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Article

Assessment of phytochemicals,antioxidant, and anti-inflammatorypotential of Boerhavia procumbensBanks ex Roxb

Jasia Bokhari1, Muhammad Rashid Khan1

and Ihsan Ul Haq2

AbstractBoerhavia procumbens is traditionally used in the treatment of various disorders including jaundice andgonorrhea, is a refrigerant, and exhibits anti-inflammatory and antispasmodic activitiesAQ1 . The purpose of thisstudy was to determine the phytochemical classes, antioxidant and anti-inflammatory activities of methanolextract (BPME) and different fractions (n-hexane (BPHE), ethyl acetate (BPEE), n-butanol (BPBE), and residualaqueous fraction (BPAE)) of B. procumbens against carrageenan-induced paw edema in rats. To assess the anti-inflammatory effects of B. procumbens, 42 Sprague Dawley male rats (150–200 g) were randomly divided intoseven groups. Group I received distilled water and group II was treated with diclofenac potassium (10 mg/kg)body weight (bw) orally. Groups III, IV, V, VI, and VII were administered BPME, BPHE, BPEE, BPBE, and BPAE(200 mg/kg bw) orally, 1 h before the treatment with carrageenan (10 mg/kg bw) in rats. Anti-inflammatoryeffects of B. procumbens were determined by estimating the inhibition of edema at 1st, 2nd, and 3rd hour aftercarrageenan injection. Qualitative analysis of methanol extract indicated the composition of diverse classes,namely, flavonoids, tannins, saponins, phlobatannins, cardiac glycosides, alkaloids, terpenoids, and anthraqui-nones. Quantitative determination illustrated that BPBE and BPEE possessed the highest concentration of totalphenolic (60.45 + 2.1 mg gallic acid equivalent per gram sample) and total flavonoid content (68.05 + 2.3 mgrutin equivalent per gram sample), respectively. A dose-dependent response for antioxidant activity wasexhibited by all the samples. The sample with the highest aptitude for antioxidant activity was the BPBEfor 2,2-azobis,3-ethylbenzothiozoline-6-sulfonic acid radical scavenging and total antioxidant capacity.Carrageenan-induced paw edema was significantly (p < 0.05) inhibited by BPBE and BPME at the 1st, 2nd,and 3rd hour and was comparable to control drug diclofenac potassium. Results revealed that various frac-tions of B. procumbens manifested the antioxidant and anti-inflammatory potential and accredit the local useof B. procumbens in various disorders.

KeywordsBoerhavia procumbens, anti-inflammatory, flavonoids, edema, carrageenan

Introduction

Free radicals are produced during natural process of

metabolism, and in all cells, a balance is maintained

by virtue of antioxidant enzymes (catalase, superox-

ide dismutase, and glutathione peroxidase) or chemi-

cals (tocopherol, glutathione, and ascorbic acid).

Free radicals act as dual-edged sword, either friend

or foe, depending upon the conditions and environ-

ment of the cell. Being a friend they play an imperative

role in neurotransmission, cytotoxicity, and defense

1Department of Biochemistry, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad, Pakistan

2Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

Corresponding author:Muhammad Rashid Khan, Department of Biochemistry, Faculty ofBiological Sciences, Quaid-i-Azam University, Islamabad 45320,Pakistan.Email: mrkhanqau@yahoo.com

Toxicology and Industrial Health1–11© The Author(s) 2014Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233714567183tih.sagepub.com

against microbes. In the cell when the level of oxidant

and antioxidant tilted toward more fabrication of oxi-

dants, while acting as foe they usually become a seri-

ous threat for cellular biomoleculesAQ2 . Free radicals are

highly reactive species implicated in diverse clinical

disorders including inflammation, cardiovascular dis-

orders, cancer, and premature aging (Gulcin, 2012).

There has been serious concern to mitigate the produc-

tion and concentration of reactive oxygen species

(ROS) such as superoxide anion, hydroxyl (OH) radi-

cal, lipid peroxide, and hydrogen peroxide (H2O2).

To overcome this hazard of ROS induction, intake of

external antioxidants in the form of dietary phytochem-

ical and as herbal medicines are reported to have good

therapeutic advancement to combat the free radical

inductions (Bokhari et al., 2013; Gulcin et al., 2011).

Flavonoids and other constituents are integral and

active components of diverse herbal medicines and

constitute a vital part of human diet. Due to their

diverse properties, such as spasmolytic, antiphlogis-

tic, antiallergic and diuretic, flavonoids are widely

used as curative agents (Merken and Beecher,

2000). These diverse functions are originated from

the basic chemical structure, modulation in structure,

kind of functional group attached, number of cyclic

rings, and type of conjugated moieties (Ercan

et al., 2013). Flavonoids are suggested to be more

important among various classes of secondary meta-

bolites because of their antioxidant and/or radical

scavenging properties (Khan et al., 2012a, 2012b).

The positive outcome resulted by intake of antioxi-

dant moieties of herbal origin have been publicized

in a number of investigational and epidemiological

studies. Recently studies have been conducted to

affirm the therapeutic potential of plants that are

playing vital roles in the management of different

diseases (Saeed et al., 2012; Sahreen et al., 2014).

In the present scientific scenario, antioxidant capac-

ity of the herbals has been investigated because a

number of human ailments are directly caused by

free radicals and/or promoted by free radical hazard.

