NewBioWorld A
Journal of Alumni Association of Biotechnology (2022) 4(1):7-12
ORIGINAL
RESEARCH ARTICLE
Preliminary examination of bioactive components and
bioactivity of Hardwickia binata Roxb. extracts obtained through
conventional extraction methods
Abhilasha Tamada, Afaque Quraishi and Smriti Adil*
School of Studies in
Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India.
abhilashatamada@gmail.com,
drafaque13@gmail.com, 15oct.sadil@gmail.com
*Corresponding Author Email- 15oct.sadil@gmail.com
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ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
25 March 2022
Received in revised form
14 June 2022
Accepted
Keywords:
Antioxidant,
DPPH,
Hardwickia
binata, phytochemical,
plant
extract.
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The phytochemical constituents,
quantitative antioxidant, and total phenolic content of Hardwicikia binata Roxb. plant parts were investigated in the present study. Methanolic extracts
of H. binata leaves and stems were obtained using the traditional extraction
methods, soxhlet and maceration. Preliminary tests for phytochemicals,
revealed the presence of flavonoid, steroids, tannin and terpenoids, tannins in
the stem and, flavonoids and phenols in the methanolic extracts of the stem
and leaves, respectively. Saponin was found in the leaves and stem parts, but
no alkaloids were identified in either. The high antioxidant content of the H.
binata stem and leaf methanolic extract was proven by the total antioxidant
assay. Furthermore, total phenol quantification among the extracts revealed
that the plant's stem extract was rich in phenols. DPPH scavenging activity showed
the potential antioxidant capability of H. binata parts. Overall, the
preliminary findings revealed the bioactive components and antioxidant
properties of H. binata leaves and stem parts; further detailed examination
and screening of the plant may prove it to be a potential source of
medication in the pharmaceutical field.
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Graphical
Abstract:
OI: 10.52228/NBW-JAAB.2022-4-1-3
Introduction
The use of medicinal herbs, shrubs, and trees, that are the best
reservoirs of bioactive compounds in plants, dates back to the dawn of time to
the humankind (Asfaw et al. 2022). They have been used as therapeutic
or remedies to ailments in India since ancient times (Gunaselvi et al. 2010). With
its mega biodiversity, India has various indigenous traditional medical systems
which includes Ayurveda, Amchi, Siddha and Unani (Srivastava et al. 2021). The
traditional scriptures of Ayurveda, such as the compendia,
lexicons, pharmacopoeia, treaties, samgraha grantha and others provides
the information about the medications. According to ancient texts
such as the Charak Samhita, a specific herb has various actions to heal illness,
and every plant may have a superior effect and other
comparatively auxiliary effects (Saxena and Vikram 2004). Alkaloids,
flavonoids, tannins, and phenolic compounds are by far the most potent
bioactive constituents in plants (Edeoga et al. 2005). These phytoconstituents
derived from medicinal plants holds a promise to be used as allopathic
disease treatment alternatives, and they seem to have a great capacity for
healing and curing a variety of ailments (Dewangan and Acharya 2017). Traditional
medicine, on the other hand, were administered in the form of crude extracts or
a mixture of chemical compounds for several years. These approaches may be
unsafe because they do not isolate probable poisonous or present as an unusable
substance along with the active chemical constituent (Patel and Patel 2016). As
a result, knowledge of phytochemical constituents, pharmacological activities,
extraction, isolation, refinement, and structural elucidation of bioactive
compounds is critical for the development of potent drugs (Patel and Patel 2016).
Hardwickia
binata Roxb., commonly known as Anjan (in
Ayurved as Anjana; in Siddha/Tamil as Katudugu or Kodapalai) (Gunaselvi et al.
2010; Shingade and Kakde, 2021). It is an attractive medium or large deciduous
ornamental tree with graceful drooping branchlets. It belongs to the
Caesalpiniaceae family and is represented by a single species, Hardwickia
binata Roxb. In folk medicine, it is used to treat diarrhoea, leprosy, worm
infection, indigestion, leucorrhoea, chronic cystitis, gonorrhoea, cancer, and
microbes (Shingade and Kakde, 2021). Its parts used include roots, leaves,
bark, seed, wood, and husk. It is a medicinal plant that belongs to the endemic
biodiversity category (Deshmukh and Ghanawat, 2020). The tannins from the
bark are used in medicines to treat diarrhoea, worms, indigestion, leprosy, and
as an appetiser. H. binata leaves extract have antimicrobial activity
against Gram-positive, Gram-negative bacteria and fungi. And, the
antimicrobial agents demonstrated by bioactive substances
includes gonorrhoea, pneumonia, eye, and mycotic infections. Leaves of H.
binata has been reported to effective in relieving headache and in
constipation treatment (Gunaselvi et al. 2010). Also, the leaf and bark have been a medicinal source for
the treatment of rheumatism (Kanaka et al. 2013). The natives of the
Chhattisgarh Plains traditionally use its leaves to treat headaches (Oudhia
2003); the aqueous paste of the leaves is also applied dermally on painful
parts as a remedy. Alongside, leaves are used as purgative and in the treatment
of constipation. Therefore, the present
work aims at screening for the phytoconstituents and antioxidant potency in
methanolic extract of H. binata stem
and leaves parts, employing different extraction methods and hence has the
present examination included the phytochemical analysis and antioxidant
screening assay.
