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v1

Moringa Oleifera L. Extracts as Bioactive Ingredients That

Increasing Safety of Body Wash Cosmetics

Zofia Nizioł-Łukaszewska1, Dominika Furman-Toczek2, Tomasz Bujak1, Tomasz Wasilewski3

1
Department of Cosmetology. The University of Information Technology and Management in Rzeszow
Kielnarowa 386a, 36-020 Tyczyn, Poland, zniziol@wsiz.rzeszow.pl, tbujak@wsiz.rzeszow.pl,
2
Department of Medical Biology and Translational Research. The University of Information Technology and
Management in Rzeszow Kielnarowa 386a, 36-020 Tyczyn, Poland, dfurman@wsiz.rzeszow.pl
3
Department of Chemistry, University of Technology and Humanities in Radom, Chrobrego 27, Radom 26-600,
Poland, tomasz.wasilewski@uthrad.pl

Abstract

Extracts obtained from leaves of Moringa tree (Moringa oleifera) are a rich source of many
bioactive compounds: flavonoids, phenolic acids or carotenoids. It also contains such
components as, vitamins (A, C, niacin, pantothenic acid), alkaloids, tannins or saponins.
Extracts and plant substances derived from the leaves of Moringa oleifera L. have a strong
antioxidant, toning and anti-inflammatory effect.

The work attempts to obtain a multifunctional plant extract derived from Moringa tree leaves.
Obtained extracts was analyzed for their biochemical and physicochemical properties. The
obtained results indicate on a strong antioxidative potential of the tested extracts. The further
step was an attempt to apply the extracts in the model body wash cosmetic. The biological
activity of extracts and model cosmetic formulation were assayed by in vitro analysis on two
human cell lines: keratinocytes (HaCat,) and fibroblasts (BJ). The results showed that the tested
extracts may affect on increasing of cell proliferation and reduce oxidative stress in cells. The
addition of the tested extracts to the model cosmetic formulation, were contributed to the
reduction of their ability to irritate the skin and improve the safety of use of the product.

Key words: Moringa oleifera L., antioxidant activity, cell culture, irritant potential

© 2018 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

Introduction

In recent years, the cosmetic industry is one of the fastest growing industries in the
world. Strong competition on the cosmetic market and high consumer expectations are forcing
manufacturers to look for innovative solutions in every aspect of the product life cycle. Until
recently, cosmetic manufacturers created the innovative advantage of their products by
incorporating new raw materials and ingredients which are less common and not used by the
competition. Examples include substances such as hyaluronic acid, peptides, polysaccharides,
exotic oils and plant extracts, and snail slime. With time, however, these raw materials are used
by a growing number of cosmetic manufacturers, so the advantage of innovation is very quickly
lost and formulators must seek new solutions. The innovativeness of cosmetics can also be
generated through the form in which skin care and beauty products are offered. Increasingly,
novel forms such as foams, jellies, creams or essences, are commercially available. However,
after the launch of cosmetics in innovative forms the market tends to be almost flooded by
products of the same type offered by various manufacturers. Consequently, the innovative
advantage of such solutions is short-lived [1-5].

In the last few years, there has been a new trend on the cosmetic market, involving the
formulation of innovative products on the basis of multifunctional ingredients. Substances of
this type are characterized by multidirectional activity, combining biologically active properties
with moisturizing effects and the ability to give cosmetics an appropriate form or improve their
safety to people and the environment. The last of the properties enumerated above is particularly
sought after by present-day consumers. The strong trend for “naturalness” in cosmetics has
contributed to an increase in consumer awareness with regard to substances used in cosmetic
production. Consumers look for products which – in addition to delivering the desired usually
multifaceted activity – are safe to people and the environment, and are able to reduce adverse
environmental impacts on the skin (anti-smog, anti-pollution cosmetics). Examples of such
ingredients include plant extracts, which, as demonstrated by a number of studies, can be used
as multifunctional cosmetic raw materials with moisturizing, soothing, anti-wrinkle and
antioxidant properties, and minimize the adverse skin effects of other cosmetic product
ingredients. The above characteristics are due to the complex chemical composition of plant
extracts, which represent solutions of active substances derived from plants in a suitable solvent
[6-13].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

