Skin Lightening and Sebum Control Efficacy of a Cosmetic Emulsion Containing Extract of Tamarind Seeds on Asian Skin Type

Abstract

Cosmetic W/O emulsion containing 4% w/w tamarind (Tamarindus indica) seeds extract was formulated and investigated its dermatological effects on functional skin parameters like skin melanin and skin sebum contents. The study was performed using 11 male volunteers and consisted of a home used, single-blinded placebo controlled trial. Tamarind seeds extract was entrapped in the internal aqueous phase of the cosmetic emulsion. The test product (emulsion with tamarind seeds extract) and the placebo product (emulsion without tamarind seeds extract) was applied and gently massaged on each side of the cheeks for one minute twice daily (at night and morning) for a duration of 12 weeks and difference in skin color and skin sebum contents between test and placebo side of cheeks was measured fortnightly by using a Mexameter MPA5 and a Sebumeter MPA5 to evaluate the effects produced. We observed that signifi- cant (p ≤ 0.05) decrease in skin melanin and sebum contents when the test product was applied while the placebo showed insignificant (p > 0.05) increase in skin melanin and skin sebum contents. The newly for- mulated cosmetic emulsion containing extract of tamarind seeds can therefore be used safely as skin se- bum control and skin lightening agent on Asian skin type

Full text

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KEY WORDS: cosmetic emulsion, mexameter, sebumeter, skin lightening, Tamarindus indica.
* Author to whom correspondence should be addressed. E-mail: barki.gold@gmail.com
Latin American Journal of Pharmacy
(formerly Acta Farmacéutica Bonaerense)
Lat. Am. J. Pharm. 34 (3): 570-5 (2015)
Regular article
Received: September 5, 2014
Revised version: January 28, 2015
Accepted: January 29, 2015
Skin Lightening and Sebum Control Efficacy of a Cosmetic Emulsion
Containing Extract of Tamarind Seeds on Asian Skin Type
Muhammad K. WAQAS 1, Naveed AKHTAR 1, Sattar BAKHSH 2,
Eduardo J. CALDEIRA 3& Barkat A. KHAN 2,4 *
1Department of Pharmacy, Faculty of Pharmacy & Alternative Medicine,
The Islamia University of Bahawalpur, Punjab, Pakistan
2Department of Pharmaceutics, Faculty of Pharmacy, Gomal University D.I Khan, KPK, Pakistan
3Tissue Morphology Laboratory, Department of Morphology and Basic Pathology,
Faculty of Medicine of Jundiaí, FMJ, Jundiaí, São Paulo, Brazil
4School of Pharmacy, Kampala International University-WC, Uganda
SUMMARY. Cosmetic W/O emulsion containing 4% w/w tamarind (Tamarindus indica) seeds extract was
formulated and investigated its dermatological effects on functional skin parameters like skin melanin and
skin sebum contents. The study was performed using 11 male volunteers and consisted of a home used,
single-blinded placebo controlled trial. Tamarind seeds extract was entrapped in the internal aqueous
phase of the cosmetic emulsion. The test product (emulsion with tamarind seeds extract) and the placebo
product (emulsion without tamarind seeds extract) was applied and gently massaged on each side of the
cheeks for one minute twice daily (at night and morning) for a duration of 12 weeks and difference in skin
color and skin sebum contents between test and placebo side of cheeks was measured fortnightly by using
a Mexameter MPA5 and a Sebumeter MPA5 to evaluate the effects produced. We observed that signifi-
cant (p≤ 0.05) decrease in skin melanin and sebum contents when the test product was applied while the
placebo showed insignificant (p> 0.05) increase in skin melanin and skin sebum contents. The newly for-
mulated cosmetic emulsion containing extract of tamarind seeds can therefore be used safely as skin se-
bum control and skin lightening agent on Asian skin type.
