Bridelia retusa (B.retusa) [Euphorbiaceae] is a
shrub or a tree up to 18 m in height armed with strong spines 7 cm
long found through India up to an altitude of 1000 m, except in very
dry regions. Bark is grey to brown exfoliating in irregular flakes.
The genus Bridelia comprises shrubs, climbers or trees
distributed in Asia, Africa and Australia (Anon., 1988). The plant
is used in folk medicine to treat diabetes, rheumatism, dysentery
and diarrhoea (Pawar
et al., 2007; Kirtikar et al.,
Raju et al., 2005).
Polyphenolic compounds belong to a large heterogeneous group of
secondary plant metabolites that are widespread in the plant kingdom
and have multiple applications in food, cosmetic and pharmaceutical
industries (Strube et al., 1993; Kahkonen
et al., 1999). The antioxidant capacity possessed by phenolic
compounds is mainly due to their redox properties, which permit them
to act as reducing agents, hydrogen donors, singlet oxygen quenchers
or metal chelators. Besides their roles as antioxidants, these
compounds exhibit a wide spectrum of medicinal properties, such as
anti-allergic, anti-inflammatory, anti-microbial, anti-thrombotic,
cardio-protective and vasodilatory effects (Balasundram et al.,
A number of factors influence the concentration of the active
constituents particularly phenolic compounds present in the
herbals. Some of the notable factors are time and period of
collection, geographical origin and climatic conditions. Sometimes,
the influence of these factors may such dominating leading to even
absence of active constituents in the same plant collected from
different regions, as evidenced by several research reports (Bilia,
2002; Houghton., 1998; Marcus et al., 2002). Selection of the
appropriate solvent for extraction of phenolic compounds is a very
important aspect that needs to be addressed in a judicious way so as
to achieve the maximum concentration of desired phytoconstituents in
plant extracts. Solubility of these active constituents in a
particular solvent also plays vital role in context of selection of
solvent. Some phenolic compounds like those present in creosote bush
resin are insoluble in water but soluble in non-polar solvents. On
the other hand, some phenolic glycosides are highly soluble in water
(Waterman and Mole, 1994). Research studies have indicated that
phenolic compounds can be extracted from plant material using a
sequence of solvents with divergent polarity (Apak et al., 2007).
The basis of most of the antioxidant based assays is either electron
transfer (ET) or hydrogen atom transfer (HAT). The dielectric
constant of the solvent, intra-/inter-molecular hydrogen bonding
associations and standard redox potential of phenolics and derived
aryloxy radicals in a given solvent may be important for electron
transfer kinetics in antioxidant assays (Huang et al., 2005; Prior
et al., 2005).
Thus, the significant impact of the molecular chemical properties of
the solvents especially the dielectric constant towards the
selectivity of phenolic components in a particular part of the plant
can be of vital interest from the research point of view. The
present work is an attempt to investigate the impact of dielectric
constant on the efficiency of a particular solvent to extract total
phenolic components from the bark of B. retusa and thereby
establishing the correlation with the subsequent antioxidant
activity residing in the plant .The experimental approach
relied on the use of 2,2-Diphenyl-1-picrylhydrazyl [DPPH] radical
scavenging assay. Apart from this, the efforts have also been
directed to observe the impact of geographical location in this
Effect of geographical location
The bark of B. retusa exhibited variations in
their contents of phytoconstituents depending upon the
geographical location from where they were collected. It
is quite evident and supported from their extractive
values (Table 1) and total polyphenol content (Table 2).
The methanolic extract of the plant collected from MAH
and AP regions exhibited the highest extractive values
of 20.70 ± 0.56 %w/w and 18.43 ± 0.97 %w/w,
respectively. The effect of geographical location on the
content of polyphenol was also evaluated. The level of
polyphenol in the methanolic extract from MAH region was
found out to be 32.52 ± 0.24 mg/100 gm as compared to AP
which was found out to be 28.04 ± 0.67 mg/100 gm. In
totality, the plant collected from the region of
Maharashtra was found to be superior with respect to
extraction yield and total polyphenol content. The
impact of geographical location on the extraction of
total phenol content is also supported by the fact that
variety of diverse factors such as worldwide changes in
seasonal patterns, weather episodes, temperature
changes, biotic and abiotic stresses may affect the
production of secondary metabolites in plants. In the
context of the biotic and abiotic
Extraction yield of bark of B. retusa with
Dielectric Constant [ε]
Bark of B. retusa
The values are expressed as Mean ± Standard deviation
MAH- Maharashtra AP- Andhra Pradesh.
Polyphenol content of B. retusa extracts obtained
with solvent of different dielectric constant.
(mg/100 gm) [MAH]
(mg/100 gm) [AP]
The values are expressed as Mean ± Standard deviation
(n=3), MAH- Maharashtra AP- Andhra Pradesh.
stresses, a special mention should be made to production
of stress induced Phenyl propanoid compounds.
