““Isolation of chemical constituents from Isolation of chemical constituents from Eclipta Eclipta alba alba LinnLinn. . for achieving standardization”for achieving standardization”

By

MITHUN NMA Dissertation submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore

In partial fulfillment of the

MASTER OF PHARMACYUnder the guidance of

Dr.s.shashidhara

Department of PharmacognosyGovernment College of Pharmacy

Bangalore – 560 027,Karnataka, India

April 2011

INTRODUCTIONINTRODUCTIONBhringaraj, Eclipta alba L., belonging to family Astraceae is commonly known as False Daisy is a common weed in moist situations throughout India, ascending up to 6000 ft 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. Bhringaraj is small annual, procumbent much branched and prostrating herb, 20 to 60 cm high, grows in fields and ditches. White flowers appear in summer and are small penny size, flower stalk-elongated having white flower on its tips and flower heads are axillary or terminal.

Eclipta alba (L.) contains wide range of active principles which includes coumestans, alkaloids, flavonoids, glycosides, polyacetylenes, triterpenoids. The leaves contain stigmasterol, α-terthienylmethanol, wedelolactone, demethylwedelolactone and demethylwedelolactone-7-glucoside. The polypeptides isolated from the plant yield cystine, glutamic acid, phenyl alanine, tyrosine and methionine on hydrolysis. Nicotine and nicotinic acid are reported to occur in this plant.

INTRODUCTIONINTRODUCTION

Medicinal Forms:Medicinal Forms: Extracts ( dry and soft), Oil, Fresh leaf juice, Extracts ( dry and soft), Oil, Fresh leaf juice,

Decoction, Maka, Tincture, An herbal poultice Decoction, Maka, Tincture, An herbal poultice made with sesame oil for skin disorders. Key made with sesame oil for skin disorders. Key ingredient of herbal hair shampooingredient of herbal hair shampoo

Traditional UsageTraditional Usage:: Antihepatotoxic , Hair growth promoter, Anti-Antihepatotoxic , Hair growth promoter, Anti-

inflammatory, Antibacterial, Antispasmodicinflammatory, Antibacterial, Antispasmodic Antihaemorrhage, Lipid lowering, Antioxidant-Antihaemorrhage, Lipid lowering, Antioxidant-

Skin Disorders, Anticancer, antivenom, Skin Disorders, Anticancer, antivenom, antigiardial, trypsin inhibitor, Analgesic.  antigiardial, trypsin inhibitor, Analgesic.  

Various formulations of Various formulations of Eclipta albaEclipta alba L. L.

Bhrngamalakadi Taila, Bhrngamalakadi Taila, Bhrngaraja Taila, Bhrngaraja Taila, Nili Bhrhgadi Taila, Nili Bhrhgadi Taila, Shadbindu Taila,Shadbindu Taila, Bhrangarajadi Cxhurna,Bhrangarajadi Cxhurna, Bhrngarajasava, Bhrngarajasava, Tekaraja Marica. Tekaraja Marica.

Detail of the parts containing chemical constituentsDetail of the parts containing chemical constituents

Sl.No. Parts Chemical constituents

1 Leaves Wedelolactone[1.6%], Desmethylwedelolactone,Desmethyl-wedelolactone-7-glucoside, stigmasterol

2 Roots Hentriacontanol, Heptacosanol& Stigmasterol, Ecliptal, Eclalbatin

3 Aerial parts β-amyrin & Luteolin-7-0-glucoside, Apigenin, Cinnaroside, Sulphur compounds, Eclalbasaponins

4 Stems Wedelolactone

5 Seeds Sterols, Ecliptalbine (alkaloid)

6 Whole plant Resin, Ecliptine, Reducing sugar, Nicotine, Stigmasterol, Triterpene saponin, Eclalbatin,Ursolic acid, Oleanolic acid

Detail of Pharmacological activities of the chemical Detail of Pharmacological activities of the chemical constituentsconstituents

Sl.No Chemical constituents Pharmacological activites

1 Wedelolactone Antihepatotoxic, Antibacterial, Trypsin Inhibitor, Antivenom

2 Eclalbosaponins Hair revitalizing, Antiproleferative, Antigiardial

3 Demethylwedelolactone Antihepatotoxic, Antihaemorrhage, Antivenom, Dye (cosmetic)

4 Dasyscyphin C Antiviral, Anticancer

5 Eclalbatin Antioxidant

6 Ecliptalbine, verazine Lipid lowering, Analgesic

Important chemical structures isolated from Important chemical structures isolated from Eclipta alba Eclipta alba L.L.

AIM AND OBJECTIVESAIM AND OBJECTIVES

OBJECTIVE OF STUDYOBJECTIVE OF STUDY

Collection and authentication of the plant.Collection and authentication of the plant. Identification of major compounds using Thin Layer Chromatography (major Identification of major compounds using Thin Layer Chromatography (major

spots). spots).

