~Isolation of chemical constituents from ~Isolation of chemical constituents from Eclipta Eclipta

alba alba Linn Linn. . for achieving standardization for achieving standardization
By
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MA8TER OF PHARMACY
Under the guidance of
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Karnataka, IndIa
ArI! 2011
1^I)OI+(I1O^ 1^I)OI+(I1O^
Bhringarai, Eclipta alba , belonging to Iamily Astraceae is
commonly known as False Daisy is a common weed in moist
situations throughout India, ascending up to 6000 It on the hills
It grows commonly in moist places as a weed all over the world
It is widely distributed throughout India, China, Thailand,
and Brazil Bhringarai is small annual, procumbent much
branched and prostrating herb, 20 to 60 cm high, grows in Iields
and ditches hite Ilowers appear in summer and are small
penny size, Ilower stalk-elongated having white Ilower on its tips
and Ilower heads are axillary or terminal
Eclipta alba (L.) contains wide range oI active principles which
includes coumestans. alkaloids, Ilavonoids, glycosides,
polyacetylenes, triterpenoids The leaves contain stigmasterol, u-
terthienylmethanol, wedelolactone, demethylwedelolactone and
demethylwedelolactone-7-glucoside The polypeptides isolated
Irom the plant yield cystine, glutamic acid, phenyl alanine,
tyrosine and methionine on hydrolysis icotine and nicotinic
acid are reported to occur in this plant
1^I)OI+(I1O^ 1^I)OI+(I1O^
edicinal Forms: edicinal Forms:
xtracts ( dry and soIt), Oil, Fresh leaI iuice, xtracts ( dry and soIt), Oil, Fresh leaI iuice,
Decoction, Maka, Tincture, An herbal poultice Decoction, Maka, Tincture, An herbal poultice
made with sesame oil Ior skin disorders Key made with sesame oil Ior skin disorders Key
ingredient oI herbal hair shampoo ingredient oI herbal hair shampoo
%raditional Usage %raditional Usage: :
Antihepatotoxic , Hair growth promoter, Anti Antihepatotoxic , Hair growth promoter, Anti- -
inIlammatory, Antibacterial, inIlammatory, Antibacterial, Antispasmodic Antispasmodic
Antihaemorrhage Antihaemorrhage, ipid lowering, Antioxidant , ipid lowering, Antioxidant- -
Skin Disorders, Anticancer, Skin Disorders, Anticancer, antivenom antivenom, ,
antigiardial antigiardial, , trypsin trypsin inhibitor, Analgesic inhibitor, Analgesic
'arious formulations of 'arious formulations of Eclipta alba Eclipta alba L. L.
73amalakadi 73amalakadi Taila Taila. .
73a7aia 73a7aia Taila Taila. .
Nili Nili 7adi 7adi Taila Taila. .
Sadbi3du Sadbi3du Taila Taila..
7a3a7aiadi 7a3a7aiadi Cxu73a Cxu73a..
73a7aiasava 73a7aiasava. .
Teka7aia Teka7aia Ma7ica Ma7ica. .
etail of the parts containing chemical constituents etail of the parts containing chemical constituents
Sl.No. Parts Chemical constituents
eaves edelolactone|6|, Desmethylwedelolactone,Desmethyl-wedelolactone-7-
glucoside, stigmasterol
2 Roots Hentriacontanol, Heptacosanol& Stigmasterol, cliptal, clalbatin
3 Aerial parts -amyrin & uteolin-7-0-glucoside, Apigenin, Cinnaroside, Sulphur compounds,
clalbasaponins
4 Stems edelolactone
5 Seeds Sterols, cliptalbine (alkaloid)
6 hole plant Resin, cliptine, Reducing sugar, icotine, Stigmasterol, Triterpene saponin,
clalbatin,Ursolic acid, Oleanolic acid
etail of Pharmacological activities of the chemical etail of Pharmacological activities of the chemical
constituents constituents
Sl.No Chemical constituents Pharmacological activites
edelolactone Antihepatotoxic, Antibacterial, Trypsin Inhibitor,
Antivenom
2 clalbosaponins Hair revitalizing, AntiproleIerative, Antigiardial
3 Demethylwedelolactone Antihepatotoxic, Antihaemorrhage, Antivenom, Dye
(cosmetic)
4 Dasyscyphin C Antiviral, Anticancer
5 clalbatin Antioxidant
6 cliptalbine, verazine ipid lowering, Analgesic
Important chemical structures isolated from Important chemical structures isolated from
Eclipta alba Eclipta alba L. L.