Nutritional stress such as low iron levels can cause
increased release of phenolic acids, presumably to help
solubilize metals and thereby facilitate their uptake (Marschner,
The amount of Camptothecin was evaluated in methanolic
extract of various parts of Nothapodytes nimmoniana
collected in the month of February from different
regions (Mahabaleshwar and Patan regions of Maharashtra
state and Sirsi region of Karnataka state) of Western
Ghats, India, using high performance liquid
chromatography. The study indicated that
geographical and climatic conditions have phenomenal
influence in the content of Camptothecin in N.
nimmoniana (Namdeo et al., 2010)
The results of the present study and the references
cited above showed that the geographical topography and
climatic conditions have a profound effect on the
extraction yield and level of Polyphenols in the bark of
Impact of solvent on the TPC and antioxidant activity
Several studies have indicated that phenolics are
responsible for the variation in the antioxidant
activity of the plant (Cai et al., 2004). They possess
antioxidant activity by there ability to inactivate
lipid free radicals or by preventing decomposition
of hydroperoxides into free radicals (Pokorney et
al., 2001 and Pitchaon et al., 2007). During the process
of oxidation of phenol, Folin-Ciocalteu reagent which is
a mixture of phosphotungstic (H3PW12O40)
and phosphomolybdic (H3PMo12O40)
acids is reduced to blue oxides of tungstene (W8O23)
and molybdene (Mo8O23). This
reaction occurs under alkaline condition provided by
sodium carbonate. The intensity of blue colour is
indicative of the quantity of phenolic compounds, which
can be measured using spectrophotometer (Conforti et
al., 2006). In the present investigation, the
content of total phenolics in extracts of B.retusa
obtained from solvents with varying dielectric constant
values was experimentally determined. The polyphenol
content in B. retusa extracts was found to be in
decreasing order as per the
extractive solvents used that is,
(Table 2). The highest concentration
of total phenolics was 32.52 ± 0.24 mg/100 gm obtained
from methanol as solvent of B.retusa species
collected from MAH and the lowest concentration of 1.57
± 0.24 mg/100gm was observed in the chloroform.
Also, the extracts showed a positive correlation between
the polyphenol contents and solvents relative polarity
(R=0.842, P<0.05), and between the polyphenol
contents and solvents dielectric constant
The results suggest that extraction by methanol could
give higher phenolic content. The findings were likely
to be in accordance with (Pérez et al., 2007) who found
that methanol was the most efficient solvent as compared
to ethanol and water for extracting phenolic compounds
from control rosemary leaves and from those
decontaminated by gamma irradiation. In addition, (Yang
et al., 2007) reported that methanol extract of lotus
rhizome had the highest yield and total phenolic
recovery. The ability to inhibit polyphenol oxidase that
causes the oxidation of phenolics and its ease of
evaporation compared to water makes methanol a suitable
choice of solvents for extraction of variety of phenolic
compounds (Yao et al., 2004).
However, (Moure et al.,
2000) suggested that both methanol and ethanol offered
the best results to extract phenolic compounds from
Gevuina avellana hulls as compared to acetone.
Similar findings proving the effectiveness of both
methanol and ethanol for extracting phenolic compounds
was shown by (Siddhuraju and Becher, 2003; Azlim Almey,
2010). Among the various phenolics exhibiting
antioxidant properties, certain classes of compounds
such as phenolic acids, hydroxycinnamic acids,
flavonoids, and carotenoids require a decreasing order
of solvent polarity for extraction, respectively,
although, suitable solvent combinations may be adopted
for specific purposes. Due to the diversity of phenolic
antioxidant phytochemicals in botanicals, certain
compromises have to be made in solvent selection (Prior
et al., 2005; Apak et al., 2007).
Antioxidant activity-DPPH and its correlation with TPC
The methanolic and the ethanolic extracts at various
concentrations ranging from 0.020-0.100 mg/ml were
tested for their antioxidant activity using DPPH radical
scavenging assay method keeping in view the suggestion
from the studies discussed in the earlier sections of
this paper. DPPH radical scavenging activity assay
assesses the capacity of the extract to donate hydrogen
or to scavenge free radicals. DPPH radical is a stable
free radical and on reacting with an antioxidant
compound which can donate hydrogen, it is reduced to
diphenylpicrylhydrazine (DPPHH). The switch in colour
(that is, from deep-violet to light-yellow) can be
Figures 1 and 2 depict the DPPH radical
scavenging activity of the methanolic and ethanolic
extract of both plant species. The result revealed that
the methanolic extract of the species from MAH exhibited
the highest radical scavenging activity (%) with 80.55 ±
0.32 followed by its ethanolic extract with 66.5 ± 0.50.
In comparison to the species from MAH the species from
AP showed less scavenging activity. The value of
scavenging activity for methanolic and the ethanolic
extracts for AP species were observed to be 61.46 ± 0.37
and 54.9 ± 0.25, respectively. The DPPH
radical scavenging activities of methanolic and the
ethanolic extract increased gradually in a dose
dependent manner. Decrease in absorbance of DPPH
solution (that is, from purple to yellow) depends on
intrinsic antioxidant activity of antioxidant as well as
on the acceleration of reaction between DPPH and
Smaller IC50 value corresponds to a higher
antioxidant activity of the plant extract (Maisuthisakul
et al., 2007). The methanolic extract decolorized the
purple colour of DPPH to the yellow of DPPHH
with an IC50 value of 0.048 mg/ml in B.
retusa of MAH region and a IC50 value of
0.069 mg/ml in B. retusa of AP region. The IC50
value of the standard ascorbic acid was observed to be
0.024 mg/ml. The IC50 value observed for the
ethanolic extract of MAH and AP region were 0.057 mg/ml
and 0.086 mg/ml respectively.
It is evident from the observations that the methanolic
extract showed higher proton donating ability on DPPH
to form stable DPPHH molecules. Both methanolic
and ethanolic extracts of B. retusa from MAH and
AP region had the lowest IC50 value,
which indicated its powerful free radical scavenger
ability.The DPPH free radical scavenging activity of the
methanolic and ethanolic extract was significantly
related to their total phenolic content (RDPPH(MeOH)
= 0.9405, RDPPH(EtOH) = 0.8520).
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