Isolation of major compounds from the extract by column chromatographyIsolation of major compounds from the extract by column chromatography

Purification of the phytoconstituents using Column Chromatography, and Purification of the phytoconstituents using Column Chromatography, and Preparative HPLC.Preparative HPLC.

Characterization of the isolated compounds.Characterization of the isolated compounds.

Estimation of the isolated compounds in the extracts of Estimation of the isolated compounds in the extracts of Eclipta alba Eclipta alba LL ..

REVIEW OF LITERATUREREVIEW OF LITERATURE

REVIEW OF LITERATUREREVIEW OF LITERATURE Coumestans, wedelolactone and desmethylwedelolactone active principles isolated Coumestans, wedelolactone and desmethylwedelolactone active principles isolated

from from Eclipta alba Eclipta alba were used for evaluation of Gal-N induced cytotoxicity activity were used for evaluation of Gal-N induced cytotoxicity activity in cultured rat hepatocytes. Wedelolactone has shown significant antihepatotoxic in cultured rat hepatocytes. Wedelolactone has shown significant antihepatotoxic activity at a dose of 0.01mg/ml. apigenin has shown more than 100% in GPT activity at a dose of 0.01mg/ml. apigenin has shown more than 100% in GPT activity, where as standard silybin has shown a protective of 78% at a dose of activity, where as standard silybin has shown a protective of 78% at a dose of 1.0mg/ml. HPLC method was developed to evaluate the content of wedelolactone 1.0mg/ml. HPLC method was developed to evaluate the content of wedelolactone and desmethylwedelolactone.and desmethylwedelolactone.

Six new Oleanane triterpene glycosides were isolated from the methanolic extract Six new Oleanane triterpene glycosides were isolated from the methanolic extract of of EcliptaEclipta albaalba Hassk. Extract was evaporated under reduced pressure to afford Hassk. Extract was evaporated under reduced pressure to afford residue. The residue was partitioned between n-hexane, water, and butanol. The residue. The residue was partitioned between n-hexane, water, and butanol. The butanol phase was removed to furnish the residue which was subjected to column butanol phase was removed to furnish the residue which was subjected to column chromatography on sephadrex LH-20 eluted with methanol & silica gel eluted with chromatography on sephadrex LH-20 eluted with methanol & silica gel eluted with chloroform: methanol: water (8:2:0.2-6:4:1) to give eclalbasaponins I-VI (1-6).chloroform: methanol: water (8:2:0.2-6:4:1) to give eclalbasaponins I-VI (1-6).

Four new taraxastane triterpene glycosides eclabasaponins VII-X were isolated Four new taraxastane triterpene glycosides eclabasaponins VII-X were isolated along with eclabasaponins I-VI from the dried, whole parts of along with eclabasaponins I-VI from the dried, whole parts of EcliptaEclipta albaalba. The . The structure of eclabasaponins VII-X were characterized as 3β,20β,16β-and 3β,20β,28- structure of eclabasaponins VII-X were characterized as 3β,20β,16β-and 3β,20β,28- trihydroxy taraxastane glycosides and their sulphated saponins on the basis of trihydroxy taraxastane glycosides and their sulphated saponins on the basis of spectral data.spectral data.

The flavanoids, apigenin, luteolin, wedelolactone, apigenin-7-O-glucoside were The flavanoids, apigenin, luteolin, wedelolactone, apigenin-7-O-glucoside were isolated from the whole plant ofisolated from the whole plant of Eclipta alba. Eclipta alba. The structure of these compounds The structure of these compounds were characterized and identified by spectral data. This was the first report of the were characterized and identified by spectral data. This was the first report of the presence of presence of luteolin and apigenin-7-O-glucoside luteolin and apigenin-7-O-glucoside inin Eclipta.alba. Eclipta.alba.

For the first time luteolin, wedelolactone and desmethylwedelolactone were For the first time luteolin, wedelolactone and desmethylwedelolactone were isolated from the aerial parts Vietnamese isolated from the aerial parts Vietnamese EcliptaEclipta.alba(L.)Hassk which was .alba(L.)Hassk which was chemically investigated. The structures of these compounds were elucidated on the chemically investigated. The structures of these compounds were elucidated on the basis of spectroscopic evidencebasis of spectroscopic evidenceHypoglycemic effect of Fenugreek seed powder Hypoglycemic effect of Fenugreek seed powder ((Trigonella foenum graecumTrigonella foenum graecum) was studied in 60 non-insulin dependent diabetic ) was studied in 60 non-insulin dependent diabetic patients.patients.

