Recent Applications of Enzymes in Personal Care Products

Abstract

The field of personal care products is clearly the most inventive rapidly evolving area. As the lines between cosmetics and drugs become increasingly blurred due to advanced understanding of human physiology, so do the topical effects of these nonprescription products. As all biological processes are based upon the activity of enzymes, these biocatalysts are also essential for body care. In the skin, enzymes are responsible for uncoupling complex inactive molecules and transforming them into simpler and often more active molecules. Proteases, for example, decouple or hydrolyze proteins, glycosidases promote the enrichment of the epidermis with ceramides, and tyrosinase facilitates the synthesis of melanin. Other classes of enzymes, such as lignin peroxidase have been found to play an important role in breakdown of eumelanin, which has led to the development of products related to skin lightening. On the other hand, lipases due to their outstanding ability of metabolizing lipids have found their way into formulations involving products for nose cleansing, makeup beauty masks, and hair care. Hyaluronidases, another group of enzymes, have been most successful in cosmetic surgeries as an active component in dermal fillers. Enzymes obviously make promising natural active agents for specified personal care products belonging to the so-called “functional cosmetics,” “cosmeceuticals,o or “treatment products.” The global industrial enzymes market is very competitive, with the companies mainly competing on the basis of product quality, performance, use of intellectual property rights, and the ability to innovate; however, some limiting factors like effectiveness of enzyme formulation, application mode, and carrier vehicles must be addressed. The research in the area of microencapsulation using nanotechnology has huge potential to give enzyme-based formulations advantageous traits resulting in superior products. In this chapter, a brief account of the most efficient enzymes in enhancing the quality of personal care products has been described with special reference to a few available commercial products. The future of nanoemulsions in enhancing enzyme activities in personal care products is also discussed.

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From Sunar, K., Kumar, U., Deshmukh, S., 2016. Recent Applications of Enzymes in Personal
Care Products. In: Dhillon, G.Singh, Kaur, S. (Eds.), Agro-Industrial Wastes as Feedstock
for Enzyme Production: Apply and Exploit the Emerging and Valuable Use Options of Waste
Biomass. Academic Press, 279–298.
ISBN: 9780128023921
Copyright © 2016 Elsevier Inc. All rights reserved.
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Agro-Industrial Wastes as Feedstock for Enzyme Production, First Edition, 2016, 279-298
CHAPTER 12
Recent Applications of Enzymes
in Personal Care Products
K. Sunar, U. Kumar, S.K. Deshmukh
The Energy and Resources Institute, New Delhi, India
INTRODUCTION
Enzymes, unique protein molecules that catalyze most of the reactions in living organ-
isms, are rightly termed as catalytic machinery of the living system. Current applications
of enzymes are focused on many different markets including pulp and paper, leather,
detergents, textiles, pharmaceuticals, chemicals, food and beverages, biofuels, animal
feeds, and personal care, among others (Adrio and Demain, 2014). At the same time, the
end use market for industrial enzymes is extremely widespread with numerous industrial
commercial applications (Adrio and Demain, 2005). Over 500 industrial products are
being made using enzymes (Johannes and Zhao, 2006; Kumar and Singh, 2013). It was
in 1833 that scientists discovered that a thermolabile substance was able to convert starch
into sugar and called it “diastase,” which is now known as “amylase.” Later, in 1926 the
protein nature of enzymes were finally confirmed when Summer (1926) successfully
crystallized urease enzymes from Jack beans. Then an era of utilizing cell-free enzymes
started with renin, an aspartic protease in cheese making. In the last 20 years, the global
beauty market has grown by 4.5% a year on average (CAGR), with annual growth rates
ranging from around 3–5.5%, also known as cosmetics and toiletries or personal care
products (PCPs) (Barbalova, 2011). The world’s cosmetic industry is worth tens of bil-
lions of US dollars, and the industry is constantly seeking new products with ingredients
that have specific actions for which enzymes have been the most preferred choice for
enhancement of PCPs. The first commercial enzyme was prepared by Rohm in
Germany in 1914. The trypsin enzyme was isolated from animals; it degraded proteins
and was used in detergents. The larger-scale commercialization of enzymes started with
microbial protease derived from Bacillus, which was used in washing powders. In 1959,
Novozyme of Denmark made it a huge business when they started manufacturing deter-
gents with these microbial enzymes. In addition to the cheese industry, enzymes had
been used in various fields, such as food industries and manufacturing of fruit juice, since
1930, however, a major breakthrough started around the 1960s when enzymes were
industrially manufactured and were used in the starch industry. The traditional acid
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hydrolysis of starch was completely replaced by alpha amylases and glucoamylases that
could entirely convert starch into glucose. The starch industry became the second largest
industry to use enzymes after the detergent industry. Over the years, biotechnology has
shown that it is now possible to utilize and harness the use of these enzymes in diverse
fields. Enzymes have recently been started to be used by cosmetic scientists in developing
PCPs for wide acceptability as they have been found to have good consumer appeal and
