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Evaluation of the antioxidant efficacy of extracts/
ingredients used in skin care products
Nik Nur Azwanida Zakaria1,2, Edward Jonathan Okello1*, Olusola Clement Idowu3
1Human Nutrition Research Centre, Institute of Cellular Medicine (ICM), Newcastle University, Newcastle Upon Tyne NE2
4HH, UK
2Faculty of Agrobased Industry (FIAT), Universiti Malaysia Kelantan, 17600 Jeli, Kelantan, Malaysia
3Hexis Lab Limited, Science Central, The Core, Bath Lane, Newcastle Upon Tyne NE4 5TF, UK
Introduction
The human skin is continuously exposed to various
environmental insults (cigarette smoke, pollutants, infrared
radiation, ultraviolet radiation etc) that are damaging to
the skin, leading to skin aging. The characteristics of aged
skin such as wrinkles, pigmentation, sagging, dryness and
dullness are aesthetically undesirable.1,2 Many strategies
and therapies for skin aging have been developed and
topical application of cosmetic products with claimed
benefits (anti-aging, anti-wrinkle etc.) has been one of
the most popular interventions by consumers as they
are perceived to be cost effective, less invasive and with
minimal risk of side effects.3
Cosmetic products with claimed benefits are commonly
termed as “cosmeceuticals”, a word coined by merging
the word cosme-(tic) and (pharma)-ceutical, to describe
“active” and science based cosmetic.4,5 The actives in
the product formulation are expected to deliver their
functions especially after mixing with other ingredients
in the product formulation. Some of the commonly used
actives are antioxidants.6–9 The use of antioxidants in
product formulation is due to understanding the role of
oxidative stress in the aging of the skin.10–14 In this study,
we evaluated the antioxidant activities of 24 extracts/
compounds used in skin care formulations and their
ability to retain their efficacy in product formulation.
Methods
Samples
All the 24 commercial extracts and the cream base were
International Journal of Phytocosmetics
and Natural Ingredients 2018;5:7 Original Article
doi 10.15171/ijpni.2018.07
Please cite this paper as: Zakaria NNA, Okello EJ, Idowu OC. Evaluation of the antioxidant efficacy of extracts/
ingredients used in skin care products. Int J Phytocos Nat Ingred. 2018;5:7. doi:10.15171/ijpni.2018.07.
Abstract
The so-called active ingredients in skin care product formulations are purported to deliver the intended
functions of the product. Active ingredients, such as the antioxidants can efficiently protect the skin if
the activities are retained after incorporating into the base matrices in the product formulation. Here,
we investigated the antioxidant activities of 24 extracts/compounds that are being used in skin care
formulations and their ability to retain the activities (efficacy) after being mixed with the base matrices.
The antioxidant activities were evaluated using 2, 2’-azino-bis (3- ethylbenzothiazoline-6-sulphonic acid
(ABTS) and diphenyl-picryl hydrazine (DPPH) assays. To test the efficacy of the samples in formulation,
the samples were mixed with base cream 1%, 2% and 4% (w:w) and allowed to settle overnight. Results
were expressed as either percentage inhibition (%) or IC50 (µg/mL). Out of the 24 samples, ten exhibited
significantly high antioxidant activities with resveratrol> pomegranate> green tea> mango> amla>
bearberry> ellagic acid> tetrahydrocurcuminoid> Rhodiola rosea> kakadu plum in the ABTS assay.
In the DPPH assay, green tea> pomegranate> tetrahydrocurcuminoid> mango> amla> resveratrol>
bearberry> Rhodiola rosea>kakudu plum>ellagic acid. Four out of the ten samples (amla, green tea,
mango and pomegranate extracts) had IC50 value lower than Trolox standard and were included in
the efficacy test. Trolox standard and amla extract seemed to retain their antioxidant activities in the
formulations, while green tea and pomegranate extracts had a decrease in activities. Only mango extract
had a synergistic effect with the cream base with higher antioxidant activity observed compared with the
extract alone. This study demonstrates the potential interaction between active and vehicle compounds,
which may hinder or enhance the activities of the active ingredients in the final product. The outcome
of the research has an impact in the cosmetic product formulation particularly in the quality control,
chemistry and efficacy of the finished products.