RESUMEN. Una emulsión cosmética W/O conteniendo 4% w/w de extracto de semillas de tamarindo (Tamarin-
dus indica) se formuló e investigaron sus efectos dermatológicos en la piel con parámetros funcionales tales co-
mo contenido de melanina y de sebo de la piel. El estudio se realizó con 11 voluntarios masculinos y consistía en
un ensayo casero de simple ciego controlado con placebo. El extracto de semillas de tamarindo fue atrapado en
la fase acuosa interna de la emulsión cosmética. El producto de prueba (emulsión con semillas de de tamarindo)
y el producto placebo (emulsión sin extracto de semillas de tamarindo) se aplicó con suave masaje en cada lado
de las mejillas durante un minuto dos veces al día (por la noche y por la mañana) con una duración de 12 sema-
nas y la diferencia en el contenido de color de la piel y sebo de la piel entre la prueba y placebo a cada lado de las
mejillas se midió cada dos semanas usando un MPA5 Mexameter y un Sebumeter MPA5 para evaluar los efectos
producidos. Hemos observado una disminución significativa (p≤ 0,05) en la melanina de la piel y el contenido
de sebo cuando se aplicó el producto de prueba, mientras que el placebo mostró aumento insignificante (p >
0,05) del contenido de melanina y sebo de la piel. Por consiguiente, la emulsión cosmética de nueva formulación
que contiene extracto de semillas de tamarindo se puede utilizar de forma segura como control de sebo de la piel
y agente de aclaramiento de piel del tipo asiática.
INTRODUCTION
Emulsions are thermodynamically unstable
systems which split into two distinguishable
phases. The instability is manifested by a num-
ber of processes such as flocculation, sedimen-
tation or creaming, phase inversion or coales-
cence that would destabilize them. Oil-in-water
or water-in-oil emulsions are the examples of
colloidal systems that are frequently used now a
day in various fields as pharmaceuticals, cosmet-
ics, paints, food and petrochemicals etc. All
these emulsions evolve gradually with time 1. A
wide variety of cosmetic emulsions are used as
bases for skincare products for healthy and dis-
eased skin. These products can range in consis-
tency from a cream to a lotion or body milk and
even a fluid for normal, oily or dry skin 2. Wa-
ter-in-oil emulsions consists of the water phase,
ISSN 0326 2383 (printed ed.)
ISSN 2362-3853 (on line ed.)

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Latin American Journal of Pharmacy - 34 (3) - 2015
which is internal /dispersed phase, mixed with
oil, which is continuous phase .This emulsion
type is often more difficult to prepare and stabi-
lize since it is most often based on totally non-
emulsifiers. However recent advances in silicon
chemistry and polymer chemistry have allowed
preparation of excellent water-in-oil (W/O)
emulsions. A real benefit of these vehicle emul-
sions is that they are readily spread on to the
lipophilic skin and provide a film which is resis-
tant to water wash off 3.
Plants represent a class of frequently used
active agents in cosmeceuticals, containig ter-
penoids, alkaloids, and phenolics, which have
been chemically characterized for their biologic
effects. Herb based products usually also pro-
vide multiple functionalities and stable formula-
tions of highly reactive ingredients such as an-
tioxidants. Plant extracts have been used for
centuries and are present in today’s products ei-
ther for their own properties or as substitutes of
animal materials that may have to be removed
from products because of pressure from animal
rights associations or diseases like bovine
spongiform encephalopathy 4. Plant secrets have
been passed down through generations as
herbal folklore, and now-a-days botanical ex-
tracts are playing an increasingly important role
in cosmetics. For the cosmetics industry, isola-
tion and purification of the active ingredient
within the crude extract are sometimes not
needed because such isolation and purification
may lead to a loss in the biological activity 5.
Tamarindus indica L. seeds are important
sources of antioxidant activity as 2-hydroxy-
3’,4’-dihydroxyacetophenone, metdihydroxyben-
zoate,3,4dihydroxyphenylacetate and (-)-epicat-
echin, in addition to oligomeric proanthocyani-
dins (OPC). OPCs are potent antioxidant, anti-
inflammatory, antihistaminic agent and ultravio-
let protection. OPCs also stabilize elastin, colla-
gen and ground substances 6. The detailed study
of the composition of Tamarindus indica L.
seeds will bring contribution to health profes-
sionals not only regarding its composition and
to evaluate its antioxidant potential, fatty acid
profile and content of tocopherols. Biological
activity assessment of tamarind seed was report-
ed on the radical scavenging, lipid peroxidation
reducing and anti-microbial activities including
anti-inflammatory potential. These findings led
to the interest in using the extract derived from
the seeds of tamarind for cosmetics 7.
