Content uploaded by Ratan Chaudhuri
Author content
All content in this area was uploaded by Ratan Chaudhuri on Dec 21, 2015
Content may be subject to copyright.
INTRODUCTION
H
uman skin colour is quite variable
around the world. It ranges from a
very dark brown among some
Africans, Australians and Asian-Indians to a
near pinkish yellow among some northwest
Europeans. There are no people who truly
have black, white, red or yellow skin.
These are commonly used terminologies
that do not reflect biological reality.
Pigmentation of the skin, due to synthesis
and dispersion of melanin in the
epidermis, is of great cosmetic and
societal significance. It is also the key
physiological defence against sun-induced
damage, such as sunburn, photo ageing
and photocarcinogenesis.
Skin lightening agents have been widely
used to either lighten or depigment the
skin. Preparations in the European market
tend to be used to treat age spots and
freckles or to obtain even-toning effects,
whereas the Asian market uses them to
change or modify skin colour. A wide
variety of skin-lighteners, namely
hydroquinone and its derivative arbutin,
kojic acid and its dalpmitate, ascorbic acid
and its derivatives such as sodium and
magnesium ascorbyl phosphates, ascorbyl
2-glucoside have been used for these
purposes.1,2
Due to the increasing use of skin
lightening agents among the global
population, it is apparent that both the
efficacy of a skin lightening formulation,
as well as safety and mildness of the skin
lightening ingredient, are equally
important. Many current skin lighteners
such as hydroquinone3,4 and Kojic acid5,6
have inadequate safety and stability
profiles. Based on the toxicity problems of
hydroquinone and kojic acid, it is quite
reasonable to question the safety of
arbutin and kojic acid dipalmitate for
human use.
Photo ageing of skin is a complex
biological process affecting various layers of
the skin, with major changes seen in the
connective tissue of the dermis.
The natural shift toward a more pro-oxidant
state in intrinsically aged skin can be
significantly accelerated by UV-irradiation.
The effects of UV-radiation from sun
exposure can induce or exacerbate oxidative
attack leading the generation of Reactive
Oxygen Species (ROS). The most severe
consequence of photo-damage is skin
cancer. Less severe photo ageing changes
result in wrinkling, scaling, dryness, and
uneven pigmentation consisting of
hyper- and hypo-pigmentation.7–10
The preliminary skin lightening profile of
Phyllanthus emblica fruits (trade named as
Emblica, standardised antioxidant fraction)
has been reported earlier11 (US Patent
6,649,150 and several US and foreign
pending patents). The purpose of the
present work is two-fold: (a) to show
broader applicability of Emblica as a
skin-lightener and (b) to investigate the
effectiveness of this material as an
anti-ageing ingredient.
About the product
P. emblica (syn. Emblica officinalis) is one
of the important Ayurvedic (science of life)
herbs in India, and has been used for
thousands of years for a wide variety of
human ailments. Its status ranges from
insignificant in the western world to
highly-prized in tropical Asia. The fruits are
selected, harvested and processed
according to strict criteria to ensure a
consistently high quality product. Emblica
antioxidant is extracted from premium
Anti-ageing
benefits of
Anti-ageing
benefits of
Ratan K Chaudhuri – EMD Chemicals Inc, Hawthorne, NY 10532, USA
PERSONAL CARE
January 2005 13
PHYLLANTHUS EMBLICA EXTRACT
8
6
4
2
0
3 weeksKey:
Delta ITA degree
Emblica
Hydroquinone
Emblica
Hydroquinone
Emblica
MAP
Emblica
Kojic acid
Study 1 Study 2 Study 3 Study 4
6 weeks 9 weeks
Figure 1: Results of clinical studies of Emblica and other skin lighteners.
quality fruits using a water-based process
(US Patent 6,124,268 and several US and
foreign pending patents). Emblica
antioxidant is distinctly different from
other commercially available extracts of
P. emblica fruits as it is defined to the
extent of well over 50% (typically, 60 to
70%) in terms of its key chemical
components.12,13 None of the extracts
of P. emblica in the market compare to
Emblica antioxidant in composition and
consistency of composition, aqueous
stability and colour.
