G.K. Jayaprakasha and Bhimu Patil
Vegetable & Fruit Improvement Center
Texas A&M University
College Station, TX 77845
METHODS FOR EXTRACTION, PURIFICATION AND CHARACTERIZATION
OF BIOACTIVE COMPOUNDS
OUT LINE
Brief introduction, challenges & Methods
Purification & Identification methods
CASE STUDY: Coumarins Purification &
identification
BIOACTIVE COMPOUNDS
Global market plant
derived BAV 2008 – $18 B B
$33 billion predicted
for 2013 (80%)
63% anticancer drugs from natural products
http://www.bccresearch.com/report/botanical-plan-derived-drugs-bio022e.html
BIOACTIVE COMPOUNDS
www.Drugdiscoverytoday.com, J. Nat. Products, 2009, 72, 507
Oyster mushroom
Taxus
brevifolia
Taxol
N
N
O
O
OOH
Chinese Happy tree
Camptothecin Lovastatin
Naturally derived structures of compounds used for prevention of various diseases
GROWTH OF BIOACTIVE COMPOUNDS
www.Drugdiscoverytoday.com, Newman & Cragg, JNP, 2012, 75, 311
DRUGS APPROVED IN
US FROM 1981-2010
WORLDWIDE BIOACTIVE
COMPOUNDS PATENTS
70+30
350+95
NP-Derived Patents Original NP Patents
78
40 33
BIOACTIVE COMPOUNDS IN CITRUS
Limonoids -57
Flavonoids -60
Coumarins -17
Pectin
Volatiles - >100
Carotenoids >30 ascorbic acid
Sterols - >10
8
WHY PURIFICATION NECESSARY?
Limonin 5mg - $105
5 g – 105,000
• Not available commercially
• Expensive for animal expts
• Number of positive results on biological activity
• Mechanism of these BAV by conducting various in vivo
studies
• Clinical trails are needed
• Requires high pure BAC in multigram quantities
WHY PURIFICATION &
IDENTIFICATION ESSENTIAL?
Chemical nature - simple monomer to a highly
polymerized structure
Occur in free or conjugated forms with
sugars, acids, and other organic molecules
Stability
Uneven distribution
CHALLENGES FOR THE EXTRACTION OF BIOACTIVE COMPOUNDS -RAW MATERIAL
Extraction is influenced by several factors Chemical structure, Glucosides – fruits, seeds Polarity of solvent, lycopene Sample matrix, Glucosides- dried, fresh- MeOH Degree of polymerization, grapes- phenolics Interaction with other cellular components Temperature, Pressure, solubility, part. coeff. Solid-to-solvent ratio Particle size Techniques
CHALLENGES - EXTRACTION
CHALLENGES TO OBTAIN PURE
COMPOUNDS
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 makes
isolation / separation difficult
Selection of raw material, Depends upon the targeted
compounds, For e.g. Limonin, – Grapefruits; Lyc – Rio-red;
Minor - start from large amount of raw materials to
enrich to small quantity and go for fractionation.
POSSIBLE REASONS FOR VARIATION OF BIOACTIVE COMPOUNDS
Limonin - LG
Phenolics, HPLC
Grape fruits
Blood Oranges
Rio red Marsh white
Cultivars - Lycopene
Environmental conditions
Post-harvest conditions, Maturity
Analysis, Methods
CRITICAL STEPS FOR EXTRACTION
Sampling/selection of raw material
Preservation of samples / extracts
Extraction of bioactive compounds
Separation & Detection
CRITICAL STEPS WITHIN EXTRACTION
Hydrolysis, Derivatization
Sample homogenization
Extraction (PLE, SFE, Sonication, Soxhlet, Vortex, Shaker, Stirring, Microwave, etc.)
Filtration, centrifugation
Pre-concentration (Liquid-liquid extraction, SPE, etc.)
SELECTION OF FAV FOR BIOACTIVE COMPOUNDS
APPROACHES FOR NATURAL
COMPOUNDS PURIFICATION
• Raw material selection
• Extraction: With solvent and without solvents
• Purification: Column chromatography
Flash chromatography
Prep. HPLC
Analytical Techniques: TLC, HPLC, GC
Structure elucidation: NMR, LC-MS, MS-MS
EXTRACTION METHODS
Soxhlet - Solvents
Hydrotropic extraction – Non-solvent
Supercritical fluid extraction- CO2
Microwave extraction
Ultrasound extraction
Pressurized extraction
SOLVENT EXTRACTION BY
SOXHLET
Dried sample
Powdered
Solvent
Extraction 6-8 h
Concentration
Bioactive fractions
Mandadi et al., Z. Naturforschung C, 62c, 179-188, 2007
NON-SOLVENT EXTRACTION /
HYDROTROPIC EXTRACTION
• Highly water soluble organic salts with
hydrophobic and hydrophilic moieties
• Increasing solubility of water insoluble
compounds
• Depends not only on the nature of
hydrotrope but also on the nature of
solute
Dandekar et al., Z. Naturforschung, 63c, 176-180, 2008
HYDROTROPIC EXTRACTION
Cont’d.
