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cosmetics
Article
Rice Water: A Traditional Ingredient with
Anti-Aging Efficacy
Joana Marto 1ID ,Ângela Neves 1, Lídia Maria Gonçalves 1ID , Pedro Pinto 1,2,
Cristina Almeida 1ID and Sandra Simões 1, *
1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,
Avenida Professor Gama Pinto, 1649-038 Lisboa, Portugal; jmmarto@ff.ulisboa.pt (J.M.);
angelabeatrizneves@campus.ul.pt (Â.N.); lgoncalves@ff.ulisboa.pt (L.M.G.); geral@phdtrials.com (P.P.)
calmeida@ff.ulisboa.pt (C.A.)
2PhD Trials, Rua das Murtas, nº1B, 1º-1700-309 Lisboa, Portugal
*Correspondence: ssimoes@ff.ulisboa.pt; Tel.: +351-217-500-769
Received: 1 March 2018; Accepted: 19 March 2018; Published: 1 April 2018
Abstract:
The skin healing benefits of rice have been known for centuries. Rice (Oryza sativa) water is
a food processing waste that can potentially be incorporated into cosmetic formulations. However,
no scientific evidence supports their role in skincare products. The aim of this project is to design
and develop a topical gel formulation containing rice water and to evaluate its biological properties,
namely, the anti-aging and antioxidant rice water properties. Rice water was evaluated in terms of
physico-chemical composition and in terms of
in vitro
biological antioxidant activity and elastase
inhibitory effect. Rice water was incorporated into a hydrogel and the developed formulation was
subjected to pharmacotechnical tests such as pH and viscosity. Biological and sensory effects were
evaluated on a panel of 12 volunteers for 28 days. The safety evaluation study was performed on rice
water gel, using the Human Repeat Insult Patch test protocol. Rice water presented
in vitro
biological
antioxidant activity and elastase inhibitory effect. The gel formulation containing 96% rice water was
biocompatible with the human skin and presented suitable cosmetic properties. Rice water should be
thus considered as an anti-aging ingredient to be used as raw material for skincare applications.
Keywords: rice water; anti-aging; topical application; hydrogel; skincare; waste-into-value
1. Introduction
Rice (Oryza sativa) is a fundamental food for almost half of the world’s population, supplying nearly
all the daily calories especially in Asia. Rice water retained after soaking or boiling rice is commonly
consumed but, in general, rice water is discarded in many food preparation procedures worldwide.
Rice bran oil is well known for its contents of antioxidant-rich components such as ferulic
acid, gamma-oryzanol and phytic acid and has been used in the cosmetic industry [
1
] and also in
the management of skin diseases [
2
]. Rice bran oil and rice bran extracts have been used in the
free form and nanoencapsulated for protection against UVB-radiation injuries [
3
,
4
], for skin disease
treatment [
5
,
6
]. Additionally, rice bran bioactive compounds have been found to exert anti-aging
activity [
7
,
8
] and to be efficient in the treatment of alopecia [
9
]. Nevertheless, rice water skin benefits
are supported by scarce scientific studies and there is a lack of scientific literature unveiling the skin
effects largely claimed by cosmetic manufacturers. The empiric use of rice water as a bath component,
especially among Asian women, became a tradition with a lack of scientific evidence.
The major component in rice grains, starch, is recommended to be added to bath water for the
treatment of atopic dermatitis or skin diseases associated with pruritus [
10
]. Starch is a biodegradable
polymer with safe application in the pharmaceutical industry [
11
] and it was demonstrated that glucose and
Cosmetics 2018,5, 26; doi:10.3390/cosmetics5020026 www.mdpi.com/journal/cosmetics
Cosmetics 2018,5, 26 2 of 12
mannose residues from polysaccharides could reduce inflammatory activity
in vivo
[
12
].
In a recent study
,
a formulation composed of starch-based nanocapsules incorporating an anti-inflammatory agent was
tested in a mouse model of cutaneous inflammation and it was possible to observe the synergistic action of
starch on the anti-inflammatory activity of the formulation [13].
In the last years, the search for new bioactive compounds to prevent skin aging has increased.
In parallel, there is an increasing concern regarding products from natural origin, if possible,
from organic
farming in cosmetic products. Rice water is a natural, economic and simple ingredient
that can be incorporated into skincare products. It can be obtained from different types of rice present
in the human diet and also from rice residues resulting from the rice industry, as a way to transform it
into an added-value product.
