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SUSTAINABLE ZETA FRACTIONS: ENHANCED MILDNESS WITH SUPERIOR ACTIVITIES
Michael Koganov, Li Zhang*, Artyom Duev, and Xiaowen Hou.
AkzoNobel Surface Chemistry LLC, 23 Snowden Avenue, Ossining, NY, USA, 10562
*) Corresponding author. Phone: +1 914 7627875; Fax: +1 914 7627829; email: [email protected]
IFSCC 2015 PRESENTATION
ABSTRACT
Proprietary Zeta Fraction technology produces bioactives from living plants without addition of any solvents
and with minimum energy consumption. Composition and activities of one of Camellia sinensis Zeta
Fractions (Serum Fraction) were investigated and compared with corresponding extracts obtained from the
same cultivar. Liquid chromatography (LC) with diode array detector and LC–MS (mass spectrometry) were
utilized. Bioactivities were tested using chemical, biochemical and cell culture (human epidermal
keratinocytes - HEK) assays. Serum Fraction uniquely preserved more diverse composition of constituents
than corresponding solvent extracts. Representatives of four surfactant classes were examined in HEK
cultures for ability to induce inflammatory biomarkers and cytotoxicity. Serum Fraction demonstrated potent
inhibition of surfactant-induced inflammatory biomarkers. Serum Fraction effectively mitigates key
inflammatory cytokine Interleukin-1α (IL-1α), as well as IL-6 and chemokine IL-8. Serum Fraction inhibited
all interleukins, with potency of some comparable to a pharmaceutical grade positive control. Solvent
extracts failed to show similar activities. Eco-profiles of Serum Fraction showed significantly lower
ecological impact compared to solvent extracts. Zeta Fraction technology enables effective utilization of
underexplored potential of living plants while protecting the integrity of molecular architecture existing in
living plant cells.
KEYWORDS
Zeta fraction - bioactives - surfactants - inflammation - Camellia sinensis
INTRODUCTION
Most conventional botanical extraction processes begin when plants are collected and dried. These steps
initiate oxidative stress, osmotic shock and decompartmentalization in cells, triggering unwanted catabolic
reactions. They often result in deleterious effects on biologically active complexes (BACs) and compounds,
including bioavailability, functional properties and safety. When dried plants are extracted, the resulting
compounds are limited by their affinity to particular solvents. As a result, valuable bioactive synergistic
complexes of compounds existing within living cells can be destroyed due to different solvent affinities.
Thus, desirable multi-functional activities of BACs can be diminished or lost. The energy consumption is
significant and further increased if solvent regeneration is utilized. Additionally, reproducibility of
conventional extracts is not always achieved. All these factors prompted the development of proprietary
Zeta Fraction technology, enabling more effective utilization of underexplored potential existing in living
plants and algae. In this article, one of Camellia sinensis Zeta Fractions – serum fraction is investigated to
evaluate its potential for imparting mildness to future personal care products by mitigating surfactant-
induced skin irritation and inflammation.
TECHNOLOGY
Zeta Fraction technology is based on fundamental scientific principles discovered by Kelvin [1], Van’t-Hoff
[2] and Debye [3]. Advancements in Derjaguin, Landau, Verwey, Overbeek (DLVO) theory [4], Density
Functional Theory [5] and broadband dielectric spectroscopy [6], as well as remarkable progress in the life
sciences over the last 20 years were extremely valuable for the development of the technology. Key steps
in Zeta Fraction technology include collection of living plants and algae that have maximum metabolic
activity; separation of intracellular material from fiber-enriched material; treatment of intracellular material to
engage particular organelles and BACs in specific interactions by directed alterations of the balance
between repulsive and attractive forces; and separation of organelles and BACs to different Zeta Fractions
[7 - 10].
Investigation of intracellular material obtained from selected plants of 13 different families revealed broad
variability of most of their physico-chemical characteristics as shown in Figure 1.
Figure 1. Physico-chemical parameter variability of intracellular material of selected plants
However, two characteristics: osmotic pressure and dielectric constant are almost independent from plant
source. These characteristics are used as key operating parameters in Zeta Fraction technology, leading to
uniformity and applicability for all investigated species.