improved performance. However, they have always been poorly evaluated for their func-
tionality in cosmetic science. Proteolytic enzymes like bromelain, papain, etc. have been
used in PCPs for skin peeling and smoothing for many years, however, the general prob-
lem associated with such use is the irritation caused by some enzymes on the skin sur-
faces due to their proteolytic activities. The area where the topical applications of
enzymes are widely explored and have shown significant benefits is in skin protection,
with enzymes having excellent stability. The enzymes used for skin protection have pro-
found abilities to capture free radicals caused by environmental pollution, microorgan-
isms, sunlight, radiations etc. The recent trend of application of enzymes in PCPs shows
ample variability in terms of enzymes used from different types of classes for their spe-
cific roles and function. Studies of enzyme formulations suitable for topical use have also
shown that such dosage forms are relatively easy to handle. However, the choice of base,
surface active agent, etc., is important to provide for a stable formulation, and proper
vehicle selection is also critical for the proper activity. Another futuristic approach to
cosmetics and skin care product development is to increase the efficacy of existing ingre-
dients that might improve skin functioning. Many new topical ingredients—from mush-
rooms to salmon caviar to sea urchin spines to green algae to knotweed—have been
placed in complex antiaging formulations (Draelos, 2012). Nanoparticles are revolution-
izing many areas of chemistry, physics, and possibly cosmetic formulation. The long-
term effects of nanoparticles in the oceans of the world are not currently known. Yet,
nanoparticles could be the next frontier in cosmetic dermatology (Sonneville-Aubrun
et al., 2004). Nanoparticles have great potential to create topical cosmeceutical formula-
tions that behave in ways that enable better penetration of active skin ingredients. Some-
day in the not-too-distant future we may be using nanoparticle therapy, nanoemulsions,
polymeric nanoparticle spheres, and nanoliposomes to improve the appearance of the
skin (Tadros et al., 2004). Nanotechnology may allow ingredients to exhibit new skin
effects, improving cosmetics and skin care product efficacy. We will discuss a few of the
specific and widely used enzymes, with the main focus on the nature of their activity
(Table 12.1).
Superoxide Dismutase
Superoxide dismutase (SOD) belongs to the oxidoreductase class of enzymes and cata-
lyzes oxidation and reduction reactions. SOD is one of the most effective and popular
tropical enzymes so far being used in skin care products. After its discovery as a blue/
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green protein in 1938 by Mann and Leilin and its subsequent characterization as an
enzyme and named as superoxide dismutase by McCord and Fridovitch in 1969, this
enzyme has been frequently used in various fields for its effective role in catalyzing
superoxide free radicals. Reactive oxygen species (ROS) are produced by cells during
normal metabolic activities such as mitochondrial oxidative phosphorylation; however,
levels of ROS vary with UV exposure and levels of antioxidant enzymes. Without inac-
tivation, ROS damages macromolecules including lipid, proteins, and DNA (Zastrow
et al., 2009). Numerous studies have tested the effects of solar radiation and oxidative
stress on the skin (Lan et al., 2013), and oxidative stress has been linked to age-related
loss of skin elasticity (Nylor et al., 2011), defective cellular signaling, and photoaging
(Lee et al., 2012). Antioxidant enzymes mediate the removal of ROS, with different
enzymes functioning in specific compartments thereby preventing ROS from reacting
with DNA and other cell signal proteins, impairing their function (Fig. 12.1). Function-
ally, SODs are characterized as potential oxidizing and reducing agents, and many studies
have demonstrated their applications in cosmetics and PCPs for younger looking skin.
L’Oréal, a cosmeceutical company, was the first to obtain a European Union (EU) patent
for this enzyme for its general use in cosmetics in the year 1973 (EU patent no. 2 287
889) and ever since the SOD from marine sources has been in use in developing PCPs.
The main factor involved in utilizing enzymes as an active component of any PCP is its
side effects and acceptability; many of the SODs derived from different sources were not
effective or not considered suitable in the early days because they were reported to cause
skin irritation. In 1987 Brooks Industries developed CuZn-SOD derived from yeast,
which was formulated as a powder (Biocell SOD-Yeast CuZn-SOD) containing approx-
imately 600 IU SOD activity. This form of yeast protein-bonded SOD is known to have
excellent stability at 45°C in aqueous solution in comparison to pure forms of SOD.
Table 12.1 Use of Enzymatic Activities in Cosmetic Products (Ugo Citernesi and Kathe Andersen;
www.iralab.it/download/pubblicazioni/New_trends_in_drug.pdf)
Enzyme Source Cosmetic Use
Protease Fungi Peeling/antiaging/
antiwrinkle
Lipases Bacteria Anticellulitis
Hyaluronidase Bacteria Moisturising agent
Tyrosinase Yeast (recombinant) Tanning agent
Tenebrio molitor
Fungi
Superoxide dismutase Yeast (Recombinant) Antifree radicals
Peroxidase Horseradish, bacteria and
yeast (recombinant)
Antifree radicals
Alkaline Yeast and fungi Antiwrinkle
Phosphatase (recombinant) Energetic
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This yeast SOD was also found to be nonirritating and nonsensitizing both in powder
form and as an active 1% yeast CuZn-SOD liposome. Studies have also shown that these
yeast-derived SODs are excellent antioxidants much better than commonly used anti-
oxidants, like tocopherol and polyphenols. A few of the examples of products containing
SOD as an active ingredient are presented in Table 12.2.