Materials and
Methods
The leaves and stems of H.
binata were collected for this study from a forest next to School of
Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur. The
collected fresh leaves and stems were sorted, cleaned with water to remove dirt
and were then shade dried at a constant temperature of 40°C for absolute
removal of moisture content. Dried plant parts were finely processed into
powder and used in experimental purposes (Figure 1). Table 1 describes the plant
part collection conditions and organoleptic features.
Two of the extraction methods were used to extract the
bioactive compounds present in H. binata.
For the maceration process, the powdered plant part and the solvent methanol
were mixed in a 1: 10 w/v (sample: solvent) ratio and placed in a closed
container for 72 hours (Pandey and Tripathi 2014). The extracts were collected
and filtered after the aliquot was stirred at regular intervals to ensure
adequate mixing of the sample with the solvent. The samples were mixed with the
solvent in the same ratio as prepared for the maceration process and subjected
to 10 cycles at the boiling point of the solvent in the Soxhlet extraction (or,
hot extraction) method. The extracts were collected and filtered after the
completion of the cycle.
% Yield of the extract
The yield percentage of H.
binata extracts, macerated leaf (ML), macerated stem (MS), soxhlet
extracted leaf (SL), soxhlet extracted stem (SM), were calculated on a dry
weight basis using the following formula:
Secondary
metabolites screening
For
preliminary phytochemical screening, various extracts of H. binata were qualitatively tested for alkaloids (Mayer’s test,
Wagner’s test), terpenoids, saponin (foam test), tannins, flavonoids (alkaline
reagent test), phenols (ferric chloride test), steroids (Salkowski’s test),
carbohydrates (Fehling’s test), proteins (Ninhydrin test) (Harborne 1973;
Trease and Evans 1989; Ayoola et al. 2008; Chaturvedi et al. 2011; Singh and
Saxena 2011; Yadav and Agrawal 2011; Mushtaq et al. 2014).
Antioxidant
assay of H. binata extract
2,2-diphenyl-2-picrylhydrazyl
(DPPH) assay
Shimada
et al. (1992) method was followed for the assessment of DPPH radical scavenging
activity in H. binata extract. 1 ml
of DPPH solution was added to 1 ml of different concentrations of test samples
(20, 40, 60, 80 and 100 µg/ml of H.
binata methanolic extract). Also, control (without the extract) was
prepared. The reaction mixture was incubated in dark for 30 min at room
temperature. The absorbance was measured at 517 nm and compared with the
standard ascorbic acid. The percentage DPPH decolorization of the sample was
determined according to the following equation:
Percent of DPPH inhibition
where
AbsControl and AbsSample are the absorbance values of the
control and test samples, respectively.
Determination
of total antioxidant activity
For
the evaluation of the total antioxidant activity, phosphomolybdenum method
described by Prieto et al. (1999) was employed. To the 1.0 ml of the test
extract, 1.0 ml of the phosphomolybdate reagent (0.6 M sulphuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate) was mixed. Then, the tubes were
covered and incubated at 95°C for 90 min in a thermal block. After heating, the
solution was cooled and brought to room temperature; the absorbance was read at
695 nm using a spectrophotometer along with the standard. Total antioxidant
capacity was calculated from the standard graph and expressed as milligram of ascorbic
acid equivalence (AAE) per gram of extract.
Determination
of total phenolic content
Folin
Ciocalteau's (FC) method was used to determine the total phenolic content of H. binata extracts (Singleton and Rossi
1965). FC reagent was added to 1 ml of the sample extract. After 3 min, 1 ml of
sodium carbonate was added to the above mixture, and the final volume was made
up to 10 ml with distilled water. After 90 min of incubation in dark, the
absorbance of the tubes was measured at 725 nm against a blank. A standard
calibration curve was plotted using gallic acid to quantify the total phenolic
in the extract. The results were expressed in milligram of catechol equivalence
(CE) per gram extract.
Statistical analysis
All the experiments were conducted in triplicates and
one-way analysis of variance (ANOVA) was performed for the data analysis.
Using, Duncan’s Multiple Range Test (DMRT) in SPSS version 16.0 tests of
significance at 0.05 levels were checked. Data were realized as Mean ±Standard
error (SE) of the determinants.
