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: Li.Zhang@akzonobel.com

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.

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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