IIIM-CSIR
New drug discovery from natural products
(NCEs and Botanicals) at
Indian Institute of Integrative Medicine
11thICSB, 16-19 April 2012, University of Mississippi, MS Confidential
Ram Vishwakarma
Indian Institute of Integrative Medicine
(Council of Scientific & Industrial Research)
Jammu, India
IIIM-CSIR
IIIM
Jam
mu
Confidential
IIIM
, a
Na
tio
na
l In
stit
ute
of
CS
IR
IIIM-CSIR
Res
earc
h Inte
rest
s of th
e In
stitute
Dis
cover
y o
f N
CEs and B
ota
nic
al dru
gs
from
natu
ral pro
duct
s (m
edic
inal pla
nts
and m
icro
bia
l sp
ecie
s)
Dis
ea
se A
rea
s o
f In
tere
st
Confidential
•C
an
cer
•In
fla
mm
ati
on
•A
lzh
eim
er
•D
rug
re
sist
an
t in
fect
ion
s
Dis
ea
se A
rea
s o
f In
tere
st
IIIM-CSIR
Dru
g d
isco
very
fro
m n
atu
ral
pro
du
cts
Over
vie
w o
f D
rug D
isco
ver
y a
t II
IM
Natural Resources
(Botany and
Microbiology)
Confidential
In v
ivo
act
ivit
y
(Mic
e T
um
or
Mo
de
ls)
Ide
nti
fica
tio
n o
f Ta
rge
t
ba
sed
po
ten
tia
l d
rug
Ca
nd
ida
te
(In
vit
ro)
Lea
d O
pti
miz
ati
on
(SA
R/Q
SA
R)
De
tail
ed
Me
cha
nis
m
of
act
ion
In v
ivo
act
ivit
y
PK
_P
D_
TO
X.
(Mic
e/R
at)
P
re-C
lin
ica
l
De
velo
pm
en
t
Natural and Synthetic
products
Medicinal
Chemistry/ In
Silico Studies
General
Pharm
acology
IIIM-CSIR
Iso
lati
on
of
mic
roo
rga
nis
ms
fro
m u
niq
ue
en
vir
on
me
nta
l n
ich
es
En
do
ph
yte
sC
old
de
sse
rts
Ho
t w
ate
r
spri
ng
s
Fre
sh w
ate
r la
kes
an
d s
tre
am
s
Ma
rin
e
en
vir
on
me
nts
Fu
ng
iB
act
eri
aA
ctin
om
yce
tes
Iso
lati
on
ne
w c
he
mic
al
en
titi
es
Pre
pa
rati
on
of
ext
ract
s
Bio
-pro
spe
ctin
g f
or
no
vel
mic
rob
ial
mo
lecu
les
Confidential
Iso
lati
on
ne
w c
he
mic
al
en
titi
es
Pre
pa
rati
on
of
ext
ract
s
Scr
ee
nin
g f
or
bio
act
ivit
y
An
tim
icro
bia
lA
nti
can
cer
Imm
un
e s
up
pre
ssio
n
Se
lect
ion
of
Po
ten
t st
ain
s
for
ep
ige
ne
tic
an
d g
en
eti
c
ma
nip
ula
tio
n
Se
lect
ion
of
Po
ten
t
mo
lecu
les
for
pre
pa
rati
on
of
ne
w b
ioa
ctiv
e a
na
log
ue
s
Lea
ds
for
dru
g
de
ve
lop
me
nt
IIIM-CSIR
Novel
enzy
mes
toolb
ox
for
bio
act
ives
Oxid
ore
duct
ase
Lyase
sH
ydro
lase
sTra
nsf
erase
Isola
tion a
nd c
hara
cter
ization o
f novel
enzy
mes
Confidential
Oxid
ore
duct
ase
Lyase
sH
ydro
lase
sTra
nsf
erase
Oxid
ase
Oxygen
ase
Per
oxid
ase
Ket
ore
duct
ase
Deh
ydro
gen
ase
Lacc
ase
Nitri
lase
Lip
ase
/est
erase
Pro
tease
Cel
lula
se
Ald
ola
seA
min
otr
ansf
eras
Acyltransf
erase
IIIM-CSIR
•N
ichola
s Pir
am
al
Indig
ene
(USA
)
•M
edla
y P
harm
aC
olg
ate
Palm
olive
(USA
)
•Lupin
Pharm
aPro
ctor &
Gam
ble
(U
SA
)
•Zandu P
harm
aPro
ctor &
Gam
ble(J
apan)
•O
choa P
harm
aTro
pic
al Bota
nic
s (M
ala
ysia)
•Pra
tist
ha Indust
ries
Sig
ma (U
SA
)
Indian companies
Foreign companies
OU
R IN
DU
STR
Y P
AR
TN
ER
S
Confidential
•Pra
tist
ha Indust
ries
Sig
ma (U
SA
)
•Bhara
t Bio
tech
Chro
madex
(U
SA
)
•H
igh B
iote
chN
ovaco
s (U
SA
)
•D
ootp
apes
hw
ar
National C
ance
r In
stitute
(U
SA
)
•IM
PC
L
•G
enova B
iote
ch
•Sev
eral C
RO
s
Hospitals (Collaborators)
Medanta Medicinty, Gurgaon NCR
Ramachandra Hospital, Chennai
IIIM-CSIR
Ris
ori
ne
RIS
OR
INE
A N
EW
AN
TI-
TB D
RU
G
Dis
covere
d a
nd D
evelo
ped b
y
A recent success story of IIIM
Confidential1
Dis
covere
d a
nd D
evelo
ped b
y
IIIM
-CSIR
, Jam
mu
and
Cadila P
harm
aceuticals
Launched in India
n m
arket in
Nov 2
009
IIIM-CSIR
Resorine
Integration of Indian system
of medicine
with m
odern m
edicine
•Rifam
picin is a potent inducer of Cyp P-450s and P-gp in gut and
liver leading to the auto-induction of its own m
etabolism
decreasing
its effective conc.
•IIIM
discovered that Piperine (an alkaloid from an Indian plant) as a
Confidential
•IIIM
discovered that Piperine (an alkaloid from an Indian plant) as a
potent inhibitor of CYP-450/P-gp and shown to enhance
bioavailability of rifampicin.
•PK studies showed that a rifam
picin (450 Standard) was bioequivalent
to a low dose of drug (200 m
g) in presence of piperine (10 m
g) due to
inhibition of Rif m
etabolizing enzymes by piperine, compensating
enzyme induction by Rif.
•After successful preclinical development and clinical trials, the drug
has been approved in the DCGI for marketing (Cadila Pharma, India)
IIIM-CSIR
Efflux pumps inhibition and
combination therapy for tuberculosis
(Indian Institute of Integrative Medicine, Jammu)
Confidential
MFS
(Major facilitator Super-family)
SMR
(Small multi-drug resistance subfamily)
MATE
(Multi antimicrobial extrusion subfamily)
RND
(Resistance/Nodulation/Division super-family)
ABC
(ATP Binding Cassette Transporters)
IIIM-CSIR
AY
US
H
Strategic partnership with NCNPR
Confidential
Un
iv.