Protection of body against various factors includ-

ing infections, wounds, burns, toxins, and allergens

is mediated through anti-inflammatory response of

the body. However, inflammation becomes a major

factor of etiology in many chronic illnesses (Kumar

et al., 2004). The imbalance induced by different

factors in the mediators and complex events of

defense may cause, maintain, or enhance the severity

in many ailments associated with inflammation

(Sosa et al., 2002). Innate defense of human body

may not be sufficient to cope with the perpetual

inflammatory response. Use of commercially avail-

able anti-inflammatory drugs is juxtaposed with seri-

ous harmful effects. It becomes the need of the day to

develop therapeutic agents with massive remedial

potential for inflammatory disorders and should be

void of side effects. Anti-inflammatory potential of

other plant extracts have been reported in earlier

studies (Khadse and Kakde, 2011).

Boerhavia procumbens is locally used in various

disorders. Shah and Khan (2006) reported the use

of leaves against edema, dropsy, and in dysmenor-

rhea. According to Qureshi et al. (2010) in the tradi-

tional system of medicine, its dried roots are used in

the treatment of respiratory and pulmonary diseases.

Other uses of the plant in folklore system of medi-

cine are to treat liver, eyes, stomach, urinary, and

throat disorders. Katewa et al. (2004) described the

ethnobotanical uses of the plant to treat swelling,

anemia, dropsy, and gonorrhea. Researchers have

reported that the plant is antispasmodic, astringent,

refrigerant, and gives relief from pain (Khan et al.,

2000; Singh et al., 2002). Previous research data

revealed that the plant is comprised of rutin, cate-

chin, caeffic acid, myricetin, quercetin, and kaemp-

ferol phytoconstituents. Recently there has been an

upsurge for natural products to cure oxidative stress

and its related anomalies. To our knowledge, there is

no published data regarding the antioxidant and anti-

inflammatory activity of B. procumbens; hence, in

this study, we have evaluated the phytochemical

classes, antioxidant and anti-inflammatory activity

of crude methanol extract and derived fractions of

B. procumbens.

Materials and methods

Plant collection

The plant material (whole plant) of B. procumbens

was sampled from the campus of Quaid-i-Azam

University, Islamabad, Pakistan, in May 2010. The

plant was validated and a voucher specimen with

accession no. 059133 (B. procumbens) was depos-

ited at the Herbarium of Pakistan, Museum of Natural

History, Islamabad, Pakistan.

Extract preparation

Pulverized plant sample (5 kg) was soaked in 10 l of

methanol in a large container for 5 days. At day 5,

the sample was filtered and re-extraction of the

2 Toxicology and Industrial Health

residue was done twice. The filtrate was dried under

rotary evaporator at 40�C to attain crude methanol

extract of B. procumbens (BPME). Further fractions

were obtained by suspending BPME (4 g) in 200 ml

of distilled water, and different fractions were

obtained in sequence of n-hexane (BPHE), ethyl

acetate (BPEE), n-butanol (BPBE) and residual aqu-

eous fraction (BPAE).

Determination of phytochemical constituents

The BPME and its derived fractions were evaluated for

the occurrence of flavonoids, tannins, saponins, phlo-

batannins, cardiac glycosides, alkaloids, terpenoids,

and anthraquinones using simple qualitative methods.

Salkowski test for terpenoids. To 5 ml of extract and

each fraction (1 mg/ml), 2 ml of chloroform was

added, followed by an addition of 3 ml of concen-

trated sulfuric acid (H2SO4). Formation of a reddish

brown coloration at the interface confirmed the pres-

ence of terpenoids (Harborne, 1973).

Test for alkaloids. An amount of 0.5 g of each sample

was heated with 8 ml of 1% hydrochloric acid (HCl)

and filtered. A volume of 2 ml of each filtrate was

titrated separately with (a) Mayer’s reagent and (b)

Dragendroff’s reagent. The presence of alkaloids was

indicated by the formation of cream or yellow preci-

pitate (Harborne, 1973).

Test for saponins. Crude methanol extract and derived

fractions (0.2 g) were mixed with 20 ml of distilled

water and boiled in a water bath. After filtration, an

aliquot of 5 ml of distilled water was mixed with 10

ml of the filtrate and mixed thoroughly. Presence of

saponins was confirmed by the formation of emulsion

after mixing the froth with three drops of olive oil and

shaken vigorously (Harborne, 1973).

Test for phlobatannins. In a small tube, 80 mg of crude

extract was added, and each fraction was boiled in

1% aqueous HCl; formation of red precipitate indi-

cated the presence of phlobatannins (Trease and

Evans, 1989).

Keller–Kiliani test (for deoxy sugars in cardiac glycosides).From the crude extract and each fraction, 5 ml of

aqueous extract (10 mg/ml) was mixed with 2 ml

of glacial acetic acid having a drop of ferric chloride

solution, followed by 1 ml of concentrated H2SO4 to

form a layer. The characteristic feature of deoxysugars

in cardenolides is the formation of brown ring at the

interface (Trease and Evans, 1989).

