CB et al. DOI: 10.5138/09750185.

1835

International Journal of Phytomedicine

2018;10(1):23-29

Original Research Article

In vitro antioxidant anti inflammatory and

cytotoxicity activities from Hexane extract of
Bryonopsis laciniosa fruits
Sanjeevkumar CB1, Ramesh L Londonkar1* and Umesh Madire Kattegouda1

Abstract

Bryonopsis laciniosa also known as “Shivlingi” annual climber with bright red fruits and is reported to be highly medicinal in
India. As a folk medicine, the plant is used in treatment of broad range of diseases and disorders. In the present study, Hexane
extract of B. laciniosa fruits were used to evaluate in vitro anti inflammatory, antioxidant and Cytotoxicity (towards MCF-7 cell line)
activities. In vitro anti inflammatory activity by inhibition of protein denaturation, antioxidant assays like DPPH, ABTS, H2O2 and
FRAP were used to measure the antioxidant capacity of the hexane extracts and cytotoxicity activity using MCF-7 breast cancer
cell line. Hexane extract showed the effective antioxidant activity in all assays compared to ascorbic acid and BHT. The results
for In vitro anti inflammatory activity of hexane extract and Dichlofenac drug were equivalent, hexane extract showed promising
activity for inhibition of protein denaturation assay. The cytotoxicity activity from hexane extract was noticeable against MCF-7
cell line. The overall results show potential application of Bryonopsis laciniosa fruits suggesting their potential application as a
health-promoting functional ingredient or natural preservative in foods.

Keywords: Bryonopsis laciniosa fruits; antioxidant; anti inflammatory activity; cytotoxicity

Introduction rosis, cardiovascular disease, cataracts, rheumatoid arthritis, in-

The adverse effects free radicals and oxidative stress on public flammatory disorders, anemia, asthma, cancer, and Parkinson’s
health have become a serious concern in last decades which is and Alzheimer’s diseases, as well as ageing [3] [4]. To overcome
mainly caused due to unhealthy life style and increased pollu- this problem, scientists suggest using of natural plant sources as
tion. Under stress, the concentration of reactive oxygen species a diet rich in antioxidant compounds. Medicinal plants are rich
(ROS) (e.g., superoxide anion radicals, hydroxyl radicals, singlet in antioxidants traps free radicals and therefore act as a type of
oxygen and hydrogen peroxide) in our bodies is higher than the preventive medicine [5]. The increasing research on antioxidants
concentration of enzymatic antioxidants (e.g., superoxide dis- and the protective role of phytoconstituents have grabbed full at-
mutase, glutathione peroxidase and catalase) and non-enzymatic tention and favored the development of the functional food and
antioxidants (e.g., ascorbic acid, vitamin E and glutathione) [1]. plant based medicine market. The plant kingdom offers a wide
This imbalance in the body leads to damage to bio molecules range of natural antioxidants. However, little is known about the
like lipids, proteins, carbohydrates and DNA, and consequently practical usefulness of most of them. Many herbal beverages,
induces degeneration, destruction and toxicity of various organic frequently used in folk medicine, have antioxidant and pharma-
molecules [2]. The increased accumulation of free radicals ini- cological properties linked with the presence of phenolic com-
tiate the development of major diseases, including atheroscle- pounds, especially flavonoids [6] [7].
Bryonia laciniosa syn Bryonopsis laciniosa (N.O. Cucur-
*Correspondence: [email protected] bitaceae) is a plant commonly known as ‘Shivlingi’ in India. The
1
Department of Biotechnology, Animal, Gulbarga University, leaves and fruits are reported to be highly medicinal value [8].
Pharmaceutical Biotechnology Laboratory, Kalburgi, India The whole plant has a bitter taste, and is a general tonic with mild
Full list of author information is available at the end of the article.