of
MS
(NC
NP
R)
CR
ISM
CS
IR (
IIIM
)
IIIM-CSIR
Inte
r-m
inis
teri
al
me
eti
ng
to
est
ab
lish
CR
ISM
•D
ep
t o
f A
YU
SH
(G
ov
t. o
f In
dia
)
•M
inis
try
of
Ex
tern
al
Aff
air
s (G
OI)
•M
inis
try
of
Eco
no
mic
Aff
air
s (G
OI)
•C
CR
AS
Confidential
•C
CR
AS
•C
CR
UM
•IC
AR
•C
SIR
It
wa
s d
eci
de
d t
o O
pe
n C
RIS
M i
n U
SA
wit
h
Mir
ror
Ce
nte
r a
t II
IM
Ba
sed
on
th
e p
rese
nta
tio
n b
y I
IIM
Sci
en
tist
s
an
d d
iscu
ssio
n t
he
pro
po
sal
wa
s a
pp
rov
ed
IIIM-CSIR
Lau
nch
ing
of
CR
ISM
in
US
A
Confidential
Inauguration of CRISM byMrs. S. Jalaja, Secretary(AYUSH)
on 6
thApril, 2009
IIIM-CSIR
�To
de
fin
e a
nd
un
de
rta
ke r
ese
arc
h a
nd
de
velo
pm
en
tal
act
ivit
ies
ba
sed
on
po
ten
tia
ls o
f th
e t
rad
itio
na
l sy
ste
ms
an
d la
test
sci
en
tifi
c a
dva
nce
s fo
r
pro
mo
tio
n a
nd
sci
en
tifi
c a
cce
pta
nce
of
ISM
an
d t
he
pro
du
cts
the
reo
f
�To
en
cou
rag
e a
nd
fa
cili
tate
co
op
era
tio
n f
or
rese
arc
h a
ctiv
itie
s b
etw
ee
n t
he
Ind
ian
an
d U
S a
cad
em
ic a
nd
in
du
stri
al
inst
itu
tio
ns
OV
ER
ALL
OB
JEC
TIV
ES
OF
CR
ISM
Confidential
Ind
ian
an
d U
S a
cad
em
ic a
nd
in
du
stri
al
inst
itu
tio
ns
�To
fa
cili
tate
dia
log
ue
be
twe
en
sci
en
tifi
c co
mm
un
ity
an
d r
eg
ula
tory
bo
die
s
of
the
tw
o c
ou
ntr
ies
an
d t
o e
volv
e a
pla
tfo
rm f
or
such
re
gu
lar
exc
ha
ng
e
�To
pro
vid
e a
nd
pro
mo
te a
uth
en
tic
info
rma
tio
n a
bo
ut
the
str
en
gth
s o
f IS
M
an
d t
o h
elp
de
mys
tify
th
e m
isco
nce
pti
on
s a
bo
ut
the
se s
yste
ms
of
me
dic
ine
s in
US
A
IIIM-CSIR D
isco
ver
y o
f N
CE
s fr
om
natu
ral pro
duct
s
Confidential
IIIM-CSIR
Phosphatidylinositol 3-kinase (PI3K) pathway as
clinically validated anticancer target
•Inhibition of PI-3K kinase (isoform
specific inhibitors)
•PI3Kαfor cancer and PI3Kγfor inflam
mation
Confidential
•PI3Kαfor cancer and PI3Kγfor inflam
mation
•Up-regulation of PTEN phosphatase
•Inhibition of AKT isoform
s
•Inhibition of mTOR
IIIM-CSIR
O
OH
OH
OH
HO
HO
P O
OO
OO
OO
123
45 6
-O
OH
O
OH
O
HO
P O
OO
OO
OO
123
45 6
-
PO OH
- O
POH
- O
O
O
OH
O
OH
O
HO
P O
OOH
OO
OO
123
45 6
-
PO OH
- O
POH
- O
O
HO
1,4,5-InsP
3
DAG
PK
C a
ctivation
PI-kinase
PI-PLC
Calcium
PI-3K
+
Central role of PI3K pathway in cell
Confidential
OO
PI
Recruitm
ent of a n
um
ber
of so
luble
prote
ins to
the
mem
barane a
nd their
activation (PH
dom
ain
s)
O
O
O
OH
O
HO
P O
OO
OO
OO
123
45 6
-
PO OH
- O
POH
- O
O
PO O-
O-
cell sig
naling
mem
brane fusi
on
chem
ota
xis
cell p
ola
rity
and h
ost
of oth
er
functions
anticancer
dru
g targ
et
PIP
3
PIP
2
IIIM-CSIR
Both the RTK and GPCR pathways are
involved in PI3K activation
Confidential
IIIM-CSIR
Medicinal chemistry of Liphagane scaffold
(Iso
form
sele
ctive P
I3K
inhib
itors)
O
HO
OH
CHO
p110α ααα IC50 - 100 nM
p110β βββ IC50 - ND
p110γ γγγ IC
50 - 1
p110δ δδδ IC50 - ND
Confidential
�A
me
rote
rpe
no
idis
ola
ted
fro
mm
ari
ne
spo
ng
eA
kaco
rall
iph
ag
a
Inh
ibit
ory
act
ivit
ya
ga
inst
PI3
Kα
(10
fold
sele
ctiv
ity
)
�M
ore
sele
ctiv
eth
an
the
syn
the
tic
LY2
94
00
2a
nd
Wo
rtm
an
in.
�IC
50:
10
0n
M
IIIM-CSIR
Synthesis of Liphagal
BBr 3, DCM
OK2CO3, DMF
60%
NaH, DMF
88%
30%
MeMgBr, THF
ZnI, NaBH3CN
MeO
MeO
CHO
OMe
MeO
MeO
CHO
OH
OClMeO
MeO
O
Br
MeO
MeO
MeO
MeO
MeO
CHO
OH
Br
Br, AcOH
54%
Br
Confidential
OO
O
MeMgBr, THF
82%
ZnI, NaBH3CN
O O
H
O
H
85%
40%
OH
-75 oC to rt
n-BuLi, THF
DMF, TMEDA
O
H
OH
Liphagal
+ _
0 OC - rt
AlCl 3 , DMS
85%
30%
MeO
MeO
MeO
MeO
MeO
MeO
ClSO3H
Nitropropane
MeO
MeO
HO
HO
MeO
MeO
OH
Br
Br
Br
Br
IIIM-CSIR
Alternative route for the synthesis of Liphagal
BBr 3, DCM
O2-Butanone
K2CO3, 70%
tBuOK
Toluene
84%
43%
Ph3P+ CH3 I-
Pd/CaCO3,
MeO
MeO
CHO
OMe
MeO
MeO
CHO
OH
O
Br
MeO
MeO
O
Br
MeO
MeO
MeO
Confidential
OO
O
Ph3P CH3 I
n-BuLi, THF
80%
Pd/CaCO3,
MeOH
O O
H
O
H
80%
40%
OH
0 oC to rt
n-BuLi, THF
DMF, TMEDA
O
H
OH
Liphagal
+ _
0 OC - rt
AlCl 3 , DMS
85%
30%
MeO
MeO
MeO
MeO
MeO
MeO
ClSO3H
Nitropropane
MeO
MeO
HO
HO
MeO
MeO
IIIM-CSIR
Modeling of liphagal and its analog with
catalytic domain of PI3K-α
liphagal
Confidential
liphagal a
nalo
gs
Dock score for liphagal: -8.5 and for liphagal based designer analog: more than -10
H-bond interactions with Val851 and Gln859
IIIM-CSIR
Design and synthesis of NCEs based on Liphagane scaffold
CHO
OMe
MeO
MeO
BBr 3
DCM
CHO
OH
MeO
MeO
MeO
MeO
OO
Br
O
2-Butanone
K2CO3
Br
tBuOK
Toluene
OO
MeO
MeO
84%
70%
43%
Pd on CaCO
H
2-Nitropropane
Confidential
OMeO
MeO
Ph3P CH3 I
n-BuLi
THF
80%
Pd on CaCO3
MeOH
OMeO
MeO
95%
OMeO
MeO
2-Nitropropane
Chlorosulfonic,
-78 OC, 50%
OMeO
MeO
H
BHO
OH
THF,n BuLi
Triethylborate
0 OC, 1hr, 75%
OHO
HO
H
BHO
OH
AlCl 3,DMS
0OC,50%
IIIM-CSIR
Total synthesis of Liphagal (six membered) based NCE
CHO
MeO
MeO
OMe
CHO
MeO
MeO
OH
CHO
MeO
MeO
OO
MeO
MeO
OO
OH
PPh3HBr
MeO
MeO
OO
PPh3HBr
MeO
MeO
OH
PPh3HBr
MeO
MeO
O
PPh3HBr
O
BBr 3
DCM,rt,
MOMCl
Et 3N,DCM
NaBH4
