METHODS FOR THE ISOLATION, PURIFICATION AND

CHARACTERIZATION OF BIOACTIVE COMPOUNDS

G.K. Jayaprakasha and Bhimu PatilVegetable & Fruit Improvement Center,

T A&M U i itTexas A&M University College Station, TX 77845

OUT LINE

Challenges & Methods for the isolation of

bi ti d (BAC)bioactive compounds (BAC)

Purification & Identification methods

CASE STUDY: Coumarins Purification &

identification

BIOACTIVE COMPOUNDSGlobal market plant derived N

NO

BAV - $18 B$26 billion

di t d f

O

OOH

CamptothecinLovastatinpredicted for 201163% ti

TaxolCamptothecinLovastatin

63% anticancer drugs from natural Oyster mushroomnatural products

Oyster mushroom

Taxus brevifolia

Chinese Happy tree

www.Drugdiscoverytoday.com, J. Nat. Products, 2009, 72, 507

ppy

GROWTH OF BIOACTIVE COMPOUNDS

DRUGS APPROVED IN US FROM 1981-2007

WORLDWIDE BIOACTIVE COMPOUNDS PATENTS

www.Drugdiscoverytoday.com, Science, 2009

US FROM 1981-2007COMPOUNDS PATENTS

BIOACTIVE COMPOUNDS IN CITRUSCITRUS

LimonoidsFlavonoidsCoumarinsSterolsPectinPectinEssential oilsLycopene &Lycopene & ascorbic acid

WHY PURIFICATION NECESSARY?

Limonin 5mg - $105

6

WHY PURIFICATION & IDENTIFICATION ESSENTIAL?IDENTIFICATION ESSENTIAL?

• Not available commercially

• Expensive for animal expts

• Number of positive results on biological activity

M h i f th BAV b d ti i i i• Mechanism of these BAV by conducting various in vivo

studies

• Clinical trails are needed

R i hi h BAC i lti titi• Requires high pure BAC in multigram quantities

CHALLENGES FOR THE EXTRACTION OF BIOACTIVEEXTRACTION OF BIOACTIVE COMPOUNDS -Raw Material

Chemical nature - simple monomer to a highlypolymerized structure

Occur in free or conjugated forms with sugars acids and other organic moleculessugars, acids, and other organic molecules

Stabilityy

Uneven distribution

CHALLENGES - Extraction

Extraction is influenced by several factorsChemical structure, Glucosides – fruits, seedsP l it f l t lPolarity of solvent, lycopeneSample matrix, Glucosides- dried, fresh- MeOHDegree of polymerization, grapes- phenolicsg p y , g p pInteraction with other cellular componentsTemperature, solubility, part. coeff.Pressure solubility part coeffPressure, solubility, part. coeff.Techniques, Solid-to-solvent ratioP ti l iParticle size

CHALLENGES TO OBTAIN PURE COMPOUNDSCOMPOUNDS

• Low concentrations (<0.2%) in fruits, vegetables

• Constitute to number of compounds

•Individual compound isolation in multigrams is a challenge

•Other components can have similar properties that makesOther components can have similar properties, that makes isolation / separation difficult

• Selection of raw material, Depends upon the targeted , p p gcompounds, For e.g. Limonin, – Grapefruits; Lyc – Rio-red;

• Minor start from large amount of raw materials to• Minor - start from large amount of raw materials to enrich to small quantity and go for fractionation.

POSSIBLE REASONS FOR VARIATION OF BIOACTIVE

COMPOUNDS

Cultivars - Lycopene

Grapefruits

Environmental conditionsPost-harvest conditions

Blood Oranges

MaturityAnalysis Methods

Limonin - LG

Phenolics HPLCAnalysis, Methods Phenolics, HPLC

CRITICAL STEPS FOR EXTRACTION

Sampling/selection of raw material

Preservation of samples / extracts

Extraction of bioactive compoundsExtraction of bioactive compounds

Separation & Detection

CRITICAL STEPS WITHIN EXTRACTIONEXTRACTION

Sample homogenization

Extraction (PLE, SFE, Sonication, Soxhlet, Vortex, Shaker, Stirring, Microwave, etc.)

