to standard estimation methods.

Total sugar:

0.5 g powdered material was homogenized with 5 ml
80 % ethanol and centrifuged using centrifuge (Eltek
TC 4100 F) for 15 min at 2000 rpm. The supernatant
volume obtained was adjusted to 10 ml with 80 %
ethanol. 0.1 ml sample was taken, mixed 0.1 ml
80 % phenol and 5 ml concentrated sulphuric acid
respectively and made up the volume to 10 ml using
80 % ethanol. Thereafter, prepared samples were kept
in an ice bath for 30 min. The D-glucose stock solution
of 0.1 mg/ml concentration was prepared as standard
and 1, 2, 3, 4 and 5 ml of stock solutions were taken for
dilutions. The calculation of total sugar was carried out
by measuring the absorbance at 490 nm double beam
Ultraviolet-Visible (UV) spectrophotometer (Thermo
Scientific-Evolution 201 UV spectrophotometer) [29].

Total carbohydrate:

100 mg material was taken into different boiling tubes,
added 5.0 ml of 2.5 N Hydrochloric Acid (HCl) in each
tube and boiled for 3 h on water bath. Samples were
cooled at room temperature, neutralized by adding
sodium carbonate and centrifuged. Dilutions were
prepared by mixing 0.2 ml of sample, 1 ml 5 % phenol
and 5 ml of 96 % sulphuric acid. Samples kept for 10
min at room temperature, then heated on water bath
for 15 min and cooled. Stock solution of D-glucose
as standard was prepared in 0.1 mg/ml concentration and dilutions were prepared by taking 0.2, 0.4, 0.6, 0.8
and 1 ml from stock solution. Total carbohydrate was
calculated by measuring the absorbance at 630 nm [30]
through UV spectrophotometer.

Total protein:

500 mg powdered materials was homogenized with 5
ml of phosphate buffer of pH 7.4 and centrifuged for
20 min at 4000 rpm. The supernatant was filtered and
volume was maintained to 10 ml with phosphate buffer.
0.1 ml of sample was taken and made up to 1 ml with
distilled water. Then 5 ml of Reagent-C was added
which was prepared by adding 50 ml Reagent-A, i.e. 2
% sodium carbonate in 0.1 N sodium hydroxide and 1 ml
Reagent-B, i.e. 0.5 % copper sulphate in 1 % potassium
sodium tartrate. Mixed the solutions and let stand for
10 min. Thereafter, 0.5 ml 1 N Folin-Ciocalteu Reagent
(FCR) was added, mixed the samples and kept in the
dark for 30 min. The stock solution of Bovine Serum
Albumin (BSA) as standard of 1 mg/ml concentration
was prepared. 0.2, 0.4, 0.6, 0.8 and 1 ml of BSA stock
solution taken for dilutions. Total protein was calculated
by measuring the absorbance at 660 nm [31] through UV
spectrophotometer.

Oil content:

Seed, cotyledon and endosperm powder was extracted
with 250 ml n-hexane through soxhlet apparatus for 8
h on water bath. The excess solvent from the extract
was evaporated through rotary evaporator at 40°,
dried in vacuum and oil content was gravimetrically
quantified [32].

Total starch:

0.5 g powdered material was homogenized with 5 ml of
80 % ethanol and centrifuged for 15 min at 2000 rpm.
The centrifugate was separated and added 4 ml distilled
water, heated for 15 min on water bath and macerated
using a glass rod. 3 ml of 52 % perchloric acid was
then added in the macerated sample, homogenized
and centrifuged for 15 min at 2000 rpm. Thus, the
supernatant obtained was made up to the volume with
distilled water. In 0.1 ml of sample, mixed 0.1 ml of 80
% phenol, 5 ml of concentrated sulphuric acid, make up
the volume up to 10 ml with distilled water and kept in
an ice bath for 30 min. The dilutions of standard were
prepared by taking 1, 2, 3, 4 and 5 ml of D-glucose
stock solution of 0.1 mg/ml concentration. Total starch
was calculated by measuring the absorbance at 490
nm [29] through UV spectrophotometer.

Total tannin:

1 g powdered material was extracted with 50 ml
distilled water by boiling on the water bath for 6-8 h,
filtered the extracts and volume was made up to 50 ml
in volumetric flask. Then mixed 0.5 ml of FCR, 1 ml
saturated sodium carbonate solution in 0.1 ml of sample
and made the volume to 10 ml with distilled water. The
stock solution of tannic acid at 0.1 mg/ml concentration
was used as standard and different dilutions were
prepared by taking 0.2, 0.4, 0.6, 0.8 and 1 ml of stock
solution. Total tannin was calculated through tannic
acid standard curve with the help of absorbance using
UV spectrophotometer at 760 nm [28].

