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Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
In vitro callus induction, phytochemical screening, and in
silico docking of Vitex negundo secondary metabolites
against HER2 protein, a breast cancer biomarker
Kumaresan Kowsalya1, Jaganathan Sakthi Yazhini Preetha1, Perakasam Lalitha
Jemima Aashmi2, Sivakumar Kowsalya 3, Muthukrishnan Arun1*
1Department of Biotechnology, Bharathiar University, Coimbatore-641046, Tamil Nadu, India
2Department of Integrative Biology, School of Biosciences and Technology, Vellore Institute of Technology,
Vellore - 632 014, Tamil Nadu, India
3PG and Research Department of Biotechnology, Nandha Arts and Science College, Erode-638052, Tamil
Nadu, India
*Corresponding Author Email ID: arun@buc.edu.in
Abstract
!" #
"$ %
% "%
%%
"&Vitex negundo'$(
")Verbenaceae.*
%
% " ) "
*Vitex+",
in silico ") -
Vitex negundo.
" / in silico 0%1 2%/%0%
"3 in vitro Vitex
negundo 4%0 5 67! ) "8 67! &$ " )
95"! :29";<"=
:5>"<";!"7%4<"816
! 8<"8 ?6 !
9"! >8"-!"*
Vitexnegundo %
"
Keywords: Vitex negundo .
Introduction
Vitex negundo 7" Verbenaceae. )
)*
@ 5!" ) )
Vitex negundoAsarvaroga nivaraniB$52!"")
" 7 C+
"*Vitex negundo D.E
% ) " ) .
$ F $ " *
4G5$, 54!")%
" )
%5HD") .
+")
"
)58I54=I52!"
Vitex negundo, . JK6$E
.=
"Vitex negundo
+.+
9
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
%DK$%
C"*C, 9!",Vitex
negundo "8 "% . % +G
Let al., 55!"Vitex%Ehrlich
ascitesK"F&">!")
"D%
in vitro" 0
.")
,., 5-9;= 5-92=7.$5-95!"
/ !
")
Vitex negundo
M-!"
.in
vitro + Vitex negundo. * # 5!)
Vitex negundo in silico ! In vitro
+ Vitex negundo ;! *
+ Vitex negundo CC
4! * Vitex negundo
!in vitro &$"
Materials and methods:
Molecular docking of selected Vitex secondary metabolites with HER2 target protein:
Collection of ligand and target protein:
*N OO0I)0N;OO/!
O0I."-Vitex negundo
P 5OD" 7
7"O. O."
In vitro callus induction:
Plant sourcing and preparation of explants:
Vitex negundo P@ P@!"*
0 I I @ D" )
I$)$ D D"
Vitex negundo 7" "*
*86!5")
> 5%58 " *
+ "5 6! %;
" * . 8%9 Q"9!" R
&$ "; ">
4%0"8%6!)"8%6!4%056!"*
# 8"2%8"9 55HD 58 $
58= 54!"
Culture condition:
* 8<HD ; 59
2."*58&$"
Analysis of biomass:
;"*
,3!"*8HD8
03!"
Sample extraction procedure:
5 C ")5
. . 58 ; "*
%" ; ; 5 " *
-
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
. " /
4HD" ,
56O.5-!"
Phytochemical screening:
Qualitative phytochemical screening:
,CK"54 O.
5- . .
"
Quantitative phytochemical screening of phenols:
*,%D$"*
!58S ,%D
. " 2 5"8 > "
8S6 8S6>8S6
5S6!"*-"*
.>28@L6L $"*
1C!6## 5!"
Quantitative phytochemical screening of flavonoid:
*C"5"8
-8 "8 C! " * "5 5
"*"55&"9
"* . "*
.458@L6L"
" * ?
CC!6K58!"
Antioxidant assays:
Determination of antioxidant activity using the DPPH radical scavenging method:
* " I"5
%56!."48S
*D5"5&0OO"5&67!"*;
. 85>
$+@L%52-Q!"*"7
" *
T%!6U56
## 5!"
Determination of antioxidant activity using the ferric reducing/antioxidant method (FRAP):
* , O I+ $ 5--2!
" * . ; & ;"5 D;K/" ;/ 52
D4/! ;"25&*O*M 4& D &,D;"2/"*
"*., O!8
8*O*M8,D;"2/5N5N5!"*;S
">, O"8
8-;"*.%
, O!"
Statistical analysis:
* + $O$$ 55"- )I& D!"
0<"*0E
0& *!8"
Results:
Molecular docking of selected Vitex secondary metabolites with HER2 target protein:
& .
