Phytochemical Analysis and In vitro Antioxidant activity of extracts of Justicia wynaadensis leaves.

ABSTRACT:
Free radicals are constantly formed in the cell causing oxidative stress when the precarious balance is
broken, in favour of free radicals proceeding to variety of pathophysiological disorders
Antioxidants that can scavenge free radicals are thus effective in ameliorating the progress of oxidative
damage. The present study was designed to estimate phenolics and flavonoids present an
to evaluate the antiradical activity of Justicia wynaadensis leaf extracts. Series of in vitro models like DPPH,
lipid peroxidation, reducing ability, superoxide scavenging, nitric oxide scavenging
hydroxyl scavenging and hydrogen peroxide scavenging activity were employed to assess the antioxidant
activity of different extracts of J. wynaadensis. Few fractions of J. wynaadensis lea
extracts showed antioxidant activity comparable to that of the standards such as BHT, Ascorbic acid,
Quercetin, Curcumin and Mannitol to certain extent. The in vitro assays revealed th
effective antioxidant and free radical scavenging capacity of the leaf extract of J. wynaadensis.
KEYWORDS: Justicia wynaadensis, antioxidant, 1,1-Diphenyl-2-picryl hydrazyl, free radical scavenging
activity.

I. INTRODUCTION:
A free radical is any molecular species capable of independent existence that contains an unpaired electron
in an atomic orbital [1]. Free radicals are continuously formed in the cell as
consequence of normal metabolic process and also from external sources such as exposure to radiation,
tobacco smoke, ozone, pollutants and industrial chemicals. When this delicately
maintained balance is shifted in favour of pro-oxidants results in ‘oxidative stress’. It has been implicated in
the etiology of several (>100) of human diseases and in the process of ageing
Oxidative stress plays a major role in the development of chronic and degenerative diseases such ascancer,
arthritis, aging, autoimmune disorders, cardiovascular and neurodegenerative disease
[2].
Antioxidants provide protection from damage caused by uncontrolled production of free radicals and the
concomitant lipid peroxidation, protein damage and DNA strand breaking. In curren
years, substantial attention has been directed towards credentials of plants with antioxidant ability that may
be used for human expenditure.
J.wynaadensis is a small climbing herb of the family Acanthaceae, native to Western Ghats of Karnataka
and Kerala states of India. Deep bluish purple colored aqueous extract of J.wynaadensi
leaves and stem is traditionally consumed by the local population in the monsoon season. It has been
reported that the plant extract possess cellular cholesterol and cholesteryl ester lowerin
ability [3]. It is also used for external application over rheumatic swellings [4]. Polyphenols and flavonoids
have been estimated in the plant extract. Extract is also known to possess peroxidas
and catalase [5]. GC-MS analysis depicted the presence of various phytoconstituents known to possess
various biological activities [6]. The lplant extract has been investigated for its anti
inflamatory activity [7]. With this background and abundant source of unique active components harboured
in plants, the present study was taken up to evaluate the plant potential as an
antioxidant.
II. MATERIALS AND METHODS:

The fresh plants collected in the month of July from Kushalnagar, Karnataka, India located in the southern
part of India. The leaves of J. wynaadensis were washed, shade dried and 100g o
powdered leaf was subjected to soxhlet extraction using petroleum ether, chloroform, ethyl acetate and
methanol in successive mode respectively for 48 hours. The extracts were concentrated in
rotary vacuum evaporator. Different solvent extracts of J. wynaadensis were subjected to preliminary
phytochemical screening for the presence of alkaloids, saponins, tannins, flavonoids
glycosides and terpenoids as per the standard procedures [8-10].
1. Quantitative estimation of polyphenols, flavonoids and tannins:
Total phenolic content was determined using Folin’s Ciocalteau reagent employing Bray and Thorpe method
[11] and expressed as mg of gallic acid equivalents/g of extract. The flavonoi
content was determined following the method of Chang et al [12] and expressed as mg of quercetin
equivalents/g of extract. Tannin content was estimated employing the method of Anonymou
[13] and expressed as tannic acid equivalents/g of extract.