/1^ /^I O!1(I1(1^ /1^ /^I O!1(I1(1^
O!1(I1(1 OI ^I+I O!1(I1(1 OI ^I+I
Collection Collection and and authentication authentication oI oI the the plant plant
IdentiIication IdentiIication oI oI maior maior compounds compounds using using Thin Thin ayer ayer Chromatography Chromatography
(maior (maior spots) spots)
Isolation Isolation oI oI maior maior compounds compounds Irom Irom the the extract extract by by column column chromatography chromatography
PuriIication PuriIication oI oI the the phytoconstituents phytoconstituents using using Column Column Chromatography, Chromatography, and and
Preparative Preparative HPC HPC
Characterization Characterization oI oI the the isolated isolated compounds compounds
stimation stimation oI oI the the isolated isolated compounds compounds in in the the extracts extracts oI oI Eclipta Eclipta alba alba
)1(11+ OI 1I1)/I+)1 )1(11+ OI 1I1)/I+)1
)1(11+ OI 1I1)/I+)1 )1(11+ OI 1I1)/I+)1
Coumestans, Coumestans, wedelolactone wedelolactone and and desmethylwedelolactone desmethylwedelolactone active active principles principles isolated isolated
Irom Irom Eclipta Eclipta alba alba were were used used Ior Ior evaluation evaluation oI oI Gal Gal- - induced induced cytotoxicity cytotoxicity activity activity
in in cultured cultured rat rat hepatocytes hepatocytes edelolactone edelolactone has has shown shown signiIicant signiIicant antihepatotoxic antihepatotoxic
activity activity at at aa dose dose oI oI 000 0mg/ml mg/ml apigenin apigenin has has shown shown more more than than 00 00 in in GPT GPT
activity, activity, where where as as standard standard silybin silybin has has shown shown aa protective protective oI oI 78 78 at at aa dose dose oI oI
00mg/ml mg/ml HPC HPC method method was was developed developed to to evaluate evaluate the the content content oI oI wedelolactone wedelolactone
and and desmethylwedelolactone desmethylwedelolactone
Six Six new new Oleanane Oleanane triterpene triterpene glycosides glycosides were were isolated isolated Irom Irom the the methanolic methanolic extract extract
oI oI Eclipta Eclipta alba alba Hassk Hassk xtract xtract was was evaporated evaporated under under reduced reduced pressure pressure to to aIIord aIIord
residue residue The The residue residue was was partitioned partitioned between between nn- -hexane, hexane, water, water, and and butanol butanol The The
butanol butanol phase phase was was removed removed to to Iurnish Iurnish the the residue residue which which was was subiected subiected to to column column
chromatography chromatography on on sephadrex sephadrex H H- -20 20 eluted eluted with with methanol methanol && silica silica gel gel eluted eluted with with
chloroIorm chloroIorm:: methanol methanol:: water water ( (88::22::0022- -66::44::) ) to to give give eclalbasaponins eclalbasaponins I I- -VI VI ( (- -66) )
Four Four new new taraxastane taraxastane triterpene triterpene glycosides glycosides eclabasaponins eclabasaponins VII VII- -X X were were isolated isolated
along along with with eclabasaponins eclabasaponins I I- -VI VI Irom Irom the the dried, dried, whole whole parts parts oI oI Eclipta Eclipta alba alba The The
structure structure oI oI eclabasaponins eclabasaponins VII VII- -X X were were characterized characterized as as 33, ,20 20, ,6 6- -and and 33, ,20 20, ,28 28- -
trihydroxy trihydroxy taraxastane taraxastane glycosides glycosides and and their their sulphated sulphated saponins saponins on on the the basis basis oI oI
spectral spectral data data
The The Ilavanoids, Ilavanoids, apigenin, apigenin, luteolin, luteolin, wedelolactone, wedelolactone, apigenin apigenin- -77- -O O- -glucoside glucoside were were
isolated isolated Irom Irom the the whole whole plant plant oI oI Eclipta Eclipta alba alba.. The The structure structure oI oI these these compounds compounds
were were characterized characterized and and identiIied identiIied by by spectral spectral data data This This was was the the Iirst Iirst report report oI oI the the
presence presence oI oI luteolin luteolin and and apigenin apigenin- -77- -O O- -glucoside glucoside in in Eclipta Eclipta..alba alba..