The low molecular weight (<1,000)
hydrolyzable tannins, namely Emblicanin A
and Emblicanin B, along with Pedunculagin
and Punigluconin are the key ingredients in
Emblica antioxidant.12–14 In nature,
Emblicanin A and Emblicanin B have
only been found in P. emblica plant.14,15
Emblica antioxidant has been
standardised16 by using high performance
thin layer chromatography (HPTLC).
Alternatively, the product can be
standardised by using high performance
liquid chromatography (HPLC).
Skin lightening property
Four separate clinical studies were done to
determine the potential of Emblica as a
skin lightener:
Study 1: Comparison of 2% Emblica
antioxidant vs 2% Hydroquinone
(13 Asians);
Study 2: Comparison of 2% Emblica
antioxidant vs 2% Hydroquinone
(13 Hispanics);
Study 3: Comparison of 1% Emblica
antioxidant vs 3% Magnesium Ascorbyl
Phosphate (MAP, 16 Asians);
Study 4: Comparison of 1% Emblica
antioxidant vs 2% Kojic Acid
(16 African-Americans).
Human volunteers with skin Types III and
IV (as defined by the Fitzpatrick Photo type
Scale) were selected for Studies 1-3.
For Study 4, skin types IV to VI were used.
This study was conducted comparing
baseline, pre-treatment to final post-
treatment results of the selected test sites.
The length of the study was nine weeks.
Each subject was given two products with
identical formulation ingredients (one with
Emblica antioxidant and the other one with
a competitive material as a positive
control) – one for the left upper arm and
the other one for the right upper arm.
Panelists were instructed to apply
approximately 0.5 ml of the test materials
twice a day. The selected test application
site was photographed prior to the start of
the study. Rigid control of photographic
technique, from the aspects of lighting,
distance, angles and camera/film settings
and specifications, and subsequent
development of photographs was followed.
Skin colour measurements were done
using a tristimulus instrument (CR300 –
Minolta Chroma Meter). Repeated
measures analysis of variance was used to
SKIN CARE
14 January 2005
PERSONAL CARE
Figure 2: Cause and consequences of UV-induced skin damage.
TABLE 1: ULTRAVIOLET SPECTRAL DATA OF FE3+- AND CU2+-CHELATORS.
Chelator Absorption Maxima of Complex (λ
max
in nm)
With Fe3+ N3- Induced Shift With Cu2+ N
3
- Induced Shift
EDTA 241, 283 241, 283, 410 240, 278 241, 279, 354
Emblica antioxidant 241, 294, 353, 377 241, 294, 353, 377 240, 272, 313 240, 272, 313
Pine Antioxidant 241, 294, 353, 384 241, 294, 353, 400,440 239, 279, 302, 331 239, 280, 307,430
Vitamin C 238, 262 241, 266, 295 239, 263, 239, 263, 284,364
Grape Antioxidant 247, 295, 353, 396 247, 295, 353, 415,430 240, 277, 328 240, 277, 328, 359
Green Tea Antioxidant 240, 272, 324, 390 240, 277, 325, 390 241, 276, 327, 403 240, 277, 336, 404
Trolox C 240, 284 240, 273, 284, 360 241, 288 241, 261, 352, 440
Gallic Acid 247, 295, 337 247, 295, 353,412 240, 258, 321 240, 258, 331,463
UV light
Skin damage
DNA Protein Lipids Carbohydrate
Reactive
oxygen species
Release of matrix
metalloprotease
Release of free
iron & copper
Strand breakage
Mutations
SH oxidation
Deactivation
of Enzymes
Peroxidation Depolymerization
of hyaluronic acid
SKIN CARE
determine if any significant differences
were observed in the mean Individual
Typology Angle (ITA degree).
ITA value is calculated from the mean
L* and b* Chroma Meter values using the
formula:
ITA Degree = [Arc Tangent ((L* - 50)/b*)]
180/3.1416.
Where L* value is the lightness value, a* is
the colour value in the red-green axis, and b*
is the colour value in the blue-yellow axis.
∆E of ITA degree was calculated by
subtracting the average ITA degree of the
treated site from that of the average
baseline (first day of study). Results of
these clinical studies are summarised in
Figure 1.