")
Vitex negundo *%5!.)in silico
0%12%/V0
"* N *-41;- 1>)5--
*5>") 0%12%/%%0%
5
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
") in silico
0%1 2%/%%0%%
"50%12%/%0%%
"
In vitro callus induction:
%&$"8%"64%0
) "8%6!" 1
,";!"*"
&$4%05"6!)"8
6!95"! 29";<"!
5>"<";!,"48!"
Qualitative phytochemical screening:
O
.*!"
* . " *
" *
."*
" * "*
"*
"*"K
"
Quantitative phytochemical screening of phenols and flavonoids:
) 4< "816 !
8 <"8 ?6 ! : ;9"9 <
"16!:"8<";?6!,"2!"
Determination of antioxidant activity by DPPH radical scavenging method and ferric
reducing/antioxidant method (FRAP):
)0OO 9"!
>2"! 98"9!" ,
0OO % " *
, O " * % *O*M%,W
+*O*M%,;W8-;")
, O>8"-!
2>"9!-";!,">!"
Discussion:
&.+
*5= &.#5!"*.
")
in vitro "
Vitex negundo
%%
% 0 ;!" Vitex %
. "*
Vitex negundo ."*
+"*
*-41;-1>5--*5>")0+
" & Vitex negundo
" ) in silico 0%
1 2%/%%0%% "
*. %
M-!"
* C
,#+, 5;!"* .
55
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
"" !
+FP5--!")
C
$et al., 5--!
,, 5--!" *
" * !
V. negundo
D, 55= !"D"D
;"*
"
" *
9")564%0"86)
" )
4%04%0)
" * Vitex
negundo"
/CC
C "*
%
"%
"
. % 5=
0 52= $ 55!" * Vitex negundo # C
C" ) C
.
* !" ) C
4<"86!8<"86!
:;9"9<"6!=:"8<";6!"
)0OO9"!
>2"!98"9!")
, O " ) , O >8"-!
2>"9!-";! ,"2!"*!
,>"*
Vitex negund=
-";!XVitex negundo >8"-!2>"9!5S6"
*")
Y%
++,#+
5;= 2=R59!"
Conclusion:
In silico Vitex
negundo"*0%12%/%0%%
"" )
.")
"* 4%0 567!)"867!" )
95"! :29";<"=:
5>" <";!" 3
4 < "8 16 ! 8 < "8 ?6 !
9"! >8"-!" *
in vitroin vivo%
Vitex-" , +
Vitex negundo
"
Acknowledgments
5
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
*.ID* DID* D!I
@#I@6 @$"6ID* D66ID* D%D*9654"5"!"
References
&"P"1 "F@$"5!"
D D
"ZOriental Journal of ChemistryZ28!--;"
K"F&">!" L7"
"ZAgroforestry systemsZ715-8%"
I,"$"O",$" QO"F #"54!" %
L7"ZInt. J. Adv. ResZ2;!99%9-4"
I$"I"O$"I"&$"I" Q0"$"FP.L" I"52!"
O1 !
L7"!"ZInt. J. Adv. Res. Biol. SciZ39!-4%--"
I+ )" ," F $Q" Q" 5--2!" * , O!
'(N, O"ZAnalytical biochemistryZ2395!>%>2"
D,"I"+,"$"&"&"Q,")"D" "$#$"F
&"55!"$LN" ZAmerican-
Eurasian Journal of Sustainable AgricultureZ55!2%54"
0&"1"Q.Q" ""D"11"7$"$"O"F 3"0";!"
% L"ZJournal of
ethnopharmacologyZ87%;!5--%2"
0&"L"I1". "1$"FPK"$"52!"?
C % $
P!"ZInternational Journal of Herbal MedicineZ42!99%-5"
## " 7. K" P" D R" , " " F 7. &" 5!"
&"ZRecords of Natural ProductsZ44!5-;"
,#+ "MK"M. ""F,#+&"5;!"+
ON
"ZTurkish journal of biologyZ372!29-%2->"
,.D""5--;!"/"ZHand book of plant cell cultureZ15;%95"
," "3 Q"" F " 3" 5--!" $
"ZPlant Cell ReportsZ824>%28"
PK"Q"FD"$"5--!" %
5" &+"ZApplied microbiology and biotechnologyZ33449%
485"
7.O"Q"F $ 3" " 5-95!" $V
"ZTheoretical and applied geneticsZ605->%54"
&.#O"P"I# $" F P " 5!"&.N
. ) "ZPhytochemistry
ReviewsZ202-;%>;"
KO"F L" 58!"O D
"ZInt J Pharm Pharm SciZ85!;8%;"
O. 1" F I 0" 5-!" O
L 7"ZJournal of Drug Delivery and
TherapeuticsZ94%!>8%>>"
0" K" F K 1" &" 2!" C
) L
7"!"ZIn Vitro Cellular & Developmental Biology-PlantZ422-%>;"
L"5-92!"ZEmbryogenesis in angiosperms: a developmental and experimental studyZL"5>!"