Based on spectral analysis, five compounds namely 17-tri-triacontanone, 1-Based on spectral analysis, five compounds namely 17-tri-triacontanone, 1-hentriacontanol, hirtin, wedelolactone & β- sitosterol, were isolated from the hentriacontanol, hirtin, wedelolactone & β- sitosterol, were isolated from the methanolic extract of methanolic extract of Eclipta albaEclipta alba (L.) Hassk. Water & methanolic extract were (L.) Hassk. Water & methanolic extract were evaluated against evaluated against Rhizoctonia soaniRhizoctonia soani & & collectotrichum falcatumcollectotrichum falcatum at different at different concentrations. Compared to methanolic extract at 2000µg/ml, the water extract concentrations. Compared to methanolic extract at 2000µg/ml, the water extract was found more active against both fungi.was found more active against both fungi.

Stigmasterol an ubiquitous steroid & two oleanane glycosides eclalbasaponin II(1) Stigmasterol an ubiquitous steroid & two oleanane glycosides eclalbasaponin II(1) and eclalbasaponin I(2) were isolated from an n-hexane extract of the stem bark of and eclalbasaponin I(2) were isolated from an n-hexane extract of the stem bark of EcliptaEclipta prostrata prostrata using column chromatography technique. Isolated stigmasterol using column chromatography technique. Isolated stigmasterol was colourless needle shaped from the fraction 30 of the n-hexane extract.was colourless needle shaped from the fraction 30 of the n-hexane extract.

MATERIALS & METHODSMATERIALS & METHODS

Materials and MethodsMaterials and Methods Plant materialPlant material The dried plant seeds of The dried plant seeds of Eclipta albaEclipta alba L. L. were obtained from Natural Remedies PVT LTD, were obtained from Natural Remedies PVT LTD,

Bangalore and authenticated by NISCAIR, Delhi. The whole plant were then sun dried, Bangalore and authenticated by NISCAIR, Delhi. The whole plant were then sun dried, powdered and stored in airtight containers for further use.powdered and stored in airtight containers for further use.

Extraction Extraction The whole plant of The whole plant of EcliptaEclipta albaalba were was coarsely powdered and subjected for extraction were was coarsely powdered and subjected for extraction

using static extractor. using static extractor. 30 kg of the powdered plant was refluxed with fresh methanol (1:3 drug to solvent ratio) for 30 kg of the powdered plant was refluxed with fresh methanol (1:3 drug to solvent ratio) for

one and half hour at temperature less than 65one and half hour at temperature less than 6500 C and the extract was strained through muslin C and the extract was strained through muslin cloth to obtained [1cloth to obtained [1stst extract]. extract].

The marc was again subjected for second time extraction (drug marc to solvent ratio is 1:2) The marc was again subjected for second time extraction (drug marc to solvent ratio is 1:2) and the extract obtained was strained through muslin cloth to yield [2and the extract obtained was strained through muslin cloth to yield [2 ndnd extract]. The same extract]. The same procedure was followed for third wash to obtained [3procedure was followed for third wash to obtained [3 rdrd extract]. extract].

The 1The 1stst, 2, 2ndnd, 3, 3rdrd extracts were combined together and concentrated up to total solids 20% in a extracts were combined together and concentrated up to total solids 20% in a cruzol concentrator (Temp. less than 85cruzol concentrator (Temp. less than 8500 C and pressure 550-mm of Hg). Further C and pressure 550-mm of Hg). Further concentration was carried out using vacuum tray drier (Temp. less than 70concentration was carried out using vacuum tray drier (Temp. less than 70 00 C and pressure C and pressure 500 mm of Hg) to obtain a dry powder. 500 mm of Hg) to obtain a dry powder. The dried powder was again refluxed with ethyl The dried powder was again refluxed with ethyl acetate (1:3 drug to solvent ratio) for half an hour at temperature < 65acetate (1:3 drug to solvent ratio) for half an hour at temperature < 65 00C and the extract was C and the extract was strained with muslin cloth to get the insoluble ethyl acetate extractstrained with muslin cloth to get the insoluble ethyl acetate extract

  

Fractionation/isolationFractionation/isolation

Liquid-liquid partitioning method was employed to separate the plant Liquid-liquid partitioning method was employed to separate the plant constituents with mixture of chemical components into groups of constituents with mixture of chemical components into groups of compounds sharing similar physico-chemical parameters. compounds sharing similar physico-chemical parameters.

400gm of ethyl acetate insoluble extract was dissolved in 2 liters of water 400gm of ethyl acetate insoluble extract was dissolved in 2 liters of water by warming. It was partitioned three times with 2 liters equal portion of by warming. It was partitioned three times with 2 liters equal portion of butanol. butanol.

The butanol layer was allowed to separate and the butanol soluble part was The butanol layer was allowed to separate and the butanol soluble part was collected, combined and concentrated to get dry powder (Temp. below 65collected, combined and concentrated to get dry powder (Temp. below 65OO C and vacuum pressure 300 mm/hg). Final concentration was done under C and vacuum pressure 300 mm/hg). Final concentration was done under vacuum pressure 200 mm/hg. vacuum pressure 200 mm/hg.

