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Indian Journal of Biotechnology
Vol 4, January 2005, pp 115-121
Moisturizing efficiency of silk protein hydrolysate: Silk fibroin
A V Daithankar, M N Padamwar, S S Pisal*, A R Paradkar and K R Mahadik
Department of Pharmaceutics, Poona College of Pharmacy and Research Centre, Bharati Vidyapeeth Deemed University
Pune 411 038, India
Received 19 September 2003; revised 11 February 2004; accepted 25 February 2004
A biomimetic approach of composition and natural function of natural moisturizing factor (NMF) with the amino acid
content of silk fibroin was advantageously used to reconstruct the skin moisturizing system. The isolation of silk hydrolysate
with water and sodium chloride treatment was complete in one hour. Lithium ion from LiBr effectively penetrated crystal
domains of fibroin and gave desired solubility. Silk fibroin from Bombyx mori cocoons was non-allergic and biocompatible
in skin and rabbit eye tests. The concentration dependent moisturizing efficacy of fibroin (1-5% w/v) in solution and cream
form has been demonstrated by TEWL in vitro and in volunteers. As compared to dry and normal skin the fibroin containing
cream revealed increased substantivity. The increased hydroxproline content was responsible for retaining higher moisture
in the skin. This in turn maintained the skin in soft and supple state. The significant drop in impedance was observed within
1 hr of the application of fibroin and the effect was sustained for more than 6 hrs. Thus, increased hydration level in stratum
corneum was achieved by fibroin treatment. The SEM of fibroin treated skin replicas showed a desired attribute of soft,
smooth skin texture and improved flexibility. The increased state of hydration caused interdigitating of cell edges as evident
in microphotographs. The rapid and sustained moisturizing efficiency observed with silk fibroin was well substantiated by
the results of skin substantivity and impedance tests.
Keywords: silk fibroin, natural moisturizer, in vivo, TEWL, SEM, impedance
IPC Code: Int. Cl.7 A 61 K 7/40, 7/48
Introduction
Dry skin conditions are the most frequent derma-
tological disorders of the skin. The lack of adequate
flexibility and extensibility due to environmental
changes results in cracking and flaking of the stratum
corneum. Water is the only plasticizer of the skin.
Environmental factors and use of detergents is the
prime factor responsible for excess water loss and dry
skin condition1. The hydrophilic substances lost from
the stratum corneum (natural moisturizing factor,
NMF) reduce the ability of the skin to hold the water
and thereby maintain the extensibility2. The NMF is
made up of amino acids (free, 40%), pyrrolidone car-
boxylic acid (12%), urea derivatives (8.5%), inorganic
salts (17%), lactate (12%) and unidentified peptides
(9.5%). The epidermis contains most of the twenty-
two amino acids normally occurring in living tissue.
NMF is amino-lipoidal in nature. The free amino ac-
ids content of NMF predominantly contains glycine
and histidine.
The facile addition of water does not suffice to
plasticize skin. It is bound up in protein-lipid mixture,
most probably within the dead cells of epidermis. The
oil containing preparations form an occlusive layer on
skin, which prevents the moisture loss from the stra-
tum corneum. This allows water to be accumulated in
the horny layer of the skin3. Straianse initiated the
research in the field of regulating moisture in the
skin4. A number of emollients lubricating the skin and
moisturizers enhancing the hydration of the skin have
been reported5. The significance of natural hygro-
scopic components is emphasized by researchers and
the nourishment of the skin with products containing
hydrophilic substances has been advised. Oils and
fatty acids containing preparations give temporary
softness to the skin. However, their prolonged use can
be harmful due to sealing of horney layer resulting in
edema and inflammation.
