Biotechnological

Communication

Biosci. Biotech. Res. Comm. 9(3): 567-575 (2016)

Antioxidant potential and in-silico analysis of

compounds from

Citrus sinensis

peel extracts

against TGF-β isoforms

Vineet Singh, Devender Arora and Ajeet Singh*

Department of Biotechnology, G B Pant Engineering College, Pauri Garhwal, Uttarakhand, INDIA

ABSTRACT

Malta (Citrus× sinensis) is a local variety of sweet orange which has been developed by hybridization between

orange and lemon from hills of Uttarakhand, India. Malta peel is generated in huge amount as a waste product of

food processing units. The present work is an effort to establish the potential anti oxidative, anti-carcinogenic and

anti-cancerous role of Malta peel. Analysis revealed the presence of antioxidant enzymes like super oxide dismutase

(SOD), catalase, glutathione peroxidase; which can check the reactive oxygen species (ROS) level and non-enzymatic

antioxidant activity as observed in FRAP-assay (TPPZ method). Presence of anti-oxidative and anti-allergic vitamins

in ample amount i.e. Vit. E, Vit. C, polyphenols and tumor preventive  avonoids (limonene) has been con rmed by

GC-MS analysis, these compounds are known to possess an anti-carcinogenic role as well. Further to understand the

possible anti-cancerous role, an in silico study was conducted using TGF isoforms (TGF-

and TGF-

) as ligands.

TGF signaling has a mechanistic role in tumor suppression and a previous medical study has con rmed its indul-

gence in different forms of cancers e.g. pancreatic cancer, renal cancer, breast cancer and lymphoma.

KEY WORDS:

MALTA PEEL, SOD, CATALASE, GLUTATHIONE PEROXIDASE, FRAP, TGF-



1, TGF-



2, ANTICANCER

567

ARTICLE INFORMATION:

*Corresponding Author: ajeetsoniyal@gmail.com

Received 11

Aug, 2016

Accepted after revision 5

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

Sweet oranges are one of major fruit crops which are

well distributed around the globe from North Ameri-

can continent to Asia. Most of the varieties of sweet

orange which are available today are result of mutation

and hybridization, occurring from past centuries (Khan

2007). In Indian subcontinent, many varieties of sweet

oranges which occur are quite different from each other

and have different names. Malta (Citrus sinensis or C.

var. sinensis (L.) Osbeck), a variety of sweet orange is

a delicious major fruit crop of northern hilly region of

568

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Vineet, Devender and Ajeet

Uttarakhand-India. In Uttarakhand, citrus species

occupy about 13.90 per cent (27400 ha) of total fruit

area and Garhwal region is one of the major citrus grow-

ing regions of Uttarakhand with 50.20 per cent (13755

ha) of the total citrus producing area (Gunwant et al.

2003).

Malta is enriched with an array of nutrients and mul-

tiple health bene ts. Malta fruit is used in different forms

by beverage and food industry i.e. in form of juice, squash

preparation, or as whole for other purposes. Malta pom-

ace and peel is the major bio-mass which does not get

utilized signi cantly and dumped as an agricultural waste

(Manthey and Grohmann 2001, Anagnostopoulou et al.

2006, Mallick and Rafeeq 2016, and Ra q et al., 2016).

Although, malta peel has high nutritional value as it con-

tains carbohydrate, vit. A, vit. C, vit. E, fatty acids, differ-

ent  avonoids and polyphenols

Manthey and Grohmann

2001, Anagnostopoulou et al. 2006, Mallick and Rafeeq

2016, and Ra q et al. 2016).

The present work is focused on the anti-carcinogenic,

anti-cancerous ability of malta peel as the peel extracts

were tested for biological activities such as antimicrobial

activities, enzymatic and non-enzymatic anti-oxidant

assays. Along with that, the presence of compounds

in malta peel extracts (hexane, ethanol and methanol)

were studied through GC-MS analysis and further in sil-

ico studies of compounds obtained were carried out by

docking against the targeted protein receptor to estab-

lish the anti-carcinogenic role.