In this study dermatological evaluation of
test product (emulsion with tamarind seeds ex-
tract) and placebo product (emulsion without
tamarind seeds extract) was done for its effects
on various skin functional parameters.
MATERIALS AND METHODS
Materials
Tamarindus indica seeds were obtained
from a local market of Bahawalpur, Pakistan
and authenticated by the CIDS (Cholistan Insti-
tute of Desert studies), The Islamia University of
Bahawalpur, Pakistan. For future reference, a
voucher specimen (Voucher no. TI- SD-6-15-87)
has been kept in the herbarium at CIDS, The Is-
lamia University of Bahawalpur, Pakistan. All
materials were used as received and were cos-
metics grade. ABIL EM®90 was purchased from
the Franken Chemicals Germany, n. Hexane &
paraffin oil was purchased from Merk KGaA
Darmstadt (Germany). Ethanol & acetone were
taken from BDH England. Distilled water was
prepared in the Cosmetics Laboratory, Depart-
ment of Pharmacy, The Islamia University of Ba-
hawalpur, Pakistan.
Plant material and preparation
of the extract
The crushed seeds were extracted with hex-
ane-ethanol-acetone (50:25:25) for 30 min, in a
1:3 seed: solvent ratio, under continuous agita-
tion at room temperature. The mixture was 1st
filtered through 8 layers of muslin cloth and
then filtered through Whatman No.1 filter paper.
It was then subjected to rotary evaporation un-
der pressure reduced to 40 °C for solvent re-
moval. Concentrated extract was stored in refrig-
erator for further studies.
Preparation of cosmetic water-in-oil
emulsions
Oily phase composed of paraffin oil (14%)
and emulsifier Abil-EM®90 (2.5%) were heated
together up to 75 ± 1 °C. At the same time dis-
tilled water (quantity sufficient to make 100%)
was heated at the same temperature and then
4% tamarind seeds extract was added in it. After
that, aqueous phase was added to the oil phase
drop by drop. Stirring was continued at 2000
rpm by the mechanical mixer for about 15 min
until complete aqueous phase was added, 2 to 3
drops of rose oil were added during this stirring
time to give good fragrance to the formulation.
After the complete addition of the aqueous
phase, the speed of the mixer was reduced to
1000 rpm for homogenization, for a period of 5
min and then the speed of the mixer was fur-

572
WAQAS M.K., AKHTAR N., BAKHSH S., CALDEIRA E.J. & KHAN B.A.
ther reduced to 500 rpm for 5 min for complete
homogenization until the emulsion cooled to
room temperature. For the placebo product, the
preparation process was the same as the above
except the addition of plant extract. Both test
and placebo products were found stable after
evaluating pH, centrifugation, electrical conduc-
tivity, phase separation, organoleptic and physi-
cal characteristics (color, creaming and liquefac-
tion) and the temperature stability tests by keep-
ing the emulsions to storage at 8 ± 0.5, 25± 0.5,
40 ± 0.5 °C, and at 40 ± 0.5 °C with 70% RH
(relative humidity) for the period of three
months.
Skin irritation assessment
To determine the skin irritation assessment,
Patch tests were performed on forearms of each
volunteer. An area of 5 ×4 cm was marked on
both forearms of all the volunteers. Basic values
for erythema and melanin were measured with
the help of Mexameter. Approximately 1.0 g of
test and placebo was applied to the 5 ×4 cm
marked regions separately on each forearm. Sur-
gical dressing was used to cover the marked
area of right and left forearm. After 48 h, it was
removed and the forearms were washed with
physiological saline and were observed for any
skin redness/irritation by a dermatologist. The
measurements of erythema and melanin were
repeated on both forearms.