Table 1. Collection
of plant material and organoleptic features of H. binata
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S. No.
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Plant part
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Plant part collection
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Physical appearance
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Season
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Month
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Temperature
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Pre- processing
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Post-processing
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1.
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Leaves
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Spring
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January
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30-35°C
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Small, bifoliate, sage
green
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Dark green, fine sticky
powder
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2.
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Stem
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Spring
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January-February
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30-35°C
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Thin, slight sap, brownish
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Dark brown, coarse powder
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Result
and Discussion
The present work focussed on the preliminary screening of
phytochemicals and assaying antioxidant activities in methanolic extracts of H. binata leaves and stem part,
extracted through maceration and soxhlet extraction methods. The following
sub-heads shows the obtained results:
Percentage Yield extract
Figure 1: Post-harvested and preliminary
processed H. binata plant parts (a) Leaves (b) Stems
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The percentage yield obtained from methanol-extracted MS,
ML, SL and SM of H. binata using
maceration or soxhlet method is tabulated in Table 2. From the obtained data,
the highest percentage extractive potential was found in the case of maceration
method. In addition, among the tested samples, percentage yield extract was
found to be higher in methanolic extracts of leaves and stem parts of H. binata that showed percentage yield
of 22.90% and 21.06%, respectively. Previously, yield of 31.9% was obtained in
the leaves methanolic extract of H. binata and was much higher in
comparison to petroleum ether extract (2.2 ug/ml), following maceration extraction
method (Hamid et al. 2018). In another study, only 0.9% and 1.8% yield was
obtained in methanolic and hexane extracts of H. binata plant material (Ali et al. 1999).
Phytochemical screening
Various qualitative phytochemical tests were performed on
methanolic extracts of H. binata stem and leaves parts for the identification of
active chemical constituents. In the present study, phytochemical screening
revealed that the two extraction methods inspected have potentially isolated
the various bioactive compounds present in the studied plant, H. binata. Saponin and carbohydrates
were found to be present in all of the methanolic extracts of H. binata that were screened. Bioactive
constituents screened in leaves part showed presence of phenol but not flavonoid
in SL, however, in comparison ML was found to contain phenol and flavonoid as
well. Flavonoid, steroid and tannin were obtained in MS, while flavonoid,
steroid and terpenoid were present in SS extract. Although, all the extracts showed negative
results for alkaloids.
In earlier investigation, different parts of H. binata have been investigated for the
presence of chemical constituents as well as secondary metabolites that account
for their therapeutic properties. It was reported that the ethanolic extracts
of the H. binata leaves and seeds
contained flavonoids, glycosides, phenol, saponins and, tannins in H. binata leaves, though alkaloids were
absent in both the parts (Sharanabasappa et al. 2007; Shingade and Kakde 2021).
Sharanabasappa et al. (2007) identified bioconstituents flavonoids, phenols,
saponins and tannins in leaves ethanolic extracts of H. binata. Besides, similar findings were reported among previous
results that showed saponins, tannins, steroids, coumarin, flavonoids, however,
alkaloids and anthraquinones were absent in the ethanolic extract obtained via
soxhlet method (Gunaselvi et al 2010). In similar, root bark exudates
methanolic extracts of H. binata
(soxhlet method) contained amino acids, carbohydrates, fixed oils, fats,
glycosides, proteins, alkaloids, flavonoids, phytosterols phenolic compounds,
saponins, tannins (Prabakaran et al. 2014). Also, Deshmukh and Ghanawat (2020)
reported in their paper the presence of steroids, glycosides in methanolic
extracts of H. binata leaves and
stem, however absence of carbohydrates, proteins and alkaloids revealed through
phytochemical screening. Deshmukh and Ghanawat (2020) conducted phytochemical
screening, and Fourier Transform Infrared (FTIR) of H. binata leaves extract. The investigation revealed the presence
of numerous phytochemical components including proteins, flavonoids,
glycosides, tannins, phenolic compounds, quinones, steroids, volatile oils, but
not alkaloids. In addition, FTIR spectroscopic analysis of H. binata crude powder confirmed the presence of alcohol, aliphatic
amines, alkanes, alkenes, alkyl halides alkynes, amides, amines, aromatics,
carboxylic acids, esters, ethers, phenols in leaves.
Antioxidant activity
H. binata antioxidant activity was measured using DPPH and was compared
with the activity of standard antioxidant ascorbic acid. The DPPH antioxidant
assay percentage showed varying inclination among the plant extracts. ML and SL
showed increasing antioxidant activities with increasing extract concentration
from 20-100 μg/ml with highest values at 100 μg/ml of 92.58 and 87.79,
respectively. On the other hand, the antioxidant activities of MS and SS were
found to be similar at a various range of concentrations, although the activity
was found to be greater than 90% for all the concentrations and the activities
were also comparable to the standard. The antioxidant capacities of all the
examined samples are presented in Figure 2.