TGF-

and TGF-

are chosen as molecular targets

for chemoprevention. TGF is a multifunctional cytokine

(Bierie and Moses 2006) which is synthesized by almost

every cell of eukaryotic organism and all of them have

speci c receptors for these peptides. Previous stud-

ies have suggested that, through various cell functions

i.e. cell differentiation, cell proliferation, cell migration

and apoptosis, (

Bierie and Moses 2006, Drabsch and Ten

Dijke 2012), TGF promotes different cancer metasta-

sis as lung, breast, bone, colorectal, prostate, pancreatic,

lymphoma and renal cortex metastasis by affecting sur-

rounding tumor microenvironment, (Slamon et al. 1987,

Levy and Hill 2006, Drabsch and Ten Dijke 2012, Huang

and Chen 2012, Imamura et al. 2012, Yu et al. 2014 and

Valvona et al. 2016

The TGF ligands are synthesized in the form of dimer

as raw hormones inside the cell (Gray and Mason 1990

and Massagué 2008). These are secreted into the extra-

cellular matrix, where they are activated as signaling

molecule by the cleaving action of furins and some con-

vertases. Activated TGF cytokines can then signal by

bringing together two pairs of receptor serine/threonine

kinases, the type 1 and type 2 receptors forming a het-

erodimer complex (Dubois 1995, Constam and Robert-

son 1999, Padua and Massagué 2009). Human genome

can encode seven type 1 receptors (ALK1, ALK2, ALK3,

ALK4, ALK5, ALK6, ALK7) and  ve type 2 receptors

(Padua and Massagué 2009, Pickup et al. 2013, Miles

et al. 2013) which are paired in different combination

as receptor complex for various members. The TGF-

ligand protein preferentially signals through the TR-2,

type-2 receptor and ALK-5 type-1 receptor, (Yang et al.

2013, Tazat et al. 2015).

TGF activation leads to the emergence of different

regulatory protein, inducing transcription of different

target genes whose functions are differentiation, prolif-

eration and activation of many other cells (Bierie and

Moses 2006, Siegel and Massagué 2003, Derynck and

Zhang 2003

). TGF signaling has a mechanistic role in

tumor suppression and regulates cancer through func-

tion via two mechanisms. First, within the tumor cell

itself or via host tumor cell interaction (Meulmeester and

Ten Dijke 2011, Zheng et al. 2014) which can forge the

anti-cancerous role of TGF.

MATERIAL AND METHODS

Malta peels were collected from the local juice produc-

ers of Pauri, Uttarakhand, these peels were shade dried

and converted into  ne powder by grinding in a blender

for further processing. TPTZ (2,4,6 Tripyridyl-s-triazine),

different solvents and chemicals were of analytical

grade and purchased from HiMedia Pvt. Ltd. (Mumbai,

India) and Sigma-Aldrich chemicals Pvt. Ltd. (Bengal-

uru, India). Malta peel powder (10 gm) was extracted

with methanol, ethanol and n-hexane at 60

C for 6 hours

in a Soxhelet apparatus and extraction was repeated till

solution became colorless. Extracts were collected and

concentrated in a rotary vacuum-evaporator and stored

at 4

C for further experiment. For non-enzymatic anti-

oxidant assay (FRAP-method) extracts were concen-

trated to form powder in a lyophilizer.

Antibacterial activity of extract prepared in different

solvents i.e. methanol, ethanol, hexane was examined

against microbes as broad spectrum antibacterial agent.

Two different strains of gram (+) S. aureus (MTCC 740),

B. subtilis (MTCC 441) and gram (-) E. coli (MTCC 443),

S. typhi (MTCC 733) were taken for antibacterial activ-

ity obtained from the Institute of Microbial Technology-

Chandigarh, India. The bacterial strains were grown

in MHA (Muller-Hinton Agar) at 37

C for 24 hr. FRAP

(Ferric reducing ability of plasma or, Ferric ion reducing

antioxidant power), is a novel method for quantitative

assay to analyze the antioxidant potential of the extract

at low pH (Benzie 1996, Liu 1982, Stookey 1970).

The reaction mixture was composed of dried extract,

0.3 M sodium acetate, 20 mM ferric chloride, 40 mM

hydrochloric acid and TPTZ (2,4,6 Tripyridyl-s-triazine).

Incubate the mixture at 37

C for 15 minutes, after that

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY 569

Vineet, Devender and Ajeet

absorbance were taken at 593 nm and compared with

standard curve of ascorbic acid. Fresh 1 gm peel were

taken and ground with 5 ml phosphate buffer (0.067 M)

in a pre-chilled mortar-pestle at -20

C, and was centri-

fuged at 10,000 ×g at 4

C for 10 minutes. The superna-

tant was collected and stored at 4

C for the further use,

(Giannopolitis and Ries 1977, Pandey et al. 2012).