Keywords: Antioxidant, Cosmetic, Active ingredients, ABTS, DPPH, Mango extract
Correspondence to
Edward Jonathan Okello
Email:
Edward.Okello@ncl.ac.uk
Received 27 July 2017
Accepted 17 Oct. 2018
ePublished 27 Nov. 2018
Zakaria et al
http://www.ijpni.org
International Journal of Phytocosmetics and Natural Ingredients 2018, 5:7
2
Efficacy of extracts used in skin care products
supplied by Hexis Lab Limited, UK.
Sample Preparation
Samples were dissolved in either water or ethanol at
4000 µg/mL. The samples dissolved in water were willow
bark extract, green tea extract with EGCG, fucoidan,
pomegranate extract, pullulan botanical extract, amla
extract, Rhodiola rosea extract, mango extract and galangal
extract. The samples dissolved in ethanol were bearberry
extract, kakadu plum extract, tetrahydrocurcuminoid,
sea buckthorn oil, resveratrol, Moringa oleifera extract,
Centella asiatica whole extract and Centella asiatica
with 2 % asiaticoside. The samples dissolved in
ethanol were bearberry extract, kakadu plum extract,
tetrahydrocurcuminoid, sea buckthorn oil, resveratrol,
Moringa oleifera extract, Centella asiatica whole extract,
Centella asiatica with 2 % asiaticoside, oleuropein 40%,
nobiletin, aloe extract and L-carnosine.
Antioxidant Assays
A working solution of 1000 µg/mL diluted from the
stock was used in the screening assay. The antioxidant
assays were followed from previous methods with some
modifications as described below.15
2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid
(ABTS) Assay
ABTS radical was pre-formed from the overnight 1:1
chemical reaction of 15 mM ABTS and 5 mM potassium
persulfate. The ABTS stock was diluted 1:50 with 5 mM
PBS, pH 7.4 to obtain 50 mL ABTS working solution. In
the assay, either Trolox standard solutions (10 µL), plant
samples (10 µL) or 70% ethanol (10 µL) for controls were
thoroughly mixed with ABTS working solution (290 µL)
in assay wells. The microplate was subsequently incubated
in the dark at 37°C for 6 minutes. All experiments were
performed in triplicate. The absorbance was determined
using a microplate reader, SpectraMax Plus384, Molecular
Device Corporation, at 734 nm. Background absorbance
was corrected by subtracting the absorbance value of
blank (water/ 70% ethanol).
Diphenyl-Picryl Hydrazine (DPPH) Assay
DPPH was diluted in 100% methanol at 0.24 mg/mL and
allowed to settle overnight at 4°C. The stock was diluted
at 1:5 with 50% methanol to obtain the DPPH working
solution.15 µL each of Trolox standards, plant samples
and solvents (water or 70% ethanol) as control wells were
mixed with 285 µL of DPPH working solution in the assay
wells. The 96 wells plate was incubated in the dark at 30 °C
for 30 minutes. After incubation, the microplate was read
at the wavelength of 517 nm using a microplate reader
SpectraMax Plus384 (Molecular Device Corporation).
Background absorbance was corrected by subtracting the
absorbance value of blank (water/ 70% ethanol).
The ABTS and DPPH percentage inhibition were
calculated as:
Percentage inhibition (%) = (1-(S/C)) x100;
S=corrected absorbance value for sample and
C=corrected absorbance value for control
IC50 Determination
Samples with high antioxidant activities were prepared at
lower concentrations to evaluate IC50 value. The IC50 value
was determined from the best fitted line for each sample
(R2≈1.00).
Efficacy Test
Four of the 24 tested samples were selected for the efficacy
test. The selection was based on the samples with IC50
lower than Trolox standard in both assays to ensure the
antioxidant activities exhibited were independent of the
free radicals introduced. The selected actives were mixed
with the base cream at 1%, 2% and 4% active: cream (w:w).
The mixtures were allowed to settle overnight at 4°C.
ABTS and DPHH assays were performed as described
above. The cream base supplied consisted of mixtures of
aqua, Helianthus annuus oil, cetyl alcohol, glycerin, Cocos
nucifera oil, PEG-100 stearate, cetearyl alcohol, Prunus
amygdalus oil, Simmondsia chinensis, Theobroma cacao
butter, Chondrus crispus, benzyl alcohol, phenoxyethanol,
potassium sorbate and tocopherol. The final concentration
of the active at 1, 2 and 4% formulation in the ABTS assay
were 1.3 µg/mL, 2.6 µg/mL and 5.2 µg/mL. The final
assay concentration of the active at similar percentage
formulations in the DPPH assay were 2 µg/mL, 4 µg/mL
and 8 µg/mL. For comparison, sample alone at similar
concentration for each formulation was investigated.