The partitioned 144gms butanol extract was subjected for further fractionation by The partitioned 144gms butanol extract was subjected for further fractionation by silica-gel column chromatography using Petroleum ether, ethyl acetate and silica-gel column chromatography using Petroleum ether, ethyl acetate and methanol. methanol.

The 10%MeOH/EA and 75%MeOH/EA fractions were selected for the isolation The 10%MeOH/EA and 75%MeOH/EA fractions were selected for the isolation of phytoconstituents which were identified by TLC using chloroform : methanol of phytoconstituents which were identified by TLC using chloroform : methanol (9.5:0.5)as mobile phase which was most effective for the separation and (9.5:0.5)as mobile phase which was most effective for the separation and detection by Anisaldehyde sulphuric acid reagent which gave different colors . detection by Anisaldehyde sulphuric acid reagent which gave different colors .

10%MeOH/EA 10%MeOH/EA was dissolved in methanol and kept overnight for recrystallization was dissolved in methanol and kept overnight for recrystallization process. The extract was filtered using filter paper no: 201. The filtrate was again process. The extract was filtered using filter paper no: 201. The filtrate was again kept for crystallization overnight to get crystals. These crystals were combined kept for crystallization overnight to get crystals. These crystals were combined subjected to TLC which gave a single band compared with different standard subjected to TLC which gave a single band compared with different standard markers and hence confirmed as markers and hence confirmed as ββ- sitosterol –d- glucoside.- sitosterol –d- glucoside.

75%MeOH/EA fraction on repeated chromatography with silica-gel column , 75%MeOH/EA fraction on repeated chromatography with silica-gel column , eluting with chloroform : methanol : water mixtures in (2:1:0.1) to (1:1:0.1) eluting with chloroform : methanol : water mixtures in (2:1:0.1) to (1:1:0.1) ratios, and further purification by Diaion HP-20 yielded two compounds ,which ratios, and further purification by Diaion HP-20 yielded two compounds ,which were analytically checked for its percentage purity by HPLC and subjected for were analytically checked for its percentage purity by HPLC and subjected for spectral studies.spectral studies.

Extraction of Extraction of Eclipta alba dried whole Eclipta alba dried whole plantplant..

Partitioning scheme of the extractPartitioning scheme of the extract

Isolation/Fractionation of 144gms Isolation/Fractionation of 144gms butanolic extract using columnI butanolic extract using columnI

Rechromatography of 75% MEOH/EA Rechromatography of 75% MEOH/EA using column 2using column 2

C:M:W ratio1 (4:1:0.1)

C:M:W ratio2 (3:1:0.1)

C:M:W ratio3 (2:1:0.1)

C:M:W ratio4 (1:1:0.1)

Isolation of Ea-01 from fraction C:M:W Isolation of Ea-01 from fraction C:M:W ratio-4 using Diaion column ratio-4 using Diaion column

Isolation of Ea-02from fraction C:M:W Isolation of Ea-02from fraction C:M:W ratio -3 using Diaion columnratio -3 using Diaion column

Isolation of Ea-03 using 10% MeOH/EA Isolation of Ea-03 using 10% MeOH/EA from column Ifrom column I

Chromatographic TLC protocolsChromatographic TLC protocols

TLC profiles:TLC profiles: Sample: 1mg of the sample extract dissolved in 1ml of methanolSample: 1mg of the sample extract dissolved in 1ml of methanol Stationary phase: silica gelStationary phase: silica gel Mobile phase : chloroform: methanol ( 9.5:0.5)Mobile phase : chloroform: methanol ( 9.5:0.5) Detection: ANS sprayDetection: ANS spray

TLCTLC was performed on layers of silica-gel 60F was performed on layers of silica-gel 60F254254 ((Merck, Germany) using Merck, Germany) using

chloroform: methanol (9.5:0.5). TLC plates were sprayed with chloroform: methanol (9.5:0.5). TLC plates were sprayed with Anisaldehyde sulphuric acid and heated at 105Anisaldehyde sulphuric acid and heated at 105ooC for 5 minutes.C for 5 minutes.

