Bs 16105

International Journal of Pure and Applied Biomedical Sciences; 2016 Vol.
1(1), ISSN: 2455-474X
Evaluation of Phytochemistry, Anti-oxidant & Free Radical

Scavenging activity of a Novel Polyherbal Formulation
Shabeer S. Iqbal & Prema Gurumurthy
Frontier Lifeline Hospital & Dr. K M Cherian Heart Foundation, Chennai, India
Date of revised paper submission: 7th July 2016; Date of acceptance: 21st July 2016
Date of publication: 14th August 2016; *First Author / Corresponding Author; Paper ID: BS16105
Abstract
In this study, we determined the phytochemical constituents, antioxidant capacity and free
radical scavenging properties of an novel polyherbal formulation (PHF) consist aqueous extract of
four plants namely Syzygium cumini, Picrorhiza kurroa, Madhuca indica and Commiphora mukul.
The phytochemical study have demonstrated the presence of various bioactive secondary metabolites
such as reducing carbohydrates, protein, steroids, flavanoids, alkaloids, tannin, phenols and inulin
etc. Ascorbic acid and Gallic acid were used as standards for antioxidant and free radical scavenging
assays. The PHFexhibited the high phenolic content as 707.74.3 mg Gallic acid equivalent /g. The
total flavanoid content of PHF was 584.23.7 mg quercetin equivalent/g. Total antioxidant capacity
of PHF was found to be 166.2 1.84 g/ ascorbic acid equivalents at 100 g/mL PHF
concentrations. In FRAP assay, the reducing ability of the PHF was found to be 962.24 8.54 M Fe
(II)/g. The reducing power assay of PHF showed a dose dependent increasing from 0.117 to 0.269
g/mL. PHF could efficiently scavenged DPPH, IC50 was found to be 1.960.16 g/mL. The IC50of
lipid peroxidation inhibition assay was found to be 38.921.43 g/mL. Outcome of this study suggests
the presence of various biologically active phytochemicals in this PHF and their potential effects
against free radical and oxidative stress.
Keywords: Syzygium cumini, Picrorhiza kurroa, Madhuca indica, Commiphora mukul, DPPH,
Phenol content.
1. INTRODUCTION
42 | P a g e
International Journal of Pure and Applied Biomedical Sciences; 2016 Vol. 1(1), ISSN: 2455-474X
Plants have been an important source of medicine from ancient era itself. Plants have been an
important source of drugs and many of the currently available drugs have been derived directly or
indirectly from them. The phytochemicals identified from traditional medicinal plants present an
exciting opportunity for the development of new types of therapeutics.
The beneficial effects of phytochemicals are associated with a number of their
biologicalactivities including antioxidant and free radicalscavenging properties. Most ofthe protective
effects of plants on living cells have beenattributed to their non-nutrient constituents such as
carotenoid, flavonoids, isoflavonoid and phenolic acids(Bahorun, T et al, 1996). Different
phytochemicals have been shown to possess a range of activities which help inprotecting and by
extension many chronic disease conditions (Oyebanji et al 2011).
In Indian traditional system of medicine, combinations of several plants are commonly used
than individual. Some of these plants have shown potent antioxidant activity (Maryam zahin et al
2009). However, many plants have not yet been screened for that activity. So, in order to
contributefurther to the knowledge of Indian traditional plants, our present study is focussed on a
polyherbal formulation (PHF) consist aqueous extractof
four plants namely Syzygium cumini,
Picrorhiza kurroa, Madhuca indica and Commiphora mukul to determine their phytochemical
constituents, antioxidant capacity and free radical scavenging properties.
2. MATERIALS AND METHODS
Chemicals:
The standard L-Ascorbic acid and Gallic acid were obtained from Hi-Media lab.Ltd. Mumbai,
India. 1,1-diphenyl-2-picryl hydrazyl (DPPH) , Nitro blue tetrazolium, NADH, Phenazine metho
sulphate, 2,4,6-tripyridyl-s-triazine, quercetin and all other common chemicals and reagents were
purchased from Sigma Chemical Co, St. Louis, MO, USA.
Physiochemical and Organoleptic properties of polyherbal formulation
The organoleptic evaluation refers to evaluation of the formulation by colour, odour, taste
andtexture etc. (Siddiqui et al. 1995). Physicochemical investigations include pH, loss on drying
(LOD), fluorescent analysis etc.
Phytochemical analysis