Received: 01 Feb 2016, Accepted: 06 Oct 2016

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CB et al. International Journal of Phytomedicine.2018;10(1):23-29

laxative effect. The plant is also used against snake bite (part not Evaluation of in vitro anti inflammatory activity
specified). Leaves are commonly used for hepato-depurative dis- In vitro anti-inflammatory activity of the hexane extract was
orders (internal use) and decoctions of the whole plant for skin tested by the method of Sangita Chandra et al. (2012) [10] with a
inflammations (external and topical use). However, local heal- slight modification. The reaction mixture (5 ml) consisted of 0.2
ers and key informants avoid using plant extracts in humans for ml of egg albumin (from fresh hen’s eggs), 2.8 mL of phosphate
long periods, so as to prevent a certain degree of toxicity and buffered saline (PBS, pH 6.4) and 2 ml of varying concentra-
adverse effects, which can vary considerably according to the tions of the hexane extract (final concentrations 62.5, 125, 250,
method of preparation, doses and physical condition of the indi- 500, and 1000 µg/ml). An equal volume of double-distilled wa-
vidual. Apart from leaves, Fruits are used as aphrodisiac, tonic. ter served as a control. The mixtures were incubated at 37 ±2
Sharp, cutting, lancinating or tearing pain, inflammation with ◦
C in an incubator for 15 min and then heated at 70 ◦ C for 5
muscular tension is cured by this plant [9] . In contrast, the fruits min. After cooling, their absorbance was measured at 660 nm
of the B.laciniosa have more potential pharmacological activi- (UV-1800 spectrophotometer, Shimadzu), with the vehicle used
ties in comparison with other parts of the plants (Stem, Leaves as a blank. Diclofenac sodium was treated similarly for determi-
and Seeds). In this study, the antioxidant activity in vitro anti in- nation of absorbance and used as a standard at the final concen-
flammatory properties and cytotoxicity were evaluated and com- trations of 62.5, 125, 250, 500, and 1000 µg/ml. The percentage
pared. inhibition of protein denaturation was calculated by using the
following formula:

Material and Methods % Inhibition = (Vt / Vc − 1) × 100,

where Vt = absorbance of the test sample, Vc = absorbance of
the control.
Chemicals
The extract/drug concentration giving 50% inhibition (IC50)
Chemicals, such as 1,1 diphenyl-2-picrylhydrazyl (DPPH), 2,20-
was determined by plotting the percentage of inhibition relative
azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammo-
to the control against treatment concentrations.
nium salt (ABTS, 98% purity), potassium ferricyanide, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),
trichloroacetic acid (TCA), dimethyl sulfoxide (DMSO), ferric In vitro antioxidant activity
chloride, and aluminium chloride, were purchased from Sigma The free radical scavenging activity of the hexane extract was
Chemicals (Steinheim, Germany). Solvents such as methanol, determined by using various in vitro assays such as DPPH,
chloroform and hexane were purchased from Merck (Germany). ABTS, Reducing power and Hydrogen peroxide assay.
Butylated hydroxytoluene (BHT) and ascorbic acid were pur-
chased from HiMedia (India). All chemicals and solvents used
DPPH radical scavenging activity
in the study were of analytical grade.
Free radical scavenging activity of the hexane extracts were de-
termined by using a stable 2,2-diphenyl-1-picrylhydrazyl radical
Plant material
(DPPH). DPPH is a free radical of violet colour. The antioxi-
Fruits of B. laciniosa L plants (5 kg) were collected from the dants in the sample scavenge the free radicals and turn it into
Gulbarga University campus during the month of December yellow colour. The change of colour from violet to yellow is
2013. proportional to the radical scavenging activity.
DPPH radical scavenging activity was assayed by the method of
Extract preparation Braca et al. (2002) [11] with a slight modification. Briefly, the
The collected fruits of B. laciniosa were washed under running assay contained 0.9 ml of DPPH solution (0.004% w/v) was
tap water to remove dust particles and dried in the shade for 72 prepared in 95% methanol and various concentrations of hexane
h. The air-dried fruits were coarse powdered and stored in an extracts and standards with the stock solutions (20 mg/ml) in the
air-tight dark glass bottle at 25 ◦C for further use. The powdered same solvent and made up to 1 ml with ethanol. The contents
fruits (100 g) were successively extracted by three different sol- were mixed well immediately and then incubated for 30 min
vents (350 ml each), from non-polar to polar, i.e., hexane, chlo- at room temperature (24 –30◦ C). The degree of reduction of
roform and methanol, using Soxhlet extraction. The successive absorbance was recorded in UV–Vis spectrophotometer at 517
extracts of different solvents were dried, weighed and stored at - nm. Ethanol (95%), DPPH solution and ascorbic acid (AA) were
4◦ c for further use. used as blank, control and reference standard respectively.

