Simeon D. Stoyanov Unilever R&D Vlaardingen, The Netherlands Laboratory of Physical Chemistry and Colloid Science, Wageningen University, NL Department of Mechanical Engineering, University College London, UK Why Saponins !?! • Natural surfactants found in more than 500 plant species (can reach 10-15 % of the dry mass) -> natural /sustainable sourcing feasible • Molecules consist of hydrophobic part (triterpenoid or steroid aglycone) and hydrophilic oligosaccharide chains (1÷3). Aglycone Quillaja Saponin: • Triterpenoid aglycone. • 2 sugar chains (2÷5 residues). • One of oldest detergents (x00 years of usage in America/Asia) • In1890 saponins were used by Lord Rayleigh “solve” dispute between Maragoni & Plateau on the existence of surface viscosity Saponin stabilized emulsions QD Escin BSC TS Introduction / Why Saponis are relevant • Natural surfactants found in more than 500 plant species (can reach 10-15 % of the dry mass) -> natural /sustainable sourcing feasible • Molecules consist of hydrophobic part (triterpenoid or steroid aglycone) and hydrophilic oligosaccharide chains (1÷3). Aglycone Quillaja Saponin: • Triterpenoid aglycone. • 2 sugar chains (2÷5 residues). • One of oldest detergents (x00 years of usage in America/Asia) • In1890 saponins by Lord Rayleigh to clarify the origin of interfacial viscosity and address the dispute between Maragoni & Plateau • Have a variety of positive bio-effects: anti-viral, anti-microbial, anti- inflammatory, cholesterol-lowering, anti-cancer, adjuvant etc. So let use saponins and 1. Characterize the surface shear & dilatational behavior of adsorption layers of saponins from various sources and structures 2. Establish structure ↔ functionality relation. Quillaja (2 sugar chains) Escin (1 sugar chains) Studied saponins Horse Chestnut Sapindus Mukurossi Camellia Oleifera (Aesculus hippocastanum) Abel Panax Ginseng Quillaja Saponaria Molina Acacia concinna Sapindus Trifoliatus Tribulus Terrestris Yucca Schidigera Trigonella foenum graecum Licorice Mono/Bidesmosidics Sapindus Mukurossi (BSC Camellia Oleifera Abel (TS) Horse Chestnut (HC/ES) Panax Ginseng (Aesculus hippocastanum) (Berry Saponin) (Tea seed saponin) (GS) Quillaja Triterpenoid Saponaria aglycone Molina (QD) Oligosaccharide chains Monodesmosidic Bidesmosidic saponins saponins Purity of Studied saponins How we can make science out of the mess ? Saponins in Type of aglycone Trade Name Abbreviation extract % Horse chestnut extract HC 20 Escin ES 95 Tea Saponin TS 96.2 Berry Saponin BSC 53 Concentrate Triterpenoid Sapindin SAP 50 Quillaja Dry 100 QD 26 Ginsenosides GS 80 Ayurvedic Saponin ASC 30 Concentrate Tribulus terrestris TT 45 extract Steroid Foamation Dry 50 FD 9 Fenusterols FEN 50 Surface Tension vs Bulk Pressure Surface tension – Energy per unit area or Force per unit length Pressure - Energy per unit volume or Force per unit area Surface tension vs bulk pressure: Equivalent bulk modulus: Peq= F/ (L )~ / E= F/ (L)~Es/ E~Es/~ 1 mN/m / 1 nm~10 atm E~Es/~30 mN/m / 1 nm~300 atm =F/L Es=F/L E~Es/~ 1000 mN/m / 1 nm~1 GPa Surface tension isotherms Gibbs adsorption isotherm d d sb d kT dln( C ) 80 Escin d Gibbs pH = natural e kT 70 d ln C d2 kT d CMC dln( c )2 c dc 60 Volmer adsorption isotherm 50 e 0 kT Surface tension, mN/m tension, Surface 40 10-4 10-3 10-2 10-1 100 101 KC exp Escin concentration, mM Van der Waals adsorption isotherm U r 2 2 k T KC exp πk T 1 e kBT rdr π Urrdr 0 B kT B r0 r0 Area per molecule at CMC = 0.54 nm2 70 Tea Saponin Mw = 1000 g/mol 60 Volmer isotherm K = 1088 m3/mol 2 inf = 3.9 mol/m 50 2 CMC = 3.1 mol/m 40 Surface tension, mN/m tension, Surface 30 10-3 10-2 10-1 100 101 102 Saponin concentration, mM Surface tension isotherms for ASC, BSC and Sapindin 65 70 ASC Berry saponin 60 65 55 60 50 55 45 50 40 45 Surface tension, mN/m 35 Surface tension, mN/m 40 30 35 10-5 10-4 10-3 10-2 10-1 100 101 10-5 10-4 10-3 10-2 10-1 100 101 Saponin concentration, wt % Saponin concentration, wt % 80 Saponidin Positive Curvature !!!! 