Test for coumarins. About 0.3 g of extract and each

fraction was taken in a test tube and its mouth was

covered with a filter paper moistened with 1 N sodium

hydroxide (NaOH) solution. After placing the test

tube in a boiling water bath for few minutes, the filter

paper was examined under ultraviolet light. The pres-

ence of coumarins is indicated by yellow fluorescence

(Trease and Evans, 1989).

Test for anthraquinones. Briefly, 200 mg of extract and

each fraction was boiled in 6 ml of 1% HCl and fil-

tered. The filtrate was mixed with 5 ml of benzene,

and after shaking, 10% ammonium hydroxide was

added. Formation of pink/violet or red color in alka-

line phase indicated the presence of anthraquinones

(Trease and Evans, 1989).

Test for tannins. An amount of 50 mg of the extract and

each fraction was mixed with 20 ml of water and fil-

tered. Formation of blue black or brownish green

color after treating the filtrate with a few drops of

0.1% ferric chloride indicated the presence of tannins

(Sofowora, 1993).

Test for flavonoids. An amount of 50 mg of the extract

and each fraction was mixed with 10 ml of distilled

water and filtered. To the aqueous filtrate, 5 ml of

dilute ammonia solution and concentrated H2SO4

were added. Development of yellow coloration con-

firmed the presence of flavonoid (Sofowora, 1993).

Total phenolic content estimation

The total phenolic content of B. procumbens was

determined by employing the method reported by

Kim et al. (2003). Briefly, Folin–Ciocalteu reagent

of 400 �l was mixed with 200 �l of each sample

(1 mg/ml) in a volumetric flask. The mixture after

incubation for 5–10 min at 25�C was mixed with

0.2 ml of 7% sodium carbonate solution, and finally,

the mixture was diluted with deionized distilled water

to 10.0 ml of volumetric flask. Before taking the

absorbance at 725 nm, the mixture was incubated for

2 h at 25�C. Calibration curve was plotted for the stan-

dard of gallic acid. Total phenolics were calculated as

milligrams of gallic acid equivalents per gram of

dried sample.

Bokhari et al. 3

Estimation of total flavonoids

The total flavonoid content was estimated according

to the method of Yong et al. (2008). An amount of

50 mg of each sample was mixed with 10 ml of

80% methanol and filtered. An aliquot of 0.3 ml of the

filtrate was mixed with 3.4 ml of 30% methanol, 0.15

ml of 0.5 M sodium nitrite and 0.15 ml of 0.3 M alu-

minum chloride hexahydrate. After mixing for 5 min,

1 ml of 1 M NaOH was added to the mixture, and

absorbance was measured at 506 nm. Rutin was used

for plotting calibration curve. The amount of total fla-

vonoid was calculated as milligram rutin equivalents

per gram of dried sample.

Antioxidant assays

DPPH radical scavenging activity. 2,2-Diphenlyl-1-

picrylhydrazyl (DPPH) scavenging activity was per-

formed by the method of Brand-Williams et al.

(1995). Different concentrations (300, 250, 200,

150, 100, 50, and 25 mg) of extract and different frac-

tions were prepared in methanol and taken in test

tubes (100 ml) in triplicates. Then, 2.4 mg DPPH was

dissolved in 100 ml methanol to prepare stock solu-

tion. The stock solution was further diluted with

methanol until attaining an absorbance less than 1.0

using the spectrophotometer at 517 nm. Then, 3 ml

of working solution of DPPH was added to each test

tube and shaken vigorously. After incubation for 30

min at room temperature, absorbance was recorded

at 517 nm. Ascorbic acid and rutin were used as pos-

itive controls. The capability to scavenge the DPPH

radical was calculated using the following equation:

Scavenging effect %ð Þ ¼OD of control� OD of sampleð Þ

OD of controlð Þ

� �� 100

Superoxide anion scavenging activity

The riboflavin–light– nitroblue tetrazolium (NBT)

system was employed to assay the superoxide radical

scavenging activity (Beauchamp and Fridovich,

1971). Briefly, a reaction mixture was prepared by the

addition of 0.5 ml of 50 mM phosphate buffer (pH

7.6), 0.3 ml of 50 mM riboflavin, 0.25 ml of 20 mM

phenazine methosulfate, and 0.1 ml of 0.5 mM NBT

before the addition of 1 ml of various samples. The

reaction mixture was illuminated with fluorescent

light for 20 min and the absorbance was taken at

560 nm. The percentage of scavenging superoxide

anion generation was calculated as:

Percentage inhibition ¼

1� Sample absorbance

Control absorbance

� �� 100

Phosphomolybdate radical scavenging activity

The total antioxidant capacity of the extract and

different fractions was determined by the method

described by Umamaheswari and Chatterjee (2008).

Briefly, 0.1 ml of sample solution was mixed with

1 ml of reagent solution (0.6 M H2SO4, 28 mM

sodium phosphate, and 4 mM ammonium molybdate)

and incubated in a water bath for 90 min at 95�C. The

absorbance of the mixture was measured at 765 nm

after the mixture was cooled down to room tempera-

ture. Ascorbic acid and rutin were used as standards.