Several synthetic and semi-synthetic compounds
are used as moisturizers. The most valuable approach
to moisturization of skin is to determine the precise
mechanism of NMF and access the damage due to
deficient material. This approach is used in natural
products from animal milk, palm fruit, gingili seed
——————
*Author for correspondence:
Tel: 91-20-2543 7237; Fax: 91-20-2543 9383
E-mail: CAYT2002@rediffmail.com
INDIAN J BIOTECHNOL, JANUARY 2005
2
and Aloe vera. Such products enjoy excellent popular-
ity as moisturizers in crude and paste form6. Protein
and protein hydrolysates from natural sources are
common ingredients of skin creams. Literature re-
vealed that protein hydrolysates from natural sources
can stimulate the skin to rebuild tissue and replace
amino acids in the stratum corneum7. India produces
nearly 14,500 million tonnes of silk per annum. The
abundant amount of silk produced is predominantly
used in textile and dyeing industry. Silk is continuous
strand of two filaments of fibroin cemented together
by silk gum (silk sericin). The unique physico-
chemical properties of fibroin have been successfully
evaluated as enzyme inhibitor, immunostimulant, ma-
terial of construction of contact lens and for con-
trolled release of drugs in gel forms8-11. Researchers
have reported that silk fibroin has, improved affinity
of eye shadows, long lasting effect for deodorants,
reduced colour bleeding from lipsticks and as anti-
wrinkle agent by promoting the collagen formation12.
Fibroin, a glycoprotein, is composed of two
equimolar protein subunits of 370 and 25 kDa,
respectively. The amino acid composition of silk
fibroin has components resembling to NMF of the
skin13. The crystalline domains of fibroin have high
percentage of glycine (44%) and alanine (30%). Silk
fibroin shows excellent water binding and absorbing
capacity. This property of a non-toxic and
biocompatible natural protein hydrolysate is well
suited for moisturizing effect. The biomimetic
approach based on knowledge of natural function of
NMF can be used to reconstruct a completely new
moisturizing system using silk fibroin. The isolation
of silk fibroin has been optimized and the safety
profile established. The moisturizing efficiency of silk
fibroin in solution and cream form was evaluated
using transepidermal water loss (TEWL) technique as
well as in volunteers. The effect of silk fibroin as
moisturizer was also investgated by hydroxyproline
assay and impedance measurements method to
support the hypothesis. The average changes of
surface of stratum corneum skin replicas were
analyzed using scanning electron microscopy.
Materials and Methods
Silk (Bombyx mori) cocoons were obtained from
Sericulture Institute, Pune. Lithium bromide, L-
hydroxyproline and p-dimethyl amino benzaldehyde
were procured from Loba Chemicals, Mumbai.
Speedex (silicon rubber fluid) was kindly provided by
Medinova Diagnostic Centre, Pune. Freshly shaded
snakeskin was obtained from Rajiv Gandhi Snakes
Park, Pune. Silk-Pro-100 (pure silk fibroin, L10027)
was received as a gift sample from Collaborative
Laboratories, New York. Steric acid, cetyl alcohol
and isopropyl myristate of spectral grade were pur-
chased from Pure Chem Ltd., Mumbai. All other
chemicals of ultra pure grade were used.
Isolation of Silk Fibroin
Silk cocoons (2 g), cut into small pieces (1 cm2)
were boiled in water for 30, 60 and 90 min separately.
The mixture was treated separately with 300 ml of
0.5% w/v aqueous sodium carbonate solution at 90-
100°C for 30, 60 and 90 min. Silk fibres were washed
with hot water till neutral pH, air dried and defatted
with petroleum ether (25 ml). Purified fibres were
dissolved in 5-8 ml of aqueous solution of lithium
bromide (9.3 M) separately. The thick paste of silk
fibres was dialysed with water for 3 days to remove
lithium bromide. The residual lithium bromide con-
tent was analyzed using atomic emission spectros-
copy14.
Safety Evaluation of Silk Fibroin
The primary skin irritation was measured by open
patch, closed patch and abraded skin tests in albino
rabbits15. Eye irritation test was performed in rabbits.
Ophthalmic and skin irritation tests of 0.1ml of 5%
w/v aqueous solution each of silk fibroin and silk-Pro-
100 were carried out separately using six rabbits (2.5-
3 kg) against water as control. The observations were
scored numerically after 24, 48 and 72 hrs. Intensity
of the skin reaction was graded in accordance to the
data used by National Institute of Occupational
Safety.
