2nd International Conference and Expo on
Separation Techniques
Development of a liquid-liquid extraction method
of resveratrol from cell culture media using
solubility parameters
Mohamad Houssam AL BALKHI
September 26 - 28, 2016
Valencia, Spain
Introduction
 stilbene: plant secondary metabolites
Resveratrol, a well-known polyphenolic compound which possesses
potent antioxidant and antiaging activities as well as a protective action
on skin
 Chemical synthesis is not profitable
 Extraction from : vine stems and rhizomes of Japanese knotweed
 their extraction remains difficult.
 Therefore grapevine cell culture systems (hairy roots)
could represent interesting systems for the large-scale
bioproduction of those compounds
Resveratrol
Plant Cell and tissue Culture
Hairy root culture, also
called transformed root culture
Secretion
Hairy roots
Bio-production
Culture medium
Ethanol
Methanol
Ethyl acetate
Ethyl acetate
SLE
LLE Chloroform
Resveratrol
 These active compounds are produced by these culture systems and are sometimes excreted in their culture
media
SLE & LLE
Destroying plant cells
Hairy roots
Secretion
Natural
Artificial
Bio-production
Culture medium
Energy
High volume of solvents
Liquid-liquid Extraction 1:1 (v/v)
Time
Instrumentations et manipulations
Pollution
Cost
The efficient and economical separation of resveratrol from culture media is always a critical
challenge.
Objective
Culture medium of grapevine hairy roots containing resveratrol
?!
Extraction of Resveratrol by a method:
Continuous extraction
 efficient  selective  rapid
preservation of plant cell viability
Economical
small volume of solvent
Ecological
Choice of extraction solvent
The three-dimensional solubility parameters of Hansen *
internal energy of the solvent
Solubility
d
internal energy of the solute
Theory of Hildebrand (1916)
Forces of dispersion
(London)
(Apolar molecules

polar
h
p
Forces of hydrogen bonding
The situation in the space of the two
substances is close
Solubility parameters of Hansen
 t      
2
d
2
p
solubility parameters are close
[*] C. Hansen, Hansen Solubility Parameters: A User’s Handbook, CRC Press, Boca Raton, FL, USA. (2007).

2 0.5
h
Physico-chemical proprieties of extraction solvent
(Small volume of solvent)
 Affinity
 Insolubility in water
 Density ˃ 1
 High viscosity
 Low boiling or high melting point
 Toxicity and volatility
Hansen solubility parameters (HSPs)
calculated using the combined group contribution methods of Van Krevelen–Hoftyzer* and Fedors**
d 
 Fdi
 F2 

pi 
i


p 
 Vi
i
 Vi
i
0 .5
  Eh
i

h   i
  Vi
 i
i
 t      
2
d
2
p

2 0.5
h
δD
δp
δH
δt
MPa1/2 MPa1/2 MPa1/2 MPa1/2
Resveratrol
23,1
6,1
20,5
31,5
 solubility parameters of Hansen for Resveratrol
 solubility parameters of Hansen for 270 solvents
* Van Krevelen and oftyzer, 1976
** Fedors, 1974