Chromatography HPLC Chromatography HPLC protocolsprotocols Analytical HPLCAnalytical HPLC Instrument : Instrument : Shimadzu (Kyoto, Japan) model LC-8A pump, an SPD-M10ADVP Shimadzu (Kyoto, Japan) model LC-8A pump, an SPD-M10ADVP

photodiode. photodiode. Column : Column : Kromosil ODS column(250 X 4.6 mm ) protected with CKromosil ODS column(250 X 4.6 mm ) protected with C1818 guard guard

column, using a gradient of acetonitrile: water (30:70) as the mobile phase. column, using a gradient of acetonitrile: water (30:70) as the mobile phase. Mobile phase : 30% acetonitrile in HPLC waterMobile phase : 30% acetonitrile in HPLC water Flow rate : 0.5 ml/minFlow rate : 0.5 ml/min Injection volume : 20µlInjection volume : 20µl Run time : 0-25minRun time : 0-25min Detector: Photodiode array detector at 270nm.Detector: Photodiode array detector at 270nm. The elution program was 0-25min from 30:70(Acetonitrile : Water) to 40:60, the The elution program was 0-25min from 30:70(Acetonitrile : Water) to 40:60, the

flow rate was 1ml/min and detection was at 270nm.flow rate was 1ml/min and detection was at 270nm. The fractions corresponding to each peak showing >95% purity (by area The fractions corresponding to each peak showing >95% purity (by area

normalization) were selected and subjected to normalization) were selected and subjected to 1313C-NMR and H-NMR spectroscopy.C-NMR and H-NMR spectroscopy. Two compounds were isolated and characterizedTwo compounds were isolated and characterized

ESTIMATION OF Ea-01 And Ea-02 IN BUTANOL ESTIMATION OF Ea-01 And Ea-02 IN BUTANOL EXTRACT BY HPLC METHODEXTRACT BY HPLC METHOD

Sample preparation (extract)Sample preparation (extract): : 10 mg of the butanol soluble fraction of 10 mg of the butanol soluble fraction of the plant was weighed and dissolved separately in 2.5ml of HPLC grade the plant was weighed and dissolved separately in 2.5ml of HPLC grade methanol with the help of sonicator for the estimation of fraction with methanol with the help of sonicator for the estimation of fraction with respect to Ea-01, and Ea-02 respectively. respect to Ea-01, and Ea-02 respectively.

Standard preparation: Standard preparation: Ea-01 (1 mg) and Ea-02 (1 mg) were dissolved in Ea-01 (1 mg) and Ea-02 (1 mg) were dissolved in 1.5ml of HPLC methanol and used as standards for the estimation . 1.5ml of HPLC methanol and used as standards for the estimation .

Procedure: Procedure: HPLC analysis of the standard and the samples was carried out HPLC analysis of the standard and the samples was carried out using above-mentioned protocol and the chromatograms were recorded. using above-mentioned protocol and the chromatograms were recorded. The retention time and peak area of the standard (Ea-01) and (Ea-02) was The retention time and peak area of the standard (Ea-01) and (Ea-02) was noted. And peak area of the corresponding peaks in the sample was also noted. And peak area of the corresponding peaks in the sample was also noted. noted.

The estimated percentage was calculated by using the formula :The estimated percentage was calculated by using the formula : Peak area of sample X standard dilution X purity of compoundPeak area of sample X standard dilution X purity of compound

Peak area of standard sample dilutionPeak area of standard sample dilution

RESULTSRESULTS

RESULTSRESULTSDetails about the physical nature and percentage yield of Methanolic and Ethyl

acetate insoluble extracts Eclipta alba :

Data of Partition chromatography of ethyl acetate insoluble extract of Eclipta alba with n-Butanol, and their yield data

TLC data for optimization of the butanol extractTLC data for optimization of the butanol extract

TLC data of partition chromatography of ethyl TLC data of partition chromatography of ethyl acetate insoluble extract & water extract with n-acetate insoluble extract & water extract with n-

butanol extract:butanol extract:  

TLC of Butanol, Ethyl acetate insoluble and Water extract using chloroform: methanol

( 9.5:0.5)

TLC data for column eluents of Butanolic soluble fractions (column I)

TLC profile of fractions collected from column 2

   TLC profile of fractions collected from column 3

TLC of the recrystallized compound Ea-03 with the standard TLC of the recrystallized compound Ea-03 with the standard markersmarkers

Data of Solubility profiles of the isolated compoundsData of Solubility profiles of the isolated compounds

HPLC analysis of the isolated compo

Analytical HPLC profile of the isolated compoundsAnalytical HPLC profile of the isolated compounds

TLC of the isolated compounds with butanol extractTLC of the isolated compounds with butanol extract

Characterization of Isolated compound: The two isolated Characterization of Isolated compound: The two isolated compounds were characterized using I.R, NMR (compounds were characterized using I.R, NMR (11H and H and 1313C) C)

Mass SpectroscopyMass SpectroscopyInterpretation of compound Ea-01: From the 1H and 13C NMR spectra’s, structure of Ea-01 was interpreted and it was proposed to have a chemical name 3-O[ β-D-glucopyranosyl] echinocystic acid 28-O-β-D-glucopyranosly methyl ester. The 1H and 13C NMR spectrum in Pyridine revealed the presence of seven tertiary methyl groups ( 0.89, 1.00, 1.01, 1.05, 1.13, 1.29 and 1.85 ppm) corresponding to one olefinic proton [ 5.62] and two anomeric protons [ 4.94(d, J=8.4 hz)]. The 13C exhibited 42 carbon signals ascribable to two β-glucopyranosyl residues consisting of one ether glucoside and one ester glucoside, which were attached to the 3-OH and 28-COOH of echinocystic acid signals suggested the presence of glucopyranosly methyl ester structure. Based on this, the structure was found to have Molecular formula: C42H68O14 and Molecular weight: 796.98092.