Protein content of the crude-enzymatic extract was

determined by the Bradford method, where Bovine albu-

min serum (BSA) was taken as standard (Bradford 1976).

To check the level of ROS, cells have many of antioxidant

enzymes. Three of the major antioxidant enzymes that

are supposed to be in a living system to prevent or repairs

such damage caused by ROS are superoxide dismutase

(SOD), catalase and glutathione peroxidase (GPx).

SOD activity was determined by the modi ed method

of (

Giannopolitis and Ries 1977). Reaction mixture con-

tains 50 mM phosphate buffer (pH 7.8), 13 mM methio-

nine, 75μm NBT, 0.1 mM EDTA, 100 μl of enzyme

extract, at last 2 mM Ribo avin and reaction was ini-

tiated by exposing the tubes to the  uorescent lamp.

Absorbance was taken at time interval of 5 minute and

15 minute at 560 nm.

The catalytic activity of SOD was calculated by the

following formula.

SOD (unit/ml) = [(V/v) – 1] × dilution factor

Where, V and v were absorbance in absence and in pres-

ence of enzyme respectively.

The assay was carried out by standardized method of

(

Sandhir and Gill 1995). The enzyme extract 40μl were

taken in reaction mixture containing 3ml H

-Phos-

phate buffer (pH 7.0) and change in absorbance were

read against the buffer blank at 240 nm for 60 seconds.

The catalytic activity was calculated by given for-

mula.

Units in the sample mixture = 17/T

Where, T was the time required to decrease the absorb-

ance by the factor of 0.05.

The assay was carried out by modi ed method of

(

Kaur et al. 2006), which contains 2 ml 0.5 M phosphate

buffer (pH 7.0), 0.1 ml 1mM EDTA, 0.1mM sodium azide,

0.10 ml 1mM GSH, 0.1 ml 2mM NADPH, 0.01 ml 0.25

mM H

and 0.1 ml 10% PMS in a total volume of 3 ml,

50 μl of sample was added at last except blank and the

absorbance was taken at 340 nm.

The GPx activity was estimated as nmol NADPH

oxidized/minute/mg protein with the molar extinction

coef cient of 6.22 X 10

−1

by using the formula.

GPx = (Δ O. D × Vol. Of assay × 1000) / (6.22 × Vol.

of enzyme × protein content)

Shimadzu QP2010 ultra series gas chromatograph-

mass spectrometer was used for the purpose of GC-MS

analysis of sample extract. The compounds were iden-

ti ed by comparison of their mass spectral data with

PubChem and other available literature data. The data-

base of nearly 200 compounds were obtained after anal-

ysis of sample extract. 3-D model of each compound

was obtained from PubChem in SDF  le format and

unknown structure were drawn with the help of Marvin

bean and converted into 3-D, SDF  le format. Further,

obtained geometrics were optimized according to stand-

ard protocol for docking.

The three dimensional structure of TGF-

was

obtained from the Protein Data Bank (.pdb) and structure

of TGF-

was retrieved through homology modeling as

complete structure was not available. For homology

modeling FASTA sequence of TGF-

is obtained from

the Uniprot with accession number (P36897) which was

allowed to blast with human sequence. The sequence

with maximum resemblance was selected and converted

into a pdb structure with the help of SWISS-MODEL;

a protein structure homology modeling server. Differ-

ent parameters as protein quality (ProQ) (

Wallner and

Elofsson 2003), z-score (ProSA) (Wiederstein and Sippl

2007, Sippl 1993), veri ed 3-D structure, Ramachandran

plot, protein and active site stability (Saves) are exam-

ined (Liithy et al. 1992, Bowie et al. 1991) to validate the

stability of retrieved structure of TGF-

. After stability

validation of the protein (TGF-

) other docking pre jobs

were done according to standard protocol.

Molecular docking is a computational procedure that

attempts to predict noncovalent binding of macromole-

cule or macromolecule and a ligand molecule ef ciently.

AutoDock structure  les PDBQT which can be seen as an

extension of the PDB  le format of all the compounds

present in extract and target proteins were generated

with the help of mgltool and docked at the binding site

of the receptor proteins and simulation was performed

on AutoDock Vina. AutoDock Vina work on Lamarckian

algorithm and geometric algorithm, calculates its own

grid map of dock site (

Trott and Olson 2010). Grids were

prepared for each protein with the same exact center and

the size of the bounding box set. This way it combines

a rapid grid based method for energy evaluation and 10

maximum negative energy possessing compounds were

obtained.

