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Original Article
PHYTOCHEMICAL SCREENING AND EVALUATION OF LEAF PARTS OF HYPOESTES
PHYLLOSTACHYA ROSEA FOR IN VITRO ANTIOXIDANT ACTIVITIES
JENSON JACOB
1*
, MEENU JOHN
2
1,2
Department of Biochemistry, Pazhassiraja College, Pulpally, Wayanad, Kerala 673579
Email: jensonjacobs@rediffmail.com
Received: 13 Jun 2020, Revised and Accepted: 16 Aug 2020
ABSTRACT
Objective: The aim of this study was focussed on phytochemical analysis, in vitro antioxidant activities of different solvent extracts of Hypoestes
phyllostachya leaf.
Methods: The leaves were washed efficiently, shade dried and coarsely powdered. The powdered leaf material of Hypoestes phyllostachya was
successively extracted with hexane, chloroform, methanol and water using Soxhlet apparatus. Preliminary phytochemical screenings for
polyphenolics, proteins, phytosteroids, flavonoids, tannins, glycosides, alkaloids carbohydrates, polyphenolics, saponins were done by following
standard procedure. In vitro antioxidant activities of various solvent extracts were assessed using D PPH, ABTS, Hydroxy radical, total antioxidant
capacity and reducing power assay. Polyphenolics and flavonoids were also estimated.
Results: The results showed that most of the phytochemical compounds such as carbohydrates, proteins, steroids, fixed oils and fatty acids, flavonoids,
alkaloids, saponins, tannins, polyphenolics and glycosides present in the methanolic and aqueous extract of Hypoestes phyllostachya. The in vitro antioxidant
activity of Hypoestes phyllostachya leaf clearly showed that it has better antioxidant activity, which was confirmed by different assays.
Conclusion: From this work, it can be concluded that Hypoestes phyllostachya has the potential to be a good antioxidant but have only a lesser
amount of flavonoids and polyphenolic contents. This was the first report on the pharmacological activity of the Hypoestes phyllostachya.
Keywords: Oxidative stress, Polyphenolics, Flavonoids, Inflammation, Medicinal plants, Herbal medicine
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ ijcpr.2020v12i5.39766. Jour nal homepage: https://innovareacademics.in/journ als/index.php/ijcpr
INTRODUCTION
Herbal medicine contains rich varieties of free radical scavenging
phytochemicals such as flavonoids, anthocyanins, carotenoids, dietary
glutathione, vitamins and endogenous metabolites and they have
antioxidant properties [1]. The free radicals induce oxidative damage
to lipids, proteins, nucleic acids, which leads to atherosclerosis, ageing,
cancer, diabetes mellitus, inflammation, AIDS and other degenerative
disorders. The living body produces free radicals naturally like
pollution exposure, smoke, fat-rich diet, rich sugar content, alcohol
consumption, which make an individual unhealthy. Oxidative stress is
defined as a “state in which oxidation exceeds the antioxidant systems
in the body secondary to a loss of the balance between them. It causes
dangerous effects such as peroxidation of lipids, oxidative DNA
damage, but also physiologic adaptation phenomena and regulation of
intracellular signal transduction.
Antioxidants are substances that inhibit oxidative stress to a target
molecule [2]. Antioxidants scavenge these free radicals due to
singlet oxygen quenchers and redox hydrogen donators [3]. They
prevent cellular damage by reducing oxidative stress and provide a
beneficial effect on human health. The free radicals formed in the
body are scavenged by natural and synthetic antioxidants [4]. But
the synthetic antioxidants are very toxic having side effects and are
now replaced by natural ones for their safer needs [5]. As the
medicinal plants contain several phytochemicals with biological
activities. Phytochemicals represent a potential source of new
compounds with antioxidant activity as it contains large amounts of
antioxidants such as Ascorbic acid, Tocopherol, flavonoid and
polyphenolic compounds.
Hypoestes phyllostachya Rosea is a tropical sub shrub usually called
polka dot plants from Acanthaceae family. It is grown as an indoor
ornamental house plant and as an accent plant in dish gardens to
add colour in partially shaded areas. The plant leaves are spotted
with pink and white colour with small violet flowers. The species of
hypoestes used as folkloric medicine for a variety of diseases and
health cares as it has antimicrobial antileishmanial and antioxidants
properties. The bioactive compounds isolated from the plant can be
used to treat various disorders. The phytochemical investigation
carried out on various hypoestes species reveals the presence of
phytochemicals as diterpenoids, alkaloids, lignans, triterpenes etc.