The main aim of this study was thus to develop a semi-solid dosage form incorporating rice water
and to test such formulation on human skin to evaluate its biological and sensory effects. To carry
out this proposal, rice water was prepared, physico-chemically characterized and tested
in vitro
for
antioxidant activity and anti-aging effects. In this study paddy rice, with peel, was used to produce the
rice water, in order to maintain all the rice components. The rice water presenting the best antioxidant
activity was incorporated in a hydrogel formulation with very few ingredients in order to incorporate
almost 96% of the rice water in the composition. After the characterization of the hydrogel, biological
and sensory effects and the safety of the formulation were evaluated in vivo, in human volunteers.
2. Materials and Methods
2.1. Materials
Paddy rice, guadiagran variety, (batch n. 0539) was a gift from “Casa Agricola Quinta das Barracas
da Rainha” (Coruche, Portugal). Purified water was obtained by reverse osmosis and electrodeionization
(Millipore, Elix 3, Darmstadt, Germany) being afterwards filtered (pore 0.22
µ
m). LecigelTM (sodium
acrylates copolymer and lecithin) and Dermosoft
®
OMP (methylpropanediol, caprylyl glycol and
phenylpropanol) were obtained from Dr. Straetmans (Hamburg, Germany). Ascorbic acid and MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide) were purchased from Sigma (St. Louis,
MO, USA). 2
0
,7
0
-dichlorodihydrofluorescein diacetate (H2-DCFDA) was obtained from Life Technologies
(Paisley, UK). Spontaneously immortalized human keratinocyte cell lines HaCaT were from CLS
(Eppelheim, Germany). Folin–Ciocalteu reagent was prepared according the requirements of Portuguese
Pharmacopoeia 9.0. Human Neutrophil Elastase (HNE) was purchased from Merck (Darmstadt, Germany).
The conductivity standards (84, 147, 1413 and 12,880
µ
S/cm, 25
◦
C) were supplied by Reagecon (Shannon,
Co. Clare, Ireland). The pH standards (pH 4, pH 7, pH 6 and pH 9) were supplied by Crison (Barcelona,
Spain). All other reagents were of analytical grade and were used without further purification.
2.2. Rice Water Preparation by Three Different Procedures
Water prepared by the boiling process (RWB):
400 g of paddy rice whole grains were boiled in
1 L
of deionized water for 30 min. Rice water was filtered through cotton gaze and frozen at
−
30
◦
C
until used.
Water prepared with the intact grain (RWM):
400 g of paddy rice whole grains were mixed with
1 L of deionized and left to shake, at room temperature, for 24 h. Rice water was then filtered through
cotton gaze and frozen at −30 ◦C until used.
Water prepared with the crushed grain (RWS):
400 g of paddy rice grains were grinded into
smaller pieces using a kitchen robot, for 10 s, mixed with 1 L of deionized water and left to shake,
at room temperature
, for 24 h. Rice water was then filtered through cotton gaze and frozen at
−
30
◦
C
until used.
Cosmetics 2018,5, 26 3 of 12
2.3. Rice Water Characterization
2.3.1. Physical Characterization
The pH was determined using a combined glass electrode from Crison GLP 21 pH meter
(Barcelona, Spain), with automatic temperature compensation. The conductivity was measured using
the CyberScan CON 200 Conductivity meter (Eutech Instruments, Ayer Rajah Crescent, Singapore).
The turbidimetry was determined using the TN 100 Infra-Red Turbidimeter (Eutech Instruments,
Ayer Rajah Crescent, Singapore).
2.3.2. Dry Residue Assay
Five millilitres of sample were weighted to a porcelain capsule with 25 g of treated sea sand
(granulometry: 0.25 a 0.35 mm). The capsule and its contents, including a small glass rod, were dried
in the oven at 102
±
2
◦
C for 2 h. Then the capsule and its contents were cooled in the desiccator
to room temperature and weighted again. After drying the systems in the oven, for one more hour,
it was cooled
in the desiccator and weighted again. The process was carried out with successive
dryings at the same temperature, followed by cooling in the desiccator, until two successive weighings
did not differ more than 0.5 mg.
2.3.3. Carbohydrates Determination by Phenol Sulphuric Acid Method
Carbohydrates determination was performed according to the methods published elsewhere [
14
].
