METHODS FOR THE ISOLATION,

PURIFICATION AND
CHARACTERIZATION OF BIOACTIVE
COMPOUNDS

G.K. Jayaprakasha and Bhimu Patil

Vegetable & Fruit Improvement Center,
T
Texas A&M U University
i it
College Station, TX 77845
 OUT LINE
„ Challenges & Methods for the isolation of
bi
bioactive
ti compounds
d (BAC)

„ Purification & Identification methods

„ CASE STUDY: Coumarins Purification &

identification
 BIOACTIVE COMPOUNDS
„ Global market O

plant derived N
N

BAV - $18 B O

$26 billion
OH O

„
Lovastatin Camptothecin
predicted
di t d for
f Taxol
2011
„ 63% anticancer
ti
drugs from
natural Oyster mushroom
products Taxus Chinese
brevifolia Happy
ppy tree

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

 GROWTH OF BIOACTIVE
COMPOUNDS

WORLDWIDE BIOACTIVE DRUGS APPROVED IN

COMPOUNDS PATENTS US FROM 1981-2007

www.Drugdiscoverytoday.com, Science, 2009

BIOACTIVE COMPOUNDS IN
CITRUS

„ Limonoids
„ Flavonoids
„ Coumarins
„ Sterols
„ Pectin
„ Essential oils
„ Lycopene &
ascorbic acid
WHY PURIFICATION NECESSARY?

Limonin 5mg - $105

6
 WHY PURIFICATION &
IDENTIFICATION ESSENTIAL?

• Not available commercially

• Expensive for animal expts
• Number of positive results on biological activity
• Mechanism
M h i off these
th BAV b
by conducting
d ti various
i i vivo
in i
studies
• Clinical trails are needed
• Requires
R i hi
high
h pure BAC in
i multigram
lti quantities
titi
 CHALLENGES FOR THE
EXTRACTION OF BIOACTIVE
COMPOUNDS -Raw Material
¾ Chemical nature - simple monomer to a highly
polymerized structure

¾ Occur in free or conjugated forms with

sugars acids,
sugars, acids and other organic molecules

¾ Stability
y

¾ Uneven distribution
 CHALLENGES - Extraction

¾Extraction is influenced by several factors

9 Chemical structure, Glucosides – fruits, seeds
9 Polarity
P l it off solvent,
l t lycopene
l
9 Sample matrix, Glucosides- dried, fresh- MeOH
9 Degree
g of p
polymerization,
y ,ggrapes-
p phenolics
p
9 Interaction with other cellular components
9 Temperature, solubility, part. coeff.
9 Pressure,
Pressure solubility,
solubility part
part. coeff.
coeff
9 Techniques,
9 Solid-to-solvent ratio
9 Particle
P ti l size
i
 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
Other components can have similar properties,
properties that makes
isolation / separation difficult
• Selection of raw material,, Depends
p upon
p the targeted
g
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
Grape
fruits
¾ Cultivars - Lycopene
Blood
¾ Environmental conditions Oranges
¾ Post-harvest conditions
¾ Maturity Limonin - LG
¾ Analysis,
Analysis Methods Phenolics HPLC
Phenolics,
 CRITICAL STEPS FOR
EXTRACTION

Sampling/selection of raw material

Preservation of samples / extracts

Extraction of bioactive compounds

Separation & Detection

 CRITICAL STEPS WITHIN
EXTRACTION

™ Sample homogenization

™ Extraction (PLE, SFE, Sonication, Soxhlet,

Vortex, Shaker, Stirring, Microwave, etc.)

™ Filtration, centrifugation
™ Hydrolysis Derivatization
Hydrolysis,

™ Pre-concentration (Liquid-liquid
extraction, SPE, etc.)
 SELECTION OF FAV FOR
BIOACTIVE COMPOUNDS
 APPROACHES FOR NATURAL
COMPOUNDS ISOLATION

• Raw material selection

• Extraction:
E i With
Wi h solvent
l and
d without
ih solvents
l
• Purification: Column chromatography
Flash chromatography
P
Prep. HPLC

Analytical
y Techniques:
q TLC, HPLC, GC
Structure elucidation: NMR, LC-MS, MS-MS
EXTRACTION METHODS

Soxhlet - Solvents
Hydrotropic extraction – Non-solvent
Supercritical
p fluid extraction- CO2
Microwave extraction
Ult
Ultrasound
d extraction
t ti
Pressurized extraction
 SOLVENT EXTRACTION BY
SOXHLET

Cit
Citrus fruits
f it

Powdered

Solvent

Extraction 6
6--8 h

Concentration

Bioactive fractions
Mandadi et al., Z. Naturforschung C, 62c, 179-188, 2007
 NON-SOLVENT EXTRACTION /
HYDROTROPIC EXTRACTION

O Na
O Na
O S O O S O

• Highly water soluble organic salts with

h d h bi and
hydrophobic dh
hydrophilic
d hili moieties
i i
H3C CH3

• Increasingg solubilityy of water insoluble N B t lb

H3C

Na-Butylbenzene sulphonate
l h t (NBBS) Na-Cumene sulphonate (Na-CuS)
compounds
O O Na