The purpose of the four clinical studies
was to compare the skin lightening effect
of Emblica with other well-known and
well-documented skin lighteners, such as
hydroquinone, Magnesium ascorbyl
phosphate (MAP) and Kojic acid. Each
comparative study consists of two products
with identical formulation ingredients (one
with Emblica antioxidant and the other with
a competitive material, positive control).
No attempt has been made to optimise
the formulation and no skin penetration
enhancers were used in the formulations.
In all four studies, Emblica antioxidant-
containing product shows a significant
increase in ITA degree (meaning skin
lightening) versus the corresponding
baselines. Studies 1 and 2 clearly show
2% Emblica antioxidant has a comparable
skin lightening effect to that of 2%
Hydroquinone in Asian as well as Hispanic
subjects. Study 3 shows 1% Emblica
antioxidant is three times better in skin
lightening efficacy on a percentage-active
basis over the 3% MAP product.
Study 4 shows 1% Emblica antioxidant is
comparable to 2% Kojic Acid.
Anti-ageing property
The unifying pathogenic agents responsible
for photo-damage are UV-generated
Reactive Oxygen Species (ROS) that
deplete and damage the enzymatic and
non-enzymatic antioxidant defence
systems of the skin, and the release of
matrix metalloproteases (MMPs) such as
MMP-1 and MMP-3, that damage the
extracellular matrix proteins.9,17
Cause and consequences of UV-induced
skin damage can be schematically
represented in Figure 2.
Emblica antioxidant has been found to
have broad-spectrum antioxidant activity,
excellent iron- and copper-chelating activity
and MMP-1 and MMP-3 inhibitory activity.
While most antioxidants go from an active
to an inactive form. Emblica antioxidant
utilises a multilevel cascade of antioxidant
compounds resulting in a long-lasting and
stable antioxidant activity18. These
multifunctional attributes are described in
the following sections.
Effects on Reactive Oxygen
Species (ROS)
Singlet oxygen, superoxide anion and
hydroxyl radicals are the three key ROS
responsible for skin damage. In-vitro
studies done with Emblica antioxidant and
a few commercially available antioxidants
clearly show superiority of Emblica
antioxidant over commercially available
antioxidants. Results are described below:
Singlet oxygen quenching: Singlet oxygen
quenching ability of Emblica antioxidant has
been evaluated by photooxidation of a
sensitiser (N,N-dimethyl-p-nitrosoaniline)
using a published method.19,20 Results show
that Emblica antioxidant (IC
50
= Inhibitory
concentration 50%; IC
50
61 µg/ml) is an
excellent singlet oxygen quencher and is
superior to Trolox C, a Vitamin E
water-soluble analog (IC
50
84 µg/ml). In
this test, vitamin C was found to be a
strong enhancer of singlet oxygen
(pro-oxidant).
Superoxide anion radical quenching:
Superoxide anion quenching ability of
Emblica antioxidant has been evaluated by
generating superoxide anion using a
hypoxanthine-xanthine oxidase test21
followed by reduction of nitroblue
tetrazolium (NBT) into nitroblue diformazan
by monitoring the light absorption of NBT at
560 nm. Results of this study show that
Emblica antioxidant is an excellent
quencher for superoxide anion. IC
50
values
for Emblica antioxidant is 12 µg/ml,
compared to IC50 26 µg/ml for vitamin C
and IC50 360 µg/ml for Trolox C, a vitamin
E water-soluble analog.
Hydroxyl radical quenching: Hydroxyl
radical quenching ability of Emblica
antioxidant has been evaluated by the
deoxyribose test method described by
Halliwell.22 Results show that Emblica
antioxidant is a significantly better hydroxyl
radical quencher than all other antioxidants
tested in this study. IC
50
values for Emblica
antioxidant is 0.92 mMol, compared to IC
50
of 2.10, 2.70 and 2.78 mMol for pine
antioxidant, Trolox C and grape antioxidant,
respectively. Vitamin C and green tea
antioxidant were found to be pro-oxidants.