Non-invasive techniques
The evaluation of skin sebum and skin
melanin contents was done by using Mexameter
and Sebumeter MPA 5 (Courage + Khazaka
Electronic GmbH, Germany.)
Study design
The study was designed as a home-use, sin-
gle-blind, randomized and placebo controlled
trial. The study was conducted in accordance of
Helsinki Declaration and was consistent with
Good Clinical Practice guidelines. The protocol
was approved by the Board of Advanced Stud-
ies and Research (BASR) and institutional ethical
committee for in vivo studies (Reference No.
4710/Acad.). Written informed consent was ob-
tained from all participating volunteers.
Subjects
Eleven volunteers were selected whose ages
were 25-35 years. Exclusion criteria included ex-
posure to topical steroid, volunteers who had
skin disease or wounds on the face, a history of
atopic dermatitis, skin hypersensitivity reaction
or a history of allergic reactions to cosmetic in-
gredients.
Application of the products
Each volunteer received two products (test
and placebo). Prior to the tests, a dermatologist
for any serious skin disease or damage especial-
ly on cheeks and forearms examined the volun-
teers. They were asked to apply one type of
product on one side of their cheeks and the
other on another side of cheek twice daily for
12 weeks. Volunteers were asked to use ap-
proximately 1 g (i.e., 0.5-1.5 g) of each product,
gently massaging it in for one minute. During
the study period they were not allowed to con-
tinue using their personal cosmetic products.
Values for different parameters were taken in
controlled room temperature 25 ± 1 °C and 45 ±
2 % relative humidity. The site of measurement
was the whole cheek. Every volunteer was in-
structed to come for measurement on 2nd, 4th,
6th, 8th, 10th, and 12th week. Values for each pa-
rameter was measured three times (n = 3) and
average of the values has been noted.
Mathematical analysis
The percentage changes for the individual
values of different parameters, taken every
week of volunteers were calculated by the fol-
lowing formula: Percentage Change = [(A – B) /
B] ×100, where A = individual value of any pa-
rameter of 2nd, 4th, 6th, 8th, 10th and 12th week
and B= zero hour value of that parameter
Statistical analysis
The evaluation of data was done by using
SPSS version 19.0 according to two-way ANOVA
for variation between different time intervals
defining a 5% level of significance and Paired
samples t-test for variation between the two
preparations. Standard error of means (SEM)
was calculated for every mean value.
RESULTS AND DISCUSSION
Skin melanin contents
Melanin is responsible for the diversity in hu-
man skin tones. Darker skin does not contain
more melanocytes, the cells are simply more ac-
tive. Variation in human skin colour is mainly
due to the presence of four pigments, namely,
Melanin, Haemoglobin, Carotene and Melanoid.
Pigmentation of the skin is controlled by hor-
mones which are synthesized and distributed by
the pituitary gland 8.

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Latin American Journal of Pharmacy - 34 (3) - 2015
The metabolic pathway involved in melanin
synthesis is extremely complicated involving
several intermediate steps. It starts with the
amino acid tyrosine oxidized by the copper
containing enzyme tyrosinase to dihydrox-
yphenylalanine (DOPA) and then to
dopaquinone. Dopaquinone undergoes a series
of non-enzymatic reactions and rearrangements
forming the different molecules that are copoly-
merized to make one of the two types of
melanin, Eumelanin, which is the dark brown/
purple/ black compound found in skin/hair and
phaeomelanin, which is yellow to reddish
brown pigment present in red hair and rarely in
human skin. Both forms of melanin combine
with other proteins to form the melanosome
that is distributed from the melanocytes to the
surrounding cells.
Maturation is categorized in four stages:
namely stages I and II include un-melanized im-
mature pre-melanosomes, while melanized
melanosomes are classified as stages III and IV.
