Antioxidant Properties of Methanolic Extracts of Boerhavia diffusa
Natural antioxidants present are responsible for inhibiting or preventing the
deleterious consequences of oxidative stress. Free radical scavengers are compounds
like polyphenols, flavonoids and phenolic compounds. The present study was carried
out to evaluate the antioxidant activity and phytochemical constituents of Boerhaavia
diffusa L., a herbaceous member of the family Nyctaginaceae from methanolic
extracts of different plant parts. Methanolic extracts of leaves, stems and
roots were prepared and screened for in vitro antioxidant activities
by using peroxidase assay, Ferric Reducing Ability of Plasma (FRAP), lipid peroxidation
assay (LPO), ABTS and the quantitative estimation of the total phenolics as
Gallic Acid Equivalent (GAE) per gram dry weight and total flavonoid as Quericitin
Equivalent (QE) per gram dry weight. Maximum total phenolic content was recorded
in leaves (24.5±1.703 mg GAE g-1 DW) While maximum total flavonoid
content was found in leaves (79.86±3.751 mg QE g-1 DW). Leaves
methanolic extract showed maximum radical scavenging acitvity for FRAP (96.664
Mm L-1 g-1), while ethyl acetate and hexane extract of
stems showed minimum activity for FRAP. Leaves showed maximum radical scavenging
activity for LPO (26.66 MDA g-1 DW) and peroxidase (0.383 Mm min-1
g-1 DW), a good correlation with the total phenolic and flavonoidal
content of plant is seen. It signifies that the plant-derived phenolics and
flavonoids represents good sources of natural antioxidants. It is seen that
this plant exhibits significant antioxidant activity.
Received: January 23, 2014;
Accepted: April 30, 2014;
Published: June 19, 2014
Plants not only provide food for man but also give a number of active compounds
with potent and varied therapeutic value. Boerhavia diffusa (Family Nyctaginaceae)
is a herbal plant which is common in the tropics in both dry and rainy seasons
and subtropics in a wild perennial herb which may be encountered in different
terrestrial habitats (Banjare et al., 2012).
Genus Boerhaavia, consisting of 40 species is distributed in tropical
and subtropical regions and warm climate. It is found in Ceylon, Australia,
Sudan and Malay Peninsula, extending to China, Africa, America and Islands of
the Pacific. Among 40 species of Boerhaavia, 6 species are found in India, namely
B. diffusa, B. erecta, B. rependa, B. chinensis,
B. hirsute and B. rubicunda. Boerhaavia diffusa in India is
found in warmer parts of the country (Scientific Name: Boerhaavia diffusa
Linn. Syn. B. repens; B. repens, Family: Nyctaginaceae, Family
Name: Hog weed, Horse Purslane, Common Indian Names Gujarati: Dholia-saturdo,
Moto-satoda, Hindi: Snathikari Kannada: Kommegida Marathi: Tambadivasu Sanskrit:Punarnava,
Raktakanda, Shothaghni, Varshabhu Bengali: Punurnava Tamil: Mukaratee-Kirei
Telugu: Punernava) and grows as common weed its, useful parts are root, leaves
and seeds (Oudhia, 2003).
The root, leaves, aerial parts or the whole plant of Boerhaavia diffusa
have been employed for the treatment of various disorders in the Ayurvedic
herbal medicine daily used by millions of people in India, Nepal, Sri Lanka
and indirectly through it being the major influence on Unani, Chinese and Tibetan
medicines. The root is mainly used to treat gonorrhea, internal inflammation
of all kinds, dyspepsia, oedema, jaundice, menstrual disorders, anaemia, liver,
gallbladder and kidney disorders, enlargement of spleen, abdominal pain, abdominal
tumours and cancers. It cures corneal ulcers and night blindness and helps restore
virility in men. People in tribal areas use it to hasten childbirth. The juice
of Boerhaavia diffusa leaves serves as a lotion in ophthalmia. It is
also administered orally as a blood purifier and to relieve muscular pain, also
is administered in diabetes (Rajpoot and Mishra, 2011;
Chude et al., 2001).