Chemical composition of the essential oil separated from leaf and
stem of Nigerian phyllostachya showed the presence of volatile
constituents. Based on the ethnobotanical reviews, the present work
was focused on the phytochemical analysis and pharmacological
properties of various solvent extracts of Hypoestes phyllostachya
Rosea. This was the first report on the activities of Hypoestes
phyllostachya leaves.
MATERIALS AND METHODS
Collection and extraction of plant material
The leaves of Hypoestes phyllostachya Rosea was collected from the
rural area of Wayanad district. The leaves are washed thoroughly,
shade dried and coarsely powdered. The powdered leaf material of
Hypoestes phyllostachya Rosea was successively extracted with
hexane, chloroform, methanol and water using soxhlet apparatus
and is stored for further in vitro assays.
Phytochemical screening of various solvent extracts of the leaf
sample
Phytochemical analysis was done to analyze the presence of
phytochemicals found in different solvent extracts of Hypoestes
phyllostachya leaf [6, 7].
In vitro antioxidant activities of various solvent extracts of leaf
DPPH radical scavenging activity
1,1-Diphenyl-2-picryl hydrazyl (DPPH) is a free radical for
measuring antioxidant activity. The reaction mixture include 2.8 ml
100µM DPPH in methanol and was added with 0.2 ml leaf extract at
different concentrations. The mixture was incubated for 30 min and
International Journal of Current Pharmaceutic al Research
ISSN- 0975-7066 Vol 12, Issue 5, 2020
Jacob et al.
Int J Curr Pharm Res, Vol 12, Issue 5, 54-58
55
the optical density was taken at 517 nm. Ascorbic acid is used as a
reference standard and methanol without sample along with DPPH
was taken as control [8].
% of Scavenging = (A control-A Test)/A control X 100.
Hydroxyl radical scavenging assay
The assay is based on the capacity of phytochemicals to compete
with salicylic acid for hydroxy radicals. The mixture include 1 ml 1.5
mmol FeSO4 added with 0.7 ml 6 mmol hydrogen peroxide, 0.3 ml
20 mmol sodium salicylate and 1 ml of different concentrations of
three solvent extracts of the leaf material. The mixture is then
incubated for 1 hour at 37 °C and the optical density of the
hydroxylated salicylate complex was measured at 562 nm. The
standard used for the assay is ascorbic acid [9].
Total antioxidant capacity (Phospho molybdenum assay)
The principle behind assay is the reduction of Mo(VI) to Mo(V) by
the leaf extract and forms a green coloured phosphate Mo(V)
complex at acidic pH. 0.3 ml leaf extract was added with 3 ml
reagent solution which contains 0.6 M Sulfuric acid, 28 mmol
Sodium phosphate 4 mmol Ammonium molybdate. Incubate the
mixture for 90 min at 95 °C and sample was cooled. The optical
density was taken at 695 nm [10].
Reducing power assay
Different concentrations of leaf extract (0.2–1 mg/ml) dissolved in
methanol was added with 2.5 ml 0.2 M phosphate buffer (pH 6.6)
and 2.5 ml 1% potassium ferricyanide. Incubated the mixture for 20
min at 50 °C and add 2.5 ml 10% TCA. Then centrifuge mixture at
3000 rpm for 15 min. 0.5 ml of the upper layer is added with 2.5 ml
of distilled water and add 0.5 ml 0.1%. ferric chloride. Read the
absorbance at 700 nm. An increased absorbance value show high
antioxidant potential [11].
Estimation of total polyphenolics
The reaction mixture contain 50 μl of sample, 3 ml water, 0.25 ml FC
reagent and 0.75 ml 20 % Na
2
CO
3.
The total volume make up to 5 ml
using water. Mixed well and incubated the mixture at 50 °C for 2 h.
Read the absorbance at 765 nm using spectrophotometer [12]. Here
gallic acid was used as the standard. The concentration of total
polyphenolic content was obtained from gallic acid standard curve.
Estimation of total flavanoid content
In this method Quercetin is used as standard. Here quercetin in
methanol is diluted to different concentrations. The diluted
querscetin (0.5 ml) were added to 1.5 ml methanol, 0.1 ml 10%
aluminum chloride, 0.1 ml 1M potassium acetate and 2.8 ml of
distilled water. Incubated the mixture for 30 min. Optical density
was determined at 415 nm. 10% aluminum chloride replaced by
distilled water was taken as blank. Similarly, 0.5 ml of different
solvent extacts of leaf material reacts with aluminum chloride for
estimating flavanoid content. The results were expressed as mg
Quercetin equivalents/g sample [13].