Effects on iron and copper
Recognising the crucial role iron plays in
initiating oxidative stress23,24 to skin, we
envisioned that an antioxidant can be a
true photoprotective agent provided it
chelates all the coordination sites in iron or
copper.25 Using the UV spectrophotometric
method developed by Graf et al,25 we
have determined the presence of free
coordination site(s) in the Fe3+- and Cu2+-
antioxidant complexes comparing their
azide-induced shift. Table 1 shows that all
complexes except Emblica antioxidant
contain at least one coordinated water
molecule as shown by the presence of
spectral shift(s) induced by sodium azide.
Effects on Matrix
Metalloproteases (MMPs)
Collagenase (MMP-1) inhibitory activity:
A dose-dependant inhibition of
gelatinase/collagenase activity by about
55 to 70% was observed with Emblica
antioxidant at 150-300 µg/ml.13
Quantification of gelatinase/collagenase
PERSONAL CARE
January 2005 15
Figure 3: Collagenase (MMP-1) inhibitory activity of Emblica antioxidant.
(Phenan = 1,10-Phenathroline).
100
80
60
40
20
0
MMP-1 activity in % of control
Control
Concentration of Emblica in microgm/ml
150 200 250 300 Phenan
inhibitory activity of Emblica antioxidant
was determined by using EnzChek
gelatinase/collagenase kit (E-12055) from
Molecular Probe by measuring the
substrate fluorescence emission at 515
nm. 1,10-Phenanthroline was used as a
positive control and collagenase without
inhibitor was used as a negative control.
Results of this study are summarised in
Figure 3.
Collagen synthesis in sun damaged skin
appears to remain similar to that of sun-
protected sites, although collagen content
decreases.17 Thus, evidence suggests that
the decrease in collagen content in photo
aged skin results from increased collagen
degradation without significant changes in
production. It seems that the protection of
existing collagen is more important in
controlling the human ageing process than
stimulating collagen production.
Stromelysin 1 (MMP-3) inhibitory
activity: An inhibition of Stromelysin 1
activity by over 50% was observed with
Emblica antioxidant at 100 µg/ml13.
Quantification of MMP-3 inhibitory activity
of Emblica antioxidant was determined by
using Chemicon MMP-3/Stromelysin
Activity Assay Kit (ECM 481). The principle
of the assay is based upon fluorescence
measurement of substrate fragments
released upon cleavage of a substrate by
MMP-3. Fluorescence intensity of the
resulting product is measured and
correlated with MMP-3 activity. Result of
this study is summarised in Figure 4.
Clinical trials: Reduction in
UV-induced erythema
Reduction in UV-induced erythema was
used as a criterion for photoprotection and
reversal of photo damage by Emblica. The
following protocol was followed:
Human volunteers
– Eleven (Protocol A)/Ten (Protocol B).
Test sites
– Subject’s back, 4 x 2.5 cm area.
Test substances
– 0.2 and 0.5% levels (creams).
Application frequency
– 2 mg/cm2once a day.
Evaluation criteria – Represented by using
the individual typology angle (ITA˚);
Measured by chromometric measurement.
∆E ITA˚ was calculated by subtracting
treated irradiated site from the untreated
irradiated site (Fig. 5).
Protocol A (Prevention) – Products were
applied for eight days and then on day
nine, pigmentation were induced by UV
light on 11 humans. Product applications
once a day were continued for 10 days.
Comparisons of untreated irradiated
control vs the sites with products were
done on day 10.
Protocol B (Reversal) – Pigmentation was
induced by UV light on 10 humans and
then products were applied and continued
the application of product once a day
for 10 days. Comparisons of untreated
irradiated control versus the product-
treated sites were done every day.
A statistically significant difference in ITA˚
was observed on day five.
Results: The results are shown in Figure 5.
For protocol A, 0.2% Emblica antioxidant
and 0.5% Vitamin E showed statistically
significant (p<0.05) reduction in
erythema. For Protocol B, only 0.2%
Emblica showed statistically significant
(p<0.05) reduction in erythema on day
five. Vitamin E and MAP failed to show any
statistically significant reversal in erythema.