In the course of research in this area, much attention has been focused on the Moringa
tree (Moringa oleifera L.), also called the tree of life, as a source of active ingredients valuable
for the cosmetic industry. Owing to the presence of a broad spectrum of bioactive compounds,
the plant has powerful antioxidant, antibacterial, toning, astringent and anti-inflammatory
properties [14-19]. Leaves of the Moringa tree have been found to contain flavonoids including
myricetin, quercetin, kaempferol, isorhamnetin or rutin, as well as phenolic acids. Fresh leaves
are, at the same time, a good source of carotenoids such as lutein, β-carotene and zeaxanthin.
In addition, the Moringa tree is characterized by a high content of vitamins C and A. The active
substances contained in the plant have been shown to have beneficial effects on human skin
and successfully replace synthetic ingredients [20-23].

The present study was an attempt to evaluate the effect of Moringa oleifera leves
extracts on the irritant potential of body wash gels. For the purpose of the study, a technology
for obtaining extracts in the process of solvent extraction was developed. Water and mixtures
of water with glycerin were used as natural extraction solvents. The extracts obtained this way
were used for further analysis to determine their basic biochemical properties: the ability to
neutralize free radicals, and the content of polyphenols and flavonoids. Analyzes of cytotoxicity
as well as the intracellular level of reactive oxygen species were performed on in vitro model:
keratinocytes (HaCaT,) and fibroblasts (BJ) cell lines. In addition, the model preparations with
applied extracts also was analyzed on cell lines.

Material and Methods

Extract derivation method

Moringa tree leaves (Moringa oleifera L.) were dried with warm air at 40° C for two days. The
samples obtained were later used for extraction method. Extraction of bioactive compounds
from dried leaves was carried out using ultrasound-assisted extraction method. 5 g of grounded
plant material and 100 mL of water with glycerine in various proportions (50/50, 60/40, 80/20)
were used to obtain extract. Extraction was carried out for 10 cycles of 10 min at room
temperature. Then, obtained extracts were collected and filtered through Whatman filter paper
No. 10. The material was stored in the dark at 4 ° C for further analysis.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

Total Phenolic Content Determination

The total phenolic content of leaves Moringa oleifera L. extracts were determined
spectrophotometrically by the Folin-Ciocalteu method according to the procedure reported by
Singleton et al with some modifications [24]. The 300 μL of leaves extract solutions and 1500
μL of 1:10 Folin-Ciocalteau reagent were mixed and after 6 minutes in the dark, 1200 μL of
sodium carbonate (7.5%) was added. After 2 h of incubation in the dark at room temperature,
the absorbance at 740 nm was measured spectrophotometrically by Aquamate Helion (Thermo
Scientific). The total phenolic concentration was calculated from a gallic acid (GA) calibration
curve (10-100 mg·L-1). Data were expressed as gallic acid equivalents (GA)·g-1 of extract
averaged from three measurements.

Total Flavonoids Content Determination

The total flavonoid content of plant extracts were evaluated using aluminium nitrate
nonahydrate according to the procedure reported by Woisky and Salatino with modifications
[25]. The 600 μL of plant extracts solutions and 2400 μL of mixture (80% C2H5OH, 10%
Al(NO3)3 × 9 H2O and 1M C2H3KO2) were mixed. After 40 min of incubation at room
temperature, the absorbance at 415 nm was measured spectrophotometrically by FilterMax F5
(Aquamate Helion). The total flavonoids concentration in extracts were calculated from a
quercetin hydrate (Qu) calibration curve (10-100 mg·mL-1) and expressed as quercetin
equivalents (Qu)·g-1 of extract averaged from three independent measurement

DPPH Radical Scavenging Assay

Antioxidant activity of plant extract was analysed using DPPH free radical scavenging assay,
according to the method described by [26]. 167 µL of 4mM ethanol solution of DPPH was
mixed with 33 µL analysed samples in different concentrations (250 µg ml-1 – 5000 µg ml-1).
The absorbance was measured at λ=516 nm in every 5 minutes for 30 minutes using UV-Vis
spectrophotometer Filter Max 5 (Thermo Scientific). DPPH solution mixed with equal volume
of distilled water was served as a control. The percentage of the DPPH radical scavenging were
calculated using the equation:

%DPPH• scavenging = [Abscontrol – Abssample]/Abscontrol × 100%

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

Cell culture

HaCaT (ATCC®, normal human keratinocytes and BJ fibroblasts (ATCC® CRL-2522™) was
obtained from the American Type Culture Collection (Manassas, VA 20108, USA). HaCaT
cells were maintained in a DMEM (Dulbecco’s modified essential medium, Gibco) with L-
glutamine, supplemented with 5% (vol/vol) FBS (fetal bovine serum, Gibco), and 1% (vol/vol)
antibiotic (100 U·mL-1 Penicillin and 1000 µg·mL-1 Streptomycin, Gibco). Fibroblast were
maintained in a MEM (Minimum Essential Medim, Gibco) contains Earle’s salt and L-
glutamine, supplemented with 5% (vol/vol) FBS (fetal bovine serum, Gibco), and 1% (vol/vol)
antibiotic (100 U mL -1 Penicillin and 1000 µg mL -1 Streptomycin, Gibco). All cultured cells
were kept at 37◦C in a humidified atmosphere of 95% air and 5% of carbon dioxide (CO2).
When the cells reached confluence, the culture medium was removed from the flask (VWR)
and cells were rinsed two times with sterile PBS (Phosphate-Buffered Saline, Gibco). The
confluent layer was trypsinised using Trypsin/EDTA (Gibco) and then resuspended in fresh
medium.

Cell Viability Assay

The resazurin sodium salt reduction assay, was used to assess cell viability. The assay was
performed according to Ivanov et al. with some modifications [27]. Cells were placed in 96-
well plates at a density of 1×104 cells/well with fresh medium. After 24 h of pre-culture,
medium was aspirated and varying concentrations (10, 5, 3, 1%) of tested extracts were added
into each well and cultured for another 24 h. The control group were non-treated cells. After
time of exposure, resazurin salt solution (Sigma, R7017) was transferred into the plates for a
final volume 250 µL/well and concentration of 60 µM in medium and incubated for 3 h at 37◦C
in darkness. The absorbance was measured at the wavelength λ=570 nm using a microplate
reader FilterMax F5 (Thermo Fisher Scientific). The experiments were performed in triplicates
for each extract concentration and presented as percentage of control values.

To evaluate if model cosmetic formulation (1% SCS) containing various concentrations (10, 5,
3, 1%) of Moringa oleifera leaves extract can affect on cells viability, the resazurin sodium salt
reduction assay was used. BJ fibroblasts and keratinocytes were seeded in transparent 96- well
plates at a density of 1×104 cells/well with fresh medium. After 24 h of pre-incubation, cells
were exposed to 30 min of model cosmetic with different concentrations of Moringa tree leaves
extract. The non-treated cells were a control group. After time of exposure, resazurin salt
solution (Sigma, R7017) was transferred into the wells for a final volume 250 µL/well and
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

concentration of 60 µM in medium and incubated for 3 h at 37◦C in darkness. The absorbance

was measured at the wavelength λ=570 nm using a microplate reader (FilterMax F5, Molecular
Devices). The experiments were performed in triplicates for each tested substance concentration
and presented as percentage of control values.

Measurement of DCF fluorescence

To measure the intracellular level of reactive oxygen species in HaCaT and fibroblasts cells,
the fluorogenic dye H2DCFDA was used. After passively diffusion into the cells, H2DCFDA
was deacetylated by intracellular esterases into the non-fluorescent compound, that upon
oxidation by ROS is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) [28].
The fluorescence was measured according to protocol previously described by [29]. Cells were
seeded in 96-well plates at a density of 1×104 cells per well and initially cultured before the
experiment for 24 h. After this, culture medium was changed on 10 µM H2DCFDA (Sigma) in
serum-free medium (DMEM or MEM for HaCaT and fibroblasts respectively). Cells were
incubated at H2DCFDA for 45 min before treatment. After this time, an intracellular oxidative
stress as well as production of ROS was induced by addition hydrogen peroxide (H2O2) to the
cells at a final concentration of 1mM in PBS for 1h. Then medium were changed and cells were
exposed into different M. oleifera leaves extract concentrations (10, 5, 3, 1%). The unexposed
cells were control group and cells treated with 1mM hydrogen peroxide (H2O2) was used as a
positive control. The DCF fluorescence was measured after 90 min of incubation, using a
microplate reader FilterMax F5 (Thermo Fisher Scientific) at maximum excitation of 485nm
and emission spectra of 530 nm.