Zeta Fraction technology considers intracellular material as relatively stable intracellular colloidal dispersion
comprised of continuous phase (cytoplasm and vacuole contents) and dispersed phase (suspended
organelles and their fragments). Stability of this dispersion is maintained by the sum of van der Waals
attractive and electrical double layer repulsive forces.Energy barrier resulting from the repulsive force
prevents particles of the dispersed phase from approaching each other unless they have sufficient energy
to overcome that barrier, in which case the attractive force will pull them into contact where they will
irreversibly adhere. DLVO theory describes interaction and potential energy of the particles based on their
parameters, distance from each other and characteristics of the continuous phase. Altering these
characteristics, especially the variables affecting the repulsive force, affects stability of the dispersion, as
displayed in Figure 2.
Figure 2. Interactions between particles in colloidal dispersion and effect of dielectric constant on
stability, where:
A12 Hamaker constant, J
r Radius, m
h Distance, m
kB Boltzmann constant, J/°K
T Temperature, °K
e0 Free space permittivity, F/m
ε Dielectric constant
φ Potential, V
e Electron charge, C
I Ionic strength, mol/m3
Top equation represents double-layer repulsive force (VD), displayed as thin dotted lines. Bottom equation
represents van der Waals attractive force (VW), displayed as thin dashed line. Sum of forces is displayed as
thick solid lines with teal and red colors indicating effect of different values of dielectric constant. Sum of
forces displayed in red clearly shows energy barrier preventing agglomeration of particles, while teal shows
removal of the energy barrier when dielectric constant is altered.
Dielectric constant ( ε ) depends on frequency ( ω ) of electromagnetic waves as shown by the Debye
equation [3] in Figure 3:
Figure 3. Debye equation.
Measurements of dielectric spectra of intracellular colloidal dispersions from selected plants of 13 different
families investigated for parameter variability revealed remarkable similarity (displayed as 95% confidence
interval) at all frequencies from 0.3 to 50.0 GHz as displayed in Figure 4.
Figure 4. Dependence of dielectric constant of intracellular colloidal dispersion on frequency of
electromagnetic waves.
Zeta Fraction technology explores dependence of repulsive force on frequency of electromagnetic waves,
enabling destabilization of intracellular colloidal dispersion and separation of its components to various Zeta
Fractions.
Zeta Fractions based bioactive ingredients are currently used in numerous personal care products by
multinational companies. Among these fractions is multifunctional parthenolide-free Tanacetum parthenium
(Feverfew) serum fraction [11], with functional properties and safety superior to “substantially-free from
parthenolide" feverfew extract produced by conventional methods [12]. Another example is protein-free and
pheophorbide-free multifunctional composition of Ficus indica, Trifolium pratense and Nelumbo nucifera
serum fractions [13, 14] having superior efficacy and functional properties compared to corresponding
extracts that require large volumes of solvents.
EXPERIMENTAL SECTION
Conventional green and black tea preparations were obtained as described in [15] and the serum fraction
as described in [10]. Analytical methods used were described in [15, 16]. Cell culture testing and evaluation
of samples were performed as described in [17]. IL-8 was induced by 6 µg/mL Sodium Dodecyl Sulfate
(SDS), IL-6 by 12.5 µg/mL SDS, IL-1α by 25 µg/mL SDS. Kallikrein 5 (KLK5) was measured without
induction. IL-18 was induced by pPD at 15 µg/mL. ORAC, DPPH, elastase, and trypsin tests were
performed as described in [15, 18].
Statistical analysis and IC50 calculations were performed using SigmaPlot 10.0 (Systat Software).
Life Cycle Assessment (LCA) of resource consumption and environmental impact was conducted for
comparison of Camellia sinensis product created with Zeta Fraction technology versus solvent extraction.
The cradle-to-grave analysis followed ISO 14040 and ISO 14044 standards, used EcoInvent database, and
was done using GaBi LCA software package (PE International).
RESULTS AND DISCUSSION
Effectiveness of surfactants is the result of amphiphilic structure which includes a hydrophobic hydrocarbon
“tail”, and hydrophilic “head”, which may be negatively charged (anionic surfactants) e.g. Sodium Dodecyl
Sulfate, positively charged (cationic surfactants) e.g. C12-C18 Ethoxylated Amine, lack a charge (nonionic
surfactants) e.g. Ethoxylated Alcohol, or have both positively and negatively charged groups within head
structure (amphotheric or zwitterionic surfactants) e.g. Cocamidopropyl Betaine.