RESULTS AND DISCUSSION
Phytochemical screening of different solvent extracts of the leaf
material
The hexane, chloroform and methanolic and aqueous leaf extract of
Hypoestes phyllostachya were screened for the presence of
phytochemical compounds. The qualitative analysis of methanolic
and aqueous leaf extract showed the presence of carbohydrates,
proteins, steroids, fixed oils and fatty acids, flavonoids, alkaloids,
saponins, tannins and polyphenolics. The hexane extract of the leaf
contains only carbohydrates and proteins whereas the chloroform
extract contains carbohydrates, proteins and saponins. When related
to other solvent extracts, the aqueous and methanolic leaf extract
had rich content of phytochemicals with higher precipitation. The
results of the analysis were represented in table 1. Even though the
phytochemical constituents are reported to have many therapeutic
and biological properties, Hypoestes phyllostachya is expected to
have certain medicinal uses [14-16]. From this phytochemical
screening, the results showed that methanolic and aqueous extract
of Hypoestes phyllostachya contains rich phytochemical constituents,
which may be due to high polar index of water and methanol solvent
than the other solvents [17].
Table 1: Phytochemical analysis of Hypoestes phyllostachya leaf
Phytochemicals
Hypoestes phyllostachya leaf
Aqueous extract Hexane extract Chloroform extract Methanol extract
Carbohydrates +++ +++ +++ +++
Proteins
+++
+++
+++
+++
Steroids +++ - - +++
Fixed oils and fatty acids
+++
-
-
+++
Volatile oils - - - -
Alkaloids
+++
-
-
+++
Flavanoids
+++
-
-
+++
Saponins
+++
-
+++
+++
Tannins
+++
-
-
+++
Poly phenolics +++ - - +++
Terpenoids
-
-
-
-
Glycosides - - - -
In vitro antioxidant activities of different solvent extracts of leaf
material
DPPH radical scavenging activity
In this assay, the percentage of scavenging of hexane extract is
31.28±0.67 at a maximum concentration of 2500μg/ml whereas the
chloroform extract showed inhibition of 39.09±1.22. But the methanolic
extract of the leaf showed the highest significant radical scavenging
effect of 64.29±0.89 on DPPH radicals at 2500 μg/ml. In the present
study, there was increased scavenging property of the DPPH radicals
with increased concentration of various extracts of Hypoestes
phyllostachya. This may specify an increased ability to provide hydrogen
ions resulting in a lighter solution which is proportional to electrons
gained. Therefore, it may be suggested that the methanolic extract of
Hypoestes phyllostachya, has DPPH scavenging activity, by decreasing the
DPPH radical to hydrazine due to its hydrogen ion-donating ability.
Hydroxy radical scavenging assay
In this assay, scavenging of hydroxy radicals increases with
increased concentrations of the Hypoestes phyllostachya leaf
extract. Here hexane extract of the leaf showed a percentage
inhibiti on of 37±1.5 whereas chloroform extract shows 62±3.2 %
inhibiti on at 2500 µg/ml. Th e methanolic extract of the leaf
showed the highest percentage of inhibition of 72.22±1.3 at 2500
µg/ml. The hydroxyl radicals are highly active of the reactive
oxygen species, which cause severe injury in adjacent
biomolecules or they cause oxidative stress to nucleic acids,
proteins and lipids. The hydroxyl scavenging activity of the
methanolic extract of Hypoestes phyllostachya was determined by
its capacity to c ompete with salicylic acid for •OH radicals in the
•OH generating system. Hence the methanolic extract can be
regarded as a better scavenger of hydroxy radicals.
Jacob et al.
Int J Curr Pharm Res, Vol 12, Issue 5, 54-58
56
Fig. 1: DPPH radical scavenging activity of various solvent extracts of Hypoestes phyllostachya
Fig. 2: Hydroxy radical scavenging activity of Hypoestes phyllostachya
Fig. 3: Total antioxidant capacity of various solvent extracts of Hypoestes phyllostachya
Total antioxidant capacity (Phospho molybdenum assay)
The total antioxidant activity of the plant extracts was determined
by the Phospho molybdenum assay. Here the methanolic extracts of
Hypoestes phyllostachya leaf showed increased total antioxidant
capacity with increasing concentration when compared to
chloroform and hexane extracts of the sample. 500μg/ml of the
methanolic extract of Hypoestes phyllostachya leaf was equivalent to
284 μg/ml of the ascorbic acid standard. When related to the hexane
and chloroform extracts, the methanolic extract showed higher
antioxidant capacity. It is a method which evaluates the reduction of
Phosphate-Mo (VI) to Phosphate Mo(V) by the methanolic extract
and presence of a bluish-green coloured Phosphate Mo(V) complex.