To assay the capacity of model cosmetic formulation (1% SCS) containing various
concentrations (5, 3, 1%) of M. oleifera leaves extract to generate intracellular level of reactive
oxygen species, fluorogenic dye H2DCFDA also was used. HaCaT and BJ fibroblast were
seeded in 96-well plates at a density of 1×104 cells per well and initially cultured for 24h. After
this, culture medium was changed on 10 µM H2DCFDA (Sigma) in serum-free medium
(DMEM or MEM for HaCaT and fibroblasts respectively). Cells were incubated in H2DCFDA
for 45 min before treatment. After this, medium were changed and cells were exposed into 1%
SCS with different extract concentrations. The control group were untreated cells and cells
treated with 1% of Sodium Coco Sulfate (SCS) was used as a positive control. The DCF
fluorescence was measured every 30 min for a total 90 min using a microplate reader FilterMax
F5 (Thermo Fisher Scientific) at maximum excitation of 485nm and emission spectra of 530
nm.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

Zein test

Irritant potential of the products was measured using zein test. To 40 mL of the samples solution
(10 wt%) was added 2 ± 0.05 g of zein from corn. The solutions with zein was shaken on a
shaker with water bath (60 min at 35◦C). The solutions were filtered on Whatman No. 1 filters
and then centrifuged at 6720 g for 10 min. The nitrogen content in the solutions was determined
by Kjeldahl method. 1 mL of the filtrate was mineralized in sulfuric acid (98%) containing
copper sulphate penthahydrate and potassium sulphate. After mineralization the solution was
transferred (with 50 mL of MiliQ water) into the flask of the Wagner–Parnas apparatus. 20 mL
of sodium hydroxide (25 wt%) was added. The released ammonia was distilled with steam.
Ammonia was bound by sulfuric acid (5 mL of 0.1 N H2SO4) in the receiver of the Wagner–
Parnas apparatus. The unbound sulfuric acid was titrated with 0.1 N sodium hydroxide. Tashiro
solution was used as an indicator. The zein number (ZN) was calculated from the equation:
𝑚𝑔𝑁⁄
𝑍𝑁 = (10 − 𝑉1) × 100 × 0.7( 100𝑚𝐿 ),
where, V1 is the volume (cm3) of sodium hydroxide used for titration of the sample. The final
result was the arithmetic mean of five independent measurements.

Statistical Analysis

Each value is the mean of three replicates. Values of different parameters were expressed as the
mean ± standard deviation (SD). The two-way analysis of variance (ANNOVA) and Bonferroni
posttest between groups were performed at the level P value of <0.05 to evaluate the
significance of differences between values. Statistical analyses were performed using GraphPad
Prism 5.0 (GraphPad Software, Inc., Sand Diego CA).

Results and Discussion

The major group of phytochemicals contributing to the antioxidant capacity of plant material
include polyphenols, carotenoids and vitamins such as vitamin C and E. There is a several
previous study, which shows that M. oleifera leaves are rich source of bioactive compounds
with antioxidant properties. It has been reported that Moringa tree leaves have sufficient
amounts of quercetin, kaempferol, β-carotene, α-tocopherol and Vitamin A [30-31].
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

With regard to this findings, the present investigation was undertaken to evaluate the
antioxidant capacity of aqueous/glycerin extracts of M. oleifera leaves. The biological activity
of these extracts was also determined on in vitro cell line model. Afterwards, an attempt was
made to develop the technology of a base cosmetic, which then was tested for its toxicity to in
vitro cell model. The cytotoxicity of tested substances was assessed on BJ fibroblast and normal
human keratinocytes (HaCaT) cell lines.