Moisturizing Efficiency of Silk Fibroin
In vitro Evaluation
Shaded snakeskin was washed sufficiently with
distilled water and air-dried. It was rehydrated using
100 ml Tris-HCI buffer (pH 7.4). The TEWL was
estimated using a small diffusion cell, fabricated from
cylindrical aluminum bar, as suggested by Martin and
Deems1. A small piece (1.5 cm diam) of snakeskin
was placed in between two silicon rubber gaskets. The
lower chamber contained 0.4 ml distilled water. The
gasket along with the skin was placed between the
lower chamber and covered with lid. One ml solution
of silk fibroin (1, 3, and 5% w/v) and 5% w/v silk-
Pro-100 was applied on snakeskin. The cell was kept
in constant humidity chamber at 37°C and weighed
DAITHANKAR et al: MOISTURIZING EFFICIENCY OF SILK PROTEIN HYDROLYSATE
3
every 24 hrs until constant weight. Rate of moisture
loss (mg/cm2/hr) was calculated by using equation 1,
Rate of moisture loss= Moisture loss (mg)/Area (cm2)
of SC exposed × Time in hr … (1)
In vivo Evaluation
A modified miniature desiccator was used to meas-
ure TEWL in human volunteers16. About 600 mg sil-
ica gel (10/20 mesh size) enclosed in small cotton bag
was used as a desiccant. Inner portion of the forearm
(upper and lower) was selected as a test site. Desic-
cant bags were weighed just before the test, and every
two hrs during the test. Normal TEWL of six healthy
volunteers was calculated using equation 2,
DC% = (U−L) × 100/l … (2)
where, DC% is percentage difference in moisture loss
of adjacent site of normal, U and L are the weights of
silica bags on the upper and lower sites, respectively.
One ml sample each of fibroin solution (1, 3 and
5% w/v) and silk-Pro-100 (5% w/v) was applied sepa-
rately on the upper portion of the forearm for 15 min.
The lower portion was kept blank. The TEWL (DT%)
was calculated as described above. The actual mois-
ture loss (net effect) was calculated using equation 3.
The in vitro and in vivo moisturizing efficiency of
standard cream, blank base and creams containing 5%
silk fibroin and 5% silk-Pro-100 (separately) was de-
termined similarly.
Net effect = DT% − DC% … (3)
Skin Substantivity of Fibroin
The forearms of the subjects were washed with
soap and then towel blotted. The quantity of cream
(5% silk fibroin, 5% silk-Pro-100) equivalent to 1 ml
solution was applied to 3 cm2 skin and allowed to re-
main in place for 3 hrs. Three successive scotch tape
stripping each of 1 cm2 were analyzed for hy-
droxyproline content as described by Sakamuto17.
Similarly, the hydroxyproline content of dry and nor-
mal skin was estimated.
Effect of Fibroin Treatment on Skin Impedance
An electrical equivalent circuit previously reported
by William et al18, was used to study the effect of
creams (5% silk fibroin, 5% silk-Pro-100) on the skin
impedance. Normal impedance values were measured
in five untreated volunteers after every hr. One ml of
the test sample was applied to forearms of the volun-
teers and the impedance was measured every hour for
a period of six hrs.
Scanning Electron Microscopy of Fibroin Treated Skin
The surface topography of normal skin, dry skin
and skin treated with optimized silk fibroin cream and
marketed cream was obtained by SEM19. The quantity
of cream (5% silk fibroin, marketed cream) equivalent
to one-milliliter solution was applied to 3 cm2 skin
and allowed to remain in place for 3 hrs. The skin rep-
licas were taken using silicon rubber fluid, mounted
on stub using double-sided adhesive tape. Skin repli-
cas were gold-coated (20 mm) using sputtering tech-
nique (VG Microtech, UK). The replicas were ob-
served under SEM (Cambridge Instruments, Stereo-
scan 120, UK) and photographed (50 ×).
Results and Discussion
Isolation of Silk Fibroin
Silk mainly contains fibroin (about 80%), gum-
ming agent (sericin) (18%) and fatty substances (2%).