The antioxidant capacity was estimated using the fol-

lowing formula:

Total antioxidant capacity %ð Þ ¼Absorbance of control� Absorbance of sampleð Þ

Absorbance of controlð Þ

� �

� 100

OH radical scavenging activity. OH radical scavenging of

each sample was estimated by the procedure described

by Halliwell and Gutteridge (1981). The reactive mix-

ture was prepared by mixing 100 �l of reagent (ferric

chloride, 100 mM; 250 �l of 2.8 mM 2-deoxyribose

in 50 mM phosphate buffer pH 7.4; 100 mM ethylene-

diaminetetraacetic acid, 1:1; v/v; 50 �l of 200 mM

H2O2) without or with 50 �l of the sample. Reaction

was triggered by adding 50 �l of ascorbate (300

mM). After incubation for 60 min at 37�C, to the mix-

ture, a solution of 1% thiobarbituric acid (TBA) in 500

�l of NaOH (50 mM) and 500 �l of 2.8% trichloroace-

tic acid (TCA) was added. The mixture was again incu-

bated for 15 min on a boiling water bath. After cooling

to room temperature, the absorbance was taken at 532

nm. OH radical scavenging was calculated as:

Percentage inhibiting activity ¼

1� Absorbance of sample

Absorbance of control

� �� 100

4 Toxicology and Industrial Health

ABTS radical scavenging activity. 2,2-Azobis,3-ethylbenzo

thiozoline-6-sulfonic acid (ABTS) radical scavenging

activity was determined by calculating the disappear-

ance of ABTS radical cation by following the method

of Re et al. (1999). In brief, for the development of

ABTS radicals, 7 mM ABTS solution was mixed with

2.45 mM potassium persulfate solution and incubated

overnight in the dark to get a dark-colored solution.

The standard solution of ABTS was diluted by the

addition of 50% methanol to have an absorbance of

0.70 + 0.02 at 745 nm at 30�C. A volume of 1 ml

of each sample was mixed with 1 ml of ABTS solu-

tion, and the absorbance was recorded after 1 min. For

calculating percentage inhibition, the following for-

mula was used:

Percentage inhibiting activity ¼

1� Absorbance of sample

Absorbance of control

� �� 100

H2O2 scavenging activity. H2O2 scavenging activity of

the extract and each fraction was determined by the

method of Ruch et al. (1989). Briefly, H2O2 working

solution (2 mM) was prepared in 50 mM phosphate

buffer (pH 7.4). The reaction mixture was prepared

by the addition of 100 ml of each sample to 400 ml

of 50 mM phosphate buffer (pH 7.4) and 600 ml of

H2O2 solution and was incubated for 10 min. At 230

nm, absorbance of the mixture was recorded against

phosphate buffer as a blank. H2O2 scavenging ability

was calculated using the following equation:

H2O2 scavenging activity ¼

1� Absorbance of sample

Absorbance of control

� �� 100

Reducing power estimation

The reducing power was based on ferric (Fe3þ) to

ferrous (Fe2þ) transformation in the presence of the

sample (Oyaizu, 1986). The Fe2þ ions can be moni-

tored by measuring the formation of Perls’ Prussian

blue at 700 nm. Various concentrations of the sample

(2 ml) were mixed with 2 ml of phosphate buffer

(0.2 M, pH 6.6) and 2 ml of potassium ferricyanide

(10 mg/ml). The mixture was incubated at 50�Cfor 20 min, followed by the addition of 2 ml of

TCA (100 mg/l). The mixture was centrifuged at

3000 r/min for 10 min to collect the upper layer of

the solution. A volume of 2 ml from each of the mix-

ture earlier mentioned was mixed with 2 ml of dis-

tilled water and 0.4 ml of 0.1% (w/v) fresh ferric

chloride. After 10 min reaction, the absorbance was

measured at 700 nm. Higher absorbance of the reac-

tion mixture indicates a higher reducing power.

Anti-inflammatory assay by carrageenan-inducedinflammation

Six-week-old 42 Sprague Dawley male rats (150–200 g)

were randomly divided into seven groups with six rats

in each group. The rats were maintained at the primate

facility of Quaid-i-Azam University, Islamabad,

Pakistan. The study protocol was approved by the

ethical committee of Quaid-i-Azam University, Isla-

mabad, for the animal care and experimentation.

Group I was administered with 0.3 ml of distilled

water, whereas group II was treated with 10 mg/kg

body weight (bw) of drug diclofenac potassium.

Groups III, IV, V, VI, and VII were treated at dose

of 200 mg/kg bw with BPME, BPHE, BPEE, BPBE,

and BPAE, respectively. All these treatments were

administered 1 h before the injection of carrageenan

(1 ml/kg bw, w:v) in the right paw of each rat to

develop the pedal inflammation (Winter et al.,

1960). Anti-inflammatory activity in each group was

determined by measuring the paw volume by plethys-

mograph accordingly before the treatment of carra-

geenan and after 1st, 2nd, and 3rd hour of

carrageenan injection. To determine the inhibition

of edema, the following equation is used:

Edema inhibition %ð Þ ¼ 1� D

C

� �� 100;

where D represents the percentage difference in

increased paw volume after the administration of test

drugs to the rats and C represents the percentage dif-

ference of increased volume in the control groups.

Statistical analysis

Results of all parametric assays were expressed as

mean + SD. Data of in vitro antioxidant assays were

analyzed with the help of computerized GraphPad

Prism software (GraphPad Software, San Diego,

California, USA) to determine the half maximal inhi-

bitory concentration (IC50) values. For analyzing the

differences among IC50 values of different fractions

of different antioxidant assays, analysis of variance

Bokhari et al. 5

test was used with least significance difference p <

0.05 as a level of significance.