Concentrated sulphuric acid (5 mL) was rapidly added to a solution of 1 mL of sample/standard and
1 mL of 5% (w/v) phenol was then added. Because the reaction is exothermic, the test tubes were
cooled to room temperature before spectrophometric readings. The change of the colour’s solution
was measured in the region of the visible and it was proportional to the amount of sugars present in
the sample. The absorbance was measured at 490 nm against purified water as reagent blank using
a spectrophotometer HITACHI U-2000 (Tokyo, Japan). A calibration curve ranging from 6 mg/L to
60 mg/L of glucose was used.
2.3.4. Total Protein by Kjeldahl Method
Mineralization
An amount of 20 mL of rice water, the catalyst tablet (copper catalyst) and the antifoam agent
(silicon) were introduced into a Kjeldahl digestion flask [
15
]. Twenty millilitres of concentrated sulfuric
acid (
$
20 = 1.84 g/cm
3
,
≥
98%) were carefully added. The Kjeldahl digestion flask was placed on the
heating device (digestion apparatus, Selecta, Barcelona, Spain) and heated at 400
◦
C for 45 min until
the liquid was completely clear (characteristic emerald green colour).
Distillation
The content of the Kjeldahl digestion flask was cooled to room temperature and 50 mL of purified
water were added. The Kjeldahl flask was mixed and placed on the Kjeltec System 1002 Distilling
unit (Tecator, Apeldoorn, Nederland). Eighty millilitres of 40% (w/v) sodium hydroxide solution were
added to the Kjeldahl tube and the distillation started and took place until approximately 150 mL of
the distillate were collected. The distillate was collected on 50 mL of 4% (w/v) boric acid solution with
4 drops of Kjeldahl indicator. The distillate was titrated with 0.1 N sulphuric acid. The end point was
given by the passage from green to violet. Phenylalanine was used as a mineralization control.
2.3.5. Analysis of Fat by Rose Gottlieb Method
For this analysis, the Rose Gottlieb method was used [
16
]. An amount of 15 mL of rice water was
placed into a decantation ampoule and weighted. An amount of 2 mL of ammonium hydroxide (25%
(w/w) was added and mixed, then 10 mL of 96% (v/v) ethyl alcohol were added and mixed thoroughly.
Cosmetics 2018,5, 26 4 of 12
Twenty millilitres of ethyl ether were added and stirred vigorously for 1 min and 25 mL of petroleum
ether were added and the mixture was stirred gently for 30 s. The mixture was left standing until clear
separation of the ethereal from aqueous layers. Two further extractions of the aqueous phase were
done as before but using only 15 mL of ethyl ether and 15 mL of petroleum ether and always keeping
the ethereal phase in the same flask. The contents of the flask were evaporated on a rotary evaporator
(60
◦
C) and the flask was dried in an oven at 105
◦
C for successive periods of 1 h until two successive
weighings did not differ more than 0.5 mg.
2.3.6. Total Phenolic Compounds by Folin–Ciocalteu Method
Total phenolic compounds were measured in rice waters samples by the Folin–Ciocalteu method.
Folin–Ciocalteu reagent was prepared according the requirements of Portuguese Pharmacopoeia 9.0.
Folin–Ciocalteu reagent (1 mL) was added to an aliquot (3 mL) of the target rice water diluted to
25 mL. The solution was shaken vigorously and allowed to stand for three min. Sodium carbonate
solution (4 mL) was added and the sample again was shaken and allowed to stand for thirty minutes.
The absorbance
was measured at 760 nm by UV/VIS Spectrophotometer (Hitachi U-2000, Tokyo,
Japan). A blank was also performed. The pyrogallol was used as reference standard of phenolic
content. The calibration curve for pyrogallol was found to be linear in the range of 0.5 to 8 mg/L.
The coefficient
of determination (r
2
) of 0.998 indicates the good linearity between the concentration
and absorbance. The method was validated for precision and accuracy by duplicate determinations
and standard controls.