Chemical tests for the screening and identification of bioactive chemical constituents in this
polyherbal formulation under study were carried out in aqueous extracts by standard methods
described by several authors (Sofowora EA et al 1984, Khandelwal KR et al 2005, Yadav M et al
2010, Palanisamy P et al 2012, Daniel et al 2012).
in vitroANTIOXIDANT AND FREE RADICAL SCAVENGING ASSAYS
Total phenolic content:
43 | P a g e
Total phenolic content was determined using Folin-Ciocalteu reagent according to the method
of Singleton et al., (1999). Aqueous solution of the PHF in the concentration of 20-100 g/mL was
used in the analysis. Based on the measured absorbance, the concentration of phenolics was read from
the calibration line; then the content of phenolics in extract was expressed in terms of gallic acid
equivalent (mg of GA/g of extract)
Total Flavanoid content:
Total Flavonoid content were measured according to the method of Ordon et al., (2006) .The
sample (concentration of 20-100 g/mL) was allowed to react with 2 % AlCl 3 solution. The
concentration of flavanoid calculated based on the calibration curve and expressed in terms of
querectin equivalent.
Total anti-oxidant capacity:
The total antioxidant activity of the extract was evaluated by the phosphomolybdenum
method (Prieto et al., 1999). Concentration of PHF was 20-100 g/mL used in the analysis. Ascorbic
acid was used as reference standard. Based on the measured absorbance, the total antioxidant activity
was read (mg/mL) from the calibration line; the antioxidant activity is expressed as the number of
equivalents of ascorbic acid (AscAE)
DPPH free radical scavenging assay:
The scavenging activity for DPPH free radicals was measured according to the procedure
described by Blios (1958). Concentration of PHF was 1-5 g/mL used in the analysis. Gallic acid was
used as standard.
Ferric reducing antioxidant power assay:
The determination of the ferric reducing activity is a modified method of Benzie and Strain
(1996). FRAP assay measures the reducing potential of antioxidant to react on ferric tripyridyltriazine
(Fe3+-TPTZ) complex and produce blue colour of ferrous tripyridyl triazine complex.
Reducing power assay:
Reducing power capacity of the PHF was determined using the method described by Oyaizu
(1986). Concentration of PHF was 40-200 g/mL used in the analysis. Ascorbic acid was used as
standard.
Inhibition of erythrocyte lipid peroxidation:
Lipid peroxidation was estimated calorimetrically by thiobarbituric acid reactive substances
(TBARS) using the modified method of Niehius and Samuelsson (1968). Hydrogen peroxide was
44 | P a g e
added to induce lipid peroxidaion. Concentrations of test and standard were ranging from 20-100
g/mL.
3. STATISTICAL ANALYSIS
Data were expressed as Mean SD for three parallel measurements. Graph Pad Prism version
6.0 for windows, Graph Pad Software, San Diego, California, USA was used. The 50 % inhibitory
concentration (IC50) was calculated from the dose response curve (Graph Pad Prism Version 6.0)
obtained by plotting percentage inhibition versus concentrations.
Percentage (%) inhibition was calculated as: Absorbance of control absorbance of test sample/ absorbance of control 100
4. RESULT AND DISCUSSION
Polyherbal formulation was subjected to various analytical techniques. Botanical parameters
revealed that PHF is brown in colour, with a pungent odour, bitter taste, and fine texture.Percent
weight loss ondrying or moisture content was found to be 4% w/w. The lessvalue of moisture content
could prevent bacterial, fungal or yeast growth (African Pharmacopoeia, 1986). Results shown in
Table-1
Table-1: Physiochemical and organoleptic characteristics of PHF
Appearance
Colour
Odour
Taste
Texture
pH
Loss on drying
Powder
Brown
Pungent
Slightly bitter
Fine
5.8
4%
The results of fluorescent studies of the powdered plant materialusing different chemical
reagents were studied and a given inTable2. Fluorescence is an important phenomenon exhibited
byvarious chemical constituents present in plant material (Kamil MS et al, 2010). If the substances
themselves are not fluorescent, they may often beconverted into fluorescent derivatives by reagents
hence somecrude drugs are often assessed qualitatively in this way and it is animportant parameter of
Pharmacognostical evaluation (Ansari S.H, 2006).
Table-2: Fluorescent analysis of PHF