The purification and treatment of silk is essential to be
used in cosmetic products. The time required for
complete solubilization of silk fibres with water
treatment time of 30, 60 and 90 min with the corre-
sponding sodium carbonate (0.5% w/v, 100°C) treat-
ment for 30, 60 and 90 min treatment were found to
be 40, 20 and 15 min, respectively. Insufficient treat-
ment with boiling water results in longer solubiliza-
tion time and poor yield. The precipitates observed in
such case indicate incomplete degumming. A water
boiling and sodium chloride treatment each for one hr
resulted in improved solubility and yield (3.2% w/v).
The purified silk fibroin is insoluble in water but
soluble in 9.3 M LiBr solution. The volume of lithium
bromide solution has significant effect on the product
characteristics. A sediment with large fraction of in-
soluble suspended particles was observed after one
day in 5 and 6 ml LiBr solution batch. However, a
transparent dialyzed solution without visible particles
was obtained with 7 and 8 ml of aqueous LiBr solu-
tion treatment.
Aqueous lithium bromide solution is a good
solvent for the solubilization of silk fibroin. Lithium
ions enter the crystal domains present in the fibroin
fibres by active ionic movement. The peptide chain is
broken by co-ordination between lithium ions and
polar tyrosine and serine. The simultaneous entry of
more water molecules breaks the intermolecular
hydrogen bonds in peptides and gives soluble
INDIAN J BIOTECHNOL, JANUARY 2005
4
fraction20,21. The average final concentration of silk
fibroin was found to be 8.3-8.5% w/v. The residual
lithium content was less than 0.00025 and well below
the safety limit.
Safety Evaluation of Silk Fibroin
Silk fibroin obtained from the silk cocoons is re-
ported to be non-toxic and biocompatible. The results
of safety evaluation of fibroin solution 5% and silk-
Pro-100 (5%) in the rabbit eye test showed no effect
on lacrimation and no signs of irritation to cornea, iris
and conjunctiva. The skin irritation test revealed ab-
sence of any kind of inflammatory response i.e.
edema or erythema (redness) showing the non-allergic
and non-irritant property and safety for human use.
The solution indicated a curing effect on abraded skin
showing tissue-rebuilding nature of proteins. From the
results of both the tests it can be confirmed that the
silk protein is biocompatible natural material22.
In vitro and In vivo Moisturizing Efficiency
The hydration state of stratum corneum can be de-
termined by several methods. The most widely used
and effective method is measurement of TEWL. The
rate of moisture loss from 1, 3 and 5% w/v silk fibroin
solution and 5% w/v silk-Pro-100 is shown in Fig. 1.
The average T/U ratio of 1, 3 and 5% silk fibroin so-
lution and 5% silk-Pro-100 solution was 1.99, 2.20,
2.61 and 2.78, respectively. The net moisture loss in
untreated volunteers and treated with 1, 3 and 5% w/v
silk fibroin and 5% w/v silk-Pro-100 solution for 12
hrs were found to be 1.97±0.434, 11.95±0.923, 14.38
± 0.468, 20.264 ± 1.159 and 21.88 ± 0.633 mg/cm2/2
hrs, respectively.
The 5% silk fibroin and 5% silk-Pro-100 were for-
mulated in o/w cream and evaluated for moisturizing
efficiency along with standard cream (cream base as
blank) by both in vitro and in vivo TEWL techniques.
The o/w creams containing 5% w/v silk fibroin and
5% silk-Pro-100 prepared separately had a viscosity
of 985 ± 15 and 924 ± 21cP respectively. The creams
with a pH of 5.4 ± 0.2 were physically stable on stor-
age at room temperature for three weeks. These
creams did not show interaction with excipients and
hence no gel formation or synerisis. The in vitro rate
of moisture loss from the creams is shown in Fig. 2.
The T/U ratio for cream base, 5% w/v SF cream, 5%
w/v S-P-100 cream and standard cream was found to
be 1.58, 2.88, 2.94 and 2.95, respectively. The rate of
moisture loss of the corresponding creams in volun-
teers was found to be 2.05 (untreated), 3.17 ± 0.98
(cream base), 23.954 ± 2.25 (silk fibroin 5% w/v),
24.07 ± 2.23 (silk-Pro-100 5% w/v) and 26.17 ± 1.21
mg/cm2/2 hrs.