Structure of Ea-01Structure of Ea-01

Interpretation of compound Ea-02:Interpretation of compound Ea-02: From the From the 11H and H and 1313C NMR C NMR spectra’s, structure of TF-03 was interpreted and it was proposed to have a spectra’s, structure of TF-03 was interpreted and it was proposed to have a chemical name chemical name 3-O-3-O-β-β-D- glucopyranosyl echinocystic acid D- glucopyranosyl echinocystic acid . The 1H and . The 1H and 13C NMR spectrum in Pyridine-d5 revealed the presence seven tertiary 13C NMR spectrum in Pyridine-d5 revealed the presence seven tertiary methyl groups ( 0.89, 1.00, 1.01, 1.05, 1.12, 1.30 and 1.85 ppm) methyl groups ( 0.89, 1.00, 1.01, 1.05, 1.12, 1.30 and 1.85 ppm) corresponding to one olefinic proton [ 5.62] and two anomeric protons corresponding to one olefinic proton [ 5.62] and two anomeric protons [ 3.95(d, J=7.2 hz)]. The 13C-NMR spectrum gave 36 carbon signals [ 3.95(d, J=7.2 hz)]. The 13C-NMR spectrum gave 36 carbon signals caused by one caused by one β-β-glucopyranosyl moiety and the O- glycosylated C-3 glucopyranosyl moiety and the O- glycosylated C-3 echinocystic acid moiety. echinocystic acid moiety.

These values were compared with the reported literatureThese values were compared with the reported literature2727(Shoji Yahara, (Shoji Yahara, Ning Ding, Toshihiro Nahara. Oleanane glycosides from Ning Ding, Toshihiro Nahara. Oleanane glycosides from EcliptaEclipta alba. alba. Chem pharm bull. 1994; 42(6): 1336-8.) Chem pharm bull. 1994; 42(6): 1336-8.) Based on this, the structure was Based on this, the structure was found to have found to have Molecular formulaMolecular formula: : CC3636HH5959OO99 and Molecular weight and Molecular weight: :

634.84032. 634.84032.

Structure of Ea-02Structure of Ea-02

Spectral data of isolated compoundsSpectral data of isolated compounds

HPLC Chromatogram Ea-01

HPLC Chromatogram of Ea-02

I.R. SPECTRA OF Ea-01 H-NMR SPECTRA OF Ea-01H-NMR SPECTRA OF Ea-01

Mass spectra of Ea-01 C-13 NMR spectra of Ea-01

I.R Spectra of Ea-02 H-NMR spectra of Ea-02

Mass spectra of TF-03 C-13 NMR of TF-03

ESTIMATION OF Ea-01 And Ea-02 IN BUTANOL ESTIMATION OF Ea-01 And Ea-02 IN BUTANOL EXTRACT BY HPLC METHODEXTRACT BY HPLC METHOD

Sample preparation (extract)Sample preparation (extract): : 10 mg of the butanol soluble fraction of 10 mg of the butanol soluble fraction of the plant was weighed and dissolved separately in 2.5ml of HPLC grade the plant was weighed and dissolved separately in 2.5ml of HPLC grade methanol with the help of sonicator for the estimation of fraction with methanol with the help of sonicator for the estimation of fraction with respect to Ea-01, and Ea-02 respectively. respect to Ea-01, and Ea-02 respectively.

Standard preparation: Standard preparation: Ea-01 (1 mg) and Ea-02 (1 mg) were dissolved in Ea-01 (1 mg) and Ea-02 (1 mg) were dissolved in 1.5ml of HPLC methanol and used as standards for the estimation . 1.5ml of HPLC methanol and used as standards for the estimation .

Procedure: Procedure: HPLC analysis of the standard and the samples was carried out HPLC analysis of the standard and the samples was carried out using above-mentioned protocol and the chromatograms were recorded. using above-mentioned protocol and the chromatograms were recorded. The retention time and peak area of the standard (Ea-01) and (Ea-02) was The retention time and peak area of the standard (Ea-01) and (Ea-02) was noted. And peak area of the corresponding peaks in the sample was also noted. And peak area of the corresponding peaks in the sample was also noted. noted.