Oxygen-free radicals, more generally known as Reactive Oxygen Species (ROS)
along with Reactive Nitrogen Species (RNS) are well recognised for playing a
dual role as both deleterious and beneficial species (Valko
et al., 2006). The human body has a complex system of natural enzymatic
and non-enzymatic antioxidant defenses which counteract the harmful effects
of free radicals and other oxidants (Alam et al.,
2013). Oxidative stress contributes to many pathological conditions and
diseases including cancer, neurological disorders, atherosclerosis, hypertension,
ischemia/perfusion, diabetes, acute respiratory distress syndrome, idiopathic
pulmonary fibrosis, chronic obstructive pulmonary disease and asthma (Rahmatullah
et al., 2010). Phenolic antioxidants inhibit lipid peroxidation by
trapping the lipid alkoxyl radical. This activity depends on the structure of
the molecules and the number and position of the hydroxyl group in the molecules
(Freeman and Crapo, 1982). Polyphenols are reducing
agents and together with other dietary reducing agents, such as vitamin C, E
and carotenoids, referred to as antioxidants, protect the bodys tissues
against oxidative stress and associated pathologies such as cancers, coronary
heart disease and inflammation (Tapiero et al., 2002).
During heavy metal stress, phenolic compounds can act as metal chelators and
on the other hand, phenolics can directly scavenge molecular species of active
oxygen (Apel and Hirt, 2004). Zn is an essential component
of numerous proteins involved in the defense against oxidative stress (Florence,
MATERIALS AND METHODS
Plant material: Different plant parts (leaves, stem and roots) of Boerhavia
diffusa were collected in month of October-December from University of Rajasthan
campus. It was washed with tap water, dried at room temperature and ground to
fine powder. The species specimen was submitted to herbarium, Department of
Botany, University of Rajasthan. Jaipur, Rajasthan, India and got the voucher
specimen No. RUBL211300.
Chemicals: All the chemicals used were of analytical graded and purchased
from Hi Media from Hi-media Laboratory Pvt. Ltd. Mumbai.
Total phenolic and flavanoidal content
Plant extraction: Two gram the each of dry material (leaves, stems and
roots) was extracted with 25 mL of methanol at room temperature for 48 h, filtered
through Whatman paper No. 1 filter paper, stored and used for quantification.
Total phenolic content: Total phenolic compound contents were determined
by the Folin-Ciocalteau method (McDonald et al., 2001;
Ebrahimzadeh et al., 2008a, b;
Nabavi et al., 2008). The extract samples (0.5
mL, 1:10 diluted) were mixed with Folin Ciocalteu reagent (5 mL, 1:10 diluted
with distilled water) for 5 min and aqueous Na2CO3 (4
mL, 1 M) were then added. The mixture was allowed to stand for 15 min and the
phenols were determined by colorimetric method at 765 nm. The standard curve
was prepared by 0, 50, 100, 150, 200 and 250 mg mL-1 solutions of
gallic acid in methanol. Total phenol values are expressed in terms of gallic
acid equivalent (mg g-1 of dry mass) which is a common reference
compound. Total phenolic content can be calculated from the equation:
||Total phenolic concentration
||Concentration of gallic acid from caliberation curve (mg mL-1)
||Volume of extract (mL)
||Wt. of methanol plant extract
Total flavanoidal content: Total flavonoid content was determined by
using aluminium chloride colorimetric method (AlCl3) according to
the known method (Dewanto et al., 2002; Sakanaka
et al., 2005) with slight modifications using Quercetin as standard.