Raw Material
Hydrotrope Solution
Extract Filtered Extract
Solid Residue
Water (pH 7 or pH 3)
Dilute Extract
Product
Dilute Hydrotrope Solution
Sour orange + Sodium cumen
sulfonate, 45C for 6 H
Mixtures of limonoids & Flavonoids
Dandekar et al., Food Chemistry, 109, 515, 2008
SUPERCRITICAL FLUID EXTRACTION
• Solutes can be separated without degradation of volatiles • SC-CO2 protects the substrate from oxygen, resulting in fewer decomposition products • SFE can eliminate the concentration 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 / ENHANCED SOLVENT EXTRACTION
Solid – Liquid extraction
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
24
MICROWAVE-ASSISTED EXTRACTION
Microwaves : Solid-Liquid
Non-ionising EM radiation freq. 300-
300,000 MHz
Fast and rapid method for small quantity Flammable solvents cannot be used Shu, Microchem. J., 2003, 74, 131; Fulzele and Satdive, J. Chromatogr., A, 2005, 1063, 9
25
SUMMARY /CONCLUSIONS
Type Sample size: solvent
Temp, C Pressure/ Time
Investment Inference
Soxhlet 1-2000 g; 4-8000 ml
Depends upon the solvent
Atmospheric / 6-8 h
Very low Efficient for polar & non-polar BAC
SFE 25-100 g; continuous flow
40-60 25 – 45 Mpa; 1-2 h
High Efficient for non-polar BAC
PLE 1-30 g 10-100 ml
80-200 1 – 10 Mpa; 10-30 min
High Efficient for polar & non-polar BAC , Safety
MAE 1 -20 g; 10-50 ml
80-150 Variable / 10-30 min
Moderate
Use of solvents is risky
HYDRO
10-100 g
40-60 Atmospheric / 6-8h
Low Residual hydrotrop
OUT LINE
Challenges & Methods for the isolation of
bioactive compounds (BAC)
Purification & Identification methods
CASE STUDY: Coumarins Purification &
identification
PURIFICATION TECHNIQUES
Distillation, Fractional Dist. BP
Fractional crystallization, solubility
Gel filtration, size
Affinity chromatography
Ion exchange chromatography
Flash chromatography
Preparative HPLC
Sample
Sample Characteristics
Stationary Phase
(conditions)
Low or Medium
Polarity
Basic
Properties
High
Polarity
Acidic
Properties
Acid
Sensitive Charged
With metal
reagent
Normal Phase Silica (NP)
Amine Silica (NP/RP)
Basic Alumina (NP)
Normal Phase Silica (NP)
Reversed Phase Silica (RP)
Acidic Alumina (NP)
Neutral alumina (NP)
Florisil (NP)
Cyano (NP)
Normal Phase Silica (NP)
Diol Silica (NP)
Cyano Silica (NP)
Neutral Alumina (NP)
Reversed Phase Silica (RP)
Cyano Silica (RP)
Reversed Phase Silica (RP)
Cyano silica (RP)
Metal Scavenger Silica (NP)
SCX (NP)
Neutral alumina (NP)
Cyano silica (NP)
SAX (NP)
Cyano (NP)
Reversed Phase Silica (RP)
SELECTION OF ADSORBENT
Most of the BAC
Proteins, AA, Alkaloids
Anthocyanins
Proteins, AA, Alkaloids
PURIFICATION BY ION EXCHANGE & ADSORBANT RESINS
M
O
L
A
S
S
E
S
D
I
L
+
+
+
R
E
S
I
N
A
D
S
O
R
B
A
N
T
A
D
S
O
R
B
A
N
T
Seed extract
Molasses
Prep. HPLC
Pure compounds
Flash 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, synthetic
Irreproducible
Modern flash techniques
Use of convenient disposable flash cartridges.
Speed up the purification process.
SAMPLE LOADING
Dry Loading Pre-adsorption of sample onto
silica for low solubility samples
Wet “dry loading” Pipetting sample onto
pre-packed solid load cartridge.
Liquid Injection Through valve allows for column
equilibration.
Liquid injection directly onto column Equilibration is skipped and
sample is run through dry column (for speed).
STRUCTURE ELUCIDATION
Spectroscopic methods
Stereochemistry
NMR MS
1H, 13C, DEPT, COSY, HMBC,
HMQC, TOCSY, ROESY, NOESY
ESI, EI, FAB, HRMS
Optical activity X-Ray
Crystallography
Quantitation GC, HPLC, TLC
33
ANALYTICAL TECHNIQUES
Thin Layer Chromatography (TLC)
Very sensitive, rapid, very accurate for the natural products
Sample is spotted onto TLC plate using a glass capillary
Plate is “developed” using a solvent system
ANALYTICAL 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
Jaiprakash et al., Food Chemistry, 2009, 114, 1351-1358.Jayaprakasha, G.K., Bioorganic and Medicinal Chemistry, 2008, 16, 5939-5951;. Bioorganic and Medicinal Chemistry, 15, 4923-4932, 2007; Poulose, et al., J. Science Food & Agriculture, 87, 1699-1709, 2007.