Visible light/Day light
45 | P a g e
Ultra violet light
Powder as such
Powder + HCl
Powder + NaOH
Powder + Methanol
Powder + FeCl3
Powder + H2SO4
Powder + H2O2
Powder + NH3
Powder + Acetic acid
Powder + HNO3
Brown
Light brown
Reddish brown
Yellowish green
Light brown
Chocolate brown
Pale brown
Orange brown
Light green
Dark yellow
Light brown
Greyish brown
Brown
Green
Pale brown
Dark blue
Pale brown
Dark green
Yellowish green
Yellowish green
The preliminary phytochemical screening have demonstrated the presence of various

bioactive secondary metabolites such as reducing carbohydrates, protein, steroids, flavanoids,
alkaloids, tannin, phenols and inulin etc. (Table-3). The phenolic compounds are the most ever-present
groups of plant metabolites which areconsidered to possess various biological properties such as, antiaging, anti-inflammation, anti-carcinogen and cardiovascular protection (Muthubalaji et al 2013). It
also protect against oxidative stress and degenerative diseases (Han X t al 2008).In vivo antidiabetic
activity of several plant extracts has been correlated with their total phenol and flavonoid content
(Rauter et al 2009, Aslan et al 2007). Flavonoids are the potent water soluble free radical scavengers
useful in preventing oxidative cell damage and have anti-cancer activity (Khattak et al
2008).Alkaloids, glycosides, carbohydrates, and steroids have demonstrated activity consistent with
their possible use in treatment of type-2 diabetes (Kumar et al, 2009). Alkaloids is one of the key
metabolites in plants possess anti-inflammatory and anti-asthmatic properties also (Muthubalaji et al
2013).Terpenoids have been shown to decrease blood glucose levels in animal studies (Kumar et al,
2009). Terpenoids are also the effective antioxidant and potent anti-cancer agents.Tannins are known
to be helpful in the treatment of ulcerated tissues and they have significant activity in cancer
prevention. Glycosides are known to lower the blood pressure (Muthubalaji et al 2013).
Table-3: Results of phytochemical screening of the PHF extracts

No
TEST
Carbohydrates
1
II
III
46 | P a g e
Molischs test
RESULT
Positive
Reducing substance
1
Benedicts test
Positive
Fehlings test
Positive
Protein
Biuret test
Positive
Xanthoprotein test
Positive
Ninhydrin test
Positive
IV
Steroids
1
Salkowskis test
Positive
Libermann-Burchard test
Positive (Terpenoids)
Flavanoids
1
Alkaline reagent test
Positive
FeCl3 test
Positive
Shinoda test
Positive
Lead acetate test
Positive
VII
Tannins and phenol

1
FeCl3 test
Positive
Potassium dichromate test
Positive
Dil.HNO3
Positive
Lead acetate test
Positive
IX
XI
Alkaloids
1
Mayers test
Positive
Wagners test
Positive
Inulin
Positive
TOTAL PHENOLIC CONTENT:

In general, the anti-free radical and antioxidant activities of plant extracts are associated to the
phenolic content(Khattak et al., 2008).The determination of total phenolics, was based on the
absorbance value of PHFsolution that react with Folin-Ciocalteu reagent and followed by comparing
with the standard solution of gallic acid equivalents. The standard curve (Figure 1) was plotted by
using gallic acid concentration ranging from 20-100 g/mL. The following equation expressed the
absorbance of gallic acid standard solution as a function of concentration: - Y = 0.1596 x + 0.0442, R 2
=0.9992 Where; x was the absorbance and Y was the Gallic acid equivalent. PHFexhibited the greater
phenolic content as mg gallic acid equivalent/g weight (mg GAE/g) for a value of 707.74.3 mg
GAE/g .
47 | P a g e
Total phenolic content