Peroxidase
There are two different types of hydroxyl free radical–scavenging enzymes, known as
peroxidase and catalase belonging to the oxidoreductase class of enzymes. Plants are
known to have heme-containing peroxidases, which are nonspecific peroxidases and
are capable of acting on a variety of substrates including hydrogen peroxide. Similar
nonspecific enzymes in animals are lactoperoxidase (thiocyanate ion oxidation), myelo-
peroxidase (phagocytosis), and thyroid peroxidase (iodine ion oxidation). However, the
Figure 12.1 A schematic representation of dierent antioxidant enzyme functioning, in specic cell
compartments for removal of reactive oxygen species. ECSOD, extracellular superoxide dismutase; Cu/
Zn SOD, copper/zinc superoxide dismutase; MnSOD, manganese superoxide dismutase. Reproduced
with input from Amaro-Ortiz, A., Betty Yan, B., D’Orazio, J.A., 2014. Ultraviolet radiation, aging and the skin:
prevention of damage by topical cAMP manipulation. Molecules 19 (5), 6202–6219.
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most studied one is the horseradish peroxidase obtained from the roots of horseradish.
These free radical–scavenging enzymes are been extensively used in PCPs. For instance,
fennel seed extracts containing peroxidase are being used in cosmetics because of their
high-lipid peroxidation activities and low odor. The pale yellow/green liquid extract
is also shown to have nonirritating and nonsensitizing activity and has shown much
better protection activity than tocopherol. Lignin peroxidase, a novel skin-lightening
active agent derived from a fungus is being studied with some interest for developing
as an ingredient in products to treat pigmentation disorders. Some pigmentation dis-
orders resulting from excessive sun exposure leading to solar lentigo are notoriously
Table 12.2 Commercial Products Containing Superoxide Dismutase as an Active Ingredient and Their
Use (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
Dismutin BT Skin care:antiaging,
antiinflammatory
DSN Nutritional Products,
LLC
Dismutin BT 5000 Skin care- antiaging and
antioxidant
DSN Nutritional Products,
LLC
Liposystem complex Skin care- IRA Istituto Ricerche
Applicate
Antioxidant
Moisturizer
Zymo Radical Skin care- IRA Istituto Ricerche
Applicate
Smoothing
Moistening
Antiwrinkling
Zymo Radical MD Skin care- IRA Istituto Ricerche
Applicate
Antiwrinkling
Moistening
Smoothing
Detox Duo Skin care MD Skincare
Protection
Antioxidant
Arch-Biocell SOD Body lotion –Skin care
antiinflammation
LONZA
Protective
Antioxidant
Soothing agent
Brookosome SOD Body lotion –Skin care
antioxidant
LONZA
Chronosphere SOD Body Lotion-skin care LONZA
Antioxidant
ProCircul8 Skin care LONZA
Protective
Antioxidant
Antiinflammatory
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difficult to treat. Melanin is a very durable compound, and researchers have been
largely unsuccessful in finding ways to break down melanin to reduce unwanted skin
pigment. The existing topical treatments for skin lightening focus on the prevention
of melanin formation by blocking tyrosinase and inhibiting its biosynthesis; by pre-
venting the stimulation of melanocytes by UVA; or by blocking the transfer of mela-
nosomes to keratinocytes via the PAR-2 receptor. The enzyme lignin peroxidase (LIP)
was first identified by Gold et al. in 1984 and has been researched for many years as a
potential agent to break down lignin to whiten wood pulp in paper production (Fig.
12.2). It was later found to break down eumelanin, which has a chemical structure
similar to lignin. The development of lignin peroxidase as a skin-lightening agent
resulted from these discoveries (US Patent and Trademark Office Patent Application
20060051305). This novel skin-lightening active ingredient is produced extracellularly
during submerged fermentation of the fungus Phanerochaete chrysosporium (Woo et al.,
2004) and then purified from the fermented liquid medium (Lonza of Switzerland).
The LIP enzyme (trademarked as Melanozyme) identifies eumelanin in the epidermis
and specifically breaks down the pigment without affecting melanin biosynthesis or
blocking tyrosinase. Although there are other types of lignin peroxidase enzymes, at
Figure 12.2 The crystal structure of lignin peroxidase at 1.70 Å resolution obtained from Protein Data
Base and catalytic cycle of lignin peroxidase. Adapted with permission from Gold, M.H., Wariishi, H., Valli,
K., 1989. Extracellular peroxidases involved in lignin degradation by the white rot basidiomycete Phanero-
chaete chrysosporium. In: Whitaker, J., Sonnet, P. (Eds.), Biocatalysis in Agricultural Biotechnology. Toronto,
Ontario, Canada: American Chemical Society, 127–140.