24
 CB et al. International Journal of Phytomedicine.2018;10(1):23-29

DPPH radical scavenging activity (%) = (Ac − As)/Ac × Hydrogen peroxide solution (1 mm) was prepared with 50 mm
100, phosphate buffer (pH 7.4). Different concentrations of the hex-
Where Ac is the absorbance without the samples and As is the ane extract (1 ml) were allowed to react with 0.6 ml of the hy-
absorbance in the presence of the samples. drogen peroxide solution. Absorbance was determined at 230
nm after 10 min against a blank solution containing phosphate
ABTS radical scavenging activity buffer without hydrogen peroxide. The scavenging activity of
chloroform extract was compared with Ascorbic acid and BHT.
ABTS radical cation decolonization activity was assayed by
The hydrogen peroxide scavenging activity was calculated ac-
the method of Thoo et al. (2013) [12] and Surveswaran et al.
cording to the following equation:
(2007) [13] with a slight modification. ABTS radical cations
were generated by reacting 7 mm ABTS with 2.45 mm potas- Hydrogen peroxide scavenging activity, % = (Ac — As)/Ac)
sium per sulfate (1:1). The mixture was left to stand for 12 to × 100,
16 h in the dark at room temperature. The ABTS radical cation Where Ac is the absorbance without the samples and As is the
solution (100 µL) was then diluted with ethanol (3.9 ml) to give absorbance in the presence of the samples.
an absorbance of 0.700 ±0.02 at 734 nm. Different concentra-
tions of the hexane extract were mixed with diluted ABTS rad- Preparation of cell line
ical cation solution (1 ml). The mixture was vortexed and left The cytotoxicity assay was carried out using MCF-7 cells (hu-
to stand at room temperature for 6 min. The absorbance of the man breast carcinoma). The cell line was procured from the
resulting solution was measured at 734 nm using a UV-visible National Centre for Cell Sciences (NCCS, Pune, India). Stock
spectrophotometer. Ascorbic acid and BHT were used standard cells were cultured in DMEM supplemented with 10% inacti-
reference for ABTS radical scavenging activity. The ABTS radi- vated foetal bovine serum (FBS), penicillin (100 IU/ml), strep-
cal scavenging activity was calculated according to the following tomycin (100 mg/ml) and amphotericin B (5 mg/ml) in a humid-
equation: ified atmosphere at 5% CO2, 37 ◦ C until reaching confluence.
ABTS radical scavenging activity, % = (Ac − As)/Ac × 100, The cells were dissociated with TPVG solution (0.2% trypsin,
0.02% EDTA, 0.05% glucose in PBS). The stock cultures were
Where Ac is the absorbance without samples and As is the
grown in 25 cm2 culture flasks, and all experiments were carried
absorbance in the presence of the samples.
out in 96-well microtitre plates (Tarsons India Pvt. Ltd., Kolkata,
India).
Ferrous reducing-antioxidant power (FRAP)
Total reducing power of the hexane extract was estimated ac- MTT cell viability screening
cording to Oyaizu (1986) [14] and Oh et al. (2013) [15] with Cell viability was assessed by measuring the amount of insoluble
a slight modification. In FRAP, potassium ferric cyanide is re- formazan formed in live cells based on the reduction of 3-(4,5-
duced to ferrous cyanide by antioxidants present in the sam- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
ple. The hexane extract of varying concentration (50 to 800 µl) salt [17] (Francis and Rita, 1986). Cell suspensions at 1 × 105
were mixed with 0.25 ml phosphate buffer (0.2 mm, pH 6.6) and cell/ml were seeded in 96 well microtiter plates. Hexane ex-
1% potassium ferricyanide (0.25 ml). The reaction system was tract in different concentration were added into the wells. MTT
closed and incubated at 50◦ C in a water bath for 20 min. After reagent was added after 72 h exposure followed by dissolution
the incubation period, 0.25 ml of 10% trichloroacetic acid was of formed formazan crystal using DMSO. Optical density was
added to the assay system and the contents were mixed well. The read with micro plate reader at 540 nm. The dose-response curve
mixture was centrifuged at 5,000 rpm for 5 min. The supernatant is plotted and concentration which gave 50% inhibition of cell
(0.5 ml) was mixed with an equal volume of distilled water and growth (IC50) is calculated. Concentration that inhibits 50% of
0.1 ml of ferric chloride solution (0.1%, w/v). The colour devel- cell viability was used as a parameter for cytotoxicity.
oped was read at 700 nm using UV-Visible spectrophotometer
and the results were compared with Ascorbic acid and BHT as Statistical analysis
a standard reference, the readings were expressed as the mean Data were expressed as mean ± SD. Analyses of variance
absorbance value. (ANOVA) were done for the comparison of results using Fis-
cher’s test. Statistical significance was set at p< 0.05.
Hydrogen peroxide-scavenging activity
The Hydrogen peroxide-scavenging activity of hexane extract
was determined by the method of Cetinkaya et al. (2012) [16] .