Hydrolysis Derivatization

Filtration, centrifugation

Hydrolysis, Derivatization

Pre-concentration (Liquid-liquid extraction, SPE, etc.)

SELECTION OF FAV FORSELECTION OF FAV FOR BIOACTIVE COMPOUNDS

APPROACHES FOR NATURAL COMPOUNDS ISOLATIONCOMPOUNDS ISOLATION

• Raw material selection

E i Wi h l d i h l• Extraction: With solvent and without solvents

• Purification: Column chromatography

Flash chromatography

P HPLCPrep. HPLC

Analytical Techniques: TLC, HPLC, GCy q

Structure elucidation: NMR, LC-MS, MS-MS

EXTRACTION METHODSEXTRACTION METHODS

Soxhlet - Solvents

Hydrotropic extraction – Non-solvent

Supercritical fluid extraction- CO2p

Microwave extraction

Ult d t tiUltrasound extraction

Pressurized extraction

SOLVENT EXTRACTION BY SOXHLETSOXHLET

Cit f itCit f itCitrus fruits Citrus fruits

PowderedPowderedPowdered Powdered

SolventSolvent

Extraction 6Extraction 6--8 h8 h

ConcentrationConcentration

Bioactive fractionsBioactive fractions

Mandadi et al., Z. Naturforschung C, 62c, 179-188, 2007

NON-SOLVENT EXTRACTION / HYDROTROPIC EXTRACTION

• Highly water soluble organic salts with h d h bi d h d hili i i

S OO

O Na

S OO

O Na

hydrophobic and hydrophilic moieties

• Increasing solubility of water insoluble CH3 CH3

CH3

N B t lb l h t (NBBS)g y

compounds

• Depends not only on the nature ofO O Na

Na-Butylbenzene sulphonate (NBBS) Na-Cumene sulphonate (Na-CuS)

Depends not only on the nature of hydrotrope but also on the nature of solute

OH

Na-Salicylate (Na-Sal)

Dandekar et al., Z. Naturforschung, 63c, 176-180, 2008

HYDROTROPIC EXTRACTIONCont’dCont d.

Raw Material

Hydrotrope Solution

Extract Filtered ExtractWater (pH 7 or pH 3)

Solid ResidueSour orange + Sodium cumen sulfonate, 45°C for 6 H

Dilute ExtractDilute Hydrotrope Solution

Product Mixtures of limonoids & Flavonoids

Dandekar et al., Food Chemistry, 109, 515, 2008

SUPERCRITICAL FLUID EXTRACTIONEXTRACTION

• Solutes can be separated without loss of volatiles

• SC-CO2 protects the substrate from oxygen, resulting in fewer d iti d tdecomposition products

SFE can eliminate the concentration processconcentration process

25-40 MPa – 3000- 7000 PSI

Jun et al., J. Agric. Food Chem., 2006, 54 (16), pp 6041–6045, Food Chemistry, 105, 1026, 2007

PRESSURIZED / ACCELERATED / ENHANCEDACCELERATED / ENHANCED

SOLVENT EXTRACTION

Solid – Liquid extractionextraction

50 200 °C /50-200 °C / 1500- 2500 PSI

Ong, et al., J. Chromatogr., A, 2000, 904, 57; G. W. Schieffer, J. Liq. Chromatogr. Relat. Technol., 2002, 25, 3033

MICROWAVE-ASSISTED EXTRACTION

Microwaves : Solid-Liquid

Non-ionising EM radiation freq. 300-

300,000 MHz

Fast and rapid method for small titquantity

Flammable solvents cannot be used

22

Shu, Microchem. J., 2003, 74, 131; Fulzele and Satdive, J. Chromatogr., A, 2005, 1063, 9