2. In vitro antioxidant activity:

Different solvent extracts of J.wynaadensis leaf was tested for its antioxidant efficacy by a range of in vitro
models and % inhibition in each assay was using the formula:
Inhibition (%) = [(AControl – ASample)/( AControl)]× 100

In vitro antioxidant activity:

Different solvent extracts of J.wynaadensis leaf was tested for its antioxidant efficacy by a range of in
vitro models and % inhibition in each assay was using the formula:
Inhibition (%) = [(AControl – ASample)/( AControl)]× 100

i. DPPH radical scavenging assay:

Total antioxidant activity of J.wynaadensis leaf extracts were estimated using stable 1,1-Diphenyl-2-picryl
hydrazyl (DPPH) radical scavenging assay following the method given by Cotelle et al. [14]. Butylated
hydroxyl toluene (BHT) was the reference standard used. To 200 µL 100 µM DPPH (1, 1-diphenyl-2-picryl
hydrazyl), 2.8 mL methanolic extracts (20-100 µg/mL) were added. After 20 minutes absorbance was read at
517 nm and percentage inhibition.

ii. Anti-lipid peroxidation assay:

The measurement of TBARS is a well established method of evaluation and monitoring lipid peroxidation and
was assayed using the method given by Prashanth et al [15]. Freshly excised wistar rat liver was homogenized
in ice cold 40 mM Tris-HCl buffer (pH 7.0) using glass teflon homogenizer and centrifuged at 3000 rpm for
10 min at 4oC. The reaction mixture containing 0.5 mL of supernatant, 1 mL J.wynaadensis leaf extracts (50-
250 µg/mL), 0.1 mL 0.15 M KCl, 0.1 mL 15 mM FeSO4, and 0.1 mL 6 mM ascorbic acid was incubated for
1 hour at 37oC. 1 mL 10% TCA (trichloro acetic acid) was added to the mixture and centrifuged at 3000 rpm
for 20 min at 4oC. 1 mL 0.8% TBA (thio barbituric acid) was added to supernatant followed by heating at
90oC for 20 minutes. Absorbance was measured at 532 nm using BHT as reference standard and inhibition of
lipid peroxidation was calculated.

iii. Reducing power assay:

The Fe3+-reducing ability of J. wynaadensis leaf extracts was determined based on the power of plant
antioxidants to form coloured complex with potassium ferricyanide as described by Oyaizu [16]. 1 mL of
different extracts of J. wynaadensis leaf (200-1000 µg/ml) was mixed with 2.5 mL 1% potassium ferricyanide
and 2.5 mL phosphate buffer (pH 6.6). The mixture was incubated at 50oC for 20 minutes. 10% TCA was
added and centrifuged at 3000 rpm for 10 minutes. The supernatant was mixed with 2.5 mL distilled water
and 0.1% FeCl3. Absorbance was measured at 700 nm using ascorbic acid as reference standard.

iv. Super oxide radical scavenging assay:

Superoxide anion radical scavenging activity was determined by NBT reduction method of Nishimiki et al.
with slight modifications [17]. The assay mixture contained 1 mL NBT (nitro blue tetrazolium) in phosphate
buffer (pH 7.4), 1 mL 468 µM NADH (β-nicotine amide adenine dinucleotide) and 0.1 mL Justicia
wynaadensis leaf extracts (200-1000 µg/mL). The reaction was initiated by adding 100 µL 60 µM PMS
(phenazine methosulphate). The reaction mixture was incubated at 30oC for 15 minutes and the absorbance
was measured at 560 nm. Quercetin was used as reference compound. Percent inhibition was calculated.

v. Nitric oxide radical scavenging assay:

Nitric oxide radical generated was assayed using Green et al. method [18]. 1 mL 10 mM sodium nitroprusside
was mixed with 1 mL of various extracts of J. wynaadensis leaf (50-250 µg/mL). The mixture was incubated
at 25oC for 150 minutes. To 1 mL incubated solution, 1 mL Griess reagent (1% sulphanilamide, 2% o-
phosphoric acid and 0.1% naphthyl ethylene diamine dihydrochloride) was added. Absorbance was measured
at 546 nm and percent inhibition was calculated. Curcumin was used as reference compound.