To confirm previous result, in this work an attempt was made to determine total phenolic
content (TPC) and total flavonoid content (TFC). The amounts of these compounds were
assayed from the calibration curves of gallic acid (y = 0.0046x+0.0452, R2 = 0.9989), and
quercetin (y = 0.0153x-0.0053, R2=0,9996), respectively. The analysis were performed for three
different dilutions (50%, 25% and 12,5%) of each extract. Obtained results shows, that the
highest amount of phenols and flavonoids was characterised by 50:50 (vol/vol)
aqueous/glycerin extract, while the lowest concentration of these compounds showed 80:20
(vol/vol) aqueous/glycerin extract (Figure 1). The difference between the highest and the lowest
TPC value for different types of 50% extract dilution it was about 24%. Considering the TFC
value, it was nearly 37%. It also was observed, that the TPC and TFC were increasing in a dose-
dependent manner for all tested types of Moringa oleifera leaves extracts.

Figure 1. Total phenolic content (TPC) (A) and total flavonoid content (TFC) (B) in 50:50 (vol/vol), 60:40 (vol/vol) and 80:20
(vol/vol) aqueous/glycerin extract of Moringa oleifera leaves. Values are mean of three replicate determinations (n=3) ± SD.
The TPC and TFC ant were calculated from the calibration curve (R2=0.9989 and R2=0.9996, respectively)

As it was mentioned above, the most important properties of phenolic compounds derived from
plant material are their antioxidant properties. The phenols consists of a hydroxyl group and
plays an essential role in the antioxidant ability by donating hydrogen and forming stable radical
intermediates [32]. The mechanism of action of phenols mainly relies on neutralization of free
radicals, chelation of metal ions and induction of dismutase enzymes, as well as peroxidases
[33]. The next stage of our research was to evaluate ability of M. oleifera leaves extracts to
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

scavenge free radicals. For thus, we used DPPH• reducing assay, where changes of color
solution are directly linked with decreasing of absorption values and the number of formed
DPPH radical [34]. To determine antioxidant properties of all tested extracts we used three
different concentrations ranging from 12,5%, 25%, to 50%, the measurements were performed
every five minutes over 30 min time period. Based on obtained data, it was shown, that each
extract concentration has a different ability to reduce free radicals (Figure 2). The highest ability
to scavenge DPPH radical was showed by 50:50 (vol/vol) aqueous/glycerin extract at the
highest tested concentration (50%). After 30 min of incubation, the level of reduced DPPH•
was above 56%, while in the lowest concentration (12.5%), the level of scavenged radicals were
oscillated 26%. Moringa tree 60:40 (vol/vol) aqueous/glycerin leaves extract was characterized
by middling antioxidant ability in comparison to other extracts. The highest reducing power of
this extract was observed for 50% of concentration and it was about 41% after 30 min of
incubation. Considering the 80:20 (vol/vol) aqueous/glycerin extract, it has the lowest
antioxidant potential. In the highest concentration, the ability to scavenge DPPH radical was
only on 35% level. Furthermore, there was observed relationship between used concentration
and antioxidant potential of extracts. When the concentration was increasing, the free radical
reducing power was higher. The order of free radical scavenging capacity was as follows: 50:50
(vol/vol) > 60:40 (vol/vol) > 80:20 (vol/vol) aqueous/glycerin.

Figure 2. Kinetics of the absorbance changes in DPPH• solutions in the presence of various concentrations of 50:50 (vol/vol)
(A), 60:40 (B), 80:20 (vol/vol) (C) aqueous/glycerin extract of Moringa oleifera leaves. Values are mean of three replicate
determinations (n=3).
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Results from DPPH• scavenging assay obtained in this paper corresponds to the other research.
[30] in his paper indicated on strong antioxidant activity of different parts M. oleifera ethanolic
extracts. Using the 2-deoxyguanosine assay model, he has shown that the highest free radical
scavenging activity was characterized by leaves. Remarkable antioxidant activity of drumstick
leaves might be possessed due to presence of quercetin and kaempferol as well as chlorogenic
acids and their derivatives, which were detected in this part of plant. In addition, [35] was
hypotised that antioxidant activity of M. oleifera leaves extracts could be accomplished though
donating protons as well as reductones, which exert activity by breaking the free radical chain.