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CB et al. International Journal of Phytomedicine.2018;10(1):23-29

Results & Discussion dent increase in percentage inhibition. The results revealed that
the fruit extracts had the higher DPPH radical scavenging abil-
ity similar to Ascorbic acid and BHT. The scavenging activity
In vitro anti inflammatory activity
of Hexane extract, Ascorbic acid and BHT were 87.22 ± 1.33,
In the present study the protein denaturation bio assay was se- 99.05 ± 0.29 and 95.08 ± 0.57 respectively are shown in Graph
lected for in vitro assessment of anti inflammatory properties 2. The IC50 values (concentration of the extract that was able to
of Hexane extract of B. laciniosa fruits. Denaturation of tissue scavenge half of the DPPH radical) for the hexane extract, ascor-
proteins is one of the well-documented causes of inflammation bic acid and BHT were 223.5 ±1.2, 31.66 ±2.3 and 28.3 ±2.4
and arthritic diseases. The production of auto-antigens in certain µg/ml respectively are presented in Table 1. Thus, by comparing
arthritic diseases may be due to denaturation of tissue proteins IC50 values of DPPH assay, BHT showed an effective antioxi-
in vivo [18] [19]. The in vitro anti-inflammatory effect of the dant activity followed by ascorbic acid and Hexane extract. By
Hexane extract was evaluated for its ability to prevent denatu- comparing overall results, hexane extract has the potential free
ration of egg albumin. The results are summarized in Graph 1. radical trapping ability by donating protons and can serve as a
The hexane extract compared with Diclofenac sodium showed source of free radical inhibitors or scavengers possibly acting
similar anti inflammatory activity. The difference was further as primary antioxidants, along with the reference standards that
confirmed by comparing the respective IC50 values. The hexane have similar scavenging activities.
extract showed an IC50 value of 362.53 ±0.67 µg/ml, whereas
that of diclofenac sodium was found to be 320.83 ±1.17 µg/ml.
Therefore, from the findings of this preliminary experiment it
can be concluded that hexane extract of B.laciniosa have a no-
ticeable anti inflammatory effect against inhibition of protein de-
naturation in vitro.

Figure 2 DPPH radical scavenging activity of Hexaneextract,

Ascorbic acid and BHT.Values are means ± SD (n = 3).

ABTS radical scavenging activity

Figure 1 In vitro anti-inflammatoryactivity of Hexane extract and To determine free radical scavenging activity hexane extract
Standard (Diclofenac sodium) Values are means ± SD(n = 3). ABTS radical scavenging assays are performed to estimate the
free radical scavenging activity of a sample and are based on
Antioxidant assays the reduction of these radicals. The hexane extract exhibited the
highest radical scavenging activity when compared to Ascorbic
Phenolic compounds exhibit their antioxidant activity through
acid and BHT. The ABTS radical scavenging activity of the hex-
their radical scavenging effects. Radical scavenging activity is
ane extract, Ascorbic acid and BHT was 79.77 ± 0.44, 69.21±
very important owing to the deleterious role of free radicals in
biological systems and generally proceeds via hydrogen atom 0.34 and 54.83 ± 0.52 % respectively are shown in Graph 3. IC50
values for hexane extract, ascorbic acid and BHT were 342.81
transfer or donation of electrons [20] .
± 0.67, 507.6 ±2.05 and 401 ±2.35 µg/ml, respectively (Ta-
ble 1). The results clearly imply that the hexane extracts inhibit
DPPH radical scavenging activity
ABTS radical or scavenge the radical in a dose dependent man-
In this study, the hexane extracts showed tendency to quench ner. ABTS•+ radical is generated from oxidation of ABTS•+ by
the DPPH free radicals, as indicated by the concentration depen- potassium persulphate, is a good tool for determining the an-

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CB et al. International Journal of Phytomedicine.2018;10(1):23-29

Table 1 IC50 values of DPPH, ABTS, Hydrogen Peroxide andAbsorbance of Reducing power of hexane extract of B.laciniosa fruits.