For For the the Iirst Iirst time time luteolin, luteolin, wedelolactone wedelolactone and and desmethylwedelolactone desmethylwedelolactone were were
isolated isolated Irom Irom the the aerial aerial parts parts Vietnamese Vietnamese Eclipta Ecliptaalba( alba()Hassk )Hassk which which was was
chemically chemically investigated investigated The The structures structures oI oI these these compounds compounds were were elucidated elucidated on on the the
basis basis oI oI spectroscopic spectroscopic evidence evidenceHypoglycemic Hypoglycemic eIIect eIIect oI oI Fenugreek Fenugreek seed seed powder powder
( (T7i43ella T7i43ella f4e3um f4e3um 7aecum 7aecum) ) was was studied studied in in 60 60 non non- -insulin insulin dependent dependent diabetic diabetic
patients patients
Based Based on on spectral spectral analysis, analysis, Iive Iive compounds compounds namely namely 7 7- -tri tri- -triacontanone, triacontanone, - -
hentriacontanol, hentriacontanol, hirtin hirtin,, wedelolactone wedelolactone && - - sitosterol sitosterol,, were were isolated isolated Irom Irom the the
methanolic methanolic extract extract oI oI Eclipta Eclipta alba alba ( () ) Hassk Hassk ater ater && methanolic methanolic extract extract were were
evaluated evaluated against against Riz4ct43ia Riz4ct43ia s4a3i s4a3i && c4llect4t7icum c4llect4t7icum falcatum falcatum at at diIIerent diIIerent
concentrations concentrations Compared Compared to to methanolic methanolic extract extract at at 2000 2000g/ml, g/ml, the the water water extract extract
was was Iound Iound more more active active against against both both Iungi Iungi
Stigmasterol Stigmasterol an an ubiquitous ubiquitous steroid steroid && two two oleanane oleanane glycosides glycosides eclalbasaponin eclalbasaponin II( II() )
and and eclalbasaponin eclalbasaponin I( I(22) ) were were isolated isolated Irom Irom an an nn- -hexane hexane extract extract oI oI the the stem stem bark bark oI oI
Eclipta Eclipta p74st7ata p74st7ata using using column column chromatography chromatography technique technique Isolated Isolated stigmasterol stigmasterol
was was colourless colourless needle needle shaped shaped Irom Irom the the Iraction Iraction 30 30 oI oI the the nn- -hexane hexane extract extract
^/I1)1/^ c ^1I1OI^ ^/I1)1/^ c ^1I1OI^
^vciv[. vnv ^cnov. ^vciv[. vnv ^cnov.
Plant material Plant material
The dried plant seeds oI The dried plant seeds oI Eclipta alba Eclipta alba were obtained Irom atural Remedies PVT were obtained Irom atural Remedies PVT
TD, Bangalore and authenticated by ISCAIR, Delhi The whole plant were then sun TD, Bangalore and authenticated by ISCAIR, Delhi The whole plant were then sun
dried, powdered and stored in airtight containers Ior Iurther use dried, powdered and stored in airtight containers Ior Iurther use
traction traction
The The whole whole plant plant oI oI Eclipta Eclipta alba alba were were was was coarsely coarsely powdered powdered and and subiected subiected Ior Ior
extraction extraction using using static static extractor extractor
30 30 kg kg oI oI the the powdered powdered plant plant was was reIluxed reIluxed with with Iresh Iresh methanol methanol ( (::33 drug drug to to solvent solvent
ratio) ratio) Ior Ior one one and and halI halI hour hour at at temperature temperature less less than than 65 65
00
CC and and the the extract extract was was strained strained
through through muslin muslin cloth cloth to to obtained obtained | |
st st
extract| extract|
The The marc marc was was again again subiected subiected Ior Ior second second time time extraction extraction (drug (drug marc marc to to solvent solvent ratio ratio is is
::22) ) and and the the extract extract obtained obtained was was strained strained through through muslin muslin cloth cloth to to yield yield | |22
nd nd
extract| extract|
The The same same procedure procedure was was Iollowed Iollowed Ior Ior third third wash wash to to obtained obtained | |33
rd rd
extract| extract|
The The
st st
,, 22
nd nd
,, 33
rd rd
extracts extracts were were combined combined together together and and concentrated concentrated up up to to total total solids solids
20 20 in in aa cruzol cruzol concentrator concentrator (Temp (Temp less less than than 85 85
00
CC and and pressure pressure 550 550- -mm mm oI oI Hg) Hg)