2.3.7. In Vitro Studies
Cell Viability
The cytotoxicity of rice water was evaluated using general cell viability endpoint MTT reduction
and it was performed according to a previous reported work [
17
]. Cell viability was assessed after 24 h
of incubation of a spontaneously immortalized human keratinocyte cell line HaCaT (CLS, Eppelheim,
Germany) with different dilutions (up to 1:100) of rice water. HaCaT cells were seeded in sterile
flat-bottom 96-well tissue culture plates (Greiner, Kremsmünster, Austria), in RPMI 1640 culture
medium (Life Technologies, Paisley, UK), supplemented with 10% Fetal serum bovine, 100 units of
penicillin G (sodium salt) and 100
µ
g of streptomycin sulfate and 2 mM L-glutamine (Life Technologies,
Paisley, UK), at a cell density of 2
×
10
5
cells/mL, 100
µ
L/well. Cells were incubated at 37
◦
C and 5%
CO
2
. Negative control was the culture medium and positive control sodium dodecyl sulfate (SDS)
at 1 mg/mL. After the time of exposition, the MTT assay was performed. Medium was replaced by
medium containing 0.25 mg/mL MTT. The cells were incubated again for 3 h. Afterwards,
the medium
was removed and the intracellular formazan crystals were solubilized and extracted with 100
µ
L
dimethylsulfoxide (DMSO). After 15 min in continuous stirring at room temperature, the absorbance
was measured at 570 nm in FLUOstar Omega BMGLabtech Microplate Reader (FLUOstar BMGLabtech,
Offenburg, Germany).
The relative cell viability (%) compared to control cells was calculated for the MTT assay using
the following equation:
% cell viability =
Abs sample
Abs control ×100 (1)
Reactive Oxygen Species (ROS) Production Measurement
The capacity of rice water to reduce the ROS production was evaluated through a fluorimeter
technique that uses 2,7’ dichlorodihydrofluorescein diacetate (H2-DCFDA) to quantify the intracellular
ROS production, according to the method described elsewhere [
17
]. Briefly, for this measurement,
HaCaT sub-confluent cells grown in 96-well plates, which were incubated for 30 min with 20
µ
M of
H2-DCFDA in the dark, at 37
◦
C, were used. Later, the medium was removed and fresh medium
Cosmetics 2018,5, 26 5 of 12
was added to the cells before being exposed to different concentrations of extracts and ascorbic
acid for
1 h
. In the case of the hydrogen peroxide (H
2
O
2
) assay, H
2
O
2
in a final concentration
of 500
µ
M was added simultaneously with the samples in study and it was used as a positive
control,
once it induces
the intracellular ROS production in cells. For the UV assay, three UV-B
lamps (Sankyo Denki G8T5E, Kanagawa, Japan) with
a peak
emission at 312 nm were used as the
UV-B source,
and measured
with
a VLX
312 radiometer equipped with a UV-B sensor (Vilber Lourmat,
Marne-la-Vallée France).
The cells
with the rice water were irradiated with a UV-B single dose of
26 mJ/cm2
for 15 min.
In each assay
,
ROS levels
were determined at 485 nm and 520 nm, as excitation
and emission wavelengths, respectively, using a florescence microplate reader (FLUOstar BMGLabtech,
Offenburg, Germany). Data collected from at least six replicates is expressed as percentage of reduction
of ROS.
Enzymatic Inhibition Assay
Fluorometric assays for the HNE inhibition activity were carried out in 200
µ
L assay buffer
(0.1 M HEPES pH 7.5 at 25
◦
C) containing 20
µ
L of 0.17
µ
M HNE (stock solution 1.7
µ
M in 0.05 M
acetate buffer, pH 5.5), 155
µ
L of assay buffer and 5
µ
L of each concentration of tested inhibitors.
After 30 min
of incubation at 25
◦
C, the reaction was initiated by the addition of 20
µ
L of fluorogenic
substrate to a final concentration of 200
µ
M (MeO-Suc-Ala-Ala-Pro-Val-AMC, Merck, Darmstadt,
Germany).
The Michaelis–Menten
constant (Km) of this substrate of HNE was previously determined
to be
185 µM
(data not shown). For all assays, saturated substrate concentration was used throughout
in order to obtain linear fluorescence curves. Controls were performed using enzyme alone, substrate
alone, enzyme with DMSO and a positive control (Sivelestat sodium salt hydrate, Sigma Aldrich
Quimica S.L., Lisbon, Portugal) [18].
2.3.8. Antimicrobial Activity
The antimicrobial activity was screened against Pseudomonas aeruginosa ATCC 9027, Staphylococcus
aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404 and Escherichia coli
ATCC 87394. The microorganism inoculum was prepared from 18 h broth culture and the suspension
was adjusted to a turbidity of 0.5 McFarland (~1.5
×
10
8
colony-forming units (cfu/mL). Then,
the Mueller–Hinton
agar (MHA) (Thermo Scientific
™
Oxoid
™
, Hampshire, UK) was poured into petri
dishes and inoculated with 100
µ
L of the suspension containing ~1.5
×
10
8
cfu/mL of microorganisms.