Samples DPPH (µg/mL) ABTS (µg/mL) H2O2 (µg/mL) Reducing power (Abs at 700 nm)
Hexane Extract 223.5 ±1.2 342.81 ± 0.67 186.91±0.53 0.827±0.02
Ascorbic acid 31.66 ±2.3 507.6 ±2.05 73.66 ±0.94 1.33±1.24
BHT 28.3 ±2.4 401 ±2.35 66.6 ±0.9 2.03±1.06

tioxidant activity of hydrogen-donating and chain breaking an-

tioxidants [21]. This assay is applicable for both lipophilic and
hydrophilic antioxidants. The radical scavenging activity of the
hexane extracts of B.laciniosa were estimated by comparing the
percentage inhibition of formation of ABTS•+ radicals with that
of Ascorbic acid and BHT. The extracts exhibited the highest
radical-scavenging activities when reacted with the ABTS radi-
cals.

Figure 4 Hydroxyl radical scavenging activity of Hexaneextract,

Ascorbic acid and BHT. Values are means ± SD(n = 3).

Ferrous reducing-antioxidant power (FRAP)

In the reducing power assay, the presence of antioxidants in
the extract of B.laciniosa would result in the reduction of
Fe3+/ferricyanide complex to its form. The reducing power
of compound may serve as a significant indicator of its potential
Figure 3 ABTS radical scavenging activity of Hexaneextract,
antioxidant activity [22] . The ferric reducing power of the hex-
Ascorbic acid and BHT. Values are means ± SD(n = 3). ane extract was determined by comparing with that of Ascorbic
acid and BHT. The increased absorbance values of the extracts
at 700 nm indicate an increase in reductive ability. Absorbance
Hydrogen peroxide scavenging activity values of hexane extracts are presented in Graph 5. The reduc-
The hydrogen peroxide radical-scavenging activity of the hex- ing power of BHT was found to be significantly higher than
ane extracts of B.laciniosa was estimated by comparing the per- those of Ascorbic acid and hexane extract. The reducing powers
centage inhibition of formation of peroxyl radicals with that of of hexane extract, Ascorbic acid and BHT were 0.827±0.02,
Ascorbic acid and BHT. Hydrogen peroxide scavenging activ- 1.33±0.04 and 2.03±0.06 respectively. The reducing power in-
ity of hexane extracts, Ascorbic acid and BHT are presented in creased with the increase of their concentrations. This data imply
Graph 4. Hexane extracts showed moderate inhibition against that these extracts have significant ability to react with free radi-
peroxyl radical which was less in comparison with Ascorbic cals to convert them into more stable nonreactive species and to
acid and BHT (78.82 ± 0.53, 93.94 ± 0.09 and 95.54 ± 0.73). terminate radical chain reaction.
The results clearly indicate that hexane extracts of B. lacin-
iosa are highly potent in neutralizing hydrogen peroxide radi- Cytotoxicity assay
cals. IC50 values of hexane extract, ascorbic acid and BHT were The search for new potential bio-active compounds in medici-
186.91±0.53, 73.66 ±0.94 and 66.6 ±0.9 µg/ml respectively are nal plant is a realistic and promising strategy for prevention and
shown in Table 1. H2O2 itself is not very reactive, but it can cure of many deadly diseases. In the present study B.laciniosa
sometimes be toxic to cell because it may give rise to hydroxyl fruits was evaluated for cytotoxic activity on MCF-7 cell line
radical in the cells. The results showed that B. laciniosa fruit using MTT (3-(4, 5-dimethylythiazol-2- yl)-2, 5-diphenyl-2H-
extract have an effective H2O2 scavenging activity. tetrazolium hydrobromide) assay. The ability of the cells to sur-
vive a toxic insult has been the basis of most Cytotoxicity assays.

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CB et al. International Journal of Phytomedicine.2018;10(1):23-29

ing activity and Cytotoxicity against Breast cancer. Hexane ex-

tract can be explored for its applications in the prevention of free
radical related diseases. The overall activity is due to the type of
phenolics groups present and may be some unidentified antioxi-
dants anti inflammatory and anti cancer compounds. The hexane
extract of B.laciniosa allows us to conclude that extracts is good
candidates for further studies of activity-monitored fractionation
to identify their active components.

Conflict of interest statement

The authors have declared that there is no conflict of interest.
Figure 5 Reducingpower of Hexane extract, Ascorbic acid and
BHT. Values are means ± SD(n = 3).

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