The biological activity of leaves of Moringa oleifera was also investigated on cells as in vitro
model. The cytotoxicity of tested extracts was assessed on HaCaT and BJ fibroblas cell lines
using resazurin method. Both cell types were treated with various extract concentrations,
ranging from 1% up to 10% in cultured medium. Obtained results indicate on cell specific effect
of analyzed extracts on cell viability (Figure 3). In HaCaT cells, all tested concentrations as
well as types of extracts showed stimulating effect on cell viability. The highest difference
between control and treated cells was observed for all types of 1% concentration of extracts.
The best ability on increasing cells viability were showed for 60:40 (vol/vol) aqueous/glycerin
extracts. It also has been observed that, the cell proliferation was increasing in dose-dependent
manner. In turn, in BJ fibroblast, extracts showed both, inhibitory and stimulatory effect. The
highest concentrations (10% and 5%) of all tested extracts showed anti-proliferative effect. For
10% of extracts concentration, the fibroblast viability was observed on 20%, 37% and 45%
level for 80:20 (v/v), 60:40(v/v) and 50:50(v/v), respectively with comparison to the control.
As well as, in HaCaT the 1% of extracts concentration have shown the most positive effect on
cell viability. The highest response was observed for 50:50 (v/v) aqueous/glycerin extract and
it was about 157%, regards to the untreated group. The less cytotoxic potential was noted for
80:20 (v/v) M. oleifera leaves extract. There also was noticed, that when concentration of
extract was decreased, the viability of cells was increased.
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Figure 3. The effect of different concentrations of Moringa oleifera leaves extracts (10, 5, 3, 1 %) on resazurin salt reduction
assay in cultured keratinocytes (A) and BJ fibroblast (B) after 24h of exposure. Data are the mean ± SD of three independent
experiments, each of which consists of three replicates per treatment group. ***p < 0.001, **p < 0.01, *p < 0.05 versus the
control.

The equal connection in cell viability with response to increasing concentration of M. oleifera
leaf extracts was observed by other researchers. The anti-proliferative effect was shown in KB
(tumor cells), HEK-293 (human lung carcinoma) and A549 cells (human embryonic kidney
cells). These papers also indicate, that anti-proliferative effect was associated with induction of
apoptosis, morphological changes and DNA fragmentation [31, 36-37].

In the next stage of this experiment it has been evaluated if M. oleifera leaves extracts could
inhibit H2O2 induced ROS production on in vitro cell models. As a substrate to determine the
intracellular formation of ROS generation, H2DCFDA assay was used. As is recommended
[38], we examined whether plant extracts without cells affected the fluorescence of the
H2DCFDA. Additionally, the separated experiment showed that there were no interactions
between plant extracts and H2DCFDA substrate in DMEM or MEM medium. After
preincubation cells with 1mM H2O2, the intracellular level of produced reactive oxygen species
level robustly increased in both, HaCaT and BJ, compared to the non-treated cells. When after
90 min of cells treatment with various concentration of all types extracts, the level of ROS
significantly decreased to the level oscillating to the control, group which were untreated cells
(Figure 4). The high ability of extracts to reduce oxidative stress might be correlated with rich
content of phenols and flavonoids. According to [36], it were directly indicated, that major
bioactive substances present in leaf extracts which provides to strong inhibition of H2O2-
induced oxidative stress are crypto-chlorogenic acid and isoquercetin.
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Figure 4. The effect of various concentrations of Moringa oleifera leaves extracts (10, 5, 3 and 1%) on the DCF fluorescence
in keratinocytes (A) and BJ fibroblast (B) cells preincubated with hydrogen peroxide (H2O2). Medium with 1mM hydrogen
peroxide (H2O2) was used as a positive control. The data are expressed as the mean ± SD of three independent experiments,
each of which consisted of three replicates per treatment group.