Activation of the melanocortin I receptor pro-
motes the synthesis of eumelanin at the expense
of phaeomelanin, although oxidation of tyrosine
by tyrosinase is needed for synthesis of both the
types of pigments. Both the light and the dark
skinned individuals have similar number of
melanocytes for the same body region, but
melanosomes that contain the pigment are more
numerous and more pigmented in darker peo-
ple than in light skinned people 9. Melanin
biosynthesis can be inhibited by avoiding ultra-
violet (UV) exposure by the inhibition of
melanocyte metabolism and proliferation. Appli-
cation of tyrosinase inhibitors may be the least
invasive procedure for maintaining skin white-
ness; such agents are increasingly used in cos-
metic products. Thus, the inhibition of melano-
genesis has been the focus on medicinal and
cosmetic treatments for skin depigmenting and
lightening.
Ultraviolet (UV) radiation can increase the
melanization and the proliferation of
melanocytes by acting on melanocytes directly
or indirectly through the release of keratinocyte
derived factors. UV radiation also induces the
formation of reactive oxygen species (ROS) in
the skin; these ROS assist melanin biosynthesis
and damage DNA and then may induce the pro-
liferation and/or apoptosis of melanocytes.
H2O2, which is one of the ROS generated, caus-
es an increase in the level of tyrosinase mRNA.
The dose of UV irradiation seems to regulate
and control the UV-induced proliferation of
Figure 1. Percentage changes in skin melanin con-
tents after application of placebo and test product.
melanocytes 10. It is reported that UV radiation
induced proliferation and melanogenesis of
melanocytes was reduced by the topical appli-
cation of antioxidants such as vitamins C and E
to the skin of hairless mice. UV irradiation in-
duced 8-OHdG (a representative DNA base-
modified product generated by ROS) within
DNA of cultured mouse keratinocytes. It also in-
duced the proliferation of keratinocytes in hu-
man skin 11. Therefore, ROS are considered to
play important role in regulating the prolifera-
tion of melanocytes as well as keratinocytes and
melanogenesis of melanocytes, while ROS scav-
engers and inhibitors of ROS production, such
as antioxidants, may reduce hyperpigmentation
or prevent new UV-induced melanogenesis 12.
In this study, the placebo improves the skin
melanin contents but in case of test product
there is regular decline in skin melanin values
throughout the study duration (Fig. 1). With the
help of two-way ANOVA test defining a 5% lev-
el of significance, it was observed that the
placebo insignificantly (0.05 < p) increased the
skin melanin values while in case of test prod-
uct there was significant (p ≤ 0.05) decrease in
skin melanin contents with respect to time. By
applying paired sample t-test, it was observed
that test product presented significant (p≤ 0.05)
effects with respect to placebo. The increase in
melanin content by the placebo may be credited
to presence of paraffin oil. The preparations
containing paraffin oil found to increase the
melanin level in the skin when applied topical-
ly.
The reduction in the skin melanin content
by the formulation may be attributed to the
presence of phenolic antioxidants, such as 2-hy-
droxy-30, 40-dihydroxyacetophenone, methyl
3,4-dihydroxybenzoate, 3,4-dihydroxyphenyl ac-
etate and epicatechin. Extracts exhibit antioxi-

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WAQAS M.K., AKHTAR N., BAKHSH S., CALDEIRA E.J. & KHAN B.A.
dant potential by reducing lipid peroxidation in
vitro. The content of tamarind seeds comprised
only procyanidins, represented (%) mainly by
oligomeric procyanidin tetramer (30.2), pro-
cyanidin hexamer (23.8), procyanidin trimer
(18.1), procyanidinpentamer (17.6) with lower
amounts of procyanidin B2 (5.5) and (-)-epicate-
chin (4.8) and possesses antioxidant activity. It
has been reported that antioxidant activity of
these phenolic compounds is stronger than vita-
min C and vitamin E .So they may reduce free
radical and indirectly decrease melanin produc-
tion 13. Proanthrocyanidines inhibited the activi-
ty of tyrosinases obtained from mushroom and
from B16 mouse melanoma cells and decreased
the melanin content of the cells 10. The further
study needs to conduct to test the extract with
melanoma cell line.