To analyze the toxicity of the docked compound

this had good negative energy and could have poten-

tial drug like qualities. An online server mcule.com had

been used for the prediction, for this smile  le format

of the compounds were uploaded on the server and

after analysis server gives the result (

Kiss et al. 2012).

Complex generated from AutoDock are visualized using

LigPlot+ v.1.4.5 (software), which shows the presence of

H-bond between ligand and target protein (Wallace et al.

1995).

570

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Vineet, Devender and Ajeet

FIGURE 1. Antibacterial activity of unripen ma lta

peel-hexane extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Hexane

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

RESULTS AND DISCUSSION

Reactive oxygen species (ROS) or free radical can damage

DNA, induces mutation and negatively affects the DNA-

repair, which results in the inactivation of the many of

tumor suppression gene. GC-MS analysis revealed that

extract contains ample amount of antioxidant content

as whole like as vit. E, vit. C which have been shown

to be anti-carcinogenic against doxorubicin induced

chromosomal aberration (Antunes and Takahashi 1998)

along with that vit. C had been reported for its role in

decreasing the frequency of induced mutation (Khan

and Sinha 1993). Extract also shows high concentration

of limonene, a  avonoid which reduces the in amma-

tory response by suppressing the production of reactive

oxygen species (ROS).

Antibacterial activity of the different extracts was

studied on both gram (+); S. aureus, B. subtilis and

gram (-); E. coli, S. typhi to examine the presence of any

possible bio-active compound which have capability to

check the growth of cell, while taking tetracycline and

respective solvents as control. Effective antibacterial

activity of unripen malta peel extract (Figure.1-3) con-

 rmed the presence of certain bioactive molecule which

have capability to check the cellular growth whereas,

there is signi cant loss of antimicrobial activity was

observed in ripen malta peel extract (Figure.4-6). This

denotes the loss of certain bio active compounds during

the due period of fruit ripening (Table 1-2).

Reactive oxygen species (ROS) are produced as a nec-

essary evil in several metabolic reactions. ROS i.e. super-

oxide, hydrogen peroxide, are cytotoxic and carcinogenic

in nature while their presence in lower concentration is

also required for the regulation of many of physiologi-

cal activities such as apoptosis, cell differentiation and

redox-sensitive signal transduction (

Allen and Balin

1989, Shibanuma et al. 1988 and Hockenbery et al. 1993).

FIGURE 3. Antibacterial activity of unripen malta

peel-methanol extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Methanol

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

FIGURE 2. Antibacterial activity of unripen malta

peel-ethanol extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Ethanol

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

FIGURE 4. Antibacterial activity of ripen malta

peel-hexane extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Hexane

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

Antioxidant activity of extracts were determined to

analyze the presence of antioxidant species which plays

crucial role against the ROS and minimizes the pres-

ence of free radicals which can damage cellular struc-

ture, DNA mutation and alteration in transcription

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY 571

Vineet, Devender and Ajeet

FIGURE 5. Antibacterial activity of ripen malta

peel-ethanol extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Ethanol

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

FIGURE 6. Antibacterial activity of ripen malta

peel-methanol extract was examined on (a.) E. coli

(b.) B. subtilis (c.) S. aureus (d.) S. typhi. Methanol

was taken as negative control and tetracycline as

positive control where volumes (Vol.) were made

in μl.

Table 1: Zone of inhibition of different strains of bacteria in presence of unripen malta peel extract (hexane, ethanol, methanol).

Tetracycline was taken as positive control and respective solvent as negative control. Where no antibacterial activity was shown

by any of negative control

Name

of strain

Hexane extract Zone of

inhibition (c.m.)

Positive

control

(c.m.)

Ethanol extract Zone of

inhibition (c.m.)

Positive

control

(c.m.)

Methanol extract Zone

of inhibition (c.m.)

Positive

control

(c.m.)

Vol.

(50μl)

Vol.

(100μl)

Vol.

(150μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(100μl)

Vol.

(150μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(50μl)

S. aureus 1.54 1.94 2.14 2.24 1.44 1.54 2.04 2.04 0.00 0.00 0.00 2.3

B. subtilis 1.64 2.04 2.34 3.84 1.34 1.64 1.84 3.64 1.42 1.60 2.00 2.9

E. coli 1.14 1.44 1.54 2.64 1.04 1.24 1.96 2.04 0.00 0.00 0.00 2.4

S. typhi 1.54 1.84 2.04 3.34 1.54 1.76 2.04 2.24 0.00 0.00 0.00 2.3

Table 2: Zone of inhibition of different strains of bacteria in presence of ripen malta peel extract (hexane, ethanol,

methanol). Tetracycline was taken as positive control and respective solvent as negative control. Where no antibacterial

activity was shown by any of negative control.