Further Further concentration concentration was was carried carried out out using using vacuum vacuum tray tray drier drier (Temp (Temp less less than than 70 70
00
CC
and and pressure pressure 500 500 mm mm oI oI Hg) Hg) to to obtain obtain aa dry dry powder powder The The dried dried powder powder was was again again
reIluxed reIluxed with with ethyl ethyl acetate acetate ( (::33 drug drug to to solvent solvent ratio) ratio) Ior Ior halI halI an an hour hour at at temperature temperature
65 65
00
CC and and the the extract extract was was strained strained with with muslin muslin cloth cloth to to get get the the insoluble insoluble ethyl ethyl acetate acetate
extract extract
Iv.ionvion/i.o[vion Iv.ionvion/i.o[vion
iquid iquid- -liquid liquid partitioning partitioning method method was was employed employed to to separate separate the the plant plant
constituents constituents with with mixture mixture oI oI chemical chemical components components into into groups groups oI oI
compounds compounds sharing sharing similar similar physico physico- -chemical chemical parameters parameters
400 400gm gm oI oI ethyl ethyl acetate acetate insoluble insoluble extract extract was was dissolved dissolved in in 22 liters liters oI oI water water
by by warming warming It It was was partitioned partitioned three three times times with with 22 liters liters equal equal portion portion oI oI
butanol butanol
The The butanol butanol layer layer was was allowed allowed to to separate separate and and the the butanol butanol soluble soluble part part was was
collected, collected, combined combined and and concentrated concentrated to to get get dry dry powder powder (Temp (Temp below below 65 65
O O
CC and and vacuum vacuum pressure pressure 300 300 mm/hg) mm/hg) Final Final concentration concentration was was done done under under
vacuum vacuum pressure pressure 200 200 mm/hg mm/hg
The The partitioned partitioned 44 44gms gms butanol butanol extract extract was was subiected subiected Ior Ior Iurther Iurther Iractionation Iractionation by by
silica silica- -gel gel column column chromatography chromatography using using Petroleum Petroleum ether, ether, ethyl ethyl acetate acetate and and
methanol methanol
The The 0 0 MeOH/A MeOH/A and and 75 75 MeOH/A MeOH/A Iractions Iractions were were selected selected Ior Ior the the isolation isolation oI oI
phytoconstituents phytoconstituents which which were were identiIied identiIied by by TC TC using using chloroIorm chloroIorm :: methanol methanol
( (9955::0055)as )as mobile mobile phase phase which which was was most most eIIective eIIective Ior Ior the the separation separation and and detection detection
by by Anisaldehyde Anisaldehyde sulphuric sulphuric acid acid reagent reagent which which gave gave diIIerent diIIerent colors colors
0 0 MeOH/A MeOH/A was was dissolved dissolved in in methanol methanol and and kept kept overnight overnight Ior Ior recrystallization recrystallization
process process The The extract extract was was Iiltered Iiltered using using Iilter Iilter paper paper no no:: 20 20 The The Iiltrate Iiltrate was was again again
kept kept Ior Ior crystallization crystallization overnight overnight to to get get crystals crystals These These crystals crystals were were combined combined
subiected subiected to to TC TC which which gave gave aa single single band band compared compared with with diIIerent diIIerent standard standard
markers markers and and hence hence conIirmed conIirmed as as - - sitosterol sitosterol dd- - glucoside glucoside
75 75 MeOH/A MeOH/A Iraction Iraction on on repeated repeated chromatography chromatography with with silica silica- -gel gel column column ,,
eluting eluting with with chloroIorm chloroIorm :: methanol methanol :: water water mixtures mixtures in in ( (22::::00) ) to to ( (::::00) ) ratios, ratios,
and and Iurther Iurther puriIication puriIication by by Diaion Diaion HP HP- -20 20 yielded yielded two two compounds compounds ,which ,which were were
analytically analytically checked checked Ior Ior its its percentage percentage purity purity by by HPC HPC and and subiected subiected Ior Ior spectral spectral
studies studies
1.v.ion o{ 1.v.ion o{ 1.[iv v[v vicv .no[c [vn 1.[iv v[v vicv .no[c [vn. .
!viioninv ..ncnc o{ nc c.v. !viioninv ..ncnc o{ nc c.v.