Given to the antioxidant nature of tested substances, based on previous experience and available
information, it was prepared a model formula of washing gel containing M. oleifera leaf extract.
To assay if model cosmetic formulation (1% Sodium Coco Sulfate – SCS) with various
concentration (5, 3 and 1%) of tested extracts can affect on cell viability it was used resazurin
reduction assay. Obtained data indicate, that in HaCaT cells, tested extracts did not induce any
significant decrease in cell viability in comparison to the control cells. Furthermore, it was
noticed, that base (1% SCS) plus 1% or 3% of extracts can significantly increase cell
metabolism. The highest cell viability growth was observed to 1% SCS+1% of 50:50 (vol/vol)
aqueous/glycerin extract and it was about 30% higher than a control group. It also can be noted,
that base (1% SCS) did not induce toxic effect on HaCaT cells. In turn, in BJ fibroblast cells,
tested substances were exhibited an opposite effect. There was no observed either toxic as well
proliferative effect on cell viability for all tested types of extracts and concentrations. Slightly
increased of BJ cell metabolism was noticed for 1% SCS+5% and 1% SCS+3% of 80:20
(vol/vol) aqueous/glycerin extract (104 and 106% respectively). Percent of viable cells after
treatment with 1% of SCS was little under of the control group (Figure 5). Obtained results
indicated, that use of extract in cosmetic formula is not unsafe for human skin cells and
additionally introduces desirable properties such as antioxidant activity.
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Figure 5. The effect of different model base formulation of cosmetic (1% SCS) containing various concentrations of Moringa
oleifera leaves extracts (5, 3, 1 %) on resazurin reduction in cultured keratinocytes (A) and fibroblast (b) after 30 min of
exposure. Data are the mean ± SD of three independent experiments, each of which consists of three replicates per treatment
group. ***p < 0.001, **p < 0.01, *p < 0.05 versus the control.

In order to further confirm above reports, the ability of model cosmetic formulation containing
M. oleifera leaf extract to generate intracellular ROS in keratinocytes and fibroblast was
assayed. The reactive oxygen species production was measured by the use of H2DCFDA
method. As the results showed, the 60:40 (v/v) and 80:20 (v/v) aqueous/glycerin Moringa tree
leaves extract did not significantly generate oxidative stress in HaCaT cells. The highest ROS
production was exhibited by 1% SCS. In addition, the most intracellular ROS level increase
was noticed for 1% SCS+ 5% 50:50 (v/v) extract and it was 2 fold higher than this of the non-
treated group. Values for other types of extracts and concentrations oscillates about the control.
There also was observed, when the extract concentration decreased, the amount of ROS in cells
was also lower (Figure 6).

Figure 6. The effect of different model base formulation of cosmetic (1% SCS) containing various concentrations of 50:50
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(vol/vol) (A), 60:40 (B), 80:20 (vol/vol) (C) Moringa oleifera leaves extracts on the DCF fluorescence in normal human
keratinocytes cells. Unexposed cells was used as a control. The data are expressed as the mean ± SD of three independent
experiments, each of which consisted of three replicates per treatment group.

When considering BJ fibroblast results it has been showed, that the highest intracellular level
of reactive oxygen production was for 1% SCS + 5% of 50:50 (v/v) extract compared to the
untreated group. Values for the other concentrations of this extract was similar to the unexposed
cells. For 60:40 (v/v) and 80:20 (v/v) aqueous/glycerin M.oleifera leaves extract did not
observed any changes in induction of oxidative stress. The level of intracellular ROS was
oscillate to the control group (Figure 7). Above findings correlates to the cell viability assay,
and it therefore seems that all types of tested extract are a desirable plant material with
remarkable antioxidant activity. These features can protect it from ageing and other diseases,
where the reactive oxygen plays major role.

Figure 7. The effect of different model base formulation of cosmetic (1% SCS) ) containing various concentrations of 50:50
(vol/vol) (A), 60:40 (B), 80:20 (vol/vol) (C) Moringa oleifera leaves extracts on the DCF fluorescence in fibroblast cells.
Medium with 1% of Sodium Coco Sulfate (SCS) was used as a positive control. The data are expressed as the mean ± SD of
three independent experiments, each of which consisted of three replicates per treatment group.