Surfactants are used in personal care for emulsification, solubilization, dispersion, and viscosity adjustment;
and to provide cleansing, wetting (including altering skin feel), or foaming abilities of the finished products.
Ability of surfactants to degrade barrier function of the skin and cause irritation and inflammation is widely
known. Enhancing mildness of products by mitigation of adverse effects of surfactants on skin is important.
Effects of different concentrations of representative surfactants of all classes described above on cultured
human epidermal keratinocytes (HEK) were investigated. Lactate Dehydrogenase (LDH) was selected as
marker of cell membrane disruption associated with cytotoxicity. IL-1α and IL-8 were selected, respectively,
as primary cytokine and secondary chemokine associated with irritation and inflammation. Results are
shown in Figure 5.
Figure 5. Effect of surfactants on biomarkers of cytotoxicity and irritation / inflammation in HEK.
In this figure, “suppress” means an increased concentration of surfactant causing decreased concentration
of biomarker; “interfere” means adverse interaction with assay system. The beginning of induction or
interference/suppression is shown at the concentration first detected. Due to wide range of concentrations
used, actual beginning of the effect could lie between concentration indicated by the figure, and the next
lowest. In cases where suppression/interference appear immediately following lack of effect, it is possible
that range of concentrations effective for induction is narrower than 10-fold.
This graph illustrates complexity of the response of skin cells to surfactants of different classes, and the
significantly different patterns of induction, suppression and cytotoxicity.
Further tests were performed using Sodium Dodecyl Sulfate (SDS), a surfactant commonly used as
benchmark irritant in studies of human skin. In addition to IL-1α and IL-8, a pleiotropic inflammatory
cytokine IL-6 was also measured. Resulting data points and approximate trend lines are shown in Figure 6.
Figure 6. Effects of SDS on interleukin levels in HEK.
Irritation and inflammation are commonly viewed [19] as a “cascade” proceeding from necessary release of
IL-1α to induction of downstream cytokines and chemokines such as IL-6 and IL-8 or other signaling
molecules such as Kallikrein 5 (KLK5), a protease also important in desquamation.
The data suggest that “cascade” view might not be a comprehensive model. Even as ubiquitous a
benchmark as SDS can trigger different portions of the irritation and inflammation process without
significantly affecting release of a primary cytokine, depending on concentration. The complexity of irritation
response of skin cells to surfactants implies that signaling “network” model is a more adequate analogy
than a signaling “cascade” model. This indicates that mitigation of such a complex signaling process using
a single bioactive ingredient must affect more than one pathway, and thus requires the ingredient to be
multifunctional.
An ingredient with a greater diversity of bioactive compounds has a greater chance of being multifunctional,
i.e. addressing multiple pathways, including potential synergy – a remarkably apt expression is “united they
work, divided they fail” [20]. To assess potential multifunctional benefits, Camellia sinensis products were
prepared using both conventional process (green tea and black tea) and Zeta Fraction technology (serum
fraction). To minimize any variability associated with different cultivars and growth conditions, tea leaves
harvested from identical source at the same time were used.
Marker compounds (+)-catechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin, (−)-catechin
gallate, (−)-gallocatechin gallate, (−)-epigallocatechin gallate, (−)-epicatechin gallate, and gallic acid were
selected for analysis due to common view [20] of them as key contributors to biological activities of
Camellia sinensis preparations. Three-dimensional UV chromatograms [21] were also recorded to display
the peaks indicating complexity of composition besides the quantified marker compounds. The data are
presented in Table I.
Table I. Comparative compositions of Camellia sinensis products.
This data clearly demonstrates that serum fraction obtained from Camellia sinensis fundamentally differs
from green and black teas. It not only contains a much greater abundance of known biologically active
compounds, but also displays a far greater diversity of compounds preserved from the living tea plant. This
implies potential for greater efficacy and potency, as well as multifunctionality enhanced by possible
synergies between the bioactive compounds.
To assess a fraction of this potential, further chemical, enzymatic and cell culture assay testing was
conducted: quenching free radicals like 2,2-Diphenyl-1-Picrylhydrazyl (DPPH); protecting a fluorescent
marker from damage by continuously generated oxygen radicals (Oxygen Radical Absorbance Capacity,
ORAC); inhibiting trypsin and elastase, proteases involved in inflammatory damage to skin extracellular
matrix; and HEK-based assays of mitigation of SDS-induced irritation and inflammation signaling molecules
IL-1α, IL-6, IL-8, and of baseline production of KLK5. [15]
Single-compound pharmaceutical-grade anti-inflammatory positive controls 2-acetoxybenzoic acid (aspirin)
and SB203580 were also tested. The results are presented in Table II.