D@O"
$" " F . 1" " !" O N
"ZBiotechnology advancesZ20!55%58;"
0" 0" $"F 0"&" 54!" L
7"V "ZInternational Journal of Developmental ResearchZ4
5255%5258"
$Q"3"C1" P""F 03"O"5--!"&
"ZCrop scienceZ322!5;8>%5;2"
5;
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
$ " $" P. $ P $" 58!" D in-vitro
Vitex negundoN )" Journal of Natural Products,
88!N52%"
$ $" 1 I" 0" P " F 1 L" P" 54!" $+
[%%+ !"ZJournal of
CrystallographyZ2014"
$O"&1"$$"$P"F P "55!"O
L$!"ZPharmacologyonlineZ25;88%5;98"
$K"F $ 0" 52!" O L
7"ZJournal of chemical and Pharmaceutical researchZ8!9%9>"
*C$""PO"Q" 1I"0"1L"P" F Q "P"9!"K
L %
"ZWorld journal of gastroenterology: WJGZ14;!;2-;"
*$"P"$#D"$$"P"F P"P"5!"&.?O700&
)L%5"ZMedicinal Chemistry ResearchZ214;-%485"
@&"..P"@K"$*"F$L"5!"
OL%
"ZTanzania Journal of Health ResearchZ145!"
L"L.7"$$"FI"55!"LN" ZInternational
Journal of Pharmaceutical InnovationZ18!-%"
R&","P0"RQ"&"F &"59!"
"ZWorld Scientific News555!5;%8"
M R" 7 R" " D Q" M 1" $ D" 7 R" F $ R" " -!" L %
"ZClinical Cancer
ResearchZ1552!8525%852-"
Figures and tables:
Fig.1. Molecular docking and ligand interaction of D-Glucose, 6-O-D-galactopyranosyl compound with HER2
protein
54
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.2: Callus induction and proliferation: ! Vitex negundo I
@=I!792!=D!D8=0!O
5=!O=,!O
;"&$4%0567!W)
"867!"$58"
58
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.3: Callus induction from leaf explants of Vitex negundo: !4%0"8G67!=I!
)"8G"67! 4%0 567!" O :"
6"! 5"*; " Bars
:5! " &
0E8"
52
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.4: Effect of 2,4-D (0.5– 2.0mg/L) on biomass of callus induced from leaf explants of Vitex negundo: !
,6=I!06"* ;" Bars
:5! " &
0E8"
5>
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.5: Effect of IAA (0.5 – 2.0 mg/L) in combination with 2,4-D best concentration (1mg/l) on callus
biomass induced from leaf explants of Vitex negundoN! , 6=I!06"*
; "Bars
:5! "&
0E8"
59
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.6: Comparative assessment of phytochemical assays in leaf-derived callus culture of Vitex negundo
!*=I!* "I
:8! " &
0E8"
5-
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
Fig.7: Comparative assessment of Antioxidant assays in leaf derived callus culture of Vitex negundo
DN& V.negundo $N D
N&&$ 4%056
) "8 6" I :8! "
&0E
8"
Table-1: List of Vitex negundo phytocompounds achieving best binding energy with the selected target
HER2 protein
Compound name Compound ID Docking score
0% 2%/%0%
%
445;495; %5;">
0% 59-8 %-"8
I+;% >4 %8"289
5%%5;%%8-5;%
%49
8;2>25 %8"84
4%%4% ;% %;8% 55-9;9 %8";
Book Chapter- Emerging Trends in Basic and Applied Sciences ISBN: 978-81-964972-3-1 Muthukrishnan Arun et.al.,
%2%%
%5 89>8- %4"-48
%%4%22%
%5%! %%
%5%
8;2-49 %4">>
-95 %4"42
895858 %4"4;
;%55 544-> %4";
4%%5%;%%M 8;24>4 %4";5
+ 54%%>%5%
!%
;858 %;"->5
;528 %;">-
O 894;8 %;"8;4
O55;%% 424 %;"8;4
55;%
9-2 %"55;
%-%!%M!% 8;24>5; %5"-;>
% 248-2 %5"94
I55%%;% 5-2-8 %5">>2
2-558%0
8;22> %5">;
%-5%
!%MM!%
8;282>8 %5"48
4%0 84-54 %"-5>
4-%0 %%
8422825 %"2-
5;22 "-
Table 2: Qualitative phytochemical analysis of V. negundo methanolic extract
'W(
'%(
5
Phytochemical tests Callus extract Leaves extract
O + +
, + +
. + +
* + +
* + +
1 + +
$ + +
O - -