Statistical Analysis
ANOVA was used to compare the mean differences
between samples and P < 0.05 was considered as significant.
Results
Screening
Significantly high antioxidant activities were observed
with the following extracts/ compounds; amla extract,
bearberry extract, ellagic acid, green tea with 90% EGCG,
kakadu plum extract, mango extract, pomegranate extract,
resveratrol, Rhodiola rosea extract, tetrahydrocurcuminoid
and oleuropein 40%), whereas low to intermediate
antioxidant activities were observed for: Centella asiatica
whole extract, Centella asiatica with 2% asiaticoside
and Moringa oleifera leaf extract’ and low to no activity
for fucoidan, nobiletin, galangal extract, kojic acid
dipalmitate, niacinamide, pullulan, sea buckthorn oil, aloe
extract, L-carnosine and willow bark extract (Table 1).
Comparison of IC50 (ABTS and DPPH Assay)
Ten samples with 80-90 % radical’s inhibitions for both
assays (amla extract, bearberry extract, ellagic acid, green
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Zakaria et al
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tea with 90% EGCG, kakadu plum extract, mango extract,
pomegranate extract, resveratrol, Rhodiola rosea extract
and tetrahydrocurcuminoid) were evaluated for potency.
In Figure 1, samples labelled 1, 2, 3, and 4 (amla extract,
green tea extract, mango extract and pomegranate extract)
had higher potency, observed from lower IC50 than Trolox
standard in both assays. The four samples were further
evaluated for efficacy in cream matrices.
Efficacy Test (1%, 2% and 4% Active: Cream Formulation)
1% Formulation. Figure 2 shows significant increase in
antioxidant activities for mango extract mixed with cream
in both assays. However, green tea and pomegranate
extract in cream had a decrease in activities in both
Table 1. Antioxidant Activities of Actives
Samples ABTS Percentage
Inhibition (%)
DPPH Percentage
Inhibition (%)
High Activity
1. Amla extract 99.1 ± 0.07 93.9 ± 0.20
2. Bearberry extract 99.6 ± 1.30 91.2 ± 0.35
3. Ellagic acid 98.6 ± 0.08 91.0 ± 0.34
4. Green tea with 90% EGCG 99.6 ± 0.03 93.2 ± 0.40
5. Kakadu plum extract 95.7 ± 4.50 90.6 ± 1.80
6. Mango extract 98.1 ± 1.16 88.9 ± 0.21
7. Pomegranate extract 99.1 ± 0.07 93.3 ± 0.08
8. Resveratrol 98.5 ± 0.76 90.3 ± 0.87
9. Rhodiola rosea extract 86.9 ± 1.48 89.4 ± 0.15
10. Tetrahydrocurcuminoid 98.7 ± 0.44 90.8 ± 1.14
11. Oleuropein 40% 99.1 ± 0.04 53.4 ±1.67
Low-Intermediate Activity
12. Centella asiatica whole
extract 12.0 ± 0.91 10.5 ± 0.20
13. Centella asiatica with 2%
asiaticoside 14.5 ± 0.87 5.72 ± 1.02
14. Moringa oleifera leaf extract 43.3 ± 3.22 17.2 ±4.30
Low to no Activity
15. Fucoidan (brown seaweed) 10.2 ± 2.37 -5.00 ±1.10
16. Nobiletin 9.76 ± 0.41 -4.22 ± 0.94
17. Galangal extract 2.36 ± 4.71 7.12 ±4.85
18. Kojic acid dipalmitate 0.37 ± 1.91 -2.00 ± 1.27
19. Niacinamide -1.61 ± 1.06 -2.77 ± 5.28
20. Pullulan 2.51 ± 2.33 -4.37 ± 2.17
21. Sea buckthorn oil 4.43 ± 8.36 -14.88 ± 2.61
22. Aloe extract 2.82 ± 2.59 0.91 ± 2.64
23. L-carnosine 4.95 ± 0.9 -2.94 ± 7.36
24. Willow bark extract 1.35 ± 1.17 -6.90 ± 4.90
The samples were grouped into high, low-intermediate and low to no
activities. The samples were tested at 1000 µg/ mL with final concentrations
of 3.3 and 2.0 µg/mL for ABTS and DPPH assay respectively. High = 80%-
90% inhibition, low-intermediate = 10-50 % and low-no activity = below
10%. Data are mean ± SD (n = 4).