ACN, Reflux
2hr
MeOH
HF/Pyridine
THF, rt, 1hr
DCC,DMAP
DCM,rt,18hrs
OH
O
O
Confidential
Isoform selective: PI3K-α; IC50: 66nM
MeO
MeO
OMeO
MeO
OMeO
MeO
O
HH
CHO
HO
HO
O
H
CHO
THF,Et 3N
Reflux,5hr
ClSO3H
2-Nitropropane
-780C,0.5hr
n-BuLi,THF
TEMDA,DMF
0OC,1.5hr
AlCl 3,DMS
0OC-rt, 1hr
OH
NaOH,Br 2
Dioxane,rt,4hrs
IIIM-CSIR
Design for NCEs of Liphagal for diversity
OHO
HO
H
O
H
OMe
O
H
N
HH
R
R
R
Confidential
O
HO
HO
H N
O
H
N
OMeO
MeO
H
OH
OHO
HO
CF3
H
O
H
BOH
HO
R
R
R
R
IIIM-CSIR
Ev
en
ts o
r Ta
rge
tsS
am
ple
cod
e
SIP
-10
03
SIP
10
04
En
zym
e b
ase
d I
C5
0P
I3K
α-
14
0 n
M
β-
100 nM
γ−
ΝD
δ−
ΝD
PI3
Kα
-1
02
nM
β-
30 µM
γ−
25 µΜ
δ−
45 µΜ
Ce
ll b
ase
d I
C5
0C
aco
-2 =
10
.0µ
MC
aco
-2 =
7.6
µM
IC50of two best compounds from Liphagal scaffold
Confidential
Ce
ll b
ase
d I
C5
0C
aco
-2 =
10
.0µ
M
HC
T-1
16
=8
.12
µM
Ca
co-2
=7
.6µ
M
HC
T-1
16
=5
.2µ
M
An
ne
xin
-V3
5-5
7%
(1-9
µM
)
50
-58
%
(1-9
µM
)
Ce
ll C
ycle
50
-60
% G
1 a
rre
st
(1-9
µM
)
64
-70
% G
1 a
rre
st
(1-9
µM
)
Wo
un
d H
ea
lin
gE
ffe
ctiv
eH
igh
ly e
ffe
ctiv
e
Ph
osp
ho
-Akt
50
-55
% D
ow
n-r
eg
ula
tio
n6
0-6
8%
Do
wn
reg
ula
tio
n
IIIM-CSIR
Tis
sue
Lun
gLe
uk
em
iaP
rost
ate
Co
lon
Ca
nce
r
Ce
ll T
yp
eA
54
9T
HP
-1
PC
-3
Co
lo 2
05
Ca
co 2
HC
T-1
16
S.N
oC
CL
Co
de
IC5
0(µ
M)
1.
SIP
-10
04
7.2± ±±±
1.3
1.9
3± ±±±
1.
0
7.3
7± ±±±
2.
8
5.1± ±±±
1.
6
7.6± ±±±
2.5
5.2
5± ±±±
2.
2
Calculated IC50Value
PI3K Isoform selectivity
PI3K αIC50100 nM
PI3K βIC5030 µM
PI3K γIC5025 µM
Confidential
PI3K ELISA Assay
Immunoflorescence
SIP-1004
-ve control
Liphagal (4µM)
(3µM)
pA
kt
DN
AM
erge
PI3K δIC5045 µM
IIIM-CSIR
Expression Studies
Annexin–V Apoptotic Assay
Confidential
IIIM-CSIR
Cell cycle Analysis
Cell Migration Assay Confidential
IIIM-CSIR
Rohitukine and Flavopiridol: CDK inhibitors
Criteria/Parameters
Ideal:
anticancer
molecule
(pre-clinical
candidate)
EnzymePotency(IC50)
CDKinhibitor(IC50<100nM)
100nM(CDK2–CYCAandCDK2–CYCE),
Proposedcandidate:<25nM
Selectivity
~100foldmore
overotherkinases(Lipid
kinases,VEGFR,IGFR,etc)
CellbasedAssay(IC50)
~50-100nM;ProposedCandidate5-10nM
Solubility
≥2mg/[email protected],Storageat(2-8
oC);
ProposedCandidate≥0.05-1mg/ml;Storageat
roomtemperature
Invitro
ADME
AllCyp’s≥10µM
OOOH
N
HO
Me
HO Rohitukine
Isolated from Indian medicinal
plant
Dyso
xylu
m b
ine
cta
rife
rum
Ho
ok
TPP as
CDK
inhibitor
Confidential
Invitro
ADME
AllCyp’s≥10µM
StableinHLM,MLM
Permeability:High
NoHERGliabilityat10µM
InvivoPK
Oralbioavailability≥20%,Halflife≥2.5-3hrs
(rat/mice)
ProposedCandidate:Oralbioavailability>40%
andHalflife>5-6hrs.
Invivo
Mousexenograftmodel-tumorregression
Safety
Nocardiac/Hepatotoxicity
IPPatentable
InitialSAR
Established
MW
Lessthan500
InSilicoPredictedTox
NIL
Cytotoxicity
10fold
Dyso
xylu
m b
ine
cta
rife
rum
Ho
ok
OOOH
N
Cl
OH
OH
N
OOOH
OH
OH
Cl
P276-00
Nicholas Piramal
Phase II
Flavopiridol
Sanofi-FDA approved
orphan drug for CLL
15
IIIM-CSIR
OOOH
N
Cl
OH
OH
OOOH
N
OH
OH
Hydrogen acceptor/donor group
OOOH
Cl
OH
OXOH
N
Cl
OH
OH
Five/Six member
sugar with
non-hydrolytic
bond
Increases further interaction with amino
acid residue of catalytic site of CDKs
Medicinal chemistry of Rohitukine/Flavopiridol
Synthesis of NCEs and derivatization based on SAR,
chemo-informatics and patent space
Confidential
N
OXOH
N
Cl
OH
OH
OXOH
N
Cl
OH
OH Presence of chloro group increases
6 fold activity compared to deschloroflavopiridol
OOOH
N
HO
Me
RY
XR1
Fla
vopir
idol analo
gs
OO
R
OH
N
HO
Me
R
Rohitukin
e a
nalo
gs
Biological assay standardization
&
screening is underway
16
�PfPK5-homologues to human cdk1(plays the role in DNA
synthesis)
�Pfmrk-homologues to human cdk7 (plays the role in DNA
replication
and transcription control)
�PfPK6
�PfPK7
10 NCE prepared
8 NCEs prepared
IIIM-CSIR
Medicinal chemistry of Rohitukine scaffold
O
HO
OOH
O
A
CBImportant structural features
required for CDK inhibition
Key sites for semi-synthetic
modifications
•Rohitukine is a flavone alkaloid isolated from plant
Dysoxylu
m b
ine
cta
rife
rum
(Meliaceae)
•This flavone led to discovery of two clinical candidates –Flavopiridol and P-276-00
•These flavones are potent inhibitors of CDKs –1,2,4 and 9
This scaffold has
shown promising
activity against
several kinases
implicated in
pathogenesis of
Confidential
Series of NCEs have been synthesized by
functionalization at these three positions
N
HO
=CH2-Ar, COAr, CH2-Heterocyle, CO-Heterocycle
A
=CH(X)-Ar, CH(X)-Heterocycle
B
=CH=CH-Ar, CH=CH-Heterocycle
C
pathogenesis of
cancer/ Alzheimers
disease/ diabetes
IIIM-NPC-08:
CDK-2: IC50= 8 nM
CDK-9: IC50= 0.32 nM
VEGFR: IC50= 12.5 nM
Dryrk-1A: IC50= 36 nM
AMPK: IC50= 21.8 nM
IIIM-CSIR
Meridianins: Marine-derived Indole alkaloid
•MeridianinsA-G are indole alkaloids isolated from tunicate A
plid
ium
meridia
num
•Potent inhibitors of CDK1 and CDK5 (Meridianin E: IC50180 and 150 nM resp.)