70 For these saponins we cannot 60 determine the characteristics of 50 adsorption layer (slope of the 40 curves contradicts Gibbs isotherm) Surface tension, mN/m 30 Mixture of components and 10-4 10-3 10-2 10-1 100 Saponin concentration, wt % presence of aggregates?! Molecular packing mono vs bi desmosides A 0.4-0.5 nm2 A 0.8-1.0 nm2 Is there a link between molecular packing & surface modulus Shear Surface Rheology • Interactions between the molecules ( ), ( ) • Kinetics of adsorption / desorption Surface Shear Rheology: bicone tool Lateral view Top view M, 2 Shear stress: МR/2 1 Shear deformation: (RRRR2 1 ) / 2( 2 1 ) The Boussinesq number surface viscous drag Bo S bulk viscous dragB .L • At Bo < 200: − Surface and sub-surface flows coupling. − Numerical procedure needed to calculate the surface viscosity. • Surfactant layer regarded as a 2D body at Bo > 200. Oscillatory Amplitude sweep (tA = 30 min) GS GS 3 10 ESC 103 ESC TS TS BSC BSC QD QD 102 102 101 101 Elastic modulus, mN/m modulus, Elastic Viscous modulus, mN/m Viscous 100 100 10-2 10-1 100 101 10-2 10-1 100 101 Strain amplitude, % Strain amplitude, % The bidesmosidic saponins have lower elastic and viscous modulus. Creep Relaxation Experiments (1) Deformation at constant torque (1 N.m). (2) Strain relaxation. Visco-elastic (some of triterp/mono) Viscous (main steroidal) 0.35 160x103 Group EV Group LV FD 3 0.30 BSC 140x10 FEN 120x103 0.25 100x103 ASC 0.20 80x103 0.15 Strain, % Strain, 3 Strain, % Strain, 60x10 QD TT 0.10 TS 40x103 0.05 20x103 ESC GS LIC 0.00 0 0 50 100 150 200 0 50 100 150 200 Time, s Time, s Rheological model (compound Voigt) G1 G2 G0 = 1/J0 0 1 2 120 0.5 wt % QD 100 10 mM NaCl • Quillaja saponin. 0.1 g/L NaN J 3 • Maxwell + Kelvin (1) + Kelvin (2). 80 0 • 6 parameters (3 viscous and 3 elastic). 60 = 0.94 mN/m 40 • 2 relaxation times. Compliance, m/N 20 t / J0 CR 0 0 0 20 40 60 80 100 120 Time, sec Molecular interpretation • Molecules aggregated in domains. • Burger Element: [Maxwell + 1st Kelvin element] – deformation and re-arrangement of domains. • 2nd Kelvin Element – re-arrangement of molecules within the domains. Viscoelasticity of triterpenoid saponins • Highly elastic surface layer, G`>>G``. • G` increases for more than 12 hours of aging of the layer. • G`` decreases or stays constant. • Saponins with one sugar chain exhibit much higher elasticity. G` ~ 1000 mN/m G` ~ 100 mN/m Oscillatory dilatational deformations Langmuir trough 302LL/D1 5 % deformation -1 135 (Nima Technology Ltd, UK) 0.016 s-1 0.024 s 130 2 125 120 115 50 40 Surface area, mm area, Surface 30 Surface pressure, mN/m pressure, Surface 20 180 190 200 210 220 230 240 250 Time, sec ( Deformation created by the moving barrier(s) in Petkov the LM is uniaxial, i.e., it is a superposition of well defined dilatation and shear et al. 2000) 1 || K s s 2 K s s K – dilatational elasticity α=ln(A/A0) - relative dilatation – shear elasticity S – dilatational viscosity Boundary effects are neglected! S – shear viscosity Adsorbing system: 0.5 wt % saponins + 10 mM NaCl Oscillatory experiments done after equilibration has been reached (>30 min) Expansion Parallel plate 2000 0.5 wt % ES parallel || 1500 K s s 1000 From the best fit 500 perpendicular 0 K+ = 204 mN/m -500 S+S = 163 mN.s/m -1000 -1500 Perpendicular plate -2000 -15 -10 -5 0 5 10 15 K s s For expanding Escin layer From the best fit K = 154 mN/m; = 50 mN/m; K- = 103 mN/m S-S = 91 mN.s/m s = 127 mN.s/m; s = 36 mN.s/m Analysis of experimental data from dilatation stress-relaxation experiments 14 Burger model 12 E2 10 E1 1 8 6 4 Surface stress, mN/m stress, Surface 2 2 0 0 50 100 150 200 250 300 350 Time, sec tR1 = 1/E1 11E t t 1RR 1 1 tR2 = 2/E2 1 E1 tRRRRRR1 E 2 t 2 t 2 t 1 t 2 t 2 From the best fit of deformation and relaxation stages we determine E1, E2, tR1 and tR2 Surface rheological properties, as determined by oscillating drop method ASC 40 28 2 mN/m Storage modulus 30 20 11 1 mN/m Loss modulus 10 Surface moduli, mN/m moduli, Surface T = 10 s 0 0 1 2 3 4 5 6 Deformation, % From this experiment we determine the surface dilatational moduli, as functions of surface deformation. Expansion and contraction of large pendant drop QS initial state contraction final state equilibrium contraction expansion final state – very state after 100 s close to the initial one The formation and destruction of elastic membrane is reversible! Isotropic vs. Anisotropic Interfaces Fluid Interfaces: - Zero surface shear elasticity; - Isotropic: Single surface tension, , which is the same along the “meridians” and “parallels”; - Uniform: The surface tension is the same in all points of the interface; - Method: DSA based on fit of meniscus profile by Laplace equation; and p – adjustable parameters. Solid Interfaces (Membranes): - Nonzero surface shear elasticity; - Anisotropic: Two different surface tensions, s and φ , along the “meridians” and “parallels”; - Noniform: The surface tensions s and φ vary from point to point throughout the interface; - Method: CMD (capillary meniscus dynamometry) based on fit of data for meniscus profile and p. Force Balances per Unit Area of a Curved Interface Balance of linear momentum per unit surface area: s σ psn ps – pressure difference across the interface; n – running unit normal to the surface; n Surface stress s 0 (tension) tensor σ 0 (axial symmetry) s const.