Sterile paper discs (6 mm; Thermo Scientific
™
Oxoid
™
, Hampshire, UK) were loaded with 15
µ
L
of rice waters. After incubation (37
◦
C, 24 or 48 h), the mean of the inhibition zone diameters were
calculated. Each assay was performed in triplicate.
2.4. Rice Water Gel (RWG) Preparation
Based on the results obtained from several physico-chemical characterization assays, RWB was
selected for the preparation of the rice water gel (RWG). The composition of the RWG and the control gel
is presented in Table 1. Dermosoft
TM
OMP (Evonik Dr. Straetmans GmbH, Hamburg, Germany) was
firstly dispersed in the rice water and the gelling agent, Lecigel
®
(Lucas Meyer Cosmetics, Champlan,
France), was added and mixed with vigorous stirring to allow for the formation of
a hydrogel
.
A control
gel containing deionized water instead of rice water was also prepared to compare the results of
in vivo
performed studies.
Cosmetics 2018,5, 26 6 of 12
Table 1.
Qualitative and quantitative composition of the prepared rice water gel (RWG) and control
hydrogel (HG).
Quantitative Composition (%, w/w)
Composition RWG HG
Sodium acrylates copolymer and lecithin 12.0 2.0
Methylpropanediol, caprylyl glycol and phenylpropanol
22.0 2.0
Water prepared by the boiling process (RWB) 96.0 -
Deionized water - 96.0
1LecigelTM;2Dermosoft®OMP
2.5. Rice Water Gel Physico-Chemical Characterization
Macroscopic organoleptic characteristics of the gel (colour, odour and appearance) were analysed.
The pH was determined using a 713 pH meter from Metrohm (Filderstadt, Germany), at a temperature
of 20
◦
C. The rheological profiles of RWG and control gel were evaluated at room temperature
using a Brookfield Rotation Viscosimeter
®
, RV DV-II, SSA with a spindle 07 (Brookfield Engineering
Laboratories, Middleborough, Massachusetts, USA). The shear rate [1/s] versus shear stress [Pa] plots
(flow curves) were obtained by submitting the samples to a shear rate sweep from 0.61 to 122/s up
and down. This means that the angular velocities varied from 0.5 rpm to 100 rpm and each one was
read after 30 s, and then reversed the velocity to the initial.
2.6. In Vivo Studies
2.6.1. Local Compatibility Tests on Human Skin (HRIPT)
The compatibility evaluation study was performed on RWG, using the Marzulli and Maibach
method [
19
]. Human Repeat Insult Patch Test (HRIPT) protocol, as described in detail elsewhere [
17
],
was performed to evaluate the RWG irritant capacity. This protocol was approved by the local Ethical
Committee and respected the Helsinki Declaration and the French Agency for the Safety of Health
Products regulations on performed HRIPT studies on cosmetic products. The study was conducted
under the supervision of a dermatologist who participated in the evaluation of irritation/allergic
reactions to the tested formulations.
2.6.2. Biological Effects of Rice Water Gel
Twelve healthy volunteers, aged between 21 and 46, were selected, and provided informed written
consent. The protocol was approved by the local Ethical Committee (Lisbon, Portugal). Volunteers
applied the RWG formulation on the forearm during 28 days and the results were compared with
a defined
control area (anatomically equivalent and without product). A placebo formulation (Control
Hydrogel-HG) was applied for the same period on the other forearm of the volunteers. The products
were applied in an amount of 2 mg/cm2.
The epidermal capacitance and skin surface lipids were evaluated with a Corneometer CM820
and a Sebumeter SM810 (C+K Electronics GmbH, Cologne, Germany), respectively, at day 0, 14 and 28.
Measurements were performed under standardized conditions, at room temperature according to the
Good Clinical Practices rules [20].
2.6.3. Rice Water Gel Sensory Analysis
A sensory analysis was conducted according to ISO 11136:2014, using a panel of 12 volunteers.