The in vitro evaluation revealed that 5% silk fib-
roin solution showed significant increase in the
TEWL values than 1 and 3% w/v silk fibroin concen-
tration. This trend was confirmed in higher T/U ratio
also. The result indicated that increase in concentra-
tion of silk fibroin, increased the water uptake from
the reservoir of the cell into the snakeskin, which then
evaporated into the atmosphere. Thus, the rate of epi-
dermal water turnover was increased. Silk-Pro-100, a
purified silk protein (source and composition not dis-
closed) was found to be equally effective for longer
period of treatment. In vivo evaluation closely ap-
proximates the use and perception of efficiency of the
Fig. 1—In vitro moisture loss through snakeskin (76% RH)
(-◊-Control, -- SF solution 1% w/v, -U- SF solution 3% w/v,
-×-SF solution 5% w/v, -o-SP solution 5% w/v)
Fig. 2—In vitro moisture loss through snakeskin (76% RH)
(-- Control, -- Blank cream -U- SF Cream 5% w/v, -×- SP
Cream 5% w/v, -o- Standard Cream)
DAITHANKAR et al: MOISTURIZING EFFICIENCY OF SILK PROTEIN HYDROLYSATE
5
material as a moisturizing agent. A trend confirming
the in vitro effect was observed in in vivo moisturiz-
ing efficacy in volunteers. This also confirms the suit-
ability of the in vitro evaluation technique. The in vi-
tro evaluation of creams revealed the supportive role
of the cream base to the moisture control. The rate of
moisture loss from the silk fibroin, silk-Pro-100
creams and standard (marketed) product was higher
than the corresponding solutions. Their in vitro as
well as in vivo evaluation showed insignificant differ-
ence in the moisturizing ability.
The behaviour of the free amino acids in the stra-
tum corneum reflects the physical well being of the
skin. The mechanism of increase in water transport by
silk fibroin lies in the fact that amino acid components
of silk fibroin are similar to natural moisturizing fac-
tor of the stratum corneum. The high molecular
weight (68000 D) of the silk fibroin forms a thin film
over the stratum corneum. This film is porous and is
permeable to water7,23,24. It drags the water from the
reservoir beneath the skin and increases the water cir-
culation across stratum corneum. The desiccant took
up more moisture, thus there was increase in weight
of desiccant showing moisturizing effect of silk pro-
teins. The resulting net effect was the continuous con-
tact of stratum corneum with the water and hence
moisturization.
Skin Substantivity of Fibroin
The water absorption capacity of protein and pep-
tides is measured by the extent of substantivity to stra-
tum corneum. The binding capacity of the silk fibroin
on stratum corneum is estimated by hydroxyproline
assay25. Fig. 3 shows significant increase in the hy-
droxyproline content of the stratum corneum after
application of the 5% w/v silk fibroin and 5% w/v
silk-Pro-100 solution within 3 hrs. The average hy-
droxyproline content for dry and normal skin and skin
treated with fibroin and silk-Pro-100 were 0.0142,
0.627, 1.734 and 1.927 µg/cm2, respectively. In com-
parison with dry and normal skin it revealed increased
substantivity. Skin substantivity measures the amount
of protein, which after extended exposure resist to
extraction by water due to formation of weaker link-
ages with the skin components26. However, the in-
creased protein binding can be analyzed in scotch tape
stripping. The increased hydroxyproline content is
responsible for higher moisture content. This in turn
maintains the skin in soft and supple state. The ob-
served results support skin moisturizing mechanism
of silk fibroin.
Effect of Fibroin Treatment on Skin Impedance
Measurement of skin impedance is sensitive means
to access the hydration state of the skin. Impedance
between two fixed points of the skin depends upon the
hydration state of stratum corneum. Higher moisture
retained in the skin causes drop in the impedance. The
effect of silk fibroin and silk-Pro-100 on skin imped-
ance in volunteers is shown in Fig. 4. The significant
drop in impedance was observed within 1 hr of appli-
cation of fibroin and the effect was sustained for more
than 6 hrs.