Increase in optical density indicates the higher antioxidant activity
of Hypoestes phyllostachya leaf extract.
Reducing power assay
In reducing power assay, the different extracts of Hypoestes
phyllostachya leaf showed reducing power ability. Her 2500μg/ml
of the methanolic extract of Hypoestes phyllostachya was found to
be equivalent to 46 μg/ml of ascorbic acid standard. From these
results, it can be substantiated as the absorbance of leaf extracts
increases , the reductive ability increases. The methanolic leaf
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57
extract has natural antioxidants to donate electrons and reduce of
Fe+3 to Fe+2 ions. The reduci ng ability of extract depends on the
presence of reductones which has antioxidant activity by breaking
the free radical chai n and donate a hydrogen atom. Hence the
reducing power leads to the termination of the radical chain
reactions that may otherwise be very damaging. The presence of
antioxida nt reductants in the methanolic extract of Hypoestes
phyllostachya of causes the reduc tion of the Fe3+c omplex to
ferrous form, which indicates the significant reducing power of the
plant extract.
Fig. 4: Reducing power assay of various solvent extracts of Hypoestes phyllostachya
Estimation of total polyphenolics
The total phenolic content was found to be 10 mg Gallic acid
equivalents present per gram Hypoestes phyllostachya. The results
were obtained from the calibration curve of gallic acid standard.
Among the different phytocompounds, Polyphenolics are widespread
in the plant kingdom as part of our daily diet and are considered as
natural antioxidants. Poly phenolics have gained great attention due to
their radical scavenging, anti-mutagenic, anti-carcinogenic and anti-
inflammatory properties. The antioxidant property of phenolics is due
to their redox activities which make it as hydrogen donors, metal
chelators, reducing agents and as well as singlet oxygen quenchers. In
this study, there is an only lesser amount of polyphenolics present per
gram of the methanolic extract of Hypoestes phyllostachya.
Fig. 5: Determination of total polyphenolic content-Calibration curve of gallic acid
Fig. 6: Determination of total flavanoid-calibration curve of quercetin
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Estimation of total flavanoid content
The flavonoid of methanol extract was found to be 13 mg quercetin
equivalents per gram of the leaf material. The results were obtained
from the standard plot of quercetin. Flavonoids are plant secondary
metabolites, the antioxidant property of which depends on the
presence of free OH groups, especially 3-OH. Plant flavonoids have
both in vitro and in vivo antioxidant activity [19]. As this is the first
report on the antioxidant activity of Hypoestes phyllostachya, the
flavonoid contents present in the plant leaf is very less.
CONCLUSION
In this study, Hypoestes phyllostachya leaf was tested to analyse the
presence of phytochemicals and their antioxidant activities.
Successive extraction of the leaf was done using Soxhlet apparatus
and the extract was partitioned using hexane, chloroform and
methanol. The analysis of different solvent extracts of the leaf
material showed the presence of several phytochemicals. The
antioxidant activity was measured by DPPH, ABTS, Hydroxy radical,
Total antioxidant capacity and reducing power assays. The flavonoid
and polyphenolics present in the leaf sample were also estimated.
The phytochemical analysis of methanolic and aqueous leaf extract
showed the presence of carbohydrates, proteins, steroids, fixed oils
and fatty acids, flavonoids, alkaloids, saponins, tannins and
polyphenolics, which are the perfect sources of antioxidants. The
findings of these studies suggested that Hypoestes phyllostachya leaf
could have a major role as a therapeutic agent in reducing oxidative
stress-related disorders. But there is lesser amounts of
polyphenolics and flavanoids present in the leaf sample. The work
regarding the isolation of active compounds responsible for
antioxidant capacity will be carried out for the future.
ACKNOWLEDGMENT
The authors are thankful to Pazhassiraja College, Pulpally, Wayanad,
Kerala, India for providing the facilities for this work.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
All the authors have contributed equally.
CONFLICTS OF INTERESTS
The authors declare that there was no conflicting interest
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