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this point, Melanozyme is the only one that has been developed and proved to be
effective for skin lightening. Melanozyme is a glycoprotein active at pH 2–4.5. Mela-
nozyme is currently proprietary and is available only in a new skin-lightening product
known on the market as Elure. The safety of lignin peroxidase as a skin-lightening
active ingredient has been demonstrated in preclinical studies with doses that are
17,000 times the recommended dose without prompting any side effects. LIP is non-
mutagenic and nonirritating to eyes. The potential for skin irritation is very low, and
in studies of 50 subjects each, there were no reports of skin irritation during acute
sensitivity or cumulative sensitivity, or when used in sensitized skin. A few examples of
products containing peroxidase as active ingredient are presented in Table 12.3.
Tyrosinase
Tyrosinase, an oxidase that is the rate-limiting enzyme for controlling the production
of melanins is mainly involved in two distinct reactions of melanin synthesis: (1) the
hydroxylation of a monophenol, and (2) the conversion of an o-diphenol to the cor-
responding o-quinone. o-quinone undergoes several reactions to eventually form mel-
anin (Hideya et al., 2007; Kumar et al., 2011) (Fig. 12.3). Melanin synthesis in
melanocytic cells is ultimately regulated by tyrosinase, a membrane-bound copper-
containing glycoprotein, which is the critical rate-limiting enzyme. Tyrosinase is pro-
duced only by melanocytic cells, and following its synthesis and subsequent processing
in the endoplasmic reticulum and Golgi, it is trafficked to specialized organelles,
termed melanosomes, wherein the pigment is synthesized and deposited. In the skin
and hair, the melanosomes are transferred from melanocytes to neighboring keratino-
cytes and are distributed in those tissues to produce visible color (Hideya et al., 2007) .
During the past years, the cosmetic industry increasingly worked with substances
involved in natural melanin formation. The advantages here are obvious. Unlike the
Table 12.3 A Few of the Commercial Products Containing Peroxidase as an Active Ingredient and
Their Uses (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
Liposystem complex Antioxidants IRA Istituto Ricerche
Applicate
Moisturizing agents
Nourishing agents
Zymo radical MD Antiwrinkle agents IRA Istituto Ricerche
Applicate
Moisturizing agents
Smoothening agent
Zymo radical Antiwrinkle agents IRA Istituto Ricerche
Applicate
Moisturizing agents
Smoothening agent
ABS fennel extract Antiaging Active Concepts
Antistress/Relaxing agents
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melanoidin process, a natural tan is induced and protection against UV radiation also
is provided. It is a well-known fact that the enzyme tyrosinase transforms the amino
acid tyrosine into dihydroxyphenylalanine (DOPA) and into its quinoid form, the
DOPA quinone, which is the base for the formation of both the melanin types,
eumelanin (dark brown) and pheomelanin (reddish yellow). The combination of both
the types is responsible for the skin tone, which varies from skin to skin. The tyrosinase
is controlled by UV radiation and induced by the α-melanocytes stimulating hormone
(α-MSH). Further tyrosinase stimulators are the β-endorphins. Endorphin-related
substances can be found in specific vegetable extracts as, eg, the chaste berry or chaste
tree (vitex agnus castus), and together with synthetic acetyl tyrosine, a tyrosine pro-
drug, they are able to induce the UV independent formation of melanin. Additional
UV radiation will speed up and stimulate the melanin formation process after the
product has been applied. New developments concentrate on additional tyrosinase
activators and adequate transport systems to integrate the substances into the skin
(Lautenschltens, 2007). Zymo-tan complex, a tanning activator, consists of tyrosine
amino acids (precursors of melanine) and tyrosinase. Tyrosinase is an enzyme that cata-
lyzes the reaction forming the melanin in the presence of solar radiation. The enzyme,
present in several plants, has been also isolated from leucocytes, yeast, and milk. Some
of the tyrosinase-based products are presented in Table 12.4.
Figure 12.3 Schematic overview of melanin synthetic pathway and the involvement of melanogenic
enzymes. Initial melanin synthesis is catalyzed by tyrosinase and is then divided into eumelanogenesis
or pheomelanogenesis. The other melanogenic enzymes, that is, l-3,4-dihydroxyphenylalanine
(DOPA) chrome tautomerase (DCT) and tyrosinase-related protein 1 (TYRP1), are involved in
eumelanogenesis.
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Proteases
Proteases (also known as peptidases or proteinases), their substrates and inhibitors are of
great relevance to biology, medicine, and biotechnology. Proteases are referred to as a
group of enzymes that hydrolyze the protein bonds of amino acids (proteolysis). Prote-
ases have evolved multiple times, and different classes of protease can perform the same
reaction by completely different catalytic mechanisms (Gupta and Khare, 2007; Kalpana
Devi et al., 2008). Proteases constitute the largest group of enzymes in bioindustry with
an array of applications. They play an important role in industrial biotechnology, espe-
cially in detergents, foods, pharmaceuticals, and in PCPs. Proteolytic enzyme is essential
for several physiological processes like digestion of food proteins, protein turnover, cell
division, blood clotting cascade, signal transduction, processing of polypeptide hormones,
etc. (Li et al., 2013). The vast variety of proteases, with specificity of their action and
application, have attracted worldwide attention to exploit their physiological as well as
biotechnological applications (Poldermans, 1990). They are considered eco-friendly
because the appropriate producers of these enzymes for commercial exploitation are
nontoxic and nonpathogenic and are designated as safe (Gupta et al., 2002). Proteases are
used extensively in the pharmaceutical industry for preparation of medicines, such as
ointments for debridement of wounds. They are also used in denture cleaners and as
contact lens enzyme cleaners (Ogunbiyi et al., 1986). Proteases that are used in the food
and detergent industries are prepared in bulk quantities and are used as crude prepara-
tions; whereas those used in medicine are produced in small amounts but require exten-
sive purification before application (Bholay and Patil, 2012). The thermostability and
their activity at high pH and the alleviation of pollution characteristics have made pro-
teolytic enzymes an ideal candidate for laundry applications. Alkaline proteases are sup-
plemented in different brands of detergents for use in home and commercial
establishments. Enzymes have been added to laundry detergents for the last 50 years to
facilitate the release of proteinaceous material in stains, such as those of milk and blood.