Formulation guidelines
Emblica antioxidant can be used in
formulations from 0.1 to 0.5% level with
acceptable aesthetics. Skin care
formulation with higher levels of Emblica
antioxidant (>0.5%, w/w) requires the use
of light fragrance. Essential oils such as
lavender, peppermint, lemon and juniper
may be added to mask the odour of
Emblica at ≤1.0%. As a skin lightener, the
preferred use level is from 1 to 2%.
Nonionic or anionic emulsifiers can be
used for making stable emulsions. Skin
penetration enhancers like lecithin can be
included to enhance the efficacy of
Emblica. Other skin lightening agents can
also be included to exploit synergism as
long as the formulation pH is acidic. pH
requirement, therefore, precludes inclusion
of magnesium or sodium ascorbyl
phosphates. However, ascorbyl glucoside
can be included in the formulation.
Formulations containing Emblica will be
off-white to white in appearance and which
will, over time, either darken or yellow,
especially at higher temperature. This
colour shift can be reduced or eliminated
by adding a small amount of sodium
metabisulfite (≤0.1%) along with ascorbic
acid (≤0.05%). The colour of the finished
product can also be slightly improved by
the use of a small amount (~0.1%) of
Licorice Extract (with ~20% Glabridin
content). At room temperature (~25˚C),
formulation holds colour pretty well for well
over one year. pH of the formulations must
be acidic (preferably, below 5.5) to
maintain its antioxidant activity and
stability. An opaque base is preferred over
a translucent one due to the improved
colour of the finished product. Emblica
antioxidant as a suspension in water can
be added to the formulation with a
moderate agitation at around 40˚C.
Prolonged heating or exposure to sunlight
must be avoided as it causes darkening of
formulated products. Several formulations
using Emblica antioxidant have been
developed in the author’s laboratory.
Summary
A properly defined and standardised
extract of Phyllanthus emblica fruit
(Emblica antioxidant) may provide great
value as a photoprotective agent
(in combination with sunscreens and
other anti-ageing ingredients), or a skin
lightening agent (along with other skin
lighteners). In short, the multiplicity of skin
beneficial effects, formulation benefits
seen with Emblica antioxidant and
unsurpassed safety of the product make it
SKIN CARE
16 January 2005
PERSONAL CARE
Figure 4: Stromelysin 1 (MMP-3) inhibitory activity
of Emblica antioxidant.
100
80
60
40
20
0
MMP-3 activity in % of control
Control
Concentration of Emblica in microgm/ml
100 200 300
Figure 5.
5
4
3
2
1
0
∆E of ITA degree
Protocol A (Prevention) Protocol B (Prevention)
0.2% EmblicaKey: 0.5% MAP 0.5% Vitamin E
SKIN CARE
an ideal choice for a variety of skin care
products targeting young and mature
skin alike.
References
1 Zhai H., Maibach H.I. Skin-Whitening Agents,
Cosm & Toil, 116, 21-25, 2001.
2 Zuidhoff H.W., van Rijsbergen J.M. Whitening
Efficacy of Frequently used Whitening
Ingredients, Cosm & Toil, 116, 53-59, 2001.
3 Levin C.Y., Maibach H. Exogeneous ochronosis.
An update on clinical features, causative agents
and treatment options, Am J Clin Dermatol, 2,
213-217, 2000.
4 DeCaprio A.P. The toxicology of hydroquinone -
relevance to occupational and environmental
exposure, Crit Rev Toxicol, 29, 283-330, 1999.
5 Takizawa T., Mitsumori K., Tamura T., Nasu M.,
Ueda M., Imai T., Hirose M. Hepatocellular
tumor induction in heterozygous p53-deficienr
CBA mice by 1 26-week dietary administration of
kojic acid, Toxicol Sci, 73, 287-293, 2003.
6 Tamura T., Mitsumori K., Onodera H., Fujimoto
N., Yasuhara K., Takegawa K., Takgi H., Hirose
M. Dose-threshold for thyroid tumor-promoting
effects of orally administered kojic acid in rats
after initiation with N-bis (2-hydroxypropyl)
nitrosoamine, J Toxicol Sci, 26, 85-94, 2001.