Skin sebum contents
Sebaceous glands are multilobular holocrine
glands generally associated with hair follicles.
The basal sebocytes sit on a basal membrane at
the outer limits of the lobes, and as cells move
from the basal layer toward the lumen of the
gland they synthesize lipids, which accumulate
as intracellular lipid droplets. As they synthesize
lipid, the cells become larger, and the nucleus
and other internal organelles are degraded. Ulti-
mately, the entire mass of the cell is converted
into a viscous liquid phase lipid mixture 14. In
most pilosebaceous units, sebum passes from
the sebaceous gland into the hair follicle via the
short sebaceous duct and outward onto the skin
surface through the follicle. Generally, the hair
follicle is large compared with the associated se-
baceous gland; however, large sebaceous glands
Figure 2. Percentage changes in skin sebum contents
after application of placebo and test product.
are associated with vellous hairs. These units
are called sebaceous follicles and predominate
on the forehead and cheeks. Human sebum
from isolated sebaceous glands consists mainly
of squalene, wax esters and triglycerides with
small proportions of cholesterol and cholesterol
esters 15.
There is a clear positive correlation between
the occurrence and severity of acne and the se-
bum secretion rate. It has been suggested that
the development of acne may result from essen-
tial fatty acid deficiency localized to the follicu-
lar epithelium. In experimental systemic essen-
tial fatty acid deficiency, the skin becomes scaly
and more permeable. If sebaceous fatty acids
were to penetrate into the follicular epithelial
cells and compete with linoleic acid from the
circulation for incorporation into lipids, a local-
ized essential fatty acid deficiency could be pro-
duced. The resulting scaling could lead to
comedo formation, and the defective barrier
function would facilitate exchange of materials
between the follicle and surrounding tissue.
This would include an influx of water and nutri-
ents into the follicle to support bacterial growth
as well as the eflux of inflammatory mediators
16. Skin sebum contents was evaluated fort-
nightly on the 2nd, 4th, 6th, 8th, 10th, and 12th
week by Sebumeter MPA 5 and the percentage
of changes are presented in Fig. 2.
The photometric method is the measurement
principle of Sebumeter MPA 5. A porous poly-
meric tape called Sebutape is coated with a
weak adhesive sufficient to hold it in contact
with the skin. As sebum is secreted from the ori-
fice of a follicle, it is adsorbed into the pores in
the polymer, and this turns the appearance of
the tape from opaque to transparent. The light
transmission represents the sebum content on
the surface of the measuring area. A micropro-
cessor calculates the result, which is shown on
the display in values from 0-350 17. In this study
the increased sebum contents was observed by
the placebo product which can be due to oily
nature of placebo having paraffin oil in it, but in
case of test product there was decline observed
in skin sebum content throughout the study pe-
riod. By applying ANOVA test, it was found that
placebo increased sebum insignificantly (p>
0.05) while the test product had a significant (p
≤ 0.05) decrease with respect to time. Paired
sample t-test showed that test produced signifi-
cant effects in comparison with placebo. The
decline in sebum content shown by the test
product can be attributed due to the presence of

575
Latin American Journal of Pharmacy - 34 (3) - 2015
unsaturated fatty acids present in Tamarind
seeds which include linoleic acid and oleic acid
18. Topical application of linoleic acid has
shown to inhibit sebum production due to se-
lective inhibition of 5α-reductase, an enzyme
found in sebaceous glands responsible for se-
bum production. There are two isozymes, type1
and type 2 for 5α-reductase. The linoleic acid
suppresses both types 19.
CONCLUSION
A stable cosmetic water-in-oil emulsion en-
trapped with 4% tamarind seed extract exerts
skin lightening effects as it significantly reduced
the skin melanin level on topical application as
well as treatment with cosmetic formulation re-
sulted in significant sebum reduction. Decrease
in skin sebum level suggests that the formula-
tion can be helpful in conditions like acne. Out
of these reasons tamarind seed extract can be
regarded as an effective and economical cos-
metic ingredient for topical skin-whitening and
sebum reducing agent.
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