One mililiter of test material was added to 10 mL volumetric flask containing
4 mL of water. To above mixture, 0.3 mL of 5% NaNO2 was added. After
5 min, 0.3 mL of 10% AlCl3 was added. After 6 min, 2 mL of 1M NaOH
was added and the total volume was made upto 10 mL with distilled water. Then
the solutions were mixed well and absorbance was measured against blank at 510
nm. Total flavanoidal content of the extracts was expressed in milligram of
quercetin equivalents g-1 DW. Total flavanoidal content can be calculated
from the equation:
||Total flavanoidal concentration
||Concentration of gallic acid from calibration curve (mg mL-1)
||Volume of extract (mL)
||Wt. of methanol plant extract
Determination of antioxidant activity
Reducing ability (FRAP assay): The determination of the total antioxidant
activity (FRAP assay) in the extract is a modified method of Benzie
and Strain (1996). The stock solutions included 300 mM acetate buffer (0.3
M acetic acid and 0.3 M sodium acetate), pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine)
solution in 40 mM HCl and 20 mM Ferric chloride (Fecl3.6H2O)
solution. The fresh working solution was prepared by mixing 25 mL acetate buffer,
2.5 mL TPTZ and 2.5 mL Fecl3.6H2O. The temperature of
the solution was raised to 37°C before use. Plant extracts (100 μL
each of methanolic ethyl acetate and hexane) were allowed to react with 2900
μL of the FRAP solution for 30 min in the dark condition. Readings of the
coloured product (ferrous tripyridyltriazine complex) were taken at 593 nm.
The standard curve was linear between 100-1000 μM FeSO4. Results
are expressed in mM Fe (II) g-1 dry mass and compared with that of
BHT, ascorbic acid, quercetin and catechin.
Peroxidase assay: The method of assay measures the oxidation of pyrogallol
to purpurogallin by peroxidase when catalyzed by peroxidise at 420 nm and at
20°C. Plant sample (200 mg) was homogenized with 10 mL of phosphate buffer
and refrigerated centrifuged at 10000 rpm for 20 min. The clear supernatant
was taken as the enzyme extract. The activity was assayed after the method of
Chance and Maehley (1955) with following modifications.
The 2.4 mL of phosphate buffer, 0.3 mL pyrogallol (50 μM) and 0.2 mL of
H2O2 (30%) were added. The amount of purpurogallin formed
was determined by taking the absorbence at 420 nm immediately after adding 0.1
mL enzyme extract. The extinction coefficient of 2.8 mM-1 cm-1
was used in calculating the enzyme activity that was expressed in terms of mM
min-1 g-1 DW.
Lipid peroxidation assay (LPO): 0.5 g of dry material was homogenized
with 10 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged
for 5 min (15000 g, 4°C). Supernatant was collected and 1 mL of supernatant
was mixed with 4 mL of 0.5% (w/v) TBA (Thiobarbituric acid) in 20% (w/v) TCA
and then incubated in water bath at 95°C for 30 min. Reaction was quickly
ended by incubating on an ice bath. In case the solution is not clear, centrifuge
at 10000 g for 10 min and the absorbance was measured at 532 and 600 nm. OD600
values were subtracted from MDA-TBA complex values at 532 nm and MDA concentration
was calculated using the Lambert-Beer law with an extinction coefficient εM
= 155 mM-1 cm-1. Results were presented as μmols
ABTS radical scavenging assay: To determine ABTS radical scavenging
assay, the method of Re et al. (1999) was adopted.
The stock solutions included 0.002M ABTS solution and 0.07 M potassium persulphate
solution. The working solution was then prepared by mixing the 25 mL of ABTS
stock and 0.1 mL of potassium persulphate stock and allowing them to react for
12 h at room temperature in the dark. The solution was then diluted by mixing
ABTS solution with ethanol to obtain an absorbance of 0.706±0.001 units
at 734 nm using the spectrophotometer. Fresh ABTS solution was prepared for
each assay. Plant extracts (1 mL) at varying concentration were allowed to react
with 3 mL of the ABTS solution and the absorbance was taken at 734 nm after
6 min using the spectrophotometer. The ABTS scavenging capacity of the extract
was compared with that of BHT and percentage inhibition calculated as:
where, ABScontrol is the absorbance of ABTS radical+methanol, ABSsample
is the absorbance of ABTS radica +sample extract/standard.
Statistical analysis: Experimental results are expressed as (M±SD).
All measurements were replicated three times. IC50 values were also
calculated by linear regression analysis. Experiments results were further analyzed
for Pearson correlation coefficient (r) between total phenolic, flavanoid and
free radical scavenging assay using the microsoft Excel 2007 software. The values
were considered to be significantly different at p<0.05.