The proteinaceous dirt coagulates on the fabric in the absence of proteinases as a result
of washing conditions. The enzymes remove not only the stain, such as blood, but also
Table 12.4 A Few of the Commercial Products Containing Tyrosinase as an Active Ingredient and
Their Use (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
Hydrosoluble zymo tan
complex
Sunscreen agents, self-
tanning agents
IRA Istituto Ricerche
Applicate
Zymo tan complex PF Sunscreen agents, self-
tanning agents
IRA Istituto Ricerche
Applicate
Zymo tan complex Sunscreen agents, self-
tanning agents
IRA Istituto Ricerche
Applicate
Brookosome Sunscreen agents, self-
tanning agents
Lonza
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other materials including proteins from body secretions and food, such as milk, egg, fish,
and meat. An ideal detergent enzyme should be stable and active in the detergent solu-
tion and should have adequate temperature stability to be effective in a wide range of
washing temperatures (Aurachalam and Saritha, 2009). A few examples of products con-
taining different types of proteases as active ingredient are presented in Table 12.5.
Lipases
Lipases belong to hydrolases and exert their activity on the carboxyl ester bonds of tria-
cylglycerols and other substrates. Their natural substrates are insoluble lipid compounds
prone to aggregation in aqueous solution. Lipases are ubiquitous enzymes present in all
types of living organisms. In eukaryotes they may be confined within an organelle (ie,
the lysosome), or they can be found in the spaces outside cells and play roles in the
metabolism, absorption, and transport of lipids. In lower eukaryotes and bacteria, lipases
can be either intracellular or be secreted in order to degrade lipid substrates present in
the environment, and in some pathogenic organisms (Candida albicans, Staphylococcus and
Pseudomonas species, Helicobacter pylori) they can even act as virulence factors. Most bacte-
rial lipases are sourced from Pseudomonas, Burkholderia, Alcaligenes, Acinetobacter, Bacillus,
and Chromobacterium species; widely used fungal lipases are produced by Candida, Humi-
cola, Penicillium, Yarrowia, Mucor, Rhizopus, and Aspergillus sp. Among the lipases from
higher eukaryotes, porcine pancreatic lipase has been in use for several years as a techni-
cal enzyme (Lotti and Alberghina, 2007). Active lipases can mainly be found in cosmetics
for surficial cleansing (anticellulite treatment) or overall body slimming, where they are
responsible for the mild loosening and removal of dirt and/or small flakes of dead corne-
ous skin (ie, peeling) and/or assist in breaking down fat deposits, often in combination
with further enzymes, such as proteases. Further applications have been described for
nose cleansing, makeup beauty masks, and hair care. Based on the broad variety of com-
pounds derived from fats and carboxylic acids in cosmetic products, lipases and their
hydrolytic, esterifying, and acylating activities show enormous potential for implementa-
tion in the production of cosmetic ingredients. In fact, a multitude of possible lipase-
catalyzed syntheses have been described to date, and a variety of products have actually
been commercialized. For classification, specialty esters, aroma compounds, and func-
tional actives can essentially be distinguished (Marion et al., 2013). A few examples of
products containing lipase as active ingredient are presented in Table 12.6.