Name of

strain

Hexane extract Zone of

inhibition (c.m.)

Positive

control

(c.m.)

Ethanol extract Zone of

inhibition (c.m.)

Positive

control

(c.m.)

Methanol extract Zone

of inhibition (c.m.)

Positive

control

(c.m.)

Vol.

(50μl)

Vol.

(100μl)

Vol.

(150μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(100μl)

Vol.

(150μl)

Vol.

(50μl)

Vol.

(50μl)

Vol.

(l00μ)

Vol.

(150μl)

Vol.

(50μl)

S. aureus .00 .70 .80 2.1 .00 .00 .00 2.1 0 0 0 2.1

B. subtilis .70 .80 .80 3.10 1.00 1.00 1.10 3.2 1.10 1.2 1.2 3.1

E. coli .00 .00 .70 2.2 .00 .00 .00 2.00 .00 .00 .00 2.2

S. typhi .00 .00 .00 2.9 .00 .00 .00 2.5 .00 .00 .00 2.8

factors. For non-enzymatic antioxidant assay standard-

ized FRAP-method was used, which con rms the anti-

oxidant potential of the ripen malta peel extract (Figure.

7) as quantity of extract was increased there is fair incre-

ment of antioxidant potential had been observed. Pro-

tein concentration was calculated by Bradford method

and it found to be 0.00809 mg/ml and enzymatic activ-

ity of essential antioxidant enzymes viz. superoxide dis-

mutase (SOD), catalase and glutathione peroxidase (GPx)

were 4.136 U/ml, 0.66667 nmol H

decomposed/min/

mg protein and 18520.9 nmol NADPH oxidized/minute/

mg protein respectively, (Weydert et al, 2011).

In silico study of possible role of compounds present

in malta peel as shown by GC-MS analysis was carried

out, considering TGF-

and TGF-

molecular protein

as target. Structure of TGF-

was obtained through

molecular homology and stability of the retrieved struc-

ture was veri ed. As saves result shows, veri ed 3-D

structure of the retrieved structure as 93.94% of the

residue had an average 3D-1D score more than 0.2,

Vineet, Devender and Ajeet

572

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 7. Non enzymatic antioxidant assay by FRAP

method, signi cant increase in antioxidant activity of

extract was observed as amount of extract increased as

compared with standard curve of ascorbic acid.

FIGURE 8. Chromatogram of GC-MS analysis of ripen

malta peel hexane extract, presence of 66 different com-

pounds were con rmed

FIGURE 10. Chromatogram of GC-MS analysis of ripen

malta peel methanolic extract, presence of 19 different

compounds were con rmed

FIGURE 11. Results of ProSA shows the stability of TGF-

b1 model by analyzing NMR and X-ray crystallography

analysis.

FIGURE 9. Chromatogram of GC-MS analysis of ripen

malta peel ethanolic extract, presence of 35 different

compound were con rmed.

FIGURE 12. Phenol 3,5-bis (1,1-Dimethylethyl) with

TGF-

, attached at dock site and LigPlot of TGF-

with

Phenol,3,5-bis(1,1-Dimethylethyl), showing H-bond for-

mation between ligand and protein

whereas required score is 80%. where result of ProSA

were also con rmed the stability of the structure

(Figure 11) as, z-score of the structure was -8.13 and

required to be a stable structure was -5.0 along with

them Ramachandran plot of TGF-1 results also veri es

the stability of structure as 98% residues are in favored

region and 2% residues are in allowed region.

Result of ProQ also favored the retrieved struc-

ture TGF-

as Predicted LGscore: 5.689 (LGscore ≥ 4

extremely good model) and Predicted MaxSub: 0.545

FIGURE 13. (+) Alpha-Tocopherol with TGF-

attached at dock site and LigPlot of TGF-

with

(+) Alpha-Tocopherol, showing H-bond formation

between ligand and protein

Vineet, Devender and Ajeet

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY 573

(MaxSub > 0.5 very good model). Docking simulation

of peel compounds obtained after GC-MS analysis (Fig-

ure.8-10) was done with TGF-

and TGF-

and ten most

stable compounds were selected. After ADME-T predic-

tion two most favorable compounds on the basis of non-

cytotoxicity Phenol 3,5-bis (1,1-Dimethylethyl) and (+)