The panel
was questioned to scale characteristics of the RWG. Each volunteer answered a few questions
about the RWG sensory attributes, such as texture (consistency and stickiness), skin feel during
application (ease of application, spreadability and oiliness), skin feeling after application (hydration,
softness of the skin and freshness). Responses were given on a scale from 1 to 5. Sensory parameters
Cosmetics 2018,5, 26 7 of 12
were evaluated by a small amount of each formulation applied between the fingertips and rubbed into
the skin.
2.7. Statistical Analysis
Each value is the average of three different experiments
±
standard deviation, except when
referred within the text. Statistical analysis was assessed by one-way analysis of variance (ANOVA)
using the Sigma Plot 11.0 software
®
(Systat Software GmbH, 11.0, Erkrath, Germany). The differences
were considered statistically significant when p< 0.05.
3. Results and Discussion
3.1. Preparation and Characterization of Rice Water
In this work, we prepared and characterized a liquid generally named as Rice Water that could be
obtained by different methods but always involves the contact of water with rice grains.
The preparation
is empirically obtained in different regions of the globe and with different health purposes. In order to
investigate which method would lead to a rice water with higher capacity of reducing the ROS production,
three types of rice water were prepared: one was obtained by boiling the whole rice grain, another one
by stirring the water in contact with crushed rice grains, and another one by stirring the water in contact
with intact rice grain. After preparing the rice water, we observed higher turbidity for water prepared by
the boiling process (RWB) and with the crushed grain (RWS) than for the water prepared with the intact
grain (RWM). On the other hand, the water obtained from the crushed rice was milkier than the rice water
obtained by boiling the rice. These observations were proven by the conductivity and turbidimetry assays,
which results are present in Table 2.
Table 2.
Physical characterization results of water prepared by the boiling process (RWB), water prepared
with the crushed grain (RWS) and water prepared with the intact grain (RWM).
Parameters RWB RWS RWM
pH at 20 ◦C 6.8 5.4 6.0
Conductivity (µs/cm 25 ◦C) 947 1654 492
Turbidimetry (TNU) 91 283 100
Dry Residue (%) 0.45 2.59 0.10
The pH values obtained are adequate to be incorporated in a formulation for skin application
(Table 2). The dry residue results (Table 2) showed that the water obtained from crushed rice presented
more solids in suspension that were not removed by the filtration process through cotton gaze.
3.2. Evaluation of Rice Water In Vitro Anti-Aging Properties
The total phenol contents of the rice waters are 3.33
±
0.36, 3.15
±
0.41 and 0.23
±
0.01 mg/L for
RWS, RWB and RWM, respectively. The total polyphenol content of rice waters was higher for RWS
and RWB than for RWM, due to the water production method used.
Rice has been extensively studied due to the great quantity of bioactive compounds, due to the
beneficial effects of these compounds on human skin, namely their antioxidant and other biological
activities, such as enzyme inhibition. According to literature, cooking processes reduce only the
average content of total phenolics in the pigmented rice; on the other hand, in non-pigmented rice,
total phenolics compounds were not significantly affected by cooking, which is our case [21].
Phenolic compounds may act as antioxidants by different mechanisms, including free radical
scavenging and inhibition of pro-oxidant enzymes, such as tyrosinase and elastase [
7
]. Thus,
we further
tested the rice water capacity of reducing intracellular generation of ROS induced both by a chemical
compound (H
2
O
2
) or by UV light, assessed
in vitro
in human keratinocytes. The percentage of
reduction of ROS by RWB is approximately 80% for both methods comparable to the values obtained
Cosmetics 2018,5, 26 8 of 12
for the positive control, ascorbic acid (Figure 1). All tested rice water samples presented a positive
reduction, the RWM being the one presenting a lower effect.
Figure 1.
Reactive Oxygen Species reduction after exposure to H
2
O
2
and UVB radiation of HaCaT
cells, containing rice water samples and ascorbic acid. The data are expressed as the mean of at least
6 replicate experiments
±
standard deviation. Significance: (*) p< 0.05 versus positive control cells
(ascorbic acid).
This antioxidant activity might be due to the transference to the water of several phenolic compounds
identified in rice, tocopherols, tocotrienols and
γ
-oryzanol [
22
]. These results are also in accordance with
the phenolic content in rice water samples obtained in this study.
Very high anti-elastase activities were exhibited by RWB and RWS, which inhibited over 89.0 and
57.9% of enzyme activity respectively. A relatively moderate anti-elastase activity was exhibited by
RWM (24.2%).
In the MTT assay, the viable cells with active metabolism are capable of converting the MTT into
a purple compound, the formazan