Hyaluronidase
Hyaluronidase, enzymes that catalyze the hydrolysis (chemical decomposition involving
the elements of water) of certain complex carbohydrates, such as hyaluronic acid (HA)
and chondroitin sulfates, have been found in insects, leeches, snake venom, mammalian
tissues (testis being the richest mammalian source), and in bacteria. HA has gained much
importance in cosmetics for its popularity in cosmetic facial augmentation. HA is a
naturally occurring glycosaminoglycan disaccharide present in skin, joint synovia,
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Table 12.5 A Few of the Commercial Products Containing Protease as an Active Ingredient and Their
Uses (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
Protease Antiaging agents Green Tech
Antiwrinkle agents
Nourishing agents
Prozymex HBT LS
9142
Antiaging agents Laboratories
Serobiologiques
Lightening and whitening agents
Exfoliants/peeling agents
Smoothing agents
Zymo acids Conditioning agents IRA Istituto Ricerche
Applicate
Depil enzyme Depilatory agents IRA Istituto Ricerche
Applicate
Antihair regrowth agents
Zymo hair MD Moisturizing agents IRA Istituto Ricerche
Applicate
Zymo lift MD Antiwrinkle agents IRA Istituto Ricerche
Applicate
Moisturizing agents
Okoumyrrhine Antiaging agents Naturactiva
Antiwrinkle agents
Smoothing agents
Antiinflammatory
Dub karite Antioxidants Stearinerie dubois
Antiaging agents
Antiwrinkle agents
Smoothing agents
Antiinflammatory
Moisturizing agents
Healing agents
Shining agents
Regenerating agent
Healing agent
PromaCare TA Lightening and whitening agents Uniproma chemical
Prozymex HBT LS
9142
Antiaging agents Laboratoires
Serobiologiques
Whitening agents
Exfoliants/peeling agents
Smoothing agents
Bromelain Lightening and whitening agents Spec-chem Industry
Smoothing agents
BioNatural enzyme
SK 320 P
Moisturizing agents Exfoliants/peeling
agents
Bio-organic concepts
Smoothing agents
Antiwrinkle agents
Conditioning agents
Eperuline PW LS
9627
Firming agents BASF
Toning agents
Antiaging agents
Antiinflammatory
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cartilage, and vitreous (Kablik et al., 2009). For its use as dermal filler, HA is chemically
cross-linked to achieve the manufacturer’s desired composition, which determines the
filler’s structure, longevity, and other properties. The properties of HA are adjusted in the
manufacturing of different commercially available HA fillers, leading to their differing
Table 12.6 A Few of the Commercial Products Containing Lipase as an Active Ingredient and Their
Uses (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
CycloLipase Slimming agents Sederma Croda
International Group
Zymo hair MD Moisturizing agents IRA Istituto Ricerche
Applicate
Sopholiance Antimicrobial and deodorants Soliance
Zymo cell MD Moisturizing agents IRA Istituto Ricerche
Applicate
Slimming agents
Zymo clear MD Moisturizing agents IRA Istituto Ricerche
Applicate
Antiacne agents
Uncaryl Antiaging agents Cobiosa
Antiinflammatory
Antiacne agents
Slimming agents
Antioxidants
Sunscreen agents/UV filters
Pheoslim Slimming agents Codif
Pheoslim G Slimming agents Codif
Lipocel-ErasePB Slimming agents PROTEOS Biotech
Lipocleansing-Erase
HydraPB
Antiacne agents PROTEOS Biotech
Peeling agents
Smoothing agents
Lipocleansing-SensitivePB Antiacne agent PROTEOS Biotech
Antiallergenic agent
Spec-Chem-Climbazole Antidandruff agents SpecChem Industry
Antimicrobials
Lipocel-Erase HYDRA PB Slimming agents PROTEOS Biotech
Facial cleaner Antimicrobial JUJU Cosmetics
Anticellulite Treatment/
Surfacial cleansing
Revue SebumSoap Antimicrobial Kanebo Cosmetics
Anticellulite Treatment/
Surfacial cleansing
Silhouette Sculptant
Exfoliating Mousse 402
Anticellulite treatment Mar ia Galland
Double Minceur Cible’e Anticellulite treatment Guinot
Bath additive with fat
dissolving enzymes
Slimming agents Ishizawa Laboratories
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structural properties. These varying properties may inform clinicians as to which HA
filler would be most appropriate for a specific clinical use. For example, a more highly
cross-linked HA filler would likely be resilient in its ability to hold its form, making it
suitable for the correction of deep wrinkles. In addition, a more monophasic filler might
cleanly retain its form and clinically have a smoother appearance. Hyaluronidase is a
naturally occurring enzyme capable of local degradation of HA (Fig. 12.4), thereby pro-
viding a means for correction or alteration of injected fillers. It is US Food and Drug
Administration (FDA) approved as a temporary dispersion agent for injectable fluids,
typically local anesthetics during retrobulbar blocks. It has been used clinically for over
60 years (Silverstein et al., 2012). In the event of complications with HA fillers, hyal-
uronidase has been used in attempt to reverse HA fillers (Lambros, 2004). Hyaluronidase
hydrolyzes HA by splitting the bond between C1 of an N-acetylglucosamine moiety
and C4 of a glucuronic acid moiety. It is FDA approved as an agent to increase tissue
Figure 12.4 Schematic overview of hyaluronic acid (HA) endocytosis and processing. HMW- HA
(∼106 Da) is rst degraded by hyaluronidase 2 (HYAL2) into smaller 104 -Da-sized fragments before it is
taken up by a cell. The cell can either utilize surface HA receptors for receptor-mediated endocytosis or
macropinocytosis. Once internalized the HA is degraded by hyaluronidase 1 (HYAL1) into small 102 Da
fragments and then exocytosed. Reproduced with input from Racine, R., Mummert, M.E., 2012. Hyaluro-
nan endocytosis: mechanisms of uptake and biological functions. In: Brian, C. (Ed.), Biochemistry, Genetics
and Molecular Biology – Molecular Regulation of Endocytosis. ISBN:978-953-51-0662-3.