Previous studies on H.
binata leaves methanolic extracts, tested for antioxidant activity ranging
from 3.12 μg/ml -50 μg/ml. DPPH free radical scavenging assay that was measured
after 40, 80 and 120 min showed highest scavenging activity against DPPH at 50
μg/ml concentration of the extract (Hamid et al. 2018). The activity was noted
to be greater than 65 μg/ml after 40 and 80 min and 70 μg/ml after 120 min. In
addition, the activity of H. binata
leaves extracts was higher compared to other plant species leaves extracts that
included Acaia siberiana, Bahunia rufescens, Pongamia pinnata
and Prosopis juliflora.
Quantitative assay of antioxidant and phenolic content
Methanolic extracts of different parts, extracted via
different extraction process resulted in contrasting total antioxidant activity
among the various extracts. ML and SS showed the highest values of 16.23 and
16.56 mg/ml for the assay, on the other hand, total antioxidant activity was
found to be low in the case of SL and MS. Data represented in Figure 3. High
total phenol values were detected in the stem part of H. binata i.e., MS and SS with values 0.52 and 0.51, while less
phenolic content was found to be present in leaves extract SL and ML, being the
values 0.29 and 0.19, respectively (Figure 4). Previous work reported the total
phenolic content in 80% methanolic extracts of H. binata was 0.94 μg/ml (Hamid et al. 2018)
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ANOVA (μg/ml concentration)
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df
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F
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p
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20
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4
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148.69
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<.0001
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40
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4
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1.167
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<.0001
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60
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4
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390.55
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<.0001
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80
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4
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128.476
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<.0001
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100
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4
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7.151
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.007
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Figure 2: DPPH scavenging activity of H. binata plant part
extracts at different concentrations. Data expressed as Mean ±Standard error
(SE) and different letters denotes significant differences from each other compared
at p< 0.05. ML, macerated leaf; MS,
macerated stem; SL, soxhlet-extracted leaf; SS, soxhlet-extracted stem; AA,
ascorbic acid (Standard).
Total antioxidant content (mg AAE/
gram extract)
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ANOVA
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df
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F
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p
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Total Antioxidant
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3
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915.65
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<.0001
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Figure 3: Total antioxidant content in
methanolic extract of H. binata plant part. Data expressed as Mean
±Standard error (SE) and different letters denotes significant differences from
each other compared at p< 0.05. ML, macerated leaf; MS, macerated stem; SL,
soxhlet-extracted leaf; SS, soxhlet-extracted stem; AAE ascorbic acid equivalence
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ANOVA
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df
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F
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p
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Total Antioxidant
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3
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26.079
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<.0001
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Figure 4: Total phenolic content in methanolic extract of H.
binata plant part. Data expressed as Mean ±Standard error (SE) and different
letters denotes signifcant differences from each other compared at p< 0.05. ML, macerated leaf; MS, macerated stem; SL,
soxhlet-extracted leaf; SS, soxhlet-extracted stem; CE, catechol equivalence.
Table
2. Yield percentage of methanolic extract of the plant parts
of H. binata
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Sl.
No.
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Plant part
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Initial weight (g)
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Final weight (g)
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Dried extract weight (g)
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% Yield extract (g)
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1.
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MS
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46.01
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43.70
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2.31
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15.4
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2.
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ML
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49.57
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44.99
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4.58
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22.9
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3.
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SL
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57.21
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50.89
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6.32
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21.06
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4.
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SS
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44.26
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40.91
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1.35
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6.75
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Note: ML, macerated leaf; MS, macerated stem; SL,
soxhlet-extracted leaf; SS, soxhlet-extracted stem
Conclusion
The
current study included phytochemical analysis, antioxidant screening assays to
determine the preliminary pharmacological potentialities of various parts of
the studied plant H. binata. The
qualitative phytochemical content of dried methanolic extracts of selected
plant leaves and stem parts was determined. The percentage yield extract
obtained was found to be greater in the case of leaves extract. The results
showed that the soxhlet extraction method extracted more biologically important
phytoconstituents than the maceration method. Furthermore, methanolic extract revealed
the presence of bioactive compounds in the stem much more than in the leaves.
The total antioxidant assay revealed that the methanolic extracts of the stem
and leaves of H. binata have high
antioxidant activity.
Conflict
of Interest
Authors
have no conflict of interest to declare.
Acknowledgement
Authors
would like to express deep gratitude to the Head of the Department, School of
Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur,
Chhattisgarh for providing laboratory and other facilities to conduct present research
work.
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