vi. Hydroxyl radical scavenging assay:

The preventive effects of extracts on deoxyribose damage, imposed by hydroxyl radicals were determined by
the method given by Kunchandy et al [19]. The reaction mixture containing 500 µL J. wynaadensis leaf
extracts (50-250 µg/mL), 100 µL 28mM 2-deoxy-2-ribose, 1.04 mM EDTA (ethylene diamine tetra acetic
acid), 0.2mM FeCl3 and 1mM ascorbic acid was incubated at 37oC for 1 hour. Scavenging activity was
determined colorimetrically at 532 nm using mannitol as reference compound. Percent inhibition was
calculated.

vii. Hydrogen peroxide scavenging assay:

Hydrogen peroxide scavenging capacity was determined according to Gulcin et al [20]. 1 mL of various
extracts of J. wynaadensis leaf in 0.1 mM phosphate buffer (pH 7.4) at various concentrations (200 - 100
µg/ml) were mixed with 2 mL 100 mM H2O2 (Hydrogen peroxide). The concentration of hydrogen peroxide
was measured at 230 nm after 10 minutes. Ascorbic acid was used as reference standard. The percentage
inhibition was calculated.

Statistical analysis:
Experimental results were presented as the mean ±standard deviation (SD) of three parallel measurements.
Statistical analyses were performed by one-way ANOVA, followed by student t-test. The difference was
considered to be statistically significant when the p value was less than 0.05.

III. RESULTS AND DISCUSSION:

Methanolic extract and ethyl acetate extract of J. wynaadensis leaf showed the presence of anthocyanins,
flavonoids, phenolics and tannins which indicates that the phytochemicals present in J. wynaadensis is
soluble in polar solvents. Successful determination of biologically active compounds from plant material is
largely dependent on the type of solvent used in the extraction procedure [21].

Table 2 depicted the amount of polyphenols, flavonoids and tannins estimated. The results showed that the
methanol extract is rich in polyphenols flavonoid and tannin content. Phenolics have an array of health-
promoting benefits; they are of current interest due to their important biological and pharmacological
properties, especially the antioxidant activity [22]. Flavonoids being a group of polyphenolic compounds are
capable of effectively scavenging the reactive oxygen species because of their phenolic hydroxyl groups. The
presence of high phenolic and flavonoid content in the fractions may be attributed directly to the antioxidant
activity.
i. DPPH radical scavenging assay:
The largest capacity to neutralize DPPH radicals was found for methanolic extract followed by the standard
BHT with IC50 value of 158.103±1.56 µg/mL and 197.653 µg/mL respectively. However, the petroleum ether,
chloroform and ethyl acetate extracts showed higher IC50 value of 370.451±2.18, 336 µg/mL, 169±0.76
µg/mL and 212.191±1.21 µg/mL compared to the standard BHT. The DPPH radical scavenging potential of
the different extracts is represented in Fig. 1.
The relatively stable organic radical DPPH is widely employed modelling systems to investigate the
scavenging activity of the plant extracts. Thus, an extract which shows good activity in DPPH antioxidant
assay, may also serve as a good antioxidant in vivo. The methanol fraction of J. wynaadensis showed
promising activity. DPPH has the advantage of being unaffected by certain side reactions, such as metal ion
chelation and enzyme inhibition [23]. This result corroborates previous study which demonstrated that DPPH
scavenging properties of plant [24].