Table II. Activities of Camellia sinensis products and positive controls.
Asterisks ( * ) indicate testing in progress.
These results show high potency and efficacy of Camellia sinensis serum fraction in decreasing levels of
irritation and inflammation markers, as well as the ability to mitigate free radicals and proteases.
Conventional green and black tea prepared from same source showed no effect in HEK-based in vitro
assays, and over 30 times less potency in antioxidant and free radical assays. Furthermore,
pharmaceutical-grade single-compound controls were highly effective in addressing their specific targets
while being completely ineffective against other interleukins involved in irritation and inflammation. Notably,
aspirin, although able to mitigate IL-1α release, had no effect on downstream markers IL-6 and IL-8.
Additionally, Camellia sinensis serum fraction was exceptionally effective at mitigating SDS-induced IL-8 in
HEK. It was capable of bringing IL-8 level down to baseline, which suggests potential for normalization of
other pathways.
A simplified view of irritation and inflammation process in the skin and its possibility of self-reinforcement
are shown in Figure 7, also suggesting the ability of a novel multifunctional ingredient to address it
comprehensively as demonstrated by Camellia sinensis serum fraction.
Figure 7. Simplified diagram of irritation and inflammation processes impacted by Camellia sinensis
serum fraction
While diverse composition of an ingredient increases potential for multifunctionality and synergy, risk of
adverse effects due to uncharacterized components or unforeseen interactions is also considered greater.
Therefore, initial safety testing was done in parallel with HEK-based assays of potency and efficacy.
Camellia sinensis serum fraction showed lack of induction of skin sensitization [22] marker IL-18 in
concentrations up to 0.1%, as well as lack of cytotoxicity as measured by LDH release.
A comprehensive safety evaluation of Camellia sinensis serum fraction followed, with results presented in
Table III.
Table III. Safety evaluations results.
Since Zeta Fraction technology is not based on solvent extraction, no energy is spent on regenerating
organic solvents as may be the case in conventional processes. Furthermore, this means that all by-
products are trivially biodegradable or recyclable. Results from a cradle-to-grave Life Cycle Assessment
analysis are provided in Table IV. Same results, with data from solvent extraction process normalized to 1,
are shown in Figure 8.
Table IV. Ecological impact assessment data.
Figure 8. Comparative graph of ecological impact assessment.
This demonstrates superior sustainability and minimal ecological impact of Zeta Fraction technology and its
products.
CONCLUSION
Skin irritation and inflammation are common problems caused by surfactants in personal care products,
and ability to impart mildness is desirable. Given the complexity of irritation and inflammation processes, an
effective solution requires coordinated effect on multiple mechanisms and pathways, which is not always
achievable by a single compound. Plant-derived multifunctional bioactive ingredients may be a better
prospective solution as demonstrated by Camellia sinensis serum fraction, a product of Zeta Fraction
technology. Products of this technology demonstrate safety, efficacy and multifunctionality due to
preserving diversity of molecular architecture of living plants and algae. This technology can serve as
important tool in discovery of novel biologically active complexes and compounds that exist in living plants
and algae. Minimal environmental impact of the sustainable Zeta Fraction technology makes it superior to
technologies utilizing organic solvents, and allows its use as a volume reduction platform, for example, in
combination with advanced conventional technologies.
ACKNOLEDGEMENTS
We are grateful to Dale Steichen and Olga Dueva-Koganov for their valuable support.
REFERENCES
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Appearance of Hyperpigmented Skin Using a Synergistic Composition Comprising Banyan Tree, Lotus,
and Clover Serum Fractions.
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Appearance of Aging Skin Using a Composition Comprising Banyan Tree, Lotus, and Clover Serum
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15. Koganov, M., Zhang, L., Duev, A., Dueva-Koganov, O., Hou, X., Biological Activities of Novel
Ingredients from Living Tea Plant (Camellia sinensis), Household and Personal Care Today, 9 (2015)
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17. Koganov, M., Zhang, L., Duev, A. Imparting Mildness with Living Tea Plant Ingredient, Personal Care,
14 (2013) 31-34.