NMR is the most powerful tool available for organic structure determination. Variety of nuclei:
1H, 13C, 15N, 19F, 31P
1940 1950 1960 1970 1980 1990 2000 2010
NMR signals first detected
Chemical shift, J-coupling
used for structural ID
NMR becomes common
Introduction of FT approach
Use of NOE for structure
Superconducting magnets
Multidimensional NMR
Solid-state NMR, imaging
Field gradients
New probe designs
NMR TIMELINE
1000 MHz
Nucleus # Protons # Neutrons Spin
1H 1 0 1/2
2H 1 1 1
12C 6 6 0
13C 6 7 1/2
16O 8 8 0
17O 8 9 5/2
14N 7 7 1
15N 7 8 1/2
• Nuclei with an odd number of protons, neutrons, or
both have spin
NUCLEAR SPIN, I
Most elements can be observed (1H, 2H, 13C, 19F, 31P, 29Si, 15N, 17O, 119Sn, 207Pb, …)
CHEMICAL SHIFT
Chemical shift originates from variations in the electronic environments around chemically distinct nuclei
Chemical shift is usually expressed in units of δ (ppm)
Measured in parts per million
Called the delta scale
NMR SIGNALS/SPECTRA
12 3.6 3.4 0
Number of signals - different kinds of protons
Location how shielded or deshielded the protons
Intensity - number of protons of that type
PROTONS IN A MOLECULE
Depending 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 SIGNALS
More electronegative atoms deshield more and give larger shift values.
Effect decreases with distance.
Additional electronegative atoms cause increase in chemical shift.
Chapter 13
TYPICAL VALUES
◙ Origin of chemical shift
◙ Diamagnetic effects
◙ Paramagnetic effects
◙ Neighboring-group effects
◙ Exchange effects
◙ Other effects
◙ Calculation of chemical shift
◙ Chemical-shift compilations
CHEMICAL SHIFT
Available fields range from 200 to 1000 MHz
Modern NMR magnets are superconducting
The magnet consists of a coil of Nb alloy wire immersed in LHe (4K or -269C)
The outer cryogen dewar is filled with LN2 (77K or -196C)
Quench video:
http://www.vimeo.com/1084900
SUPERCONDUCTING MAGNETS
1D & 2D NMR SPECTROSCOPY
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 spectroscopy Proton – 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.
High vacuum/Mylar
High vacuum
LN2 vessel
Gas shield Solenoid
LHe vessel
INSIDE A SUPERCON MAGNET
SUMMARY / CONCLUSIONS
Adsorption / affinity chromatography – Good separation technique for BAC Flash chromatography is rapid, and reproducible TLC is more actuate for the confirmation of the purity, fast rapid, reliable HPLC - good tool for the quantification 2D NMR will help for the accurate assignments of NMR signals
OUT LINE
Challenges & Methods for the isolation of
bioactive compounds (BAC)
Purification & Identification methods
CASE STUDY: Coumarins Purification &
identification
OBJECTIVE
Extraction of bioactive compounds from
limes
Purification of coumarins by flash
chromatography
Identification of isolated compounds by NMR
and mass spectral analysis
Extract Spent
EXTRACTION OF COUMARINS
Extraction with chloroform
Concentrated under vacuum
Whole limes, juiced & freeze dried
FLASH SEPARATION
FLASH FRACTIONS & COMPOUNDS
Compound 1
Compound 2
Compound 3
HPLC ANALYSIS
A). 3mM Phosphoric acid
B). ACN
max.- 254 nm, 0.7ml/min
Time
(min)
3mM
Phosphor
ic acid
(%)
Acetonitri
le (%)
0 45 55
20 10 90
25 25 75
30 25 75
HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE (HMQC) SPECTRA OF COMPOUND 2 (DIMETHOXYCOUMARIN)
Aromatic region Aliphatic region
MULTIPLE-BOND CH CORRELATION (HMBC) SPECTRA OF DIMETHOXYCOUMARIN
1H NMR
13C N
MR
IDENTIFICATION EI/MS
Jaiprakash et al., Food Chemistry, 2009, 114, 1351-1358.Jayaprakasha, G.K., Bioorganic and Medicinal 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-dimethoxycoumarin
C11H10O4
MW-206.19
Exact mass -206.08
CRITICAL APPROACHES FOR THE ISOLATION & IDENTIFICATION
Identifying the bioactive compounds (BAC) of our interest Understanding the interaction of BAC with sample matrix
Stability of the BAC
Optimization of extraction solvents
Selection of purification methods, adsorbent, elution solvents
Nature of compounds, MW, volatality, polarity of BAC
USDA-CSREES
ACKNOWLEDGEMENTS
Vegetable & Fruit Improvement Center | Texas A&M University System
