METHODS FOR THE ISOLATION, PURIFICATION AND CHARACTERIZATION OF BIOACTIVE COMPOUNDS G.K. Jayaprakasha and Bhimu Patil Vegetable & Fruit Improvement Center, T A&M U i it Texas A&M University College Station, TX 77845
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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
ANYANY
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