N HN H
O
N Ts
O
AcCl,SnCl 4
Toluene,
0 oC, 2 h
TsCl, DIPEA
DMAP/ DCM,
rt, 20 h, 95%
N H
N
N
H2N
N Ts
O
N
DMF-DMA
DMF,1100C
H2N
NH.HCl
NH2
OMe
HO
reflux, 24 h, 55-60%
4h, 85%
RR
RR
R
90%
Confidential
Meridianin G: R = H (Amount = 1 g)
Meridianin C: R = 5-Br (Amount = 500 mg)
•Series of analogs have been synthesized
•Analogs with promising activity against cancer targets have been
identified
IIIM-MCD-110:
IGF-1R: IC5050 nM
IIIM-CSIR
Fascaplysin: Marine-derived Indole Alkaloid
•Fascaplysin
isaindole
alkaloid
isolatedfrom
marinesponge
Fa
sca
ply
sin
op
sissp.
•Shown potent and selective CDK4 inhibition (over CDK2)
New route for
total synthesis
N H
NH2
O
H
OCl
N H
N O
Cl
a
Confidential
total synthesis
N H
N O
Fascaplysin
b
Cl
Series of analogs have been synthesized
Few analogs showed promising activity against cancer targets
IIIM-MCD-203:
PI3K-a: IC506.8 mM
IIIM-CSIR
Lead optimization of Bergenin scaffold
•Bergeninoccursinvarietyofspeciesof
Berg
enia.
•Traditionally,thisplantisusedintreatmentofrheumatoidarthritisandother
inflammatorydiseases
•Weisolatedbergeninfromrhizomesof
Berg
enia
str
acheyiandplanned
semisyntheticmodificationsforitsimmunomodulatoryproperties.
HO
OCH3 OH
OHO
HO
OCH3 OH
OHO
NR1
HCHO +
N H
R1
R2
DMSO, RT, 10 h
Mannich reaction
Confidential
Shreyans Jain, Samdarshi
OO
HO
OH
OO
HO
OH
NR1
R2
H
NR1
R2
=
N
HO
NO
NN
N
NN
N
HO
O
N
HO
O
OH
N
N
HO
O
O
Bergenin
Mannich reaction
Analogs with IC50= 100 nM against TNF-a and IL-6 production have been identified
IIIM-NPC-018: IC50 = 100 nM (TNF-α)
IIIM-CSIR
NCEs based on Colchicine scaffold
NH
O O
H3CO
H3CO H3CO
OCH3
Deprotection and
then modification
around free'NH2'
Modifications
on 'NH'
Series of NCEs have been synthesized by
functionalization at these positions
Several compounds showed promising
immunomodulatory activity and anticancer
activity in cell based and enzyme based
assays
IIIM-NPC-27:
Confidential
OCH3
Labile and can be easily replaced
IIIM-NPC-27:
TNFa: 42% inhibition at 100 nM
IIIM-NPC-27:
PI3Ka: IC5015 nM
Cell lines: Showed IC50< 1 mM in all
cancer cell lines (breast, colon, pancreatic
and leukemia)
IIIM-CSIR
New anti-inflammatory leads from Flavan scaffold
•Veryfewmethodsknownforsynthesis
offlavans.
•Shortcomingsofreportedmethods:
multiplesteps,expensive,hazardous
chemicals,protection/deprotection
stepsandhomogeneouscatalytic
systems.
Flavone (65% yield)
HO
OH
O
HO
OH
OH
Silica-HClO4
ACN, 80 oC, 1-2 h
+ +Flavans (76-92% yield)
HCHO
HO
OH
O O
(i). Silica-HClO4
(ii). Dioxane (1% H2O)
DDQ (6 equiv.)
"One-pot"
ACN, 80 oC, 1.5 h
OR
R
O
RO
O
R
R
O
R
O
O
O
OPh
Ph
Ph
HO
O
O
O
HO
OH
O
O
O
HO
OH
O
CHO
OHC
4 h, 76%
HO
OH
OH
HCHO
++
HO
OH
O
O
O
Catalyst
O
O
Solvent
Confidential
Entry
Catalyst(%w/w)
Solvent
Temp.
(°C)
Time
(h)
Yieldb
(%)
1Amberlyst-15(50)
ACN
80
350
2Amberlite-IR-50(50)
ACN
80
350
3Amberlite-IR-140(50)ACN
80
350
4Amberlyst-15(50)
H2O
100
320
5Amberlyst-15(50)
Dioxane
100
630
6Silicagel(50)
ACN
80
60
7Silica-I2(50)
ACN
80
355
8Silica-FeCl 3(50)
ACN
80
360
9Silica-HClO4(50)
ACN
80
384
10
Silica-HClO4(50)
H2O
100
350
11
Silica-HClO4(50)
Dioxane
100
350
12
Silica-HClO4(20)
ACN
80
370
13
Silica-HClO4(10)
ACN
80
350
14
Silica-HClO4(10)
ACN
80
12
80
15
Silica-HClO4(5)
ACN
80
335
Ph
HO
OH
O
O
O
HO
OH
O
O
O
HO
OH
O
CHO
OHC
HO
OH
O
O
O
HO
OH
O
O
O
HO
OH
O
O
O
HO
OH
O
O
OO
18 h, 70%
6 h, 35%
6 h, 52%
1.5 h, 84%
4 h, 76%
1.5 h, 86%
1 h, 92%
3 h, 87%
1 h, 92%
1 h, 92%
1 h, 92%
Reaction
time and
yields
HO
O
OH
OH
HO
OH
CHO
OHC
CHO
Sideroxylonals: R1 = H, R2 = isobutyl
grandinal: R1 = isobutyl, R2 = H
R1
OR2
Total synthesis of
these natural flavans
is in progress
IIIM-CSIRNatural Product Leads for Alzheimers’ Disease
ClinicalProofofthetarget:
•Twoindependentclinicalstudieshaveshownthat‘ decreasedclearanceof
amyloid-bfromthebrain’istheprimeconcerninAD.
•ItistheAb-clearanceandnottheproduction,thatisimpairedinAD.
•TherateofAbproductionissameasthatinhealthyvolunteers;whereasrateof
clearanceisimpairedby25-30%(S
cie
nce,2010,
330,1774).
P-Glycoprotein is the novel target for AD
Confidential
clearanceisimpairedby25-30%(S
cie
nce,2010,
330,1774).
•Thus,ADiscausedbyanimbalancebetweenAbproductionandclearance,
resultinginincreasedamountsofAbinvariousformsintheCNS.
P-GlycoproteinandAbclearance
•PGPdeficiencyattheBBBincreasesAbdepositioninADmousemodel.
•ThusourapproachistoenhancetheclearanceofAbacrosstheBBB,via
inducingtheexpressionofPGP.