• Depends not only on the nature of OH

hydrotrope but also on the nature of

solute Na-Salicylate (Na-Sal)

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

 HYDROTROPIC EXTRACTION
Cont d.
Cont’d

Raw Material

Hydrotrope Solution

Extract Filtered Extract

Water (pH 7 or pH 3)

Solid Residue
Sour orange + Sodium cumen
sulfonate, 45°C for 6 H
Dilute Hydrotrope Solution Dilute Extract

Product Mixtures of limonoids & Flavonoids

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

 SUPERCRITICAL FLUID
EXTRACTION

• Solutes can be separated

without loss of volatiles

• SC-CO2 protects the

substrate from oxygen,
resulting in fewer
d
decomposition
iti products
d t

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

200 °C /
50-200
50
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

quantity
tit

Flammable solvents cannot be used

Shu, Microchem. J., 2003, 74, 131; Fulzele and Satdive, J. Chromatogr., A, 2005,
1063, 9
22
 SUMMARY /CONCLUSIONS
/
Type Sample Temp Pressure/ Investment Inference
size: solvent Time

Soxhlet 1-2000 g; Depends upon Atmospheric / Very low Efficient for

4-8000 ml the solvent 6-8 h polar & non-
polar
l BAC
SFE 25-100 g; 25 – 45 Mpa; High Efficient for
continuous 1-2 h non-polar
flow BAC
PLE 1-30 g 80-200 1 – 10 Mpa; High Efficient for
10-100 ml 10-30 min polar & non-
polar BAC ,
Safety
MAE 1 -20 g; 80-150 Variable / 10- Moderate Use of
10-50 ml 30 min solvents is
risky
HYDRO 10-100 g 40-60 Atmospheric Low Residual
23
/ 6-8h hydrotrop
 OUT LINE
„ Introduction

„ Challenges & Methods for the isolation of

bioactive compounds
p (BAC)
( )

„ Purification & Identification methods

„ CASE STUDY: Coumarins Purification &

identification
 PURIFICATION
TECHNIQUES
™Distillation, Fractional Dist. BP
™Fractional crystallization,
crystallization solubility
™Gel filtration, size
™Affinity chromatography
™Ion exchange chromatography
™Flash chromatography
™Preparative HPLC
 SELECTION OF ADSORBENT
Proteins, AA, Sample Proteins,
Alkaloids Anthocyanins
y AA, Most of the BAC
Sample
Alk l id
Alkaloids
Characteristics
With metal Acid Acidic Basic High Low or Medium
Charged
reagent Sensitive Properties Properties Polarity Polarity

Stationary Phase
Normal Phase Silica (NP)
(conditions)
Reversed Phase Silica (RP)
Acidic Alumina (NP)
Reversed Phase Silica (RP) Reversed Phase Silica (RP)
SAX (NP)
Cyano silica (RP) Cyano Silica (RP)
Cyano (NP)

Metal Scavenger Silica (NP) Neutral alumina (NP) Normal Phase Silica (NP) Normal Phase Silica (NP)
Florisil (NP) Amine Silica (NP/RP) Diol Silica (NP)
Cyano (NP) Basic Alumina (NP) Cyano Silica (NP)
SCX (NP) Neutral Alumina (NP)
Neutral alumina (NP) Reversed Phase Silica (RP)
Cyano silica (NP)
 PURIFICATION BY ION EXCHANGE
& ADSORBANT RESINS

+
Prep. HPLC
+ A A
M + D D
O R S S
L E O O
A S R R
I
Molasses S
S N
B
A
B
A
E N N
S T T Pure compounds
D
I
L

Flash chromatography
Seed extract
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.

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.
q

„ Liquid injection directly onto

column
„ Equilibration is skipped and
sample is run through dry
column (for speed).
 STRUCTURE ELUCIDATION

Quantification, HPLC, GC
Purity, HPLC, GC, TLC
 ANALYTICAL TECHNIQUES

Thin Layer Chromatography (TLC)

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

„ Sample is spotted onto TLC plate using a

glass capillary

„ Plate is “developed”
p using
g a solvent
system

31
 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

NMR is the most

powerful tool available
p
for organic structure
determination.

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

Jaiprakash
p et al.,, Food Chemistry,
y, 2009,, 114,, 1351-1358.Jayaprakasha,
y p , G.K.,, Bioorganic
g 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.
 NUCLEAR SPIN
‰ Nucleus with an odd atomic number or an
odd
dd mass number
b hhas a nuclear
l spin
i
‰ The spinning charged nucleus generates a
magnetic
ti field
fi ld
‰When placed in an external field, spinning
protons act like bar magnets
 CHEMICAL SHIFT
„ Dependence of nuclear magnetic energy
levels on the electronic environment in a
molecule
„ Measured in pparts p
per million
„ Called the delta scale
 NMR SIGNALS
¾ Number of signals - different kinds of protons
¾Location how shielded or deshielded the protons
¾Intensity - number of protons of that type