SUMMARY /CONCLUSIONS/Type Sample

size: solvent Temp Pressure/

TimeInvestment Inference

Soxhlet 1-2000 g;4-8000 ml

Depends upon the solvent

Atmospheric / 6-8 h

Very low Efficient for polar & non-

lpolar BAC

SFE 25-100 g; continuous flow

25 – 45 Mpa; 1-2 h

High Efficient for non-polar BACflow BAC

PLE 1-30 g10-100 ml

80-200 1 – 10 Mpa; 10-30 min

High Efficient for polar & non-polar BACpolar BAC ,Safety

MAE 1 -20 g; 10-50 ml

80-150 Variable / 10-30 min

Moderate Use of solvents is

23

risky

HYDRO 10-100 g 40-60 Atmospheric / 6-8h

Low Residual hydrotrop

OUT LINE

Introduction

Challenges & Methods for the isolation of

bioactive compounds (BAC) p ( )

Purification & Identification methods

CASE STUDY: Coumarins Purification &

identificationidentification

PURIFICATIONTECHNIQUES

Distillation, Fractional Dist. BP

Fractional crystallization solubilityFractional crystallization, solubility

Gel filtration, size

Affinity chromatography

Ion exchange chromatography

Flash chromatographyFlash chromatography

Preparative HPLC

SELECTION OF ADSORBENTSample

SELECTION OF ADSORBENT

Most of the BACProteins, AA, Alk l id

AnthocyaninsProteins, AA, Alkaloids

SampleCharacteristics

Low or MediumPolarity

BasicProperties

HighPolarity

AcidicProperties

AcidSensitiveChargedWith metal

reagent

Alkaloidsy

Stationary Phase(conditions) Normal Phase Silica (NP)

Reversed Phase Silica (RP)

Acidic Alumina (NP)Acidic Alumina (NP)Reversed Phase Silica (RP)

Cyano Silica (RP)

Reversed Phase Silica (RP)

Cyano silica (RP)SAX (NP)

Cyano (NP)

Normal Phase Silica (NP)

Amine Silica (NP/RP)

Basic Alumina (NP)

Neutral alumina (NP)

Florisil (NP)

Cyano (NP)

Normal Phase Silica (NP)

Diol Silica (NP)

Cyano Silica (NP)

Neutral Alumina (NP)

Metal Scavenger Silica (NP)

SCX (NP) Neutral Alumina (NP)SCX (NP)

Neutral alumina (NP)

Cyano silica (NP)Reversed Phase Silica (RP)

PURIFICATION BY ION EXCHANGE & ADSORBANT RESINS

++ A

Prep. HPLC

MOLAS

++RESI

ADSORB

ADSORBMolasses S

SES

D

NBANT

BANT

Molasses

Pure compounds

IL

Seed extractFlash chromatography

Jayaprakasha et al., US patent, 2007/0237885 A1

FLASH CHROMATOGRAPHY

Clark Steel (1978) – purification, Silica gel

Glass columns high flow rates – 5 ml/min, faster

Low molecular weight natural, syntheticLow molecular weight natural, synthetic

Irreproducible

Modern flash techniques

Use of convenient disposable flash cartridges.

Speed up the purification processSpeed up the purification process.

SAMPLE LOADING

Dry LoadingPre-adsorption of sample onto silica for low solubility samples

Wet “dry loading” Pipetting sample ontopre-packed solid load cartridge.

Liquid InjectionThrough valve allows for column equilibration.q

Liquid injection directly onto column

Equilibration is skipped andEquilibration is skipped and sample is run through dry column (for speed).