0 .5
Solubility parameters of Hansen
(HSPs)
δD
MPa1/2
δp
MPa1/2
δH
MPa1/2
δt
MPa1/2
Resveratrol
23,1
6,1
20,5
31,5
Methanol
14,7
12,3
Benzyle benzoate
15,8
20
8,8
5,1
19,4
5,2
Ethyl acetate
15,8
5,3
7,2
18,2
Water
12,2
22,8
40,8
48
Ethanol
22,3
29,4
26.5
21,3
Choice of the extraction solvent
Physical and chemical characteristics
C14H12O2
C14H12O3
228,2433
212,2439
Resveratrol
Methanol
CH4O
32,0419
Benzyl benzoate
Ethanol
C2H6O
46,0684
Ethyl acetate
C4H8O2
88,1051
Choice of the extraction solvent
Physical and chemical characteristics
Benzyl
benzoate
Ethyl acetate
Ethanol
Methnol
Miscibility
in water
immiscible
immiscible
miscible
miscible
Density
(g.cm-3)
1,1
0,9245
0,789
0,7910
0.998
Viscosity
(mPa.s)
8,29
0,421
1,20
0,5513
1,002
Boiling
Point
324 °C
77 °C
79 °C
65 °C
100 °C
Melting
point
21 °C
−83,6 °C
−117 °C
−98 °C
0 °C
254 °C
LogP
3,97
0,73
-0,32
-0,82/-0,66
-1
3.1
H2O
Resveratrol
~0,03-0.05
mg·ml-1
Extraction protocol
Solvent A
Solvent B
1st LLE
2d LLE
Evaporation
Dry extract
1 ml/100 mL
1:100 v/v
Resveratrol-containing
medium
Solvent A
Containing resveratrol
1 ml/1 mL
1:1 v/v
Solvent B
Containing resveratrol
Choice of extraction solvent
Partition coefficient *calculations using solubility parameters
𝑙𝑛𝐾𝑆𝑊 =
𝑙𝑛𝐾𝑆𝐵 =
𝑅
𝑉𝑚
𝑅𝑇
𝑅
𝑉𝑚
𝑅𝑇
𝛿𝑡𝑊
𝛿𝑡𝐵
−
𝛿𝑡𝑅 2
− 𝛿𝑡𝑅 2
−
−
𝛿𝑡𝑆
𝛿𝑡𝑆
𝑅 2
− 𝛿𝑡
𝑅 2
− 𝛿𝑡
𝑊
𝑉𝑚
+ 𝑙𝑛 𝑆
𝑉𝑚
𝐵
𝑉𝑚
+ 𝑙𝑛 𝑆
𝑉𝑚
The HSPs were then used to calculate:
 the partition coefficients of resveratrol from water to different solvents (𝑙𝑛𝐾𝑆𝑊 )
 and from benzyl benzoate to other different solvents (𝑙𝑛𝐾𝑆𝐵 )
* Srebrenik and Cohen, 1976
Choice of extraction solvent
Partition coefficient of resveratrol between different solvents
𝛿𝑑 *
(𝑀𝑃𝑎0.5 )
Resveratrol
𝛿ℎ *
𝛿𝑡
𝛿𝑝 *
𝑙𝑛𝐾𝑆𝑊
(𝑀𝑃𝑎0.5 ) (𝑀𝑃𝑎0.5 ) (𝑀𝑃𝑎0.5 )
𝑙𝑛𝐾𝑆𝐵
23,1
6,1
20,5
31,5
Methanol
14.7
12.3
22.3
29.4
14.3
7.3
Ethanol
15.8
8.8
19.4
26.5
12.7
5.8
Benzyl benzoate
20.0
5.1
5.2
21.3
7.0
Ethyl acetate
15.8
5.3
7.2
18.2
3.4
-3.6
Conclusion (I)
Choice of solvent for the liquid-liquid extraction
Medium-benzyl benzoate extraction
 benzyl benzoate
Benzyl benzoate-solvent extraction
 Methanol
Liquid-liquid extraction
Extraction methods
shaking
Centrifugation
Vortexing
LLE methods
Extraction
Rapid
Extraction time
Efficient
[Resveeatrol]
Economical
Selective
Volume of BnB
[Resveratrol] / [total extract]
????
????
????
????
Medium-benzyl benzoate extraction
Extraction time
80
90
70
80
Recovery percentage %
Recovery percentage (%)
100
60
50
40
30
20
70
60
50
40
30
20
10
10
0
0
3
7
14
Time (hour)
24
72
0
5
10
15
Time (min)
20
Effect of extraction time on recovery percentage of resveratrol
25
30
Benzyl benzoate-medium ratio
100
90
Recovery percentage %
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
Solvent volume (mL)
3
3.5
4
Effect of benzyl benzoate volume on the extraction recovery of resveratrol (1 mg/100 mL) by:
● shaking method ▲ centrifugation method
Resveratrol-medium ratio
100
90
Recovery percentage %
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
Resveratrol (mg/100 mL)
3
3.5
4
Effect of resveratrol/medium ratio (w/v, mg/mL) on the recovery of resveratrol by 1 mL of
benzyl benzoate: ● shaking and ▲ centrifugation method
Effect of the nature of the culture medium
on resveratrol recovery
100
90
Recovery percentage %
80
70
60
50
40
30
20
10
0
1/2 SH
MS
Medium
B5
Resveratrol recovery under conditions of benzyl benzoate-medium ratio of 1:100 v/v and resveratrolmedium ratio of 1:100 mg/ml using:
Shaking for 24 h
Centrifugation for 20 min
Vortexing for 1 min
Resveratrol selectivity
[Resveratrol] / [Total extract]
92 %
HPLC chromatogram of : (A) the hairy roots extract-containing medium (B) the extractcontaining medium with resveratrol before extraction (C) the extract-containing medium
with resveratrol after extraction (D) resveratrol extracted in the organic phase
Choice of extraction solvent
Partition coefficient of resveratrol between different solvents
𝛿𝑑 *
(𝑀𝑃𝑎0.5 )
Resveratrol
𝛿ℎ *
𝛿𝑡
𝛿𝑝 *
𝑙𝑛𝐾𝑆𝑊
(𝑀𝑃𝑎0.5 ) (𝑀𝑃𝑎0.5 ) (𝑀𝑃𝑎0.5 )
𝑙𝑛𝐾𝑆𝐵
23,1
6,1
20,5
31,5
Methanol
14.7
12.3
22.3
29.4
14.3
7.3
Ethanol
15.8
8.8
19.4
26.5
12.7
5.8
Benzyl benzoate
20.0
5.1
5.2
21.3
7.0
Ethyl acetate
15.8
5.3
7.2
18.2
3.4
-3.6
Benzyl benzoate-solvent extraction
1:1 v/v
Miscible mixture of methanol with benzyl benzoate before freezing (A) and separation of benzyl
benzoate/methanol phases by freezing (B)
Recovery of resveratrol from benzyl benzoate by unique extraction
Methanol:
97 ± 3%,
ethanol: 86 ± 4%
Ethyl acetate 5% ± 2
Conclusion
Extraction
Rapid
Efficient
Economical
Selective
Extraction time
Unique extraction
~ 70 %
Volume of BnB
[Resveratrol] / [Total extract]
1 min, 20 min, 24 h
Multiple extraction (3X)
~ 100 %
1 ml/100 mL
1:100 v/v
92 %
Thank you for your
attention
Paramètre
de
solubilité
Description de la théorie
La méthode : principes de base
au sein des molécules
interaction de London
les trois forces de Van der Walls
Quatre types de forces de cohésion
interaction de Keesom
interaction de Debye
la force de liaison hydrogène.
Ces forces permettent à un solvant de rester à l’état liquide, en s’opposant
efficacement à l’agitation thermique.