Table 1: Qualitative phytochemical screening of different extract of J.wynaadensis

Sl. Phytochemical
No. test
1 Alkaloids
2 Anthocyanins
3 Flavonoids
4 Glycosides
5 Phenolics
6 Saponins
7 Steroids
8 Tannins
Note: +: Present -: Absent
Petroleum Chloroform Ethyl acetate Methanol
ether
- - - -
- - + +
- + + +
- - - -
+ + + +
- - - -
- - - -
+ + + +

Table 2: Total phenolics, flavonoids and tannins contents in different solvent extracts of J.
wynaadensis leaf.
Solvent extract Total Phenolics (mg Total Flavonoids (mg Tannins
used GAE g-1 of extract) QE g-1 of extract) (mg TAE g-1 of
extract)
Petroleum ether 177.45±1.35 123.34±1.57 144.73±0.75
Chloroform 389.68±0.08 178.37±1.34 212.58±0.88
Ethyl acetate 539.25±1.12 389.38±1.29 315.03±1.78
Methanol 768.34±0.98 421.35±0.76 499.09±0.99
Figure 1. DPPH scavenging activity of different extracts of J. wynaadensis leaf.

ii. Anti-lipid peroxidation assay:

Lipid peroxide inhibition activity of different extracts of the plant was evaluated and it was observed that
significantly higher (P<0.05) activity exists in the methanol and ethyl acetate extract of J. wynaadensis leaf
in comparison with standard BHT. IC50 value of methanol and ethyl acetate extract and BHT are 39.009±0.99
µg/mL, 46.667±1.71 µg/mL and 265.32±1.32 µg/mL respectively. And the least antilipid peroxidative
activity of 336±1.56 µg/mL and 567.18±2.91 µg/mL was expressed by petroleum ether and chloroform
respectively. Hence, the tested plant extracts showed differential capacity to inhibit lipid peroxidation as
depicted in the Fig. 2.

Lipids are highly prone to free radical damage resulting in the production of peroxide which subsequently
decomposes to form carbonyl compounds that are measured by the TBA method. Peroxidation of lipids is
particularly more damaging because the formation of lipid peroxidation products leads to a facile propagation
of free radical reactions. Effectiveness of the extracts to inhibit peroxidation of the lipids indicates its activity
by which they can diminish the induction and/or propagation of lipid peroxides and thereby reducing the
oxidative damage. Higher antilipid peroxidative effect of the extracts may be attributed to the presence of
polyphenols and flavonoids.

Figure 2. Anti-lipid peroxidative activity of different extracts of J.wynaadensis

iii. Reducing power assay:
The Fig. 3 represents the reductive potential of all the tested samples. The results showed that all the extracts
have a reductive activity, which increased proportionally with the concentration. Here, methanol extract
showed higher reducing power with an absorbance of 0.88 compared to ascorbic acid which showed
absorbance of 0.74 at 700nm followed by the ethyl acetate, chloroform and petroleum ether showing an
absorbance of 0.62, 0.60 and 0.44 at 700nm. The reducing capacity of a compound may serve as a significant
indicator of its potential antioxidant activity.

The antioxidant activities of putative antioxidants have been attributed to various mechanisms, such as the
prevention of chain initiation, transition metal ion catalyst binding, peroxides decomposition , prevention of
continued proton abstraction, and radical scavenging [25]. Many of the above mentioned mechanisms depend
upon formation of reductones. Reductones are potent antioxidants developed during the reduction reactions
and are known to terminate the chain reactions by proton donation [26].
Figure 3. Reducing ability of different extracts of J.wynaadensis

iv. Super oxide radical scavenging assay:

Fig. 4. reflects the superoxide radical quenching capability of various fractions of the plant and was found in
the following order: methanol > quercetin > ethyl acetate > chloroform > petroleum ether. IC50 values are
41.002±1.09 µg/mL, 46.50±2.03 µg/mL, 1060.367±0.99 µg/mL, 2071.908±1.76 µg/mL and 2286.536±1.09
µg/mL respectively. In the concentration range investigated, all the extracts demonstrated super oxide
quenching activity that increased linearly with concentration. Superoxide scavenging ability of plant extract
might primarily be due to the presence of flavonoids [27].