Scie
nce2010,330, 1774
Bra
in, 2011, In press (doi: 10.1093/brain/awr298)
IIIM-CSIR
Medicinal Chemistry of Natural products PGP inducers
OH
OH
O
H3CO
H3COCO
NH
O
HO
HO
NN
OOH
OO
•Medicinal chemistry around following scaffolds is under way at IIIM.
Confidential
OH
O
OO
N
Rifampicin
Hyperforin
•Further, we are also working on the identification of novel scaffolds
using in
-sili
cohigh-throughput screening of compound libraries
IIIM-CSIR
Designing hyperforin analogs as potential PGP inducers
Detailed patent search
has been done. There
is no any earlier
patent on use of
Hyperforin or analogs
acting via PGP
induction mechanism
for AD treatment
OOH
OO
A libraryof polyalkylated
acylphloroglucinols
(Polyketides)
Variation in alkyl chain
length (e.g. methyl, geranyl)
Reduction of
carbonyls
Confidential
for AD treatment
OOH
OO
Hyperforin
HO
OH
OH
O
OOH
OO
RVariation in
chain length
length (e.g. methyl, geranyl)
Naturally occurring prenylated acylphloroglucinols will be isolated from
plants like Garcinia indica, Dryopteris sp.
R
Prenylated
acylphloroglucinols
Bicyclo[3.3.1]nonanone core
based analogs
R
R = allyl, prenyl, geranyl
IIIM-CSIR
Dis
cov
ery
an
d D
ev
elo
pm
en
t o
f B
ota
nic
als
fro
m
Ind
ian
Me
dic
ina
l P
lan
ts
Confidential
IIIM-CSIR
•Natural Product IIIM-K002 derived from the Plant RJM/035 (Alcoholic
Extract) as Antihypertensive (in collaboration with CDRI, Lucknow)
•Natural Product IIIM-K003 derived from the plant RJM 024 (alcoholic
extract) as Immunostimulator
Botanicals for Pre-clinical and Clinical stage
(The leads derived from the CSIR-Net Work Project)
Confidential
•Natural Product IIIM-K003 derived from the plant RJM 0862 (alcoholic
extract) as Hepatoprotective
•Natural Product ICB-derived from the plant Anti-ulcerogenic (ICB 014,
hydroalcoholic extract) (in collaboration with with IICB,Kolkota)
•Plant Based (RJM1195) Extract (IIIMRE-4)with potent Anti-arthritic Activity.
IIIM-CSIR
Immunomostimulant: RJM/0024
Bioactive Single Molecule IIIMK003
.
O
O
O
O
HO
H
HOH
HHO
Safe
ty stu
die
s: Original extract safe at 2 g/kg, p.o. in mice at single dose.
�K003showsstim
ulationin
humoralandCMIresponse,skin
allograftrejection,
phagocytosis,lymphocytesproliferationresponsesto
T&
Bcellsin
mice,anti-
inflam
matory
activity
inrats
and
induces
IL-2
production
inmouse
spleenocytes.
�Theim
munestim
ulatory
activityconfirm
edbyseveral
invitro
andin
vivo
assays.
Confidential
Effect of RJM0024 alcoholic Extract
and IIIMK003 on graft rejection in mice
Treatment
Dose
mg/kg
p.o.
Mortality
RejectionT
ime
(Days)
Mean ± S.E.
Graft
Rejection
Results
Control
- Nil
13.00 ± 0.25
-
Cyclospo
rine
5
Nil
18.16 ± 0.16
40.00↓
K003
5
Nil
8.50 ± 0.22
35.00↑
Active
Immunostimulatory activity ofRJM0024IIIM K003 on
Humoral and Cell mediated immune response in norm
al
Balb/C mice
Sa
mp
le
H
um
ora
l (i
n-v
ivo
) C
MI
(in
-viv
o)
D
ose
mg
/k
g
p.o
.
An
tib
od
y
tit
re
Me
an
± S
.E.
Imm
un
o-
mo
du
lato
ry
Activ
ity
(%
)
Fo
ot t
hic
kn
ess
(mm
) 2
4h
M
ea
n ±
S.E
.
Imm
un
o-
mo
du
lato
ry
Activ
ity
(%
)
No
rm
al
Co
ntro
l
6.1
6 ±
0.1
6
- 0
.85
± 0
.06
-
Cy
clo
ph
osp
ha
mid
e
25
0
4.3
3 ±
0.2
1
30
↓
-
Cy
clo
sp
orin
5
-
- 0
.45
± 0
.02
4
7.0
5↓
K0
03
0
.1
7.1
6 ±
0.1
6
16
.23
↑
1.0
0 ±
0.0
3
18
.00
↑
K0
03
0
.5
7.5
0 ±
0.2
1
21
.75
↑
1.0
8 ±
0.0
3
27
.05
↑
K0
03
1
7
.00
± 0
.25
1
3.6
3↑
0
.98
± 0
.01
1
5.2
9↑
K0
03
5
6
.50
± 0
.22
5
.51
↑
0.9
3 ±
0.0
4
09
.41
↑
Le
va
mis
ole
2
.5
8.0
0 ±
0.2
5
29
.87
↑
1.2
0 ±
0.0
4
41
.17
↑
IIIM-CSIR
invivo
eff
icacy
of
K003
alo
ne
and
inco
mbin
ation
with
rifam
pic
inagain
stMycobacterium
tuberculosis
H37R
v.
Mic
esp
ecie
s:Sw
iss
alb
ino,4-6
wee
ks,
20-2
2gm
,M
ode
of
infe
ction:
Intr
anasa
l
with
50µ µµµl
volu
me
conta
inin
g1.5
x10
6C
FU
/mouse
M.T
BH
37R
v.Tre
atm
ent:
PO
xO
Dx
30
days
start
ing
from
48hrs
post
infe
ctio
n.
786.4506
786.6666
5.5695
678
Log CFU/lung
End
poin
t:M
ice
sacr
ific
ed
48hrs
aft
erth
eco
mple
tion
of
trea
tmen
t.C
FU
/lef
tlu
ng
was
det
erm
ined
A-D
isse
ctio
n:
One
wee
kaft
er
com
ple
tion
of
trea
tmen
tC
FU
/lef
t
lung
wasdet
erm
ined
.
B-D
isse
ctio
n:
One
month
after
com
ple
tion
oftr
eatm
entC
FU
/lef
t
lung
wasdet
erm
ined
.