Alpha-Tocopherol (Table-3) were selected and there Lig-

Plot were drown to analyze the presence of any possible

H-bond formation between ligand and protein. LigPlot

con rmed the presence of hydrogen bond formed

(+)

Alpha-Tocopherol also had shown the maximum energy

(-9.1 k.cal/mol.) and reported for their medicinal proper-

ties (Nelson et al., 2016, Marubayashi et al. 1986) and

one H-bond formation with TGF-

(Figure.13) in dock-

ing results and had been con rmed as best compound

for possible drug, where as Phenol 3,5-bis (1,1-Dimeth-

ylethyl) was the most stable-non cytotoxic compound

with TGF-

(-6.8 k.cal/mol.) protein (Figure.12). In the

present study we found that malta peel extracts, which

is considered as an agriculture waste shown the presence

of potential bio-active compounds with nontoxic nature

that can play a major role in cancer research. 8-hydrox-

ylinalool shown the no cytotoxicity and highest stability

against TGF- isoforms as selected target, which plays a

crucial role in cancer

CONFLICT OF INTEREST

The authors don’t have any con ict of interest to declare.

ACKNOWLEDGMENTS

This study was conducted in the Department of Biotech-

nology, G. B. Pant Engineering College (GBPEC), Pauri

Garhwal (Uttarakhand), India. VS is thankful to AICTE

and DA is thankful to TEQIP-II (Technical Education

Quality Improvement Programme, Government of India)

for  nancial assistance.

REFERENCES

Allen RG. and Balin A.K. (1989) Oxidative in uence on devel-

opment and differentiation: an overview of a free radical the-

ory of development.Free Radical Biology and Medicine Vol. 6

No 6: Pages 631-661.

Anagnostopoulou MA., Kefalas P., Papageorgiou VP., Assimo-

poulou A.N. and Boskou, D. (2006) Radical scavenging activity

of various extracts and fractions of sweet orange peel (Citrus

sinensis).Food chemistry Vol.94 No 1: Pages 19-25.

Antunes LMG. and Takahashi CS. (1998) Effects of high doses

of vitamins C and E against doxorubicin-induced chromosomal

damage in Wistar rat bone marrow cells.Mutation Research/

Genetic Toxicology and Environmental Mutagenesis Vol.419

No 1: Pages 137-143.

Benzie IF. (1996) An automated, speci c, spectrophotometric

method for measuring ascorbic acid in plasma (EFTSA).Clini-

cal biochemistry Vol.29 No 2: Pages 111-116.

Bierie B. and Moses HL. (2006) TGF-

 and cancer.Cytokine &

growth factor reviews Vol.17 No 1: Pages 29-40.

Bowie JU., Luthy R. and Eisenberg D. (1991) A method to iden-

tify protein sequences that fold into a known three-dimen-

sional structure.Science Vol. 253 No 5016: Pages 164-170.

Bradford MM. (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Analytical biochemistry

Vol.72 No 1-2: Pages 248-254.

Constam DB. and Robertson EJ. (1999) Regulation of bone

morphogenetic protein activity by pro domains and proprotein

convertases. The Journal of cell biology Vol.144 No 1: Pages

139-149.

Derynck R. and Zhang YE. (2003) Smad-dependent and Smad-

independent pathways in TGF-

 family signalling. Nature

Vol.425 No 6958: Pages 577-584.

Drabsch Y. and Ten Dijke P. (2012) TGF- signalling and its

role in cancer progression and metastasis.Cancer and Metas-

tasis Reviews Vol.31 No 3-4: Pages 553-568.

Dubois CM., Laprise MH., Blanchette F., Gentry LE. and Leduc

R. (1995) Processing of transforming growth factor 1 precursor

by human furin convertase.Journal of Biological Chemistry

Vol.270 No 18: Pages 10618-10624.

Giannopolitis CN. and Ries SK. (1977) Superoxide dismutases

I. Occurrence in higher plants.Plant physiology Vol.59 No 2:

Pages 309-314.

Gray AM. and Mason AJ., (1990) Requirement for activin A

and transforming growth factor-b1 pro-regions in homodimer

assembly.Science Vol. 247: Pages1328-1330.

Gunwant V., Raturi M., Hussain M. and Rana D. (2013) Market-

ing of sweet orange (Malta) in India. International Journal of

Emerging Research in Management and Technology Vol. 3 No

2: Pages 45-49.