18. Koganov, M., Dueva-Koganov, O., Duev, A., Recht, P., Micceri, S., Managing the Effects of Photoaging
of Skin, Personal Care, 5 (2012) 89-92.
19. Weiss, T., Basketter, A., Schröder, K. R., In Vitro Skin Irritation: Facts and Future. State of the Art
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Figure 1. Physico-chemical parameter variability of intracellular material of selected plants
Figure 2. Interactions between particles in colloidal dispersion and effect of dielectric constant on
stability, where:
A12 Hamaker constant, J
r Radius, m
h Distance, m
kB Boltzmann constant, J/°K
T Temperature, °K
e0 Free space permittivity, F/m
ε Dielectric constant
φ Potential, V
e Electron charge, C
I Ionic strength, mol/m3
ε'
ε'Potential Energy
Interparticle Distance
Figure 3. Debye equation.
Figure 4. Dependence of dielectric constant of intracellular colloidal dispersion on frequency of
electromagnetic waves.
Figure 5. Effect of surfactants on biomarkers of cytotoxicity and irritation / inflammation in HEK.
Figure 6. Effects of SDS on interleukin levels in HEK.
Figure 7. Simplified diagram of irritation and inflammation processes impacted by Camellia sinensis
serum fraction
Figure 8. Comparative graph of ecological impact assessment.
Table I. Comparative compositions of Camellia sinensis products.
Biomarker Content, µg/mL
Serum Fraction Green Tea Black Tea
Catechin (+) 160 4 3
Gallocatechin (-) 410 3 7
Epigallocatechin (-) 4450 5 90
Epicatechin (-) 2000 30 40
Catechin Gallate (-) 53 0.2 0.2
Gallocatechin Gallate (-) 18 <0.2 <0.2
Epigallocatechin Gallate (-) 1850 20 70
Epicatechin Gallate (-) 425 20 20
Gallic Acid 265 100 3
Total Catechins 9366 82 230
LC UV DAD Chromatograms
Chromatogram Parameters
RT 0 – 15 min
Wavelength 200-420 nm
Intensity 0 - 3400
RT 0 – 15 min
Wavelength 200-420 nm
Intensity 0 - 580
RT 0 – 15 min
Wavelength 200-420 nm
Intensity 0 - 480
Table II. Activities of Camellia sinensis products and positive controls.
Serum Fraction Green Tea Black Tea Aspirin SB203580
Elastase IC50 0.08 % v/v * *
Trypsin IC50 0.30 % v/v * *
KLK5 IC50 0.21 % v/v * * *
IL-8 IC50 0.08 % v/v ineffective ineffective ineffective 0.7 µg/mL
IL-6 IC50 0.20 % v/v * * ineffective < 0.08 µg/mL
IL-1α IC50 0.26 % v/v ineffective ineffective 230 µg/mL ineffective
ORAC 153 mg/g equiv. 2.4 mg/g equiv. 1.9 mg/g equiv.
DPPH 91 mg/g equiv. 2.5 mg/g equiv. *
Table III. Safety evaluations results.
Method Results
In vitro Skin Irritation Non-irritant
In vitro Eye Irritation Non-irritant
Genotoxicity
Non-mutagenic to S.typhimurium
Dermal Sensitization
(Local Lymph Node Assay)
Not a dermal sensitizer in the LLNA
Note: LLNA study completed in 2012
Phototoxicity
(OECD 432)
No phototoxic potential
Acute Toxicity to Daphnia Magna
(OECD 202)
EC 50 and No Observed Effect Concentration (NOEC)
empirically estimated as >1000mg/L and 1000mg/L
Algal Growth Inhibition Test
(OECD 201)
No observed effect
(Tested concentration 250 mg/L)
Table IV. Ecological impact assessment data.
Ecological impact per 1000 kg of Camellia sinensis product Zeta Fraction Process Extraction with Solvents
Global warming potential, kg CO2 equiv. 550 3100
Ozone depletion potential, g CFC-11 equiv. 0.02 0.2
Acidification potential, kg SO2 equiv. 1.5 3.9
Photochemical ozone creation potential, kg ethene equiv. 0.3 1.7
Eutrophication potential, kg PO43- equiv. 0.22 0.48
Primary energy demand, MJ 11000 51000
Abiotic resource use, g Sb equiv. 0.44 1.9
Land use, m2 per year 1000 1000