Total phenolics:

Total phenolic content was estimated by extracting
50 g of powdered materials with 250 ml methanol.
Extracts were filtered after 24 h, concentrated by rotary
evaporator and dried in vacuum. Stock solutions of 1
mg/ml concentration were prepared by dissolving the
extracts in methanol. 0.5 ml of stock solutions was
taken in 25 ml volumetric flask, added 10 ml of distilled
water, 1.5 ml of FCR and allowed to stand for 5 min. 4
ml of 20 % sodium carbonate solution was then added
and made up the volume to 25 ml with distilled water.
The prepared mixtures were kept in dark for 30 min. The
stock solution of gallic acid of 0.1 mg/ml concentration
was prepared as standard. Total phenolic content was
calculated by using gallic acid standard curve with the
measurement of absorbance at 765 nm through UV
spectrophotometer. The equation: y=115.9x+0.113,
correlation coefficient (r2)=0.999 was used according
to calibration curve (y=Absorbance, x=Gallic Acid
Equivalent (GAE)) [33].

Total flavonoids:

Total flavonoid content was estimated by extracting
50 g of powdered materials with 250 ml methanol.
The stock solutions of methanolic extracts at 1 mg/ml
concentration were prepared. In 1 ml sample, added 1 ml
of 2 % methanolic aluminium chloride solution, made
the volume to 10 ml using methanol and allowed to
stand the mixtures for 1 h. Similar process was repeated
for preparation of quercetin stock solution of 0.1 mg/
ml concentration as standard and prepared the dilutions
using 0.2, 0.4, 0.6, 0.8 and 1 of stock solution. Total
flavonoid content was calculated through quercetin
standard curve with the help of absorbance recorded
at 420 nm using UV spectrophotometer. The equation
74.61x+0.058, r2=0.998 was used for the calculation of total flavonoid content (y=Absorbance, x=Quercetin
Equivalent (QE) in mg/ml) [34].

Antioxidant activity:

The samples were prepared by extracting the materials
in methanol. The stock solutions of methanolic extract
of samples and 0.135 mM methanolic solution of DPPH
were prepared in 1 mg/ml concentration. Different
dilutions were prepared by taking 0.02, 0.04, 0.06, 0.08
and 0.1 ml methanolic stock solution of samples, added
1 ml DPPH solution and made the volume up to 2 ml
with methanol. The mixtures were allowed to stand for
30 min in the dark. The similar process was repeated to
prepare the dilutions of BHA of 1 mg/ml concentration
as antioxidant standard. The absorbance was measured
at 517 nm through UV spectrophotometer [35].

Statistical analysis:

The statistical analysis of data was carried out by
performing the experiments in triplicate. The data was
represented by mean±Standard Deviation (SD). The
linear correlation coefficient was observed using MS
Office Excel 2007. Regression equation was used for
phenolics, flavonoids concentrations and Half-Maximal
Inhibitory Concentration (IC50) values in DPPH radical scavenging assay calculation. The p values less than
0.05 were considered significant.

Results and Discussion

The organoleptic properties and solubility of seed,
cotyledon and endosperm powders were determined
for the quality and acceptability of powdered materials
(Table 1 and Table 2). The qualitative study showed
that phytochemicals were majorly found in alcohol and
water extracts of the seed, cotyledon and endosperm
(Table 3).







Sample Color Odour Taste Texture
Seed Creamish brown Characteristic Neutral Amorphous
Cotyledon Cream Characteristic Nutty with slight bitterness Amorphous
Endosperm Brown Characteristic Nutty with slight bitterness Amorphous

Table 1: Organoleptic Properties of A. nilotica Seed and their Parts.







Sample Hexane Chloroform Acetone Ethanol Water Boiling water
Seed + +
Cotyledon + +
Endosperm + +

Table 2: Solubility of A. nilotica Seed and their Parts.















Phytochemical groups Seed Cotyledon Endosperm
H    Chl    Ac    Al    W H    Chl    Ac    Al    W H    Chl    Ac    Al    W
Carbohydrate –       –       –       +      + –       –       –        +      + –       –       –        +       +
Protein –        –       –       +      + –       –       –        +      + –       –       –        +       +
Alkaloid –       +       +      +      + –       +       –       +      + –       –       –        +       –
Phenolic –       –       –        +      – –       –       –        +       – –       –       –        +       –
Flavonoid –       –       –        +      – –       –       –        +       – –       –       –        +       –
Tannin –       –       –        +      + –       –       –        +      + –       –       –        +       +
Saponin –       –       –         –      + –       –       –         –      + –       –       –         –       +
Glycoside –       –       –         –      – –       –       –         –      – –       –       –         –       –
Tri-terpenoid –       –       –         –      – –       –       –         –      – –       –       –         –       –
Sterols –       –       –         –      – –       –       –         –      – –       –       –         –       –

Table 3: Phytochemical Screening of A. nilotica Seed and their Parts.