3.6 3.4 0
 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
i larger
l shift
hift values.
l
„ Effect decreases with
distance.
distance
„ Additional electronegative
atoms cause increase in
chemical shift.
TYPICAL VALUES

Chapter 13
 1D & 2D NMR SPECTROSCOPY
9 INEPT (Insensitive
I iti Nuclei
N l i Enhancement
E h t by
b Polarization
P l i ti Transfer
T f )

9 DEPT (Distortionless Enhancement by Polarization Transfer)

9 SEFT (Spin-echo Fourier Transform)

) Carbon status – primary, secondary, tertiary, quaternary

9 COSY – Homonuclear correlation spectroscopy

)Proton – proton correlations

9HSQC - Heteronuclear single quantum coherence

9HMQC - Heteronuclear Multiple Quantum Coherence
)Carbon and Proton direct attachments

9HMBC – Heteronuclear Multiple Bond Correlation

)Carbon and Proton on adjacent carbon (2 or 3) attachments

9TOCSY (or) HOHAHA - Total Correlation Spectroscopy

)Carbon and Proton on neighboring carbon through hetero atom.
 SUMMARY / CONCLUSIONS

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

Flash chromatography is rapid,

rapid and reproducible

TLC is more actuate for the confirmation of the purity,

f
fast rapid,
id reliable
li bl

HPLC - g
good tool for the q
quantification

2D NMR will help for the accurate assignments of

NMR signals
 OUT LINE
„ Introduction

„ Challenges & Methods for the isolation of

bioactive compounds
p (BAC)
( )

„ Purification & Identification methods

„ CASE STUDY: Coumarins Purification &

identification
 OBJECTIVE

™ Extraction of bioactive compounds from

limes
li

™ Purification of coumarins by flash

chromatography

™ Identification of isolated compounds by NMR

andd mass spectral
t l analysis
l i
 EXTRACTION OF COUMARINS

Whole limes, juiced & freeze dried

Extraction with chloroform

Extract Spent

Concentrated under vacuum

 FLASH INSTRUMENT
CONDITIONS/CHROMATOGRAM
FLASH FRACTIONS & COMPOUNDS

Compound 1

Compound 2

Compound 3
 HPLC ANALYSIS
A). 3mM Phosphoric acid
B). ACN
λmax.- 254 nm, 0.5ml/min Compound 1

Time 3mM Acetonitri

Compound 2
(min) Phosphor le (%)
ic acid
(%)
0 45 55
20 10 90 Compound 3

25 25 75
30 25 75
HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE (HMQC)
SPECTRA OF COMPOUND 2 (DIMETHOXYCOUMARIN)

A
Aromatic
ti region
i
Aliphatic region
MULTIPLE-BOND CH CORRELATION (HMBC) SPECTRA OF
DIMETHOXYCOUMARIN
1H NMR

13C
NMR
 HETERONUCLEAR MULTIPLE-QUANTUM
COHERENCE (HMQC) SPECTRA OF COMPOUND 3
(ISOIMPINELLIN)
Aromatic region Aliphatic region
HETERONUCLEAR MULTIPLE-QUANTUM COHERENCE
(HMBC) SPECTRA OF ISOIMPINELLIN
1H NMR

13C
NMR
 IDENTIFICATION EI/MS

Jaiprakash
p et al.,, Food Chemistry,
y, 2009,, 114,, 1351-1358.Jayaprakasha,
y p , G.K.,, Bioorganic
g 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
MS 5,7 dimethoxycoumarin

JRP-SAMPLE,S3_090122140209 #1 RT: 0.04 AV: 1 NL: 7.18E7

T: + c Full ms [50.00-600.00]
206.08
100

90 178.09

80

70
Relative Abundancce

60
163.06

50 Exact mass -206.08

40
C11H10O4
135.03
30
MW-206.19
20
69.01
92.04
10

217.15 281.26 335.12 373.44 429.28 501.35 533.88

0
100 200 300 400 500 600
m/z
 MS-TOF analysis of Isopimpinellin

JRP-SAMPLE,S4 #129 RT: 0.56 AV: 1 NL: 2.20E8

T: + c Full ms [50.00-600.00]
246 07
246.07
100 231.04
OCH3
90 O O O

80

H3CO
70
ative Abundance

60

50
Rela

C13H10O5 – 246.2
40

30
175.06

20 188.03
69.05 160.03
81.08
10

256.28 341.38 386.40 460.47 494.64 536.48

0
100 200 300 400 500 600
m/z
 CRITICAL APPROACHES FOR
THE ISOLATION & IDETIFICATION
™Identifying the bioactive compounds (BAC) of our
interest

d t di
™Understanding
™U the
th interaction
i t ti off BAC with
ith sample
l
matrix

™ Stability of the BAC

™ Optimization of extraction solvents

™ Selection of purification methods, adsorbent, elution

solvents
l t

™ Nature of compounds, MW, volatality, polarity of BAC

 ACKNOWLEDGEMENTS

USDA-CSREES

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

ANY