7 Chaudhuri R.K. Role of antioxidants in suncare
products, in Sunscreens: Regulations and
commercial development, Third edition, Ed N
Shaath, Marcel Dekker, New York, in press,
2004.
8 Pinnell S.R. Cutaneous phtodamage, oxidative
stress, and topical antioxidant protection,
J Am Acad Dermatol, 48, 1-19, 2003.
PC
9 Berneburg M., Plettenberg H., Krutmann J.
Photoaging of human skin, Photodermatol
Photoimmunol Photomed, 16: 239-244, 2000.
10 Wenk J., Brenneisen P., Meewes C., Wlaschek
M., Peters T., Blaudschwun R., Ma W., Kuhr L.,
Schneider L., Scharftetter-Kochanek K.
UV-Induced oxidative stress and photoaging,
in J Thiele and P. Elsner, Eds. Oxidants and
Antioxidants in Cutaneous Biology. Current Prob.
Dermatol. Basel, Karger, 29, 83-94, 2001.
11 Chaudhuri R.K., Marchio F. Hydrolyzable tannins
from Phyllanthus Emblica: A new class of safe
and effective skin lightening agent, PCIA
Conference Proceedings, Shanghai, China,
2003, pp 7-15.
12 Chaudhuri R.K., Puccetti G. Transition metal-
induced oxidation: Implications for skin care
products, 117, 43-56, 2001.
13 Chaudhuri R.K., Hwang C., Puccetti G., Guttierez
G., Serrar M. Low molecular weight tannins of
Phyllanthus Emblica: Anti-aging effects,
Cosmetics & Toiletries, 119, 59-70, 2004a.
14 Ghosal S., Triphati V.K., Chauhan S. Active
Constituents of Emblica officinalis: Part 1
– The Chemistry and Antioxidative Effects of Two
Hydrolysable Tannins, Emblicanin A and
Emblicanin B, Indian Journal of Chemistry, 35B,
941-948 (1996) and references cited therein.
15 Chaudhuri R.K. Phyllanthus tannins, in
Cosmeceuticals: Drugs vs Cosmetics, Second
edition, Eds P Elsner and H Maibach, Marcel
Dekker, New York, in press, 2004b.
16 Monograph on Emblic, Merck KGaA, Darmstadt,
Germany, 2001.
17 Bernstein E.F., Uitto J. The effect of
photodamage on dermal extracellular matrix,
PERSONAL CARE
January 2005 17
Clinics in Dermatology, 14: 143-151, 1996.
18 Chaudhuri R.K. Emblica cascading antioxidant:
A novel natural skin care ingredient,
Skin Pharmacol Appl Skin Physiol, 15, 374-380,
2002.
19 Kraljic I., El Moshni S. A new method for the
detection of singlet oxygen in aqueous solutions,
Photochem. Photobiol, 28, 577-582, 1978.
20 Gonzalez S., Pathak M.A. Inhibition of ultraviolet
– induced formation of reactive oxygen species,
lipid peroxidation, erythema and skin
photosentization by Polypodium leucotomos,
Photodermatol. Photoimmunol. Photomed.,
12, 45-56, 1996.
21 Paya M., Halliwell B., Hoult J.R.S. Interactions
of a series of coumarins with reactive oxygen
species, Biochem Pharmacol, 44, 205-214,
1992.
22 Halliwell B., Gutteridge J.M., Aruoma O.I.
The deoxyribose method: A simple “test tube”
assay for determination of rate constants for
reactions of hydroxyl radicals, Anal Biochem,
165, 215-219, 1987.
23 Cerutti P.A. Oxy-radicals and cancer, Lancet,
344, 862-863, 1994.
24 Bissett D.L., Mcbride J.F. Iron content of human
epidermis from sun-exposed and non-exposed
body sites, J Soc Cosmet Chem, 43, 215-217,
1992.
25 Graf E., Mahoney J.R., Byrant R.G.,
Eaton J.W. Iron-catalyzed hydroxyl radical
formation, stringent requirement for free iron
coordination sites, J Biol Chim, 259,
3620-3624, 1984.
26 Kato K., Terao S., Shimamoto N., Hirata M.
Journal of Medicinal Chemistry, 31, 793, 1988.