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permeability to facilitate subcutaneous hydration, drug dispersion, and reabsorption of
radiopaque dyes, so its use to reverse HA fillers is off-label. Different formulations of
hyaluronidase are available, including a human recombinant agent and an ovine agent
(Rao et al., 2014). A few examples of products containing lipase as active ingredient are
presented in Table 12.7.
APPLICATION OF NANOPARTICLES FOR ENZYME IMMOBILIZATION
Nanotechnology plays a crucial role in developing elegant and effective cosmeceuticals
by using smaller particles that are readily absorbed into the skin and repair damage easily
and more efficiently (Singh et al., 2013a). Incorporation of nanotechnology in cosme-
ceuticals is aimed toward making incense of perfumes last longer, sunscreens to protect
the skin, antiaging creams to fight back the years, and moisturizers to maintain the
hydration of skin. Some of the innovations brought by nanotechnology intervention in
the cosmeceutical arena are nanoemulsions (which are transparent and have unique tac-
tile and texture properties), nanocapsules (which are used in skin care products), nanopig-
ments (that are transparent and increase the efficiency of sunscreen products), liposome
formulations (which contain small vesicles consisting of conventional cosmetic materials
that protect oxygen or light sensitive cosmetic ingredients), niosomes, nanocrystals, solid
lipid nanoparticles, carbon nanotubes, fullerenes, and dendrimers (Fig. 12.5). The pri-
mary advantages of using nanoparticles in cosmeceuticals include improvement in the
stability of cosmetic ingredients (eg, vitamins, unsaturated fatty acids, and antioxidants)
by encapsulating within the nanoparticles; efficient protection of the skin from harmful
ultraviolet (UV) rays; aesthetically pleasing products (eg, in mineral sunscreens, using
smaller particles of active mineral allows them to be applied without leaving a noticeable
Table 12.7 A Few Commercial Products Containing Hyaluronidase as an Active Ingredient and Their
Use (http://cosmetics.specialchem.com)
Products Purpose Manufacturer
Hyaluronidase Moisturizing agent IRA Istituto Ricerche
Applicate
Alpaflor Edelweiss B Antiaging DSM
Antimicrobial
Antioxidants
EcoCare Telmesteine Antiinflammatory Ecochem
Antiaging
Phytessence Wakame Anti-Inflammatory Croda
Antiaging
Moisturizing agent
Antioxidants
Hyaldrain PB Slimming agents PROTEOS Biotech
Hyalucorrect PB Conditioning agents PROTEOS Biotech
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white cast); targeting of active ingredient to the desired site and controlled release of
active ingredients for prolonged effect (Padamwar and Pokharkar, 2006; Mu and Sprando,
2010). LifePak Nano is a perfect example of a commercial product in which nanoencap-
sulation increases bioavailability of coenzyme Q10, protecting cells, tissues, and organs in
the body against the ravages of aging (Lohani et al., 2014). There has been considerable
interest in the development of enzyme immobilization techniques because immobilized
enzymes have enhanced stability compared to soluble enzymes, and can easily be sepa-
rated from the reaction. A few examples of nanoimmobilized enzymes are presented in
Table 12.8. Approaches used for the design of immobilized enzymes have become
increasingly more rational and are employed to generate improved catalysts for industrial
applications. There are a variety of methods used to immobilize enzymes, the three of
the most common being adsorption, entrapment, and cross-linking or covalently bind-
ing to a support. Currently, the major focus of enzyme immobilization has been in the
development of robust enzymes that are not only active but also stable and selective in
organic solvents. The ideal immobilization procedure for a given enzyme is one that
permits a high-turnover rate of the enzyme while retaining high-catalytic activity over
time. Proteins are immobilized either by physical adsorption to the surface of the
nanoparticle or by covalent bonding to previously functionalized nanoparticles. Applica-
tion of nanoparticles in formulations for PCPs has paved the way for utilizing these
Figure 12.5 Schematic overview of dierent types of nanoencapsulations.