2
1.5
Absorbance
f(x) = 0.02x - 0.04

R = 1
1
0.5
0
10 20 30 40 50 60 70 80 90 100 110
concentration g/ml
Figure 1: Total phenolic content of PHF expressed in gallic acid equivalents (mg GAE/g).
Data are presented as Mean SD.
TOTAL FLAVANOID CONTENT:
The method of Orden et al, 2006; was used to estimate the total flavonoid contents of the
extract solution based on the formation of a flavonoid-aluminium complex. The antioxidant activity of
quercetinand its derivatives have been reported in severalexperimental models.The standard curve
(Figure 2) was done by using quercetin concentration ranging from 20-100 g/mL. The following
equation expressed the absorbance of quercetin standard solution as a function of concentration: - Y =
0.0057 x + 0.0609, R2 =0.9974 Where; x was the absorbance and Y was the quercetin equivalent. The
total flavanoid content of PHF was 584.23.7 mg quercetin equvalent/g.
TOTAL FLAVANOID CONTENT
0.6
f(x) = 0.01x - 0.06
R = 1
0.4
Absorbance
0.2
0
10 20 30 40 50 60 70 80 90 100110
Concentration g/ml
Figure 2: Total Flavanoid content of PHF expressed in Ascorbic acid equivalents (mg AscE/g).
Data are presented as Mean SD
TOTAL ANTIOXIDANT CAPACITY:
48 | P a g e
The antioxidant activity of the PHF was evaluated by the phosphomolybdenum method and
followed by comparing with the standard solution of ascorbic acid equivalents. The standard curve
was done by using ascorbic acid concentration ranging from 20-100 g /mL. The following equation
expressed the absorbance of ascorbic acid standard solution as a function of concentration: - Y
=0.0125 x + 0.0401, R2 =0.9977 Where; x was the absorbance and Y was the ascorbic acid equivalent
(mg/g). PHF showed an increase in antioxidant capacity with an increase in dose. Total antioxidant
capacity of PHF was found to be 166.2 1.84 g ascorbic acid equivalents at 100 g/mL PHF
concentration (figure 3). It determines the total antioxidant capacity.
Total antioxidant capacity
1.5
1
absorbance
0.5
f(x) = 0.01x - 0.04

R = 1
0
10 20 30 40 50 60 70 80 90 100 110
concentration g/ml
Figure 3: Total antioxidant activity of PHF. Data are presented as Mean SD

DPPH FREE RADICAL SCAVENGING ASSAY:
In DPPH assay, the anti-oxidants were able to reduce DPPH to yellow coloured diphenyl
picrylhydrazone (Frankel and Meyer, 2000). The method based on the reduction DPPH in alcoholic
solution in the presence of hydrogen donating anti-oxidant due to formation of the non-radical form
DPPH-H in the reaction. DPPH is usually used as a reagent to evaluate free radical and accepts an
electron or hydrogen radical to become a stable diamagnetic molecule (Oyaizu et al, 1986).
In this study, the free radical scavenging ability of each sample was evaluated through
recording the change of absorbance produced by the reduction of DPPH . The percentage scavenging
activity of each sample against DPPH was ranging from 41.72 to 64.67 % and 43.1 to 83.3 % for PHF
and ascorbic acid respectively (figure 4). The IC 50 was found to be 1.960.16 g/mL, 1.480.08
g/mL for PHF and for standard, ascorbic acid respectively.
49 | P a g e
DPPH FREE RADICAL SCAVENGING ASSAY

100
80
% of inhibition
60
Ascorbic acid
40
PHF
20
0
1
concentration g/ml
Figure 4: DPPH free radical scavenging activity of PHF and ascorbic acid.
Data are presented as the percentage of DPPH radical scavenging, Mean SD.
FERRIC REDUCING ANTIOXIDANT POWER ASSAY:
The ferric reducing/antioxidant power (FRAP assay) is widely used in the evaluation of the
antioxidant component in dietary polyphenols. Antioxidant activity increased proportionally to the
polyphenol content. According to recent reports, a highly positive relationship between total phenols
and antioxidant activity appearsto be the trend in many plant species (Adeolu A et al, 2009).
The antioxidant potentials of the PHF and ascorbic acid were estimated from their ability to
reduce ferric tripyridyltriazine, TPRZ-Fe (III) complex to TPTZ-Fe (II).The standard curve was linear
between 200 and 1000 M FeSO4. The following equation expressed the absorbance of
FeSO4standard solution as a function of concentration: - Y =0.0115 x 0.026, R2 = 0.9967 Where; x
was the absorbance and Y was the FeSO4 equivalent M Fe (II)/g (Figure-5). PHF showed an increase
in ferric reducing power with an increase in dose. The reducing ability of the PHF was found to be
962.24 8.54 M Fe (II)/g and for ascorbic acid 1204.4 11.64 at 100 g/mL concentration.
50 | P a g e
FERRIC REDUCING ANTIOXIDANT POWER ASSAY