RESULTS AND DISCUSSION
In the present study, we have investigated the antioxidant activity of Boerhavia
diffusa by different assays and found their scavenging activity. Table
1 shows the total flavonoid content maximum in leaves (79.86±3.757
mg GAE g-1 DW) while minimum in roots (7.08±0.36 mg GAE g-1
DW) and total phenolic content was seen maximum in leaves (24.5±1.703
mg QE g-1 DW) while minimum in roots (0.25±0.243). Table
2 shows FRAP activity of ethylacetate, hexane and methanol extracts. In
ethyl acetate extract, maximum activity is seen in stem (53±2.645 Mm
L-1 g-1) while minimum in roots (27.551±1.392 Mm
L-1 g-1). In hexane extract maximum activity is seen in
stem (89±3.605 Mm L-1 g-1) while minimum in root
(16.887±1.293 Mm L-1 g-1). In methanol extract
maximum activity is seen in leaves (137.67±2.516 Mm L-1 g-1)
while minimum in roots (74.33±2.081 Mm L-1 g-1).
Table 3 shows peroxidase activity maximum in leaves (0.124±0.11
Mm min-1 g-1 DW) while minimum in roots (0.0589±0.040
Mm min-1 g-1 DW). Table 4 shows LPO
activity maximum in leaves (23.377±1.108 mg QE g-1 DW) while
minimum in root (4.481±0.357). Table 5 shows the IC50
of methanolic extracts of which leaves shows highest activity for ABTS assay
(58.721±2.460 Mm min-1 g-1) while stem show minimum
activity (732.344±9.844 Mm min-1 g-1). Table
6 shows correlation values of Total Phenolic Content (TPC) and Total Flavonoidal
Content (TFC) with the different free radical scavenging assays performed.
|| Total phenolic and flavonoidal contents in different plant
parts of B. diffusa
|| Total antioxidant (FRAP) activity of methanol, ethyl acetate
and hexane extract of different plant parts of B. diffusa
|| Peroxidase activity in different plant parts of B. diffusa
|| LPO activity in different plant parts of B. diffusa
|| IC50 values of different plant parts of B.
diffusa of ABTS radical scavenging assay
For ABTS, we find a positive correlation with the phenolic content of the plant
in all plant parts (leaves, stem and roots) while a negative correlation with
the flavonoidal content for all plant parts, this may be a reason for the highest
activity of the stem methanolic extracts for the ABTS assay. For the LPO activity
assay we see a positive correlation with both phenolic and flavonoidal content
which may explains its highest activity in leaves but in contrast to LPO, for
peroxidase assay highest activity is seen in leaves which shows positive correlation
with the phenolic and flavonoidal content. Phenolic and flavonoidal content
have shown a good correlation with antioxidant activity, this may be due to
structural differences. According to the Singleton and Rossi
(1965) various phenolic compounds have different responses in this assay.
The molar response of this method is roughly proportional to the number of phenolic
hydroxyl groups in a given substrate but the reducing capacity is enhanced when
two phenolic hydroxyl groups are oriented ortho or para. Since these structural
features of phenolic compounds are reportedly also responsible for antioxidant
activity, measurements of phenols in infusions may be related to their antioxidant
properties (Katalinic et al., 2006). Phenolic
compounds such as flavonoids, phenolic acid and tannins, possess anti-inflammatory,
anti-carcinogenic, anti-atherosclerotic and other properties that may be related
to their antioxidant activities (Chung et al., 1998;
Wong et al., 2006). Flavonoids and flavonols
are two polyphenolic compounds that play an important role in stabilizing lipid
oxidation and are associated with antioxidant activity (Yen
et al., 1993). Phenolic compounds may contribute directly to antioxidative
action (Duh et al., 1999). Polyphenolic compounds
may have an inhibitory effect on mutagenesis and carcinogenesis in humans when
as much as 1.0 g is ingested daily from a diet rich in fruits and vegetables
(Tanaka et al., 1998).
With the above study conducted, it may be concluded that the polyphenols present
in plant Boerhavia diffusa, provide an understanding of its beneficial
effects against the reactive oxygen species which in turn help in checking the
pathological conditions like cancer, degeneration, autoimmune diseases, Alzheimers
and aging caused due to them. With this study we conclude that this plant has
a great potential and put to further research would help in putting it to commercial
use as a marketed medicine.
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