Quantitative estimation revealed that total sugar was
4.43 % in seed, 4.73 % in cotyledon and 0.56 % in
endosperm. Cotyledon part was found to have 17.83
% carbohydrate which was higher than in the seed i.e.
9.66 % and in endosperm i.e. 3.16 %. Total protein was
estimated up to 30.45 % in the cotyledon part which
was significantly higher than in seed i.e. 1.23 % and in
endosperm i.e. 0.99 %. The cotyledon was also rich in
oil i.e. 11.78 % while seed had 7.16 % and endosperm
part had only 0.18 %. Starch was estimated in similar
ranges i.e. 2.86 % in the seed and endosperm part and
2.30 % in the cotyledon. Total tannin varied as 1 % in
cotyledon, 0.7 % in seed and 0.46 % in the endosperm
respectively (fig. 1).

Pharmaceutical-Sciences-Phytochemicals

Fig. 1: Phytochemicals percentage in seed, cotyledon and endosperm.

Note: Comparative phytochemicals (%) estimated in A. nilotica seed and their parts,.

The quantification of phenolic and flavonoid content
were determined through calibration curves of gallic
acid and quercetin (fig. 2 and fig. 3). The phenolic
content was 2.66 mg/g GAE in endosperm part which
was significantly higher than 1.80 mg/g and 1.26 mg/g
GAE in seed and cotyledon respectively. Endosperm
part was also rich in flavonoid content with 7.66 mg/g
QE which was noteworthy and more than in seed 4.0
mg/g QE and cotyledon 1.33 mg/g QE (Table 4).

Pharmaceutical-Sciences-Calibration

Fig. 2: Calibration curve of gallic acid

Note: Absorbance at different concentrations of gallic acid showed linear curve for which y=12.03x+0.011 and r2=0.997 and.

Pharmaceutical-Sciences-quercetin

Fig. 3: Calibration curve of quercetin.

Note: Absorbance at different concentrations of quercetin showed linear curve for which y=25.3x+0.006 and r2=0.996 and.







Sample Total phenolic content [GAE (mg/g)]±SD (n=3) Total flavonoid content [QE (mg/g)]±SD (n=3)
Seed 1.80±0.40 4.0±1.0
Cotyledon 1.26±0.11 1.33±0.57
Endosperm 2.66±0.18 7.66±1.52

Table 4: Total Phenolic and Flavonoid Content in A. nilotica Seed and their Parts.

The DPPH radical scavenging assay of A. nilotica seed,
cotyledon and endosperm in comparison to standard
BHA showed that percentage (%) inhibition of free
radicals in BHA was 82.88 % and in seed, cotyledon
and endosperm was found to be 49.02 %, 15.69 % and
74.07 % respectively (fig. 4). A. nilotica endosperm was
comparable to standard and exhibit more potentiality for free radical reducing ability than the seed and
cotyledon. The inhibitory concentration (IC50) value
and % inhibition for antioxidant activity are inversely
proportional to each other; % inhibition increases and
IC50 value decreases. % inhibition of BHA was higher
with lower IC50 value, i.e. 10.61 μg/ml. Endosperm
showed most significant antioxidant activity with
IC50 value, i.e. 25.84 μg/ml, lower than that of seed
and cotyledon, i.e. 55 and 191.10 μg/ml respectively
as compared to BHA (Table 5). Thus, endosperm was
identified as most potential inhibitor of DPPH radicals.
Further, the graph (fig. 5) of reducing power capacity
of seed, cotyledon and endosperm in methanol extracts
as compared to standard BHA showed that as the
concentration of the extracts increased, inhibition of
DPPH radicals also increased.

Pharmaceutical-Sciences-scavenging

Fig. 4: DPPH radical scavenging activity of standard (BHA) and samples.

Note: Data were taken in triplicate and means were calculated (mean±SD) i.e. significant (p<0.05); DPPH: 2,2-Diphenyl-1-Picrylhydrazyl
and BHA: Butylated Hydroxylanisole








Sample IC50 value (µg/ml)±SD (N=3)
BHA 10.61±0.63
Seed 55±0.78
Cotyledon 191.10±18.79
Endosperm 25.84±0.21

Table 5: Ic50 Values in DPPH Radical Scavenging Model in A. nilotica Seed and their Parts.

Pharmaceutical-Sciences-Inhibition

Fig. 5: % Inhibition of DPPH against different concentrations.

Note: DPPH radical scavenging activity was increased with increase in concentration of BHA and samples,.

The results revealed that the A. nilotica seed contained
nutritionally and medicinally important phytochemicals.
The part specific study of seed explored the utilization
of seed parts for specific purpose. The cotyledon part
was rich in sugar, carbohydrate, protein and oil for
nutritional applications, while the endosperm part
was useful for antioxidant property due to phenolics
and flavonoids. The antioxidants reduce the level of
Reactive Oxygen Species (ROS). ROS is a major cause
of oxidative stress diseases such as pain, inflammation,
tissue damage or injury, neurodegenerative disorder,
cardiovascular problem and cancer. Thus, the study
prospect for plant based better source materials to
develop safe and eco-friendly healthcare industrial
products.

Acknowledgements:

Authors are thankful to the Director, CSIR-National
Botanical Research Institute, Lucknow, for providing
facilities and support. Geetendra Kumar is also thankful
to University Grant Commission for financial support.

Conflict of interests:

The authors declared no conflict of interest.

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