1.o[vion/Iv.ionvion o{ )!!vn. vvno[i. c.v. v.inv 1.o[vion/Iv.ionvion o{ )!!vn. vvno[i. c.v. v.inv
.o[vnn1 .o[vnn1
)c.nonvovvn, o{ ^1O1/1/ v.inv .o[vnn 2 )c.nonvovvn, o{ ^1O1/1/ v.inv .o[vnn 2
ratio1 (411)
ratio2 (311)
ratio3 (211)
ratio4 (111)
1.o[vion o{ 1v 1.o[vion o{ 1v0) {on {v.ion (:^:+ vio 0) {on {v.ion (:^:+ vio! v.inv Iivion ! v.inv Iivion
.o[vnn .o[vnn
1.o[vion o{ 1v 1.o[vion o{ 1v02{on {v.ion (:^:+ vio 02{on {v.ion (:^:+ vio v.inv Iivion v.inv Iivion
.o[vnn .o[vnn
1.o[vion o{ 1v 1.o[vion o{ 1v0 v.inv )0 0 v.inv )0 ^cO1 ^cO1/1/ {on .o[vnn 1 /1/ {on .o[vnn 1
(nonvovvni. I( oo.o[. (nonvovvni. I( oo.o[.
%LC %LC profiles profiles: :
Sample Sample: : 11mg mg of of the the sample sample etract etract dissolved dissolved in in 11ml ml of of methanol methanol
Stationary Stationary phase phase: : silica silica gel gel
obile obile phase phase : : chloroform chloroform: : methanol methanol ( ( 99..55: :00..55) )
etection etection: : ANS ANS spray spray
%LC %LC was was perIormed perIormed on on layers layers oI oI silica silica- -gel gel 60 60F F
254 254
( (Merck, Merck, Germany) Germany) using using
chloroIorm chloroIorm:: methanol methanol ( (9955::0055) ) TC TC plates plates were were sprayed sprayed with with
Anisaldehyde Anisaldehyde sulphuric sulphuric acid acid and and heated heated at at 05 05
oo
CC Ior Ior 55 minutes minutes
(nonvovvn, 1!( oo.o[. (nonvovvn, 1!( oo.o[.
Analytical Analytical HPLC HPLC
Instrument Instrument : : Shimadzu Shimadzu (Kyoto, (Kyoto, Japan) Japan) model model C C- -88A A pump, pump, an an SPD SPD- -
M M0 0ADVP ADVP photodiode photodiode
Column Column : : Kromosil Kromosil ODS ODS column( column(250 250 X X 4466 mm mm ) ) protected protected with with CC
8 8
guard guard
column, column, using using aa gradient gradient oI oI acetonitrile acetonitrile:: water water ( (30 30::70 70) ) as as the the mobile mobile phase phase
obile obile phase phase : : 30 30 acetonitrile acetonitrile in in HPLC HPLC water water
Flow Flow rate rate : : 00..55 ml/min ml/min
Injection Injection volume volume : : 20 20l l
Run Run time time : : 00- -25 25min min
etector etector: : Photodiode Photodiode array array detector detector at at 270 270nm nm..
The The elution elution program program was was 00- -25 25min min Irom Irom 30 30::70 70(Acetonitrile (Acetonitrile :: ater) ater) to to
40 40::60 60,, the the Ilow Ilow rate rate was was ml/min ml/min and and detection detection was was at at 270 270nm nm
The The Iractions Iractions corresponding corresponding to to each each peak peak showing showing ~~95 95 purity purity (by (by area area
normalization) normalization) were were selected selected and and subiected subiected to to
3 3
CC- -MR MR and and H H- -MR MR
spectroscopy spectroscopy
Two Two compounds compounds were were isolated isolated and and characterized characterized
!'TJ!ATJCA CT !o !'TJ!ATJCA CT !oUJ A+o !o UJ A+o !oU? JA tUTAAC! U? JA tUTAAC!