STRUCTURE ELUCIDATION

Quantification, HPLC, GCPurity, HPLC, GC, TLC

ANALYTICAL TECHNIQUESANALYTICAL TECHNIQUES

Thin Layer Chromatography (TLC)

Very sensitive rapid very accurate forVery sensitive, rapid, very accurate for the natural products

Sample is spotted onto TLC plate using aSample is spotted onto TLC plate using a glass capillary

Plate is “developed” using a solvent p gsystem

31

ANALYTICAL TECHNIQUESANALYTICAL TECHNIQUES Cont.,

High Performance Liquid Chromatography (HPLC) Gas Chromatography (GC)

Vikram, et al., Analytica Chemica Acta, 590, 180-186, 2007; Tian, et al., J. Chromatography B, 846, 385-390, 2007; Perez, et al., J. Chromatography, 1190, 394-397, 2008.

IDENTIFICATION BY NUCLEAR MAGNETIC RESONANCE (NMR) ( )

SPECTRA

NMR is the most powerful tool available pfor organic structure determination.

Variety of nuclei:1H 13C 15N 19F 31P

Jaiprakash et al., Food Chemistry, 2009, 114, 1351-1358.Jayaprakasha, G.K., Bioorganic and

H, C, N, F, P

p , y, , , y p , , gMedicinal Chemistry, 2008, 16, 5939-5951;. Bioorganic and Medicinal Chemistry, 15, 4923-4932, 2007; Poulose, et al., J. Science Food & Agriculture, 87, 1699-1709, 2007.

NUCLEAR SPINNUCLEAR SPINNucleus with an odd atomic number or an

dd b h l iodd mass number has a nuclear spinThe spinning charged nucleus generates a

ti fi ldmagnetic fieldWhen placed in an external field, spinning

protons act like bar magnetsprotons act like bar magnets

CHEMICAL SHIFTDependence of nuclear magnetic energy levels on the electronic environment in alevels on the electronic environment in a moleculeMeasured in parts per millionp pCalled the delta scale

NMR SIGNALSNumber of signals - different kinds of protonsLocation how shielded or deshielded the protonsLocation how shielded or deshielded the protonsIntensity - number of protons of that type

3.6 3.4 0

PROTONS IN A MOLECULEPROTONS IN A MOLECULEDepending on their chemical

environment, protons in a molecule are shielded by different amounts

Methanol - CH3OH

3H -1 – signal -3.4 ppm 1 – signal – 3.6 ppm

LOCATION OF SIGNALSLOCATION OF SIGNALS

More electronegative atoms deshield more and i l hift lgive larger shift values.

Effect decreases with distancedistance.Additional electronegative atoms cause increase inatoms cause increase in chemical shift.

TYPICAL VALUESTYPICAL VALUES

Chapter 13

1D & 2D NMR SPECTROSCOPYINEPT (I iti N l i E h t b P l i ti T f )INEPT (Insensitive Nuclei Enhancement by Polarization Transfer)

DEPT (Distortionless Enhancement by Polarization Transfer)

SEFT (Spin-echo Fourier Transform)

Carbon status – primary, secondary, tertiary, quaternary

COSY – Homonuclear correlation spectroscopyProton – proton correlations

HSQC - Heteronuclear single quantum coherence HMQC - Heteronuclear Multiple Quantum Coherence Carbon and Proton direct attachments

HMBC – Heteronuclear Multiple Bond Correlation Carbon and Proton on adjacent carbon (2 or 3) attachments

TOCSY (or) HOHAHA - Total Correlation Spectroscopy Carbon and Proton on neighboring carbon through hetero atom.

SUMMARY / CONCLUSIONS

Adsorption / affinity chromatography – Good p / y g p yseparation technique for BAC

Flash chromatography is rapid and reproducibleFlash chromatography is rapid, and reproducible

TLC is more actuate for the confirmation of the purity, f id li blfast rapid, reliable

HPLC - good tool for the quantificationg q

2D NMR will help for the accurate assignments of NMR signalsNMR signals

OUT LINE

Introduction

Challenges & Methods for the isolation of

bioactive compounds (BAC) p ( )

Purification & Identification methods

CASE STUDY: Coumarins Purification &

identificationidentification

OBJECTIVE

Extraction of bioactive compounds from Extraction of bioactive compounds from lililimeslimes

Purification of Purification of coumarinscoumarins by flash by flash chromatographychromatography