Table 3: Result of Most favorable non cytotoxic compounds of ripen malta peel

extract docked with TGF-β

and TGF-β

Name of compound

Energy with TGF-β

(k.cal/mol.)

Energy with TGF-β

(k.cal/mol.)

Solvent

(+) Alpha-Tocopherol -9.1 -6.3 Hexane

Phenol3,5-bis(1,1-

Dimethylethyl)

-6.8 -6.8 Hexane

Vineet, Devender and Ajeet

574

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Hockenbery DM., Oltvai ZN., Yin XM., Milliman CL. and Kors-

meyer SJ. (1993) Bcl-2 functions in an antioxidant pathway to

prevent apoptosis. Cell Vol.75 No 2: Pages 241-251.

Huang F. and Chen. YG. (2012) Regulation of TGF-

 receptor

activity.Cell & bioscience Vol.2 No 1: Page 1.

Imamura T., Hikita A. and Inoue Y. (2012) The roles of TGF-

signaling in carcinogenesis and breast cancer metasta-

sis.Breast Cancer Vol.19 No 2: Pages 118-124.

Kaur G., Alam MS., Jabbar Z., Javed K. and Athar M. (2006)

Evaluation of antioxidant activity of Cassia siamea  ow-

ers.Journal of Ethnopharmacology Vol.108 No 3: Pages 340-

348.

Khan PK. and Sinha SP. (1993) Antimutagenic ef cacy of

higher doses of vitamin C.Mutation Research/Genetic Toxicol-

ogy Vol.298 No 3: Pages 157-161.

Khan, I.A. ed., 2007.Citrus genetics, breeding and biotechnol-

ogy. CABI.

Kiss R., Sandor M. and Szalai FA. (2012) http://Mcule. com: a

public web service for drug discovery.Journal of Cheminfor-

matics Vol.4: Pages 1-1.

Levy L. and Hill CS. (2006) Alterations in components of

the TGF-

 superfamily signaling pathways in human can-

cer. Cytokine & growth factor reviews Vol. 17 No 1: Pages

41-58.

Liithy R., Bowie JU. and Eisenberg D. (1992) Assessment

of protein models with three-dimensional pro les. Nature

Vol.356 No 6364: Pages 83-85.

Liu TZ., Chin N., Kiser M. and Bigler, W. (1982) Speci c spec-

trophotometry of ascorbic acid in serum or plasma by use of

ascorbate oxidase. Clinical Chemistry Vol. 28 No 11: Pages

2225-2228.

Mallick N. and Khan RA. (2016) Antihyperlipidemic effects of

Citrus sinensis, Citrus paradisi, and their combinations. Jour-

nal of Pharmacy & Bioallied Cciences, 8(2), p.112.

Manthey JA. and Grohmann K. (2001) Phenols in citrus peel

by products. Concentrations of hydroxycinnamates and poly-

methoxylated  avones in citrus peel molasses. Journal of

Agricultural and Food Chemistry Vol. 49 No7: Pages 3268-

3273.

Marubayashi S., Dohi K., Ochi K. and Kawasaki T. (1986) Role

of free radicals in ischemic rat liver cell injury: prevention

of damage by alpha-tocopherol administration. Surgery, 99(2),

Pages 184-192.

Massagué J. (2008) TGF

 in cancer.Cell Vol.134 No 2: Pages

215-230.

Meulmeester E. and Ten Dijke P. (2011) The dynamic roles of

TGF‐

 in cancer. The Journal of pathology Vol. 223 No 2:

Pages 206-219.

Miles DC., Wakeling SI., Stringer JM., Van den Bergen JA.,

Wilhelm D., Sinclair AH. and Western PS. (2013) Signaling

through the TGF beta-activin receptors ALK4/5/7 regulates

testis formation and male germ cell development. PloS one

Vol.8 No 1: Page 54606.

Nelson SM., Panagiotou OA., Anic GM., Mondul AM., Män-

nistö S., Weinstein SJ., and Albanes D. (2016) Metabolomics

analysis of serum 25-hydroxy-vitamin D in the Alpha-Tocoph-

erol, Beta-Carotene Cancer Prevention (ATBC) Study. Interna-

tional Journal of Epidemiology, dyw148.

Padua D. and Massagué J. (2009) Roles of TGF in metasta-

sis.Cell research Vol.19 No 1: Pages 89-102.