Results

Phytochemical screening of B. procumbens

In this study, the quality screening of the crude

methanol extract and various fractions of B. procum-

bens showed the presence of different classes of phy-

toconstituents. The results of this study indicated

that BPME was consisted of all the phytochemical

classes except the coumarins. Fractionation by esca-

lating polarity resulted in separation of phytochem-

icals in this study. Tannins and flavonoid were

present in all the extract/fractions of B. procumbens.

The presence of terpenoids and phlobatannins was

restricted only to BPME (Table 1). Saponins were

present in all the samples except in BPAE where

their presence was not confirmed.

Total phenolic and total flavonoid content

Results of this experiment indicated that BPEE pos-

sessed the highest (p < 0.05) concentration of total

flavonoid content with respect to other extract/frac-

tions (Table 2). The amount of total flavonoid was

ranked second in BPHE, followed by BPBE > BPME

> BPAE. The total flavonoid content of BPBE and

BPME was statistically similar (p > 0.05) to each

other; however, it was significantly higher (p <

0.05) than BPAE.

The highest amount of total phenolic content was

estimated in BPBE and was statistically similar (p >

0.05) to BPEE. However, BPHE and BPME have the

similar concentration of total phenolic content. BPAE

contained the lowest quantity of total phenolic content.

Extraction yield

The results of this study illustrated that the highest

extraction yield was estimated in BPBE as compared

to other samples. The least extraction yield was

recorded in BPME. The order of extraction yield

obtained in this experiment was BPBE, followed

by BPAE, BPEE, BPHE, and BPME.

Antioxidant assays

DPPH radical scavenging activity. The scavenging effect

of B. procumbens on DPPH radical varied significantly

(p < 0.05) with its extract/fractions. During the DPPH

radical scavenging assay, the highest DPPH scaven-

ging activity was recorded for BPME, which was man-

ifested by lower half maximal effective concentration

(EC50) values. The DPPH radical scavenging ability

of samples can be ranked as BPME > BPEE > BPBE

> BPAE > BPHE. This indicates that the kind and

quality of phytoconstituents present in different frac-

tions affects the antioxidant ability. The EC50 values

of scavenging DPPH radicals for the BPME and BPEE

were 78.3 + 2.41 and 93.3 + 2.87 mg/ml, respec-

tively, while for the BPHE, it was 193 + 3.33 mg/ml

(Table 3). However, significantly lower values of

EC50 were recorded for ascorbate and rutin. The per-

centage of DPPH scavenging activity was increased

dose dependently for all the extract/fractions and for

the standard chemicals, that is, ascorbic acid and rutin.

Superoxide anion scavenging activity. The superoxide

radical scavenging capacity of extract and different

Table 1. Phytochemical constituents of the B. procumbens.

Chemicals BPME BPHE BPEE BPBE BPAE

Alkaloids þ � þ þ �Tannins þ þ þ þ þSaponins þ þ þ þ �Flavonoids þ þ þ þ þTerpenoids þ � � � �Cardiac glycosides þ þ � þ þAnthraquinones þ þ þ � þCoumarins � � � � �Phlobatannins þ � � � �

þ: present;�: absent; B. procumbens: Boerhavia procumbens; BPME:B. procumbens methanol extract; BPHE: B. procumbens n-hexaneextract; BPEE: B. procumbens ethyl acetate extract; BPBE: B. pro-cumbens n-butanol extract; BPAE: B. procumbens aqueous extract.

Table 2. Total phenolic, flavonoid, and extraction yield ofB. procumbens.a

Extract/fraction

Total flavonoid(milligram rutinequivalent per

gram dry weight)

Total phenolic(milligram gallicacid equivalent

per gramdry weight)

Extractionyield (%)

BPME 41.68 + 3.2b 40.37 + 3.2c 3.5 + .02d

BPHE 50.98 + 4.1c 45.87 + 2.5c 5.2 + 0.21b

BPEE 68.05 + 2.3e 59.34 + 1.5e 4.9 + 0.08b

BPBE 43.96 + 1.5b 60.45 + 2.1e 12.4 + 1.2e

BPAE 6.30 + 1.01d 22.12 + 1.0b 10.1 + 1.0c

B. procumbens: Boerhavia procumbens; BPME: B. procumbens metha-nol extract; BPHE: B. procumbens n-hexane extract; BPEE: B. pro-cumbens ethyl acetate extract; BPBE: B. procumbens n-butanolextract; BPAE: B. procumbens aqueous extract.aEach value in the table is represented as mean + SD (n ¼ 3).Values not with similar superscript letter (b–e) in each column aresignificantly different at p < 0.05.

6 Toxicology and Industrial Health

fractions was compared with same doses of ascorbic

acid ranging from 25 to 250 mg/ml. The results

obtained in this experiment showed that all the sam-

ples scavenged the superoxide radicals in a dose-

dependent manner. The EC50 values for superoxide

radical scavenging were in the order of BPME >

BPAE > BPBE > BPAE > BPHE (Table 3). These

variations in scavenging of superoxide radicals could

be due to the presence of reactive concentration of

bioactive constituents in various samples. However,

when compared with ascorbic acid, the superoxide

radical scavenging activity of the extract/fraction was

significantly (p < 0.05) lower.