Skin moisture is closely related to its normal func-
tioning and its measurement helps in early diagnosis
of non-visible skin conditions. William et al18 have
applied the electrical equivalent principle to evaluate
the performance of drugs and cosmetics by recording
the skin impedance. Low frequency (l mHz) imped-
ance measurement is preferred to higher ones due to
minimum induced changes in skin components and
higher sensitivity. Any substance similar in properties
Fig. 3—Hydroxyproline content after skin treat-
ment
Fig. 4—Effect of skin treatment on skin impedance (-- Normal
skin, -- Silk fibroin cream, -U- SP100 Cream)
INDIAN J BIOTECHNOL, JANUARY 2005
6
to natural moisturizing factors of stratum corneum can
cause drop in impedance e.g. sodium salicylate. How-
ever, highly resistive substances like liquid paraffin
get incorporated in stratum corneum were reported to
increase the impedance. In the present study, increase
in hydration level in stratum corneum was achieved
after treatment with silk fibroin. This change in elec-
trical properties of stratum corneum may be attributed
to more movement of keratin chains due to plasticiz-
ing effect of retained water27. The results are in coor-
dination with the in vitro and in vivo moisturizing ef-
fects.
Scanning Electron Microscopy of Fibroin Treated Skin
The visual changes in the surface of the stratum
corneum including uniformity, number and nature of
white ridges, smoothness and scaling can be revealed
by SEM5. The SEM for normal, dry and skin treated
with silk fibroin as well as marketed moisturizing
cream is shown in Fig. 5. Although SEM was ob-
tained at a magnification of 50× and 500×, the magni-
fication at 50× revealed the changes in the skin tex-
ture. Dry skin showed more flakes and cells separated
by more white lines with massive desquamation of
stratum corneum. The normal skin cells are highly
organized with end-to-end arrangement in units of
vertical column. A desired attribute of soft, smooth
skin texture and improved flexibility was evident in
skin treated with silk fibroin (as well as marketed
product).
Epidermal cells construct superficial layer of epi-
dermis, mainly composed of 10-15 cell layers of flat-
tened keratinized dead cells. The horny layer is 10-15
µm thick in dry skin. Average daily loss of 0.5-1 g of
horny layers occurs from the normal skin. The dry
skin condition can be treated by occlusive, humec-
tancy or restoration of deficient material. In the pre-
sent study, cream containing 5% silk fibroin increased
the transepidermal water loss and the process swelled
the dry cells several times its volume. More water was
bound to protein lipid complexes of the dead cells.
This caused the cells to interdigitate their lateral edges
with adjacent cells to form a cohesive lamina. The
plasticizing effect of increased water in dead cells
enhanced mildness and imparts substantivity28. The
microphotographs reveal that the silk fibroin as mois-
turizer brought out significant improvement in the
texture of dry skin. The qualitative assessment
showed better surface texture as compared to mar-
keted product. Hence, it can be confirmed that the silk
hydrolysate helped to rebuild the skin tissues29.
Conclusion
Occlusive type of moisturizers has limitations of
temporary softness with grease filling. The resem-
blance of amino acid composition of natural moistur-
izing factor and silk fibroin (high molecular weight)
can be advantageously used as skin moisturizer. The
fibroin isolated with lithium bromide revealed the
biocompatible nature of silk hydrolysate. The mois-
turizing efficiency of the silk fibroin and silk-Pro-100
has been demonstrated and correlates well in in vitro
and in vivo techniques. The increased hydroxproline
content, rapid drop in skin impedance and a soft,
Fig. 5—Scanning electron microphotographs of skin replicas
(50×): a. Dry skin, b. Silk fibroin cream, c. Standard cream
DAITHANKAR et al: MOISTURIZING EFFICIENCY OF SILK PROTEIN HYDROLYSATE
7
smooth skin texture with improved flexibility in scan-
ning electron microscopy substantiate the effect ob-
served in transdermal water loss technique. The study
revealed silk fibroin as a promising new natural mois-
turizing agent.
Acknowledgement
SSP gratefully acknowledges All India Council for
Technical Education (AICTE), New Delhi, for pro-
viding financial assistance as research fellowship in
the form of ‘Career Award for Young Teachers 2002’.
Authors are thankful to Dr Shivajirao S Kadam, Prin-
cipal, Poona College of Pharmacy, Bharati Vid-
yapeeth Deemed University, Pune for support in the
implementation of the research scheme.
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