Pandey N., Gupta B. and Pathak GC. (2012) Antioxidant

responses of pea genotypes to zinc de ciency.Russian Journal

of Plant Physiology Vol. 59 No 2: Pages 198-205

Pickup M., Novitskiy S. and Moses HL. (2013) The roles of TGF

[beta] in the tumour microenvironment.Nature Reviews Can-

cer Vol.13 No 11: Pages 788-799.

Ra q S., Kaul R., So S A., Bashir N., Nazir F. & Nayik GA.

(2016). Citrus peel as a source of functional ingredient: A

review. Journal of the Saudi Society of Agricultural Sciences.

Sandhir R. and Gill KD. (1995) Effect of lead on lipid peroxida-

tion in liver of rats.Biological trace element research Vol.48

No 1: Pages 91-97.

Shibanuma M., Kuroki T. and Nose K. (1988) Induction of

DNA-replication and expression of proto-oncogene cis-myc

and cis-fos in quiescent balb/3t3 cells by xanthine xanthine-

oxidase.Oncogene Vol.3 No 1: Pages 17-21.

Siegel PM. and Massagué J. (2003) Cytostatic and apoptotic

actions of TGF-

 in homeostasis and cancer.Nature Reviews

Cancer Vol.3 No 11: Pages 807-820.

Sippl MJ. (1993) Recognition of errors in three‐dimensional

structures of proteins.Proteins: Structure, Function, and Bio-

informatics Vol.17 No 4: Pages 355-362.

Slamon DJ., Clark GM., Wong SG., Levin WJ., Ullrich A. and

McGuire WL. (1987) Human breast cancer: correlation of

relapse and science Vol.3798106 No 177: Page 235.

Stookey LL. (1970) Ferrozine---a new spectrophotometric rea-

gent for iron. Analytical chemistry Vol.42 No 7: Pages 779-

781.

Tazat K., Hector-Greene M., Blobe GC. and Henis YI. (2015)

RIII independently binds type I and type II TGF- recep-

tors to inhibit TGF-

 signaling. Molecular biology of the cell,

26(19), pp.3535-3545.

Trott O. and Olson AJ. (2010) AutoDock Vina: improving the

speed and accuracy of docking with a new scoring function,

ef cient optimization, and multithreading.Journal of compu-

tational chemistry Vol.31 No 2: Pages 455-461.

Valvona CJ., Fillmore HL., Nunn PB. and Pilkington GJ. (2016)

The Regulation and Function of Lactate Dehydrogenase A:

Therapeutic Potential in Brain Tumor. Brain Pathology 26, no.

1: Pages 3-17.

Wallace AC., Laskowski RA. and Thornton JM. (1995) LIGPLOT:

a program to generate schematic diagrams of protein-ligand

interactions. Protein engineering Vol. 8 No 2: Pages 127-

134.

Wallner B. and Elofsson A. (2003) Can correct protein models

be identi ed?.Protein science Vol.12 No 5: Pages 1073-1086.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

INVITRO

AND

IN SILICO

ANALYSIS OF MALTA PEEL EXTRACT FOR ANTI CANCEROUS ACTIVITY 575

Vineet, Devender and Ajeet

Weydert CJ. and Cullen JJ. (2010) Measurement of superox-

ide dismutase, catalase and glutathione peroxidase in cultured

cells and tissue. Nature protocols, 5(1), pages.51-66.

Wiederstein M. and Sippl M.J. (2007) ProSA-web: interactive

web service for the recognition of errors in three-dimensional

structures of proteins. Nucleic acids research Vol.35 No suppl

2: Pages W407-W410.

Yang L., Pang Y. and Moses HL. (2010) TGF-

 and immune

cells: an important regulatory axis in the tumor microenviron-

ment and progression. Trends in immunology Vol. 31 No 6:

Pages 220-227.

Yu Y., Xiao CH., Tan LD., Wang QS., Li XQ. and Feng YM.

(2014) Cancer-associated  broblasts induce epithelial–mes-

enchymal transition of breast cancer cells through paracrine

TGF-

 signalling. British journal of cancer Vol. 110 No 3:

Pages 724-732.

Zheng C., Fang Y., Tong W., Li G., Wu H., Zhou W., Lin Q., Yang

F., Yang Z., Wang P. and Peng, Y. (2014) Synthesis and biologi-

cal evaluation of novel tetrahydro-

-carboline derivatives as

antitumor growth and metastasis agents through inhibiting the

transforming growth factor-

 signaling pathway. Journal of

medicinal chemistry Vol.57 No 3: Pages 600-612.