The superoxide anion plays an important role in the formation of other reactive oxygen species such as
hydrogen peroxide, hydroxyl radical, or singlet oxygen in living systems [28]. The superoxide anion can react
with nitric oxide and form peroxynitrite, which can generate toxic compounds such as hydroxyl radical and
nitric dioxide

Figure 4. Super oxide radical scavenging activity of different extracts of J.wynaadensis.

v. Nitric oxide radical scavenging assay:

The results of the study on nitric oxide inhibition by the extract and the standard curcumin is shown in the fig.
5. The analysis revealed that all the extract suppressed nitroprusside from generating nitric oxide radicals in a
dose dependent manner indicating stoichiometric way of scavenging by the antioxidants. But all the extracts
showed lesser nitric oxide radical scavenging ability in comparison to that of the curcumin (IC50 value
137.565±2.21 µg/mL). IC50 value of 131.148±1.90 µg/mL, 244.163±1.33 µg/mL, 483.424±1.67 µg/mL and
661.561±2.79 µg/mL was shown by methanol, ethyl acetate, chloroform and petroleum ether respectively.
Nitric oxide is a diffusible free radical generated by the endothelial cells and macrophages [29]. Nitric oxide
reacts with oxygen and water to form nitrate and nitrite anions. Nitric oxide and superoxide anion may react
together to produce significant amounts of a much more oxidatively active molecule, peroxynitrite anion,
which is a potent oxidising agent that can cause DNA fragmentation and lipid oxidation. One of the reasons
for antiinflamatory activity of J.wynaadensis studied in vivo might be due to the nitric oxide radical
scavenging activity [7].

Figure 5. Nitric oxide radical scavenging activity of different extracts of J.wynaadensis

vi. Hydroxyl radical scavenging assay:
Different extracts of J.wynaadensis showed a considerable competition with deoxy ribose for the hydroxyl
radicals. It is worth mentioning that the chloroform and methanol extract represented higher scavenging
activity having an IC50 value of 26.817±2.09 µg/mL and 44.998±1.32 µg/mL when compared to petroleum
ether and ethyl acetate which showed 172.003±2.45 µg/mL and 132.563±1.89 µg/mL respectively while the
reference standard mannitol exhibited an IC50 value of 56.864±2.24 µg/mL. The inhibitory activity of
petroleum ether and ethyl acetate were low compared to standard. Though the phytochemical contents was
found to be low in the chloroform extract and exhibited potential hydroxyl radical inhibitory activity, it may
be due to presence of various antioxidant molecules which function synergistically and independent of the
concentration. The hydroxyl radical inhibition analysis demonstrated the consistently strong antioxidant
properties of extracts. Hydroxyl radical is the major reactive free radical which can cause oxidative damage
to lipids, DNA and proteins [30].

Figure 6. Hydroxyl radical scavenging activity of different extracts of J.wynaadensis

vii. Hydrogen peroxide scavenging assay:

In the present study carried out on scavenging of hydrogen peroxide it was observed that all the extract of J.
wynaadensis exhibited poor inhibition of hydrogen peroxide. Ascorbic acid used as a standard was effective
in scavenging hydrogen peroxide exhibiting IC50 value of 49.68±1.25. The IC50 value of the methanol, ethyl
acetate, chloroform and petroleum ether extracts are 396.66±2.33, 837.90±1.46, 837.90±.96 and 883.895±1.34
µg/mL respectively. Scavenging of hydrogen peroxide by extracts may be attributed to their phenolics, which
can donate electrons to hydrogen peroxide, thus neutralizing it to water [31,32].

Hydrogen peroxide is a weak oxidizing agent. It is highly diffusible and crosses the plasma membrane easily.
it is the least reactive molecule among reactive oxygen species and can generate the hydroxyl radical in the
presence of metal ions in living systems which may be the origin of many of its toxic effects. Hydrogen
peroxide can inactivates a few enzymes directly, usually by oxidation of essential thiol (-SH) groups.

Figure 7. Hydrogen peroxide scavenging activity of different extracts of J.wynaadensis

IV. CONCLUSION:

The present investigation on J. wynaadensis leaf extracts indicated potential anti radical activity. Methanolic
extract exhibited relatively higher activity compared to standards in all the assays except hydroxyl radicals
and hydrogen peroxide scavenging. The antioxidant activity of the extract is attributed to the presence of large
amount of phenolics and flavonoids. Hence, the plant may become a source of natural antioxidant.