The estimated percentage was calculated by using the formula :The estimated percentage was calculated by using the formula : Peak area of sample X standard dilution X purity of compoundPeak area of sample X standard dilution X purity of compound

Peak area of standard sample dilutionPeak area of standard sample dilution

In the HPLC chromatogram of the butanol fraction, the area of Ea-01 was found to be In the HPLC chromatogram of the butanol fraction, the area of Ea-01 was found to be 1459284 So the Ea-01 present in the Butanol fraction was found to be 1459284 So the Ea-01 present in the Butanol fraction was found to be 0.35%0.35%.(.(By By calculating according to the formula given in methodology).calculating according to the formula given in methodology).

Again in the HPLC chromatogram of the butanol fraction, the area of Ea-02 was found Again in the HPLC chromatogram of the butanol fraction, the area of Ea-02 was found to be 7525235. So the Ea-02 present in the Butanol fraction was found to be to be 7525235. So the Ea-02 present in the Butanol fraction was found 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 Ea-01 (1.451 min in compound and 1.440 min in butanol fraction) and observed in the Ea-01 (1.451 min in compound and 1.440 min in butanol fraction) and Ea-02 (2.324 min in compound and 2.322 min in Butanol fraction). It confirms the Ea-02 (2.324 min in compound and 2.322 min in Butanol fraction). It confirms the presence of the compounds in both the fraction of Butanol extract of the plant part. presence of the compounds in both the fraction of Butanol extract of the plant part. Hence the process can be said as standardized.Hence the process can be said as standardized.

DISCUSSIONDISCUSSION

DISCUSSION Eclipta alba Linn. is used in traditional system of medicine for treatment of many diseases. A number of studies on isolation of chemical constituents and pharmacological activities of Eclipta alba and its extracts have been reported. Coumestans have been isolated as biologically active compounds. But there are scanty reports on estimation using HPLC. Many herbal industries are using this drug for preparing different kinds of formulations.

Hence an attempt was made to isolate, purify and characterize compounds to be used as markers which constitute chemically defined constituents, which can serve as a powerful tool, for standardization of extracts and formulation of Eclipta alba.

Isolation of the phytoconstituents: The methanolic extract of Eclipta alba L. whole plant was partitioned between water and butanol to get rich fraction.

The butanol extract was then fractionated using repeated column chromatography.

Different stationary phases viz., Silica gel, Diaion HP 20 columns were used for the study. Diaion HP20 is actually used for isolation of bimolecular like proteins, but it also exerts a good separation of bigger molecules with the polar ones getting eluted first.

The butanolic extract obtained from methanolic extract was adsorbed on the silica and charged onto a column packed with the stationary phase and eluted with different solvents of varying polarities.

Six different fractions were collected from column chromatography of butanolic extract were evaluated for TLC pattern using chloroform: methanol in the ratio of 9.5:0.5 as the solvent system. TLC analysis has indicated that among all the fractions collected, fraction 2 and 5 (10%MEOH/EA and 75%MEOH/EA) showed distinct bands of constituents.

Purification of the isolated compounds:The above extracts were subjected to further purification by Diaion column to get Ea-01 and Ea-02 compounds.

Analysis of isolated compoundsTLC, Melting point analysis and HPLC method were used to analyze the isolated compounds Ea-01 and Ea-02 for their purity.

The HPLC analysis indicated that the first and second compound Ea01 and Ea-02 was 100% pure.

Compound Ea-03 which was a pure compound which was subjected toTLC analysis by comparing with different standard markers to show a single spot i.e., β- sitosterol d-glucoside.

13C NMR and 1HNMR spectroscopy were used to characterize the isolated compounds. The spectra were compared with the reported literature27.

1HNMR spectrum shows the protons and 13C NMR shows the no. of carbons in the isolated compounds.

The values of Ea-01 and Ea-02 obtained in our study were compared with those reported in the literature27 Shoji Yahara, Ning Ding, Toshihiro Nahara. Oleanane glycosides from Eclipta alba. Chem pharm bull. 1994; 42(6): 1336-8. and indicated that compound were Eclalbasaponin I and Eclalbasaponin II.

Estimation of isolated compounds in the 50% Methanolic extract In order to check the suitability of the two isolated compounds as markers, HPLC analytical method was developed. The butanol extract and isolated compounds were subjected to HPLC for estimation of the isolated compounds. Distinct peaks of both compounds at retention time 1.451 and 2.324 were observed in the chromatograms, indicating the method to be suitable and sensitive. The compounds Ea-01 and Ea-02 were found to be present in 0.35 and 1.78% 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.

Eclalbasaponin I Eclalbasaponin II

CONCLUSION /SUMMARY

Butanol extract was subjected 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 found to be suitable for fractionation/isolation of phytoconstituents at commercial scale. A TLC system with chloroform: methanol(9.5:0.5) as mobile phase and Anisaldehyde sulphuric acid as detecting agent was optimized to identify the phytoconstituents The HPLC method developed was found to be fast, simple and accurate. On the basis of purity and yield of two isolated compounds viz. Ea-01 and Ea-02 were selected for purification and characterization. The isolated, purified compounds were characterized by determining their physical properties like solubility, melting point and then subjecting them to NMR spectroscopy.