Rifam
pic
in +
RJM
0024 K
003 a
gain
st M
.tuberculosis Confidential
7.0511
5.4737
5.0563
4.2011
4.4811
2.7528
01234567
Infection control
M.tb
K003 0.5 mg/kgRif 2.5 mg/kgRif 2.5 + K003
0.5 mg/kg
Rif 5.0 mg/kgRif 5.0 + K003
0.5 mg/kg
Log CFU
3.2605
4.9221
5.551
4.0543
00
01234567
Early
Con
trol
Late C
ontro
lK00
3 0.5m
g/kg
Rif 5M
g/kg
Rif 5+
K00
3 Rif 10
mg/kg
Rif 10
+k003
0.5m
g/kg
Log CFU/lung
4.916
3.943
1.2329
3.2605
0123456
Early
Control
Late
Control
Rif
5Mg/kg
Rif
5+K003
Rif
10mg/kg
Rif
10+k003
0.5mg/kg
Log CFU/lung
Results:
Rif
10mg/kg
and
combination
ofRif
10mg/kg
with
RJM
0024IIIM
K003
(0.5mg/kg)achievessterilityin1month
treatm
ent(A)Howeverreactivationismuchless
withRif10mg/kgincombinationwithRJM0024IIIM
K003(B)
IIIM-CSIR
Bioavailability: 76.11 %
Eff
ect on B
P &
HR
in a
naes
thet
ized
SH
R (5 a
nd 1
0 m
g/k
g, i.v.):
Fall in B.P.: 14 and 22% mmHg respectively and m
ean arterial BP returned to baseline in 75 and 160
minutes respectively. The effect on heart rate was not significantly altered. T
he effect of propranolol
on BP was slightly more but it also produced significant bradycardia, which was not associated with
No anticholinergic or antihistaminic (0.05-3uM)
activity
Eff
ect on B
P &
HR
in a
naes
thet
ized
norm
ote
nsi
ve
rats
(2.5
, 5.0
& 7
.5 m
g/k
g, i.v.):
Fall in B.P.: 20-35% mmHg with a m
axim
um duration of 80 m
inutes at highest 7.5
In liter
atu
re, th
e pla
nt is
rep
ort
ed a
s ca
dio
tonic
. The
antihyper
tensi
ve
act
ion o
f R
JM
IIIM
K003 sta
ys fo
r lo
nger
dura
tion.It has no sig
nific
ant ef
fect
on h
eart
rate
. O
OO
CH3
OH
OH
Anti-H
yper
tensi
ve
candid
ate
(R
JM
0035 IIIMK002)
Confidential
Fall in B.P.: 20-35% mmHg with a m
axim
um duration of 80 m
inutes at highest 7.5
mg/kg dose tested. T
he effect on heart rate was not significant altered.
Effectin
SHR
on
heart
rate
(100
mg/kg,p.o. ):Itshowednobradycardia
&nosignificantfallinHR(N=5).
Effect on aortic ring: Acetylcholine (ACh) in a dose dependent manner produced endothelium dependent
relaxation in phenylephrine (1 ×10-6 M) precontracted isolated rat aortic ring preparation. At 300 micromole, ACh
showed maximum relaxation of 80.9 ±4.88 % (p<0.001). K002 followed the same pattern and exhibited
maximum relaxation of 54.5 ±7.84 % with equal concentration. The vasoreactivity of K002 was reduced to 10-
15% in the denuded aortic ring, indicating K002 effects are mediated via the receptor located on the endothelium
IIIM-CSIR
Eff
ect
on
BP
Effec
tin
SH
Rs
by
NIB
P(1
0m
g/k
g,i.v.):Fallin
B.P.:
19.50±
1.0%
mmHgwithin
15minutes,whichwas
sustained
upto
120
minutesand
returned
tobasal
in180
minutes.
(MAP=170.25+6.25
mmHg)
Effectin
SHRsbyNIBP(50mg/kg,p.o.):Therewasafallof11.72
±2.90%mmHgwithin60minutes.Evenafter300minutes,still11.07
±1.931%mmHgfallwasrecorded.*P<0.05Vspreadministration
BP(N=5).
Effect on Isolated Heart
It produced mild ionotropic effect. The compound also exhibited
mild anti-ischemic effect as well.
Effect on Cardiac Action Potential
Anti-H
yper
tensi
ve
candid
ate
(R
JM
0035 IIIMK002)
Confidential
Effectin
consciousSHRbytelemetry(100mg/kg,p.o.):Fallin
B.P.:Thepeakeffectof16%wasobservedat120minutes,which
persistedforover240min,followedbygradualrecovery>330
minutes.TheHRwasnotaltered.Further,theclinicallyusedanti-
hypertensivedrugatenolol(10mgp.o.)producedpeakfallof25%in
BPwithaccompanyingbradycardiaofabout17%.
50
75
100
125
030
60
90
120
150
180
210
240
270
300
330
Tim
e (min)
% Change BP
Vehicle
Compound K002 100 m
g/kg p.o.
Effect of K002 on Blood Pressure
in conscious rats by Telemetry
Effect on Cardiac Action Potential
K002 (0.2 µg to 6.4 µg/ml) does not seem to act on calcium channel to produce its
anti-hypertensive effect.
Mechanism of Action:
IIIMK002showedsignificantconcentrationdependentinhibition(invitro)of
ACE..Syntheticsubstrate:Furylacrylphenylalanylglycylglycine(FAPGG).
SourceofACE:Serum.Therefore,itislogicaltoinferthat
antihypertensiveeffectofK002seemstobemediatedin
amajorwaybyACEmechanism.
IIIM-CSIR Plant: ICB 014 Hydroalcoholic extract (Flowers)
Bioactive Single molecule (Oenothein CNatural abundance 0.008%)
In v
itro
In v
itroStudies
Studies
Anti-secretory Activity
Isolated stomach 25% inhibition at 10 mg/chamber,
Anti H+Pump activity ─ IC50∼1 µg/ml (omeprazole 5 µg/ml)
Anti-
H. pylo
riActivityMIC5025 µg/ml (clinical and standard strains) MBC 800 µg/ml
Specific against H
. pylo
ri(very high MIC with others)
Bacteriostatic, Does not develop Drug Resistance (same MIC –10 passages)
Effective at acidic pH as well.
OO
OOH
OH
O
O
OH
OH
OH
O
O OH
OH
OH
OH
OH
O
O
O
OH
In v
ivostudies
♦Cold restraint ulcer ─ Protects ~55% at 40 mg/kg; OPZL , 10 mg/kg, 68%
Anti-U
lcer
candid
ate
Confidential
OH
OH
♦Cold restraint ulcer ─ Protects ~55% at 40 mg/kg; OPZL , 10 mg/kg, 68%
♦Aspirin-induced ulcer ─ Protects ~60% at 50 mg/kg; OPZL, 10 mg/kg, 68%
♦Ethanol-induced ulcer ─ Protects ~80% at 20 mg/kg; sucralfate, 500 mg/kg 65%
♦Pylorus ligation ulcer ─ Protects 20-40% at 25-50 mg/kg; OPZL, 10 mg/kg, 50%
♦Indomethacin-induced ulcer ─ Protects 85% at 10 mg/kg; OPZL, 5 mg/kg, 65%
♦H
. pylo
riclearance and eradication ─ Showing hints for efficacy
Safety studies
♦Single dose acute toxicity: 5 g/kg (p.o.) in rats ─ No Mortality
♦One week toxicity: 1 g/kg/day(p.o.) (two divided doses) for 1 week–No Mortality
Conclusions:
♦Strong inhibitor of gastric H+Pump. Inhibition is perhaps irreversible in nature.
♦Inhibits basal acid secretion (40%) and histamine-stimulated acid secretion (25%) at 50 µg/ml
in isolated gastric parietal cells. It isSpecific against H
. pylo
ri.
♦It is more like bacteriostatic (time kill assay; MBC ~ 200 µg/ml with majority of the strains).
IIIM-CSIR
Hepatoprotective candidate
Plant: RJM0862
Bioactive Molecule : RJM0862 IIIMK003
Natural Abundance of IIIMK003 (7.0 %).
In vitro
hepatoprotective activity
•No cytotoxicity at 10 -100 µg/ml
•Restores depletion of GSH (16-34%) at 10 -100 µg/ml
•Prevents the leakage of LDH (46-71%), ALT ( 60 -89%) at 10 -
100 µg/ml in primary monolayer culture of rat hepatocytes.
In vivo hepatoprotective activity
Safetystudies:
O
O
OOH O
O
glucose
OH
OH
O
CH3
HO
HO
mannose
OH
HO
Confidential
In vivo hepatoprotective activity
Do
se m
g/k
g,
p.o
.