OH radical scavenging activity. In this study, the extract

and all the fractions showed dose-dependent response

for OH radical scavenging activity. The EC50 values

of the extract and fractions for OH radical could be

ranked as BPBE > BPAE > BPME > BPEE > BPHE.

The EC50 values for scavenging of OH radicals for the

BPBE was 43.3 + 1.32 mg/ml, while for BPHE, it

was >250 mg/ml (Table 3).

Phosphomolybdate assay (total antioxidant capacity).Table 3 depicts the total antioxidant capacity of

methanol extract and its different fractions. The

results obtained in this study clearly imply that all the

samples scavenged the phosphomolybdate radicals in

a dose-dependent way. The phosphomolybdate radi-

cal scavenging activities of the extract and fractions

can be ranked in the order of BPEE > BPME > BPHE

> BPBE > BPAE. The EC50 values of antioxidant

capacity for the BPEE and BPME were 31.7 +

1.62 and 38.3 + 0.95 mg/ml, respectively. The lowest

phosphomolybdate scavenging activity with EC50

value of 123 + 4.32 mg/ml was recorded for BPAE

(Table 3). However, the scavenging ability of ascor-

bic acid for phosphomolybdate radical was remark-

ably high as compared to the extract and fractions.

H2O2 radical scavenging activity. The scavenging effect

of the extract/fractions on H2O2 is shown in Table 3.

The results obtained in this study showed that differ-

ent extract/fractions were capable of scavenging the

H2O2 in a concentration-dependent manner. The scaven-

ging ability of various extract/fractions for H2O2 clearly

imply that BPBE and BPAE were more efficient with

EC50 values having values of 55 + 2.09 mg/ml and

61.7 + 1.07 mg/ml, respectively. On the other hand,

BPEE and BPHE were least effective with EC50 values

of >250 mg/ml and >>250 mg/ml, respectively. The

scavenging abilities on H2O2 of different fractions can

be ranked in descending order of BPBE > BPAE >

BPME > BPEE > BPHE (Table 3).

ABTS radical scavenging activity. Results of ABTS radi-

cal scavenging ability of different samples are shown

in Table 3. Various extract/fractions of B. procumbens

were capable of scavenging the ABTS radicals in a

concentration-dependent manner that can be ranked

as BPEE > BPBE > BPAE > BPME > BPHE (Table

3). In this study, EC50 value obtained for BPEE was

46.7 + 2.23 mg/ml as compared to ascorbic acid

(66.7 + 3.6). The least scavenging capacity for

ABTS radicals was determined for BPHE with EC50

values of 222 + 4.81 mg/ml.

Table 3. EC50 values of B. procumbens on different assay systems.a

EC50 (mg/ml)

ExtractDPPH radical

scavengingSuperoxide

radical scavengingH2O2

scavengingHydroxyl radical

scavengingABTS radicalscavenging

Total antioxidantcapacity

BPME 78.3 + 2.41b 55 + 1.32b 71.7 + 1.4c 85 + 3.32c 148 + 1.49d 38.3 + 0.95e

BPHE 193 + 3.33f >500f >250f >250f 222 + 4.8f 45 + 1.49c

BPEE 93.3 + 2.87e >250d >250d >250d 46.7 + 2.23b 31.7 + 1.62b

BPBE 115 + 3.45b 178 + 3.08e 55 + 2.09b 43.3 + 1.32c 115 + 3.24d 75 + 2.31f

BPAE 135 + 1.54d 162 + 1.1e 61.7 + 1.07e 65 + 2.6e 117 + 2.17c 123 + 4.32f

AA 20 + 1.03g 21.7 + 0.89g 23.3 + 2.01g 33.3 + 1.8g 66.7 + 3.6g 25 + 0.43g

Rutin 18.3 + 1.00g – 30 + 2.31g – – 30 + 0.81g

B. procumbens: Boerhavia procumbens; BPME: B. procumbens methanol extract; BPHE: B. procumbens n-hexane extract; BPEE: B. procum-bens ethyl acetate extract; BPBE: B. procumbens n-butanol extract; BPAE: B. procumbens aqueous extract; EC50: half maximal effectiveconcentration; DPPH: 2,2-diphenlyl-1-picrylhydrazyl; H2O2: hydrogen peroxide; ABTS: 2,2-azobis,3-ethylbenzothiozoline-6-sulfonicacid; AA: ascorbic acid; –: not determined.aEach value in the table is represented as mean + SD (n ¼ 3). Superscripts (b–g) show significance at p < 0.05.

Bokhari et al. 7

Reducing power estimation

In this experiment, absorbance at 720 nm was recorded

at various concentrations (25–250 �g/ml) of B. pro-

cumbens extract/fractions in the reaction mixture. High

absorbance values indicated the reducing power poten-

tial at specific concentration. The results obtained

clearly imply that all the tested samples inhibit or sca-

venge the radical in a concentration-dependent manner.

The sequence for reducing power at 250 mg/ml was

BPAE > BPME > BPEE > BPBE > BPHE.