!XT1ACT tY ]1!C !!T]C1 !XT1ACT tY ]1!C !!T]C1
Sample Sample preparation preparation (etract) (etract):: 0 0 mg mg oI oI the the butanol butanol soluble soluble Iraction Iraction oI oI the the
plant plant was was weighed weighed and and dissolved dissolved separately separately in in 2255ml ml oI oI HPC HPC grade grade
methanol methanol with with the the help help oI oI sonicator sonicator Ior Ior the the estimation estimation oI oI Iraction Iraction with with
respect respect to to a a- -0 0,, and and a a- -02 02 respectively respectively
Standard Standard preparation preparation: : a a- -0 0 ( ( mg) mg) and and a a- -02 02 ( ( mg) mg) were were dissolved dissolved in in
55ml ml oI oI HPC HPC methanol methanol and and used used as as standards standards Ior Ior the the estimation estimation
Procedure Procedure:: HPC HPC analysis analysis oI oI the the standard standard and and the the samples samples was was carried carried out out
using using above above- -mentioned mentioned protocol protocol and and the the chromatograms chromatograms were were recorded recorded
The The retention retention time time and and peak peak area area oI oI the the standard standard (a (a- -0 0) ) and and (a (a- -02 02) ) was was
noted noted And And peak peak area area oI oI the the corresponding corresponding peaks peaks in in the the sample sample was was also also
noted noted
The The estimated estimated percentage percentage was was calculated calculated by by using using the the Iormula Iormula ::
Peak Peak area area of of sample sample X X standard standard dilution dilution X X purity purity of of compound compound
Peak Peak area area of of standard standard sample sample dilution dilution
)1^+I^ )1^+I^
RSUL%S RSUL%S
etails about the physical nature and percentage yield of ethanolic and thyl
acetate insoluble etracts Eclipta alba :
ata of Partition chromatography of ethyl acetate insoluble etract of Eclipta alba
with n-Butanol. and their yield data
%LC data for optimization of the butanol etract %LC data for optimization of the butanol etract
%LC data of partition chromatography of ethyl %LC data of partition chromatography of ethyl
acetate insoluble etract & water etract with n acetate insoluble etract & water etract with n- -
butanol etract: butanol etract:
I( o{ vvno[, 1n,[ v.cvc in.o[v[c
vnv +vc c.v. v.inv .n[oo{on:
ncnvno[ ( ^.:0.)
%LC data for column eluents of Butanolic
soluble fractions (column I)
%LC profile of fractions collected from column 2
%LC profile of fractions collected from column 3
%LC of the recrystallized compound a %LC of the recrystallized compound a- -03 with the standard 03 with the standard
markers markers
ata of Solubility profiles of the isolated compounds ata of Solubility profiles of the isolated compounds
HPLC analysis of the isolated compo
Analytical HPLC profile of the isolated compounds Analytical HPLC profile of the isolated compounds
%LC of the isolated compounds with butanol etract %LC of the isolated compounds with butanol etract
Characterization of Isolated compound: %he two isolated Characterization of Isolated compound: %he two isolated
compounds were characterized using I.R. NR ( compounds were characterized using I.R. NR (
11
H and H and
13 13
C) C)
ass Spectroscopy ass Spectroscopy
Interpretation of compound a-01: From the

H and
3
C MR
spectra`s, structure oI a-0 was interpreted and it was proposed to
have a chemical name 3-O| -D-glucopyranosyl| echinocystic acid
28-O--D-glucopyranosly methyl ester The H and 3C MR
spectrum in Pyridine revealed the presence oI seven tertiary methyl
groups ( 089, 00, 0, 05, 3, 29 and 85 ppm)
corresponding to one oleIinic proton | 562| and two anomeric
protons | 494(d, .4 z)]. The 3C exhibited 42 carbon signals
ascribable to two -glucopyranosyl residues consisting oI one ether
glucoside and one ester glucoside, which were attached to the 3-OH
and 28-COOH oI echinocystic acid signals suggested the presence oI
glucopyranosly methyl ester structure
Based on this, the structure was Iound to have olecular formula:
C
42
H
68
O
14
and Molecular weight: 796.98092.
Structure of a Structure of a- -01 01
Interpretation of compound a Interpretation of compound a- -02: 02: From the From the

H and H and
3 3
C MR spectra`s, C MR spectra`s,
structure oI TF structure oI TF- -03 was interpreted and it was proposed to have a chemical 03 was interpreted and it was proposed to have a chemical
name name 33- -O O- -- -D D- - glucopyranosyl echinocystic acid glucopyranosyl echinocystic acid The H and 3C MR The H and 3C MR
spectrum in Pyridine spectrum in Pyridine- -d5 revealed the presence seven tertiary methyl groups d5 revealed the presence seven tertiary methyl groups
( 089, 00, 0, 05, 2, 30 and 85 ( 089, 00, 0, 05, 2, 30 and 85 ppm ppm) corresponding to one ) corresponding to one
oleIinic oleIinic proton | 562| and two proton | 562| and two anomeric anomeric protons | 395(d, J72 protons | 395(d, J72 hz hz)| The )| The
3C 3C- -MR spectrum gave 36 carbon signals caused by one MR spectrum gave 36 carbon signals caused by one - -
glucopyranosyl glucopyranosyl moiety and the O moiety and the O- - glycosylated glycosylated CC- -3 3 echinocystic echinocystic acid acid
moiety moiety
These values were compared with the reported literature These values were compared with the reported literature
27 27
(Shoii (Shoii Yahara Yahara, ,
ing ing Ding, Toshihiro Ding, Toshihiro ahara ahara Oleanane glycosides Irom Oleanane glycosides Irom Eclipta Eclipta alba alba
Chem Chem pharm pharm bull 994; 42(6): 336 bull 994; 42(6): 336- -8) 8) Based on this, the structure was Based on this, the structure was
Iound to have Iound to have olecular formula olecular formula: : C C
36 36
HH
59 59
OO
99
and Molecular weight and Molecular weight: :
634.84032. 634.84032.