The two isolated compounds Ea-01 & Ea-02 were characterized as 3-O[ β-D- glucopyranosyl] echinocystic acid 28-O-β-D- glucopyranosly methyl ester (Eclalbasaponin I) and 3-O-β-D- glucopyranosyl echinocystic acid (Eclalbasaponin II).

The two isolated compounds were found to be suitable candidates to be used as markers.

The two isolated compounds were estimated in the butanolic extract of the whole plant. It was found that the isolated compounds are present in the methanolic extracts in 0.35 and 1.78%.

BIBLIOGRAPHYBIBLIOGRAPHY

BIBLIOGRAPHY1) Harish Padh, Patel BV. Herbal drugs. Current science. 2001; 81(1): 15-6.

2) Daniel M. Medicinal Plants Chemistry and Properties. USA: Science Publishers; 2006.

3) Mukherjee PK. Quality control of Herbal drugs. 1st ed. New Delhi: Business Horizon Pharma Publisher; 2002.

4) Upadhyay RK, Pandey MB, Jha RN, Pandey VB. Phenolic constituents of Eclipta alba. Indian drugs. 2000; 30(2): 73-6.

5) Yahara S. Taraxastane glycosides from Eclipta alba. Phytochemistry. 1997; 44(1): 131-5.

6) Nguyen Thi Viet Thanh, Nguyen Van Dau. Bioactive principles of the Vietnamese Eclipta alba (L.) hassk(Asteraceae). J Chem. 2006; 44 (6): 777-81.

7) Murali B, Amit A, Anand MS, Samiulla. Estimation of wedelolactone and demethylwedelolactone in Eclipta alba Hassk. by improved chromatographic analysis. J Nat Remedies. 2002; 2(1): 99-101.

8) Mohammad S Rahman, Rasheduzzaman chowdhury, Chowdhury M Hasan, Mohammad A Rashid. Oleanane glycosides from Eclipta prostrata. Dhaka Univ. J. Pharm. Sci. 2005; 4(2): 1816-20.

9) Patel MB, Mishra SH. A simple spectroflurometric estimation of wedelolactone in methanol extract of Eclipta alba. Phcog mag. 2006; 2(7): 168-71.

10) Unnikrishan KP, Anu fathima. Hashim KM, Indira balachandran. Antioxidant studies & determination of wedelolactone in Eclipta alba. J Plant sci. 2007; 2(4): 459-64.

11) Rajesh kumar chawla, Rajvir singh, Kalidhar S.B. Phytochemical study & antifungal effect of extracts from Eclipta alba (L.) on phytopathogenic fungi. J med & aro plant sci. 2008; 30: 73-6.

12) Ananthi J, Prakasam, Pugalendi KV. Antihyperglycemic activity of Eclipta alba leaf on alloxan-induced diabetic rats. Yale J bio & med. 2003; 76: 97-102.

13) Karthik S, Vigneswari K, Jegatheesan K. Screening of antibacterial & antioxidant activities of leaves of Eclipta prostrata (L). Scientific research & essay.2007; 2(4): 101-4.

14) Shalini Sharma, Mukesh S Sikarwar. Wound healing activity of ethanolic extract of leaves of Eclipta alba. Phcog mag. 2008; 4(13): 108-11.

15) Otilia JF lobo, David banji, Annamalai AR, Manavalan R. Evaluation of antiaggressive activity of Eclipta alba in experimental animals. Pak J Pharm Sci. 2008; 21(2): 195-9.

16) Otilia banji, David banji, Annamalai AR, Manavalan R. Investigation on the effect of Eclipta alba on animal models of learning & memory. Indian J physiol pharmacol. 2007; 51(3): 274-8.

17) Mi Kyeong Lee, Na Ry Ha, Hyekyung Yang, Sang Hyun Sung , Gun Hee Kim, Young Choong Kim. Antiproliferative activity of triterpenoids from Eclipta prostate hepatic stellate cells. Phytomedicine. 2007; 10(1): 15-21.

18) Nahid Tabassum, Shyam. S. Agrawal. Hepatoprotective activity of Eclipta alba Hassk. against paracetamol induced hepatocellular damage in rats. JK Practitioner. 2004; 11(4): 278-80.

19) Baskaran P, Jayabalan N. Role of basal media, carbon sources and growth regulators in micropropagation of Eclipta alba – A VALUABLE MEDICINAL HERB. KMITL Sci J. 2005; 5(2): 469-81.

20) Borthakur M. Micropropagation of Eclipta alba and Eupatorium adenophorum using a single step nodal cutting technique. J plant cell tissue and organ culture. 2000; 62: 239-42.

21) Das M. Antihepatotoxic principles in tissue culture of Eclipta alba Linn. Indian drugs. 1990; 28(8): 356-8.

THANK YOU