In the next step the Moringa tree leaves extracts were tested by evaluating its ability to reduce
the irritant potential of Sodium Coco Sulphate (SCS), an anionic surfactant used in the
formulation of body wash cosmetics. For this purpose, four samples constituting model washing
systems were prepared. Each sample contained 1 wt% of SCS combined with 1, 3 and 5 wt%
of the extracts prepared with different ratio of water and glycerin as extractant. The reference
(baseline) sample contained 1 wt% solution of SCS without any extract. The pH of each sample
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

was adjusted with 25% citric acid to a value of 5.5, the physiological pH value of the skin. The
irritant potential of model body wash systems was analyzed by zein value measurements. In
the surfactants solution zein protein is denatured and then is solubilized in the solution. This
process simulates the behaviour of surfactants in relation to the skin proteins. The test results
are presented in Fig 8.

Figure 8. Irritant potential of model products containing 1.0 wt% SCS and extract from Moringa oleifera L (1%, 3%, 5%).

The test showed that the M. oleifera leaves extracts might improve the safety of use of the
model washing system on terms of its effect on the skin. The addition of analyzed extracts to
1% SCS causes a decrease in the value of the zein number, and the same reduces skin irritation
potential. The value of the measured parameter in the base sample was at 451 mgN/100 mL.
According to the literature [2-4, 7-8, 10-13, 39-40], the sample should be classified as highly
irritating, as the value of the zein number exceeds 400 mgN/100 mL. The skin irritation
potential of the samples with addition of extracts was about 10–20% lower than the baseline. It
was observed, that used extract concentrations was not significantly affect on the value of zein
number, and the differences noted between the samples are within the margin of error. It was
not observed significant influence of the extractant on the analysed parameter.

The decrease of the zein number following to the application of the extract to the model
cosmetic formula, might be the result of presence active substances in the extracts. In aqueous
solutions and at low concentrations, surfactants occur as the form of individual particles referred
as a monomers [2, 4-5, 10-13]. When specific concentration, unique due to a compound is
reached, referred as the critical micelle concentration (CMC), the micellar aggregates starts to
appears in surfactant solutions. The irritation potential is significantly correlated with the type
and concentration of the surface active agent. The highest skin irritation ability is attributed to
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 September 2018 doi:10.20944/preprints201809.0508.v1

anionic surfactants and surfactants present as the forms of monomers. They demonstrate the
capacity to form a strong and long-lasting bonds with epidermal proteins. Following to their
ability to bind with proteins, surfactants may cause denaturation and elimination (elution)
proteins from the skin, which results as a cutaneous irritation. Available data indicates that the
irritant potential might be reduced by bindings of monomers with various types of substances
including peptides, polymers, polysaccharides and mineral salts, where all of them were found
in plant extracts [2, 4-5, 10-13, 39-46]. Another factor which potentially is contributing to a
reduction of irritant potential is the stabilization of micelles formed in solutions. Micelles are
thermodynamically unstable aggregates, which constantly disintegrates and releases monomers
into the volume phase of the solution. Stabilization of micelles in the presence of plant extracts
may take place through the incorporation of such substances as proteins, polyphenols and
flavonoids, as well as solvents used in the extraction process (glycerin, glycols), into their
structure [2, 4-5, 10-13, 41-46].

Conclusions
The paper was an attempt to determine the properties and the applicability of extracts from
Moringa oleifera leaves in model products. The tested extracts were characterized by a high
content of phenolic compounds, flavonoids and high antioxidant potential. In vitro toxicity
studies showed that the tested extracts in concentrations up to 5% showed a positive effect on
cell proliferation and metabolism. It also has been shown that the extracts may contribute to the
reduction of oxidative stress in cells. It was noted, that tested model formulation of cosmetic
(1% SCS) with addition of different types of extracts in various concentrations does not affect
negatively on cell metabolism. Analyzes defining the ROS level showed that model cosmetic
formulation (1% SCS) with presence of tested extracts do not cause increasing formation of
intracellular of reactive forms of oxygen. To summarize, conducted experiments in this paper
showed, that application of Moringa oleifera leaves extracts to the model cosmetic formulation
might contribute to reduce skin irritation and improve the safety of the product.

Conflicts of Interest
The authors declare no conflict of interest.

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