Av
era
ge
%
Pro
tect
ion
(ALT
, A
ST,
ALP
, B
RB
N,
TG
, A
lb.,
LP
&
GS
H)
CC
l4A
PAP
Ga
lN
Act
eo
sid
e 0
.62
52
7.5
3±
3.9
73
7.4
3±
4.2
83
7.8
9±
2.2
4
Act
eo
sid
e 1
.25
44
.29±
4.1
75
4.2
7±
4.0
95
0.1
6±
1.9
8
Act
eo
sid
e 2
.50
59
.62±
2.6
36
6.3
0±
4.2
96
0.8
4±
2.5
6
Act
eo
sid
e 5
.00
71
.66±
1.8
78
0.9
8±
3.3
47
2.1
5±
1.6
2
Sil
ym
ari
n 5
0.0
56
.35±
0.9
95
9.5
8±
1.7
95
6.4
4±
1.8
6
Safetystudies:
Acute:>3g/kg,p.o.
Sub-acute:56Days,repeated
oraldose
2.5,5and10times
ofED50(2.5mg/kg,p.o.)
Stability studies: Stable under
accelerated conditions at 25 oC
•The Hepatoprotective activity has
been confirm
ed by several in
vitro
and in vivo assays.
IIIM-CSIR
Immunosuppressive candidate
Plant: RJM1195 (Stem/leaf). Bioactivity in Alcoholic (A001) and Modified
Alcoholic Extract (RE-4)
Extract A001 potent but Toxic.
The extract RE4 devoid of toxicityis potent TNF alpha inhibitor, anti-arthritic
activity (using CIA model in mice, Nicolas Piramal). Anti-arthritic activity
using Mycobacterium Adjuvant Induced Arthritis in rats.
Anti-inflammatory activity using Carrageenan induced edema in rats.
Confidential
Anti-inflammatory activity using Carrageenan induced edema in rats.
Six compounds isolated from RE4, (triterpenoid / steroids) are less active
than RE4. Therefore, RE4 pursued).
Conclusion:
Bioactive extract RE-4has significant TNFαinhibitory, anti-arthritic and anti-
inflammatory activities.
TNFαinhibitors are in demand for the treatment of Rheumatoid Arthritis at
present and thus the lead is important.
IIIM-CSIR
Effect on Articular index of Arthritic (CIA) DBA/1J m
ice
4.00
5.00
6.00
7.00
8.00
Articular index
AntiArthritic Activity RJM1195 IIIM RE-4
Confidential
0.00
1.00
2.00
3.00
4.00
12
34
56
78
910
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Day
Articular index
Control, Distilled water, 10 m
l/kg, p.o., twice daily
Enbrel, 3 m
g/kg, s.c., once daily
RE-4, 200 m
g/kg, p.o. tw
ice daily
IIIM-CSIR
cGMP pilot plant for Extraction, Form
ulation and Packaging of
Traditional (ISM) Herbal M
edicines
�As per W
HO guidelines
�Extraction unit : 25-30 kg of plant material (dry weight).
�Automated form
ulation &
packaging unit
�Total Project Cost: US$ 4 m
illion (funded by D
ST, A
YU
SH
& C
SIR
)
Confidential
1
Carpet A
rea 2
900 S
qm
(G
F+FF)
IIIM-CSIR
Poss
ible
Utiliza
tion o
f cG
MP faci
lity
�To be used by the institute and CSIR
labs to facilitate their R&D
activities related to drug development.
�To be run under -Public Private Partnership as per CSIR
guidelines.
�Process development, scale-up &
optimization of lab-processes
and converting them
into commercially viable technologies.
Confidential
and converting them
into commercially viable technologies.
�Generation of authentic and accurate clinically acceptable data,
which can potentially lead to new
IPR’s
�Generation of data for effective regulation and control of
herbal/traditional m
edicines m
oving in to national/international
markets.
�Producing products for clinical trials.
�Producing products for initial market survey of the industry.
IIIM-CSIR
Facilities for QC/QA for
Chemistry Manufacturing Control of Botanicals
Majo
r C
MC
info
rmation g
ener
ate
d�
Chem
ical id
entifica
tion b
y spec
trosc
opic
and /or
chro
mato
gra
phic
fin
ger
pri
nting.
�A
ssay o
f act
ive
const
ituen
ts o
r ch
ara
cter
istic
mark
ers.
�H
eavy m
etals
�R
esid
ual pes
tici
des
�A
dven
titious to
xin
s (e
.g. aflato
xin
)
�M
icro
bia
l L
oad
�R
esid
ual so
lven
ts
�R
esid
ue
on ignitio
n/w
ate
r co
nte
nt/st
rength
by d
ry w
eight
Confidential
ATOMIC
ABSORPTIO
N SPECTROPHOTOMETER) ICPMS (Aglient)
(Perkin Elm
er Analyst-800
LCMSMS (Waters -LC2695
GC
-MS/M
S (Ther
mo) G
C (Shim
adzu
)
ION
Chro
mato
gra
ph (M
ethro
m) H
PTLC
(C
AM
AA
G
IIIM-CSIR
CM
C o
f Tinospora Cordifolia:
Chromatographic fingerprinting: HPTLC//HPLC//MASS –
LCMSMS
2000
2000
1: 215 nm, 8 nm
MIXTURE OF TINO,ECD,TC,CORDIOSIDE
20-03-12-TINO6
Retention Time
Name
750
750
1: 215 nm, 8 nm
I-01067(03)
20-03-12-TINO13
Retention Time
Name
Cordiofolioside
TC-1
Tinosporaside
Ecdysone
Photodocumentation at White R
QCQA AND CMC
DIV
ISIO
NH
PT
LCP
RO
FIL
E
HP
LCP
RO
FIL
ETinospora-aq./aq-alc
Tinospora Ecdysone TC1
Cordiofolioside
Confidential
Minutes
1618
2022
2426
28
0
1000
01000
17.323 Cordifolioside
20.203 Ecdysterone
22.987 Tinosporaside
24.683 TC1
Name
Minutes
1618
2022
2426
28
0
250
500
750
0250
500
750
16.181 16.427
16.736
17.355 Cordifolioside
17.920
18.848
19.296 19.637
20.405 Ecdysterone
21.547
21.952 22.144 22.464
23.008 Tinosporaside23.360 23.691 24.011
24.384
24.821 TC1
25.323
26.677
27.552
Name
29-Mar-2012 11:37:56
VAA208
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
% 0
100
TINOSPORASIDE-MS 24 (0.846) Cm (21:27)
Scan ES+
7.23e7
515.31
313.35
73.78
160.56
87.74
294.89
413.35
331.42 383.48
510.28
515.44
1007.54
531.29
547.20
1023.52
28-Mar-2012 15:39:33
VAA208
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
% 0
100
TC-1-MS 43 (0.796) Cm (41:54)
Scan ES+
1.30e8
413.15
327.41
98.77
73.93
100.53
148.64
199.43
299.38
391.31
803.34
429.06
605.24
597.55
798.58
613.26
787.69
819.38
29-Mar-2012 12:26:19
VAA208
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
% 0
100
ECDYSTERONE-MS 19 (0.670) Cm (19:29)
Scan ES+
1.07e8
503.37
445.32
98.70
73.78
427.31
413.35
160.82
983.74
519.35 543.42 559.40
999.78 1023.78
1464.26
28-Mar-2012 12:51:59
VAA208
m/z
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
% 0
100
CORDIOFOLIOSIDE-MS 51 (0.944) Cm (33:54)
Scan ES+
1.01e8
527.19
98.44 132.99160.96
192.97
567.16
1031.52
583.20
MA
SS
-LC
MS
MS
OF
4 M
AR
KE
RS
IIIM-CSIR
CM
C o
f Tinospora Cordifolia:
Residual pesticides// solvents//metals
Pa
ram
ete
rR
esu
ltLi
mit
Ala
chlo
rB
DL
20
Aldrin
BD
L5
0
Dieldrin
BD
L5
0
Brompropylate
BD
L3
00
0
Chlorfenvinphos
BD
L5
00
Chlorpyriphos
BD
L2
00
Chlorpyriphosm
ethyl
BD
L1
00
Cypermethrin isomers
BD
L1
00
0
p-p-D
DT
BD
L1
00
0
o-p-D
DT
BD
L1
00
0
p-p-D
DE
BD
L1
00
0
Pa
ram
ete
rR
esu
ltLi
mit
Delta-HCH
BD
L300
Malathion
BD
L1
00
0
Parathion-m
ethyl
BD
L2
00
0
Pe
rme
thri
nB
DL
10
00
Ph
osa
lon
eB
DL
10
0
Pip
ero
nyl
bu
toxi
de
BD
L3
00
0
Pir
imip
ho
s m
eth
yl
BD
L4
00
0
Pyre
thri
ns
(su
mo
f)B
DL
30
00
Quintozene
BDL
1000
Pa
ram
ete
rR
esu
ltLi
mit
Endosulfan
Sulfate
BD
L3
00
0
Endrin
BD
L5
0
Fenvalerate
BD
L1
50
0
Heptachlor
BD
L5
0
Heptachlor
exoepoxide
BD
L5
0
Heptachlor
Endoepoxide
BD
L5
0
Hexachlorobenze
ne
BD
L1
00
Alpha-HCH
BD
L3
00
RT:0.00 - 46.42
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative Abundance
19.81
22.65
12.51
10.02
9.18
33.30
35.58
25.26
6.97
38.7939.40
5.40
13.87
29.25
17.55
36.96
31.72
40.74
NL:
1.29E7
TIC F: MS
SPIKEDHE
RBALAT8P
PM
Confidential
p-p-D
DE
BD
L1
00
0
Diazinon
BD
L5
00
Endosulfan (α+β)
BD
L3
00
0
Azi
no
ph
os
me
thyl
BD
L1
00
0Beta-HCH
BD
L3
00
Gam
ma HCH
BD
L6
00
05
10
15
20
25
30
35
40
45
Time (min)
05
10
15
20
15.61
42.51
28.33
2.M
eth
oxy
eth
anol
2,e
thoxy e
thanol
Pyrid
ine
Tolu
ene
Eth
yle
ne G
lycol
Form
am
ide
N,N
,Dim
eth
yl -
aceta
mid
e
Dim
eth
yl-
Form
am
ide
Chlo
rbenze
ne
O-X
yle
ne
m-X
yle
ne
P-X
yle
ne
N,N
, D
imeth
yl
Pyrrolidin
Tetr
ale
ne
Sulfola
ne
Hig
h b
oilin
g p
oin
t so
lven
ts
Meth
anol
Aceto
nitrile
Meth
yle
ne C
hlo
rid
e
Hexane
Nitrom
eth
ane
Chlo
rofo
rm
Cyclo
hexane
Carbonte
traclo
rid
e
Benze
ne
1,2
dim
eth
oxy
Eth
ane
Meth
yl C
yclo
hexane
1,4
Dio
xane
Low
boilin
g p
oin
t so
lven
ts
Aq
Ex
t.
Dry
ste
m
Lim
its
(pp
m)
Pb
1.1
65
.21
10
Cd
0.0
04
0.0
80
.3
Hg
BD
LB
DL
1.0
As
BD
LB
DL
3.0
Methods A
OA
C:I CPMS
IIIM-CSIR
CM
C o
f Tinospora Cordifolia:
Aflato
xin
//M
icro
bia
l Load
Minutes
24
68
10
12
14
16
18
20
22
24
0.00
0.02
0.04
0.06
0.08
0.10
0.00
0.02
0.04
0.06
0.08
0.10
4.825 AFLATOXINS G1
5.342
6.033 AFLATOXINS B1
7.117 7.533
8.700 AFLATOXINS G2
9.675
10.817
11.917 AFLATOXINS B2
13.092
14.292
14.992
Detector A (Ex:350nm, Em:450nm)
AFLATOXINS STANDARD
02-01-11-afla8
Retention Time
Name
Minutes
24
68
10
12
14
16
18
20
22
24
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
4.683 (AFLATOXINS G1)
5.208 5.583
5.908 (AFLATOXINS B1)
6.942
7.742 8.042
(AFLATOXINS G2)
9.400
10.050
11.167
(AFLATOXINS B2)12.175
12.708
13.817
15.700
17.008
17.867
18.500
19.533
21.683
22.308
Detector A (Ex:350nm, Em:450nm)
I-01020-B
08-11-11-AFLA2
Retention Time
Name
Re
sult
s
S.N
o.
Pa
ram
ete
rR
esu
lt
1.
AF
LAT
OX
INS
B1
BD
L
2.
AF
LAT
OX
INS
G1
BD
L
3.
AF
LAT
OX
INS
B2
BD
L
4.
AF
LAT
OX
INS
G2
BD
L
5-2
0n
g/g
(a
s p
er
US
FD
A)
BD
L=B
elo
w D
ete
ctio
n L
imit
sMixture of aflatoxins G1,B1,G2&B2
Plant extract
HP
LC P
RO
FIL
E :
A
fla
tox
in G
1,B
1,G
2&
B2
ma
rke
rs a
nd
p
lan
t e
xtr
act
Confidential
Mixture of aflatoxins G1,B1,G2&B2
Plant extract
MIC
RO
BIA
L LO
AD
S.N
oP
ara
me
ter
Re
sult
Lim
its/
cfu
/g
1.
E c
oli
Ab
sen
t1
0
2.
Sa
lmo
ne
lla
Ab
sen
tA
bse
nt
3.
Pse
ud
om
on
as
ae
rug
ino
sa
Ab
sen
tA
bse
nt
4.
Sta
ph
ylo
cocc
us
au
reu
sA
bse
nt
Ab
sen
t
5.
Tota
l Ba
cte
ria
l C
ou
nt
2.6
x10
4<
10
5
6.
Yea
st a
nd
Mo
uld
3
0<
10
3
7.
En
tero
ba
cte
rice
ae
an
d
oth
er
G-v
e b
act
eri
a
<1
0<
10
3
RE
SID
UE
ON
IG
NIT
AT
ION
/ W
AT
ER
CO
NT
EN
T
S.N
o.
Pa
ram
ete
rR
esu
ltLi
mit
s
1.
Tota
l Ash
0.5
67
3%
No
t m
ore
th
an
9%
2.
Aci
d I
nso
lub
le A
shN
ilN
ot
mo
re t
ha
n 1
.5%
3.
Mo
istu
re1
5.5
4%
-
IIIM-CSIR
Plants under captive cultivation
and other plant resources in Gene Bank
Ae
gle
ma
rme
los
Wo
od
ford
iafr
uti
cosa
Co
leb
roo
kia
op
po
siti
foli
a
Confidential
Plant name
A. marmelos C. oppositifolia W. fruticosa.
No. of Plants
87
872
288
Area covered (in acres) 0.93
0.57
0.3
Pla
ceN
o.
of
spe
cie
s
IIIM
Ja
mm
u C
am
pu
s Fa
rm2
5
IIIM
Fa
rm (
Ch
ath
a)
11
4
IIIM
Fa
rms
(Ka
shm
ir)
79
IIIM-CSIR
Symposium on
IND enabling studies
for botanical drug discovery
Announcement
Confidential
To b
e o
rga
niz
ed
by
CR
ISM
Mir
ror
Ce
nte
r
at
CS
IR-I
nd
ian
In
stit
ute
of
Inte
gra
tiv
e M
ed
icin
e,
Jam
mu
, In
dia
29-30 October, 2012
IIIM-CSIR
Th
an
k Y
ou
Confidential