1.5
1
f(x) = 0.01x - 0.03

R = 1
Absorbance 0.5
0
10
20
30
40
50
60
70
80
90 100 110
Concentration mol/L
Figure 5: Ferric Reducing Antioxidant Power Activity of PHF. Data are presented as Mean SD.
REDUCING POWER ASSAY:
In this assay, the yellow colour of the test solution changes to various shades of green and
blue, depending on the reducing power of each compound. The presence of reducers (i.e.
antioxidants) causes the reduction of the Fe3+/ferricyanide complex to the ferrous form. Therefore,
measuring the formation of Perls Prussian blue at700 nm can monitor the Fe2+concentration.
Figure 6., shown the reducing power of PHF and standards a function of their concentration.
The reducing power of PHF and ascorbic acid ranged from 0.117 to 0.269 and 0.206 to 0.739 Abs for
20 g/mL to 100 g/mL respectively. The reducing properties are generally associated with the
presence of reductones (Duh et al 1999), which have been shown to exert antioxidant action by
breaking the free radical chain by donating a hydrogen atom (Gordon MH, 1990).
Re ducing powe r assay
0.8
0.6
Absorbance
0.4
Ascorbic acid
0.2
PHF
0
20
40
60
80
100
Concentration g/ml
Figure 6: Reducing power activity of PHF and ascorbic acid. Data are presented as Mean SD.
INHIBITION OF ERYTHROCYTE LIPID PEROXIDATION:
51 | P a g e
Lipid peroxidation refers to the oxidative degradation of lipids. It is the process in which free
radicals steal electrons from the lipids in cell membranes, resulting in cell damage. This process
proceeds by a free radical chain reaction mechanism. Malondialdehyde (MDA) is the end products of
lipid peroxidation, generally considered as a marker for oxidative stress.
The percentage inhibition of erythrocyte membrane lipid peroxidation activities of the PHF
and ascorbic acid are shown in Figure 7. The percentage inhibition activities of PHFand ascorbic acid
were ranging from 42.86 to 66.23% and 44.64 to 91.07 % respectively. The IC 50 was found to be
38.921.43 g/mL and 29.331.51 g/mL for PHF and standard respectively. H2O2 induced
peroxidation level is reduced when added various concentration of PHF.
Erythrocyte me mbrane lipid pe roxidation activity
100
80
60
% of Inhibition
Ascorbic acid
40
PHF
20
0
20
40
60
80
100
Concentration g/ml
Figure 7: Inhibition of erythrocyte membrane lipid peroxidation activity of PHF and ascorbic
acid. Data are presented as the percentage of lipid peroxidation inhibition, Mean SD.
Table 4: Total phenolic content, Total anti-oxidant capacity, Total flavanoid content and
Ferric reducing activities of PHF.
Sample
Total phenolic
content
(mg GAE/g)
Total
Anti-oxidant
capacity
(mgAscAE/g)
Total flavanoid
content
(mgAscAE/g)
FRAP assay
( M Fe (II)/g
PHF
707.688.3
166.02 2.54
584.227.4
962.24 18.5
Ascorbic
----------------
-----------------
-------------------
1204.4 22.64
acid/Gallic acid
Table 5: IC50 values of in vitro DPPH and Lipid peroxidation inhibition assay
52 | P a g e
Sample
DPPH(g/mL)
Lipid peroxidation(g/mL)
PHF
1.960.06
38.921.03
Ascorbic acid
1.480.02
29.331.91
4. CONCLUSION
This polyherbal formulation was formulated by using various plant parts in a fixed
combination. The prepared formulation was screened for phytochemical content, antioxidant activity
and also for free radical scavenging property as per standardized methods. The research outcomes of
this study suggests that this PHF has very good antioxidant and free radical scavenging activity, which
might be helpful in preventing or slowing the progress of various oxidative stress induced diseases. It
also indicates that PHF possess many phytochemicals which could be beneficial to human health.
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International Journal of Pure and Applied Biomedical Sciences; 2016 Vol.

1(1), ISSN: 2455-474X

Evaluation of Phytochemistry, Anti-oxidant & Free Radical

four plants namely Syzygium cumini,

Table-2: Fluorescent analysis of PHF

Ultra violet light

The preliminary phytochemical screening have demonstrated the presence of various

Table-3: Results of phytochemical screening of the PHF extracts

Alkaline reagent test

Lead acetate test

Tannins and phenol

Potassium dichromate test

Lead acetate test

TOTAL PHENOLIC CONTENT:

Total phenolic content

f(x) = 0.02x - 0.04

f(x) = 0.01x - 0.04

Figure 3: Total antioxidant activity of PHF. Data are presented as Mean SD

DPPH FREE RADICAL SCAVENGING ASSAY

FERRIC REDUCING ANTIOXIDANT POWER ASSAY

f(x) = 0.01x - 0.03

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