Identification of isolated compounds by NMR Identification of isolated compounds by NMR d t l l id t l l iand mass spectral analysisand mass spectral analysis

EXTRACTION OF COUMARINS

Whole limes, juiced & freeze dried

Extraction with chloroform

Extract Spent

Concentrated under vacuum

FLASH INSTRUMENT CONDITIONS/CHROMATOGRAMCONDITIONS/CHROMATOGRAM

FLASH FRACTIONS & COMPOUNDS

Compound 1

Compound 2

Compound 3

HPLC ANALYSIS

A). 3mM Phosphoric acid

B). ACNCompound 1λmax.- 254 nm, 0.5ml/min

Compound 2Time Time (min)(min)

3mM Phosphor

AcetonitriAcetonitrile (%)le (%)(min)(min) Phosphor

ic acid (%)

le (%)le (%)

0 45 55Compound 3

0 45 5520 10 90

25 25 7530 25 75

HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE (HMQC) SPECTRA OF COMPOUND 2 (DIMETHOXYCOUMARIN)

A ti iAromatic regionAliphatic region

MULTIPLE-BOND CH CORRELATION (HMBC) SPECTRA OF DIMETHOXYCOUMARIN

1H NMR

13C NM

R

HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE (HMQC) SPECTRA OF COMPOUND 3

(ISOIMPINELLIN)(ISOIMPINELLIN)

Aromatic region Aliphatic region

HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE (HMBC) SPECTRA OF ISOIMPINELLIN

1H NMR

13C NM

R

IDENTIFICATION EI/MS

Jaiprakash et al., Food Chemistry, 2009, 114, 1351-1358.Jayaprakasha, G.K., Bioorganic and p , y, , , y p , , gMedicinal Chemistry, 2008, 16, 5939-5951;. Bioorganic and Medicinal Chemistry, 15, 4923-4932, 2007; Poulose, et al., J. Science Food & Agriculture, 87, 1699-1709, 2007.

MS- TOF analysis of 5,7-dimethoxycoumarinMS TOF analysis of 5,7 dimethoxycoumarin

JRP-SAMPLE,S3_090122140209 #1 RT: 0.04 AV: 1 NL: 7.18E7T: + c Full ms [50.00-600.00]

100206.08

70

80

90

ce

178.09

C11H10O430

40

50

60

Rel

ativ

e A

bund

anc

163.06

135.03

Exact mass -206.08

MW-206.19

100 200 300 400 500 6000

10

20

30

69.0192.04

217.15 335.12281.26 373.44 429.28 501.35 533.88

100 200 300 400 500 600m/z

MS-TOF analysis of Isopimpinellin

JRP-SAMPLE,S4 #129 RT: 0.56 AV: 1 NL: 2.20E8T: + c Full ms [50.00-600.00]

246 07

80

90

100246.07

231.04

OO O

OCH3

H3CO

40

50

60

70

ativ

e A

bund

ance

H3CO

10

20

30

40

Rel

a

175.06

188.03160.0369.05

81.08

C13H10O5 – 246.2

100 200 300 400 500 600m/z

0

10

256.28 341.38 386.40 460.47 494.64 536.48

CRITICAL APPROACHES FOR THE ISOLATION & IDETIFICATIONTHE ISOLATION & IDETIFICATION

Identifying the bioactive compounds (BAC) of ourIdentifying the bioactive compounds (BAC) of our interest

U d t di th i t ti f BAC ith lUnderstanding the interaction of BAC with sample matrix

Stability of the BAC

Optimization of extraction solventsOptimization of extraction solvents

Selection of purification methods, adsorbent, elution l tsolvents

Nature of compounds, MW, volatality, polarity of BAC

AACKNOWLEDGEMENTSCKNOWLEDGEMENTSAACKNOWLEDGEMENTSCKNOWLEDGEMENTS

USDA-CSREES

Vegetable & Fruit Improvement Center | Texas A&M University System

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