Correlation of EC50 values of antioxidant activitiesand phytochemical content

The amount of total flavonoid and phenolic with EC50

values for superoxide radical scavenging activity were

significantly correlated; R2 ¼ 0.7558 and R2 ¼ 0.942,

respectively (Table 4). However, weak correlation of

EC50 values was established with DPPH, OH, H2O2,

and ABTS radical scavenging activities (Table 4).

Anti-inflammatory activity

The in vivo anti-inflammatory activities of crude metha-

nol extract and various fractions of B. procumbens were

evaluated against the carrageenan-induced paw edema

in rat. The results obtained from this study indicated that

BPBE inhibited the edema development, which was

comparable to that of the diclofenac potassium, a stan-

dard anti-inflammatory drug after 1st and 2nd hour of

carrageenan treatment. Anti-inflammatory effects of

BPME and BPBE were statistically similar to each after

3rd hour of carrageenan treatment, while these were

statistically different from other groups. Inhibition of

edema development after 3rd hour of carrageenan treat-

ment can be ranked in order of BPME > BPBE > BPAE

> BPEE > BPHE (Table 5).

Discussion

Oxidative stress is induced by the perpetual genera-

tion of free radicals causing an imbalance of proox-

idants and antioxidants in the organisms and can

bring about damages to biomolecules. Nowadays,

antioxidants are gaining credit in scavenging free

radicals and are suggested to be involved in the pre-

vention and cure of many disorders including cancer,

aging, and a range of other diseases (Gulcin et al.,

2011).

In this study, a wide range of phytoconstituents

such as phenolics, flavonoids, tannins, alkaloids, and

terpenoids were detected in BPME and other fractions

of B. procumbens. Among them, flavonoids and ter-

penoids have gained a particular interest because of

their broad pharmacological activities. Similar phyto-

chemicals have been reported in other studies

(Bokhari et al., 2013). Putative therapeutic effects of

much traditional medicine may be ascribed to the

presence of flavonoid (Braca et al., 2003). Flavonoids

are the most common and widely distributed group of

plant phenolic compounds, which can be synthesized

from the amino acid phenylalanine.

Pharmacological properties of various extracts

and compounds may be attributed to the antioxidant

potential. An easiest way to assess the antioxidant

activity of extract/fraction is DPPH stable free radi-

cal method (Koleva et al., 2002). During the DPPH

scavenging activity BPME exhibited the highest

scavenging activity for DPPH radicals. However,

weak association of EC50 value of DPPH with total

phenolic and total flavonoid content suggests the

involvement of primary antioxidant compounds in

the extract/fractions other than phenolics and flavo-

noids that are capable of donating hydrogen to a free

radical in order to remove odd electron that is

responsible for radical’s reactivity.

Table 4. Correlation between the EC50 values of antioxi-dant activities and phenolic and flavonoids content of B.procumbens.

Assays

Correlation R2

Total flavonoidcontent

Total phenoliccontent

EC50 of DPPH radicalscavenging ability

0.00482 0.0585

EC50 of superoxideradical scavengingability

0.7858a 0.9421b

EC50 of antioxidantcapacity

0.0332 0.0284

EC50 of hydroxyl radicalscavenging ability

0.01153 0.1461

EC50 of H2O2 scavengingability

0.06276 0.2757

EC50 of ABTS radicalscavenging ability

0.01035 0.1129

EC50: half maximal effective concentration; B. procumbens: Boerha-via procumbens; DPPH: 2,2-diphenlyl-1-picrylhydrazyl; H2O2:hydrogen peroxide; ABTS: 2,2-azobis,3-ethylbenzothiozoline-6-sulfonic acid.ap < 0.05: significance level.bp < 0.01: significance level.

8 Toxicology and Industrial Health

Superoxide anions are among the most deadly

oxidants involved either directly or indirectly in the

damages of biomolecules (Bokhari et al., 2013). The

results of our study indicated strong correlation of

EC50 value for the superoxide anions with the total

phenolic and total flavonoid content suggesting the

crucial role of polyphenolics in eliminating the

superoxide anions. Superoxide anions are found to

be harmful for cellular components (Khan et al.

2012a). They indirectly cause lipid peroxidation of

membranes through singlet oxygen production. An

appreciable estimate of superoxide anion scavenging

ability of B. procumbens suggests that it might be

able to protect the cells from highly reactive super-

oxide anions.

Phosphomolybdenum assay is a quantitative

method to evaluate total antioxidant capacity of the

extracts. This study demonstrated that ethyl acetate

fraction exhibited the highest antioxidant capacity for

phosphomolybdate reduction. Therefore, on the basis

of IC50 values obtained in this study, it was proposed

that B. procumbens might contain antioxidants that

contribute significantly to scavenge the free radicals,

thereby protecting the cells from oxidation. The

results of this assay have shown low level of associa-

tion between the total antioxidant capacity and the

total phenolic and total flavonoid contents, suggesting

the importance of compounds other than polyphenols

to act as chain terminator, transforming relative free

radical species into more stable nonreactive products

(Ercan et al., 2013).

The evidence of OH scavenging activity by the

extract and its fractions would be obtained through

the deoxyribose system. OH radicals are produced

by the reaction of H2O2 and ferrous that would

react with 2-deoxyribose. A red color is developed

because of the reaction between the TBA and the

products of deoxyribose degradation. Scavenging

of OH radical is directly proportional to the antioxi-

dant activity of the extract/fraction and would be

observed by the low intensity of red color. The

results of this study indicated the crucial role of

BPBE in scavenging of OH radicals and H2O2 sug-

gesting the termination of chain reaction of ROS.