Structure of a Structure of a- -02 02
Spectral data of isolated compounds Spectral data of isolated compounds
HPLC Chromatogram a-01
HPLC Chromatogram of a-02
I.R. SPC%RA OF a-01 HH- -NR SPC%RA OF a NR SPC%RA OF a- -01 01
ass spectra of a-01
C-13 NR spectra of a-01
I.R Spectra of a-02 H-NR spectra of a-02
ass spectra of %F-03 C-13 NR of %F-03
!'TJ!ATJCA CT !o !'TJ!ATJCA CT !oUJ A+o !o UJ A+o !oU? JA tUTAAC! U? JA tUTAAC!
!XT1ACT tY ]1!C !!T]C1 !XT1ACT tY ]1!C !!T]C1
Sample Sample preparation preparation (etract) (etract):: 0 0 mg mg oI oI the the butanol butanol soluble soluble Iraction Iraction oI oI the the
plant plant was was weighed weighed and and dissolved dissolved separately separately in in 2255ml ml oI oI HPC HPC grade grade
methanol methanol with with the the help help oI oI sonicator sonicator Ior Ior the the estimation estimation oI oI Iraction Iraction with with
respect respect to to a a- -0 0,, and and a a- -02 02 respectively respectively
Standard Standard preparation preparation: : a a- -0 0 ( ( mg) mg) and and a a- -02 02 ( ( mg) mg) were were dissolved dissolved in in
55ml ml oI oI HPC HPC methanol methanol and and used used as as standards standards Ior Ior the the estimation estimation
Procedure Procedure:: HPC HPC analysis analysis oI oI the the standard standard and and the the samples samples was was carried carried out out
using using above above- -mentioned mentioned protocol protocol and and the the chromatograms chromatograms were were recorded recorded
The The retention retention time time and and peak peak area area oI oI the the standard standard (a (a- -0 0) ) and and (a (a- -02 02) ) was was
noted noted And And peak peak area area oI oI the the corresponding corresponding peaks peaks in in the the sample sample was was also also
noted noted
The The estimated estimated percentage percentage was was calculated calculated by by using using the the Iormula Iormula ::
Peak Peak area area of of sample sample X X standard standard dilution dilution X X purity purity of of compound compound
Peak Peak area area of of standard standard sample sample dilution dilution
In the HPC chromatogram oI the butanol Iraction, the area oI a In the HPC chromatogram oI the butanol Iraction, the area oI a- -0 was Iound to be 0 was Iound to be
459284 So the a 459284 So the a- -0 present in the Butanol Iraction was Iound to be 0 present in the Butanol Iraction was Iound to be 0.35 0.35.( .(By By
calculating according to the Iormula given in methodology) calculating according to the Iormula given in methodology)
Again in the HPC chromatogram oI the butanol Iraction, the area oI a Again in the HPC chromatogram oI the butanol Iraction, the area oI a- -02 was Iound 02 was Iound
to be 7525235 So the a to be 7525235 So the a- -02 present in the Butanol Iraction was Iound to be 02 present in the Butanol Iraction was Iound to be 1.78 . 1.78 .
Peaks were observed in the similar retention time corresponding to the values as Peaks were observed in the similar retention time corresponding to the values as
observed in the a observed in the a- -0 (45 min in compound and 440 min in butanol Iraction) and 0 (45 min in compound and 440 min in butanol Iraction) and
a a- -02 (2324 min in compound and 2322 min in Butanol Iraction) It conIirms the 02 (2324 min in compound and 2322 min in Butanol Iraction) It conIirms the
presence oI the compounds in both the Iraction oI Butanol extract oI the plant part presence oI the compounds in both the Iraction oI Butanol extract oI the plant part
Hence the process can be said as standardized Hence the process can be said as standardized
I1^(+^^1O^ I1^(+^^1O^
I1^(+^^1O^
clipta alba inn is used in traditional system oI medicine Ior treatment oI many
diseases A number oI studies on isolation oI chemical constituents and pharmacological
activities oI clipta alba and its extracts have been reported Coumestans have been
isolated as biologically active compounds But there are scanty reports on estimation using
HPC
Many herbal industries are using this drug Ior preparing diIIerent kinds oI Iormulations
Hence an attempt was made to isolate, puriIy and characterize compounds to be used as
markers which constitute chemically deIined constituents, which can serve as a powerIul
tool, Ior standardization oI extracts and Iormulation oI Eclipta alba.