Figure 1: Comparison of IC50 Values of Selected Samples. Samples
labelled 1, 2, 3 and 4 had lower IC50 than Trolox standard in both assays
and were further evaluated for efficacy.
assays. Trolox and amla extract were either retained or had
increase in antioxidant activities in the formulation.
2% Formulation. In a 2% formulation (Figure 3), Trolox
standard retained its activity, but green tea had lower
activity when mixed with cream in both assays. Amla,
mango and pomegranate extract retained their activities
in only one assay. Mango extract had significantly higher
antioxidant activity when in cream, while amla and
pomegranate had lower antioxidant activities when in
cream.
4 % Formulation. In a higher percentage formulation,
mango extract consistently had higher antioxidant
activities in both assays (Figure 4), while Trolox and
amla extracts retain their activities. Green tea with 90%
EGCG and pomegranate extracts were not included at 4%
formulation because the assay’s limit was reached for both
samples based on their IC50 value (Figure 1).
Discussion
Common approaches in the cosmetic product
development focus on the inclusion of “actives”, such as
botanical extracts or pure compounds in the product
formulation with intended effects to promote healthy skin
and/or manage the characteristics of aged skin. Actives,
such as antioxidants are believed capable of diminishing
the oxidative damages caused by free radicals, potentially
by restoring the balance between endogenous antioxidants
and the oxidants.7,9,16
In this study, we demonstrated that botanical extracts
possessed high antioxidant capacities as pure compounds
(resveratrol and tetrahydrocurcuminoid) (Table 1).
However, some of the extracts (Centella asiatica, Moringa
oleifera, galangal, willow bark, aloe, fucoidan, sea
buckthorn oil) had low or no activity, may be due to the
extraction methods used to prepare the samples.17–20 The
other samples may not be active as antioxidants, such as
kojic acid dipalmitate and nobiletin which are being used
as skin whitening agents,21,22 niacinamide as anti-bacterial
and anti-inflammatory23 and pullulan as a natural polymer
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Efficacy of extracts used in skin care products
to enhance delivery of drugs or cosmetic agents.24
Comparison of sample’s potency, expressed as IC50
(Figure 1), showed four samples (amla, green tea, mango,
pomegranate) to have lower IC50 compared with Trolox
in both assays. Of the 4 samples, we demonstrated that
the mango extract in the cosmetic formulation exhibited
a consistent synergistic interaction with the other
ingredients in the cream base. Mango extracts has been
shown to have protective effects against skin aging by being
able to reduce wrinkle formation and increase collagen
bundle.25 With an enhance activity in cream formulation,
the use of mango extract in this formulation will ensure
the ability of the product to retain claimed effect.
Although, the mechanisms involved is unclear, its active
compound (mangiferin, anthocyanin and carotenoids)
were postulated to exert synergism.26,27 Similar synergistic
effect, respective to the combinations, was observed in a
study investigating synergistic antioxidant effects of fruits,
vegetables and legumes extracts.28 However, antagonisms
effect may also occur within a formulation. Green tea
and pomegranate were observed to have significant
reduction at almost all percentage formulation. The
Figure 2: 1% Formulation. Comparison of antioxidant activities between sample alone vs. sample + cream in ABTS and DPPH assays. Only
mango extract + cream showed an increase in activity in both assays compared to extract alone (Arrow). Data are the mean ± SD (n=4) with
P < 0.05 considered as significant.
Figure 3. 2% Formulation. Comparison of antioxidant activities between sample alone vs. sample + cream in ABTS and DPPH assays. Mango
extract + cream has higher antioxidant in the DPPH assay (arrow). Data are the mean ± SD (n=4) with p< 0.05 considered as significant.
Figure 4. 4% Formulation. Comparison of antioxidant activities between sample alone vs. sample + cream in ABTS and DPPH assays. Data
are the mean ± SD (n=4) with P < 0.05 considered as significant.
International Journal of Phytocosmetics and Natural Ingredients 2018, 5:7
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observed results suggest antagonistic effect or instability
of the extracts within the formulation. For example, green
tea was found to be pH-dependent, where the green tea
and its component were unstable and easily degraded in
alkaline solution than in acidic solution.29,30 Evaluation the
potential interactions between ingredients in a cosmetic
formulation is a critical aspect to ensure product efficacy.
Conclusion
This study provided information on the antioxidant
activities of commonly used extracts/compounds and the
potential interaction between ingredients in a cosmetic
formulation. However, the reported activities of synergism
or antagonism between the studies extracts were respective
to the cream ingredients.
Conflict of Interests
None.
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