H2O2 can probably react with Fe2þ and possibly

Cu2þ ions inside the cell to form OH radical that can

damage the macromolecules in a cell. Elimination of

H2O2 and of OH radicals provides biological advan-

tage for cells to control the amount of H2O2 that is

allowed to accumulate.

ABTS free radical scavenging method is an excel-

lent means for determining the antioxidant activity of

a broad diversity of substances, such as hydrogen-

donating antioxidants or scavengers of lipid peroxyl

radicals or scavengers of chain-breaking antioxidants

(Bokhari et al., 2013). The ethyl acetate fraction in

our study was found to have an appreciable scaven-

ging activity of ABTS radical; this implies that they

may be useful for treating radical-related pathological

damage especially at higher concentrations. The

reductive ability of the antioxidant can be measured

by reducing power, which is evaluated by the trans-

formation of Fe3þ to Fe2þ ions by donating an elec-

tron in the presence of the extract. The reducing

capacity of the aqueous fraction of B. procumbens

may serve as an indicator of potential antioxidant

activities through the action of breaking the free rad-

ical chain by donating hydrogen atom (Gulcin, 2012;

Khan et al., 2012a).

Many herbal medicines are used in different sys-

tems of medicines to cure inflammation. In this

experiment, the herbal medicine B. procumbens has

Table 5. Edema inhibition relative to control at 1st, 2nd, and 3rd hour.a

Chemical/extract Doses (mg/kg)

% Edema inhibition relative to control at 1st, 2nd, and 3rd hour

1st hour 2nd hour 3rd hour

DW 0.3 ml 0 0 0DIC 10 mg/kg 44.11 + 2.4b 62.98 + 4.60b 87.36 + 4.4b

BPME 200 mg/kg 40.32 + 3.6b 54.45 + 6.1c 74.34 + 6.2c

BPHE 200 mg/kg 20.12 + 4.2d 40.23 + 3.9e 50.31 + 3.6f

BPEE 200 mg/kg 38.32 + 5.1c 55.12 + 2.7c 72.45 + 3.2d

BPBE 200 mg/kg 42.64 + 2.7b 59.09 + 1.9b 77.89 + 1.8c

BPAE 200 mg/kg 38.45 + 4.8c 48.78 + 3.8d 65.23 + 4.8e

DW: distilled water; DIC: diclofenac potassium; BPME: B. procumbens methanol extract; BPHE: B. procumbens n-hexane extract; BPEE: B.procumbens ethyl acetate extract; BPBE: B. procumbens n-butanol extract; BPAE: B. procumbens aqueous extract.aMean + SD (n ¼ 6). Different superscript alphabets in the column show significance at p < 0.05.

Bokhari et al. 9

been investigated for its phytochemical composition

and anti-inflammatory potential against carrageenan-

induced paw edema. This conventional model of pedal

edema is used widely for acute inflammatory response

(Gamache et al., 1986). A number of primary responses

are generated at the site of edema formation, which

includes enhanced capillary permeability and leuko-

cyte infiltration. This response is accompanied with

alteration in the metabolic changes along with migra-

tion of neutrophils and macrophages. Arachidonic

acid is metabolized by cyclooxygenase and lipoxy-

genase pathways (Crunkhon and Maecock, 1971),

with the consequent release of inflammatory mediators

such as histamine, serotonin, bradykinin, nitric oxide

(NO), and prostaglandins (Omote et al., 2001; Vinegar

et al., 1969). Carrageenan administration in this experi-

ment causes an acute inflammatory stage (Ishola et al.,

2011, 2012; Kumar et al., 2004). Triphasic event has

been suggested for the carrageenan-induced edema

formation in rat paw (Vinegar et al., 1969). During

the initial phase, histamine and serotonin are liber-

ated, followed by release of kinins in the second

phase, whereas prostaglandins, lysosomes, bradyki-

nins, and protease are released in the third stage of

paw edema. It has been reported that carrageenan in its

later stage of treatment causes synthesis of NO from

peripheral NO synthase isoforms (Szolcsanyi et al.,

1998). Treatment of BPME and fractions showed

anti-edematous effects at all stages of edema develop-

ment. However, BPME and BPBE of B. procumbens

depicted significantly higher anti-inflammatory

response during all the stages of inflammation com-

parable with the control drug diclofenac potassium.

It was established in earlier studies that later stage

of edema development is more prone to anti-

inflammatory active constituents (Di Rosa et al.,

1971). These results suggested that B. procumbens

might attenuate both early and delayed phases of

carrageenan-induced inflammation probably by the

inhibition of histamine and NO release.

Conclusion

The experimental results indicated that B. procumbens

possesses marked antioxidant and anti-inflammatory

capacity. These results suggested the amelioration of

ROS, which may be attributed to the marked concen-

tration of polyphenolics in B. procumbens. So it can

be inferred that these natural compounds involved in

treatment for inflammation.

Acknowledgement

We would like to thank Higher Education Commission

(HEC) Government of Pakistan for providing support (no.

20-1556/R&D/092234) to this project.

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