Isolation of the phytoconstituents:
The methanolic extract oI Eclipta alba whole plant was partitioned between water
and butanol to get rich Iraction
The butanol extract was then Iractionated using repeated column chromatography
DiIIerent stationary phases viz, Silica gel, Diaion HP 20 columns were used Ior the
study Diaion HP20 is actually used Ior isolation oI bimolecular like proteins, but it also
exerts a good separation oI bigger molecules with the polar ones getting eluted Iirst
The butanolic extract obtained Irom methanolic extract was adsorbed on the silica and
charged onto a column packed with the stationary phase and eluted with diIIerent
solvents oI varying polarities
Six diIIerent Iractions were collected Irom column chromatography oI butanolic
extract were evaluated Ior TC pattern using chloroIorm: methanol in the ratio oI
95:05 as the solvent system TC analysis has indicated that among all the Iractions
collected, Iraction 2 and 5 (0MOH/A and 75MOH/A) showed distinct bands
oI constituents
Purification of the isolated compounds:
The above extracts were subiected to Iurther puriIication by Diaion column to get a-
0 and a-02 compounds
Analysis of isolated compounds
TC, Melting point analysis and HPC method were used to analyze the isolated
compounds a-0 and a-02 Ior their purity
The HPC analysis indicated that the Iirst and second compound a0 and a-02 was
00 pure
Compound a-03 which was a pure compound which was subiected toTC analysis
by comparing with diIIerent standard markers to show a single spot ie, - sitosterol d-
glucoside

3
C MR and

HMR spectroscopy were used to characterize the isolated compounds

The spectra were compared with the reported literature
27

HMR spectrum shows the protons and

3
C MR shows the no oI carbons in the
isolated compounds
The 0 values oI a-0 and a-02 obtained in our study were compared with those
reported in the literature
27
Shoii Yahara, ing Ding, Toshihiro ahara Oleanane
glycosides Irom Eclipta alba. Cem pa7m bull. 1994, 42(6). 1336-. and indicated that
compound were clalbasaponin I and clalbasaponin II
stimation of isolated compounds in the 50 ethanolic etract
In order to check the suitability oI the two isolated compounds as markers, HPC
analytical method was developed
The butanol extract and isolated compounds were subiected to HPC Ior estimation
oI the isolated compounds
Distinct peaks oI both compounds at retention time 45 and 2324 were observed in
the chromatograms, indicating the method to be suitable and sensitive
The compounds a-0 and a-02 were Iound to be present in 035 and 78 in the
butanolic extract
Hence it can be concluded that the compounds isolated in the present study can be used
as markers to standardize Eclipta alba extracts
clalbasaponin I
clalbasaponin II
(O^(+^1O^ /^+^^/)
Butanol extract was subiected to column chromatography using Silica gel,
Diaion HP-20 columns as stationary phases and the solvents with varying
polarity as mobile phase
This method was Iound to be suitable Ior Iractionation/isolation oI
phytoconstituents at commercial scale
A TC system with chloroIorm: methanol(95:05) as mobile phase and
Anisaldehyde sulphuric acid as detecting agent was optimized to identiIy the
phytoconstituents
The HPC method developed was Iound to be Iast, simple and accurate
On the basis oI purity and yield oI two isolated compounds viz a-0 and
a-02 were selected Ior puriIication and characterization
The isolated, puriIied compounds were characterized by determining their
physical properties like solubility, melting point and then subiecting them to
MR spectroscopy
The two isolated compounds a-0 & a-02 were characterized as 3-O| -D-
glucopyranosyl| echinocystic acid 28-O--D- glucopyranosly methyl ester
(clalbasaponin I) and 3-O--D- glucopyranosyl echinocystic acid (clalbasaponin
II)
The two isolated compounds were Iound to be suitable candidates to be used as
markers
The two isolated compounds were estimated in the butanolic extract oI the whole
plant It was Iound that the isolated compounds are present in the methanolic
extracts in 035 and 78
11O)/!1 11O)/!1
11O)/!1
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I1/^1 O+