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Table 12.8 Examples of Nanoimmobilized Enzymes With Enhanced Activity (Singh etal., 2013b)
Enzyme Applications Kinetic Parameters Nanoparticles Used
α-Chymotrypsin Proteolysis (cleave peptide
amide bonds)
Immobilized enzyme: Km = 31.7 μM,
kcat = 20.0 s−1; Soluble enzyme:
Km = 47.8 μM, kcat = 17.8 s−1
General
Glucose oxidase Estimation of glucose level up
to 300 mg mL−1
Immobilized enzyme: Km = 3.74 mM,
Soluble enzyme = 5.85 mM
Gold NPs
Diastase Starch hydrolysis Immobilized enzyme: Km = 8414 mM,
Vmax = 4.92 μmol min−1
g−1; Soluble
enzyme: Km = 10,176 mM,
Vmax = 2.71 μmol min−1
mg−1
Fe impregnated silica
NPs
Keratinase Synthesis of keratin Immobilized enzyme: Specific
activity = 129.0 U mg−1; Soluble enzyme:
Specific activity = 37 U mg−1
Iron oxide NPs
Horseradish peroxidase catalyzes the conversion of
chromogenic substrates (eg,
TMB, DAB, ABTS) into
colored products
Immobilized enzyme: Km = 0.8 mM,
Vmax = 0.72 μmol min−1
mg−1; Soluble
enzyme: Km = 0.43 mM,
Vmax = 0.35 μmol min−1
mg−1
Nanoporous Cu NPs
Glucose oxidase Estimation of glucose level Immobilized enzyme: Km = 2.7 mM,
Vmax = 28.6 U μg−1; Soluble enzyme:
Km = 9 mM, Vmax = 6.2 μmol min−1
mg−1
Silver NPs
β-1,4-Glucosidase
(Agaricus arvensis)
Lignocellulose hydrolysis Immobilized enzyme: Km = 3.8 mM,
Vmax = 3347 μmol min−1
mg−1; Soluble
enzyme: Km = 2.5 mM,
Vmax = 3028 μmol min−1
mg−1
Silica NPs
Diastase α-amylase Hydrolyzing soluble starch Immobilized enzyme: Km = 10.3 mg mL−1;
Vmax = 4.36 μmol mL−1 min−1; Soluble
enzyme: Km = 8.85 mg mL−1;
Vmax = 2.81 μmol mL−1 min−1
Ag NP’s doped gum
acacia-gelatin-silica
nanohybrid
Laccase Bioremediation of
environmental pollutants
Immobilized enzyme: Km
(10−2 mM) = 10.7, Vmax
(10−2 mM min−1) = 14.0; Soluble enzyme:
Km (10−2 mM) = 5.69, Vmax
(10−2 mM min−1) = 7.7
General
α-galactosidase
(Aspergillus terreus gr.)
Animal feed Immobilized enzyme: Km = 1.40 mM,
Vmax = 20.16 U mL−1; Soluble enzyme:
Km = 4.2 mM, Vmax = 16.33 U mL−1
Calcium alginate (Beads)
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particles for developing these products with enhanced enzyme activity. For example,
LifePak Nano, a nutritional antiaging program formulated to nourish and protect cells,
tissues, and organs in the body with the specific purpose of guarding against the ravages
of aging, has developed a product (Face Gel- Pharmanex/USA) that uses nanoparticles
to enhance enzyme activity and in this product; nanoencapsulation increases bioavail-
ability of coenzyme Q10 by 5–10 times (Lohani et al., 2014).
FUTURE PERSPECTIVES
The global personal care market, estimated at about $300 billion at the retail level, is a
highly attractive segment of the consumer products space. The market has seen steady
growth of 4.5% per annum in the last few years, from low-capital-intensive asset base,
providing high return on capital to investors. Organics-based products is the fastest
growing segment of the global personal care industry. Rising concerns for health
safety, increasing go-green consciousness and growing consumer awareness toward
hazards of synthetic chemicals have fueled the demand for organic PCPs, and increas-
ing health awareness among consumers will continue to drive the growth of the
organic personal care market during the forecast period. Among the organic and natu-
ral products, enzymes derived from various sources have found their specific utility in
formulations of PCPs, which have been increasing rapidly. Application of enzymes in
a given topical application depends on the nature of product as well as the limitations
associated with the enzyme activity. Shorter shelf life of enzyme-based PCPs is a factor
limiting consumer demand. Synthetic products are loaded with a large amount of
preservatives in order to conserve their attributes. Enzyme-based PCP manufacturers
have a hard time sourcing organic ingredients as an alternative to synthetic preserva-
tives. The organic products containing enzymes as an active ingredient with natural
preservatives have a short shelf life or need to be refrigerated. With the advent of
nanotechnology, the effectiveness and durability of enzyme-based PCPs have been
enhanced. Utilization of nanoparticles for enzyme stability and action is now being
considered as an effective measure to address the problems that enzyme-based PCPs
face. The enzyme groups that have proved to be quite useful are the oxidoreductases,
proteases, and hydrolases, In addition, the search for new enzymes still continues.
According to estimates by Kline & Co, a leading consultancy firm, the antiaging seg-
ment is the single largest product type in the personal care market and is the key
growth engine. Skin care and hair care—the two largest segments of the market—are
also the fastest growing, providing sizeable growth opportunities for suppliers. Cosme-
ceuticals, which are cosmetic products with drug-like benefits, has become the fastest-
growing segment of the cosmetics and personal care industry. The global cosmeceuticals
market offers huge potential among Asian countries, like Japan, China, and India,
which are set to attract major players in the future. Though the market is at a nascent
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stage in developing countries, such as India and China, there remains a large untapped
potential, with the desire to look young and fair.
LIST OF ABBREVIATIONS
Cu/Zn SOD Copper/zinc superoxide dismutase
DOPA Dihydroxyphenylalanine
ECSOD Extracellular superoxide dismutase
HA Hyaluronic acid
HYAL1 Hyaluronidase 1
HYAL2 Hyaluronidase 2
LIP Lignin peroxidase
MnSOD Manganese superoxide dismutase
NP Nanoparticle
PCP Personal care products
ROS Reactive oxygen species
SOD Superoxide dismutase
TYRP1 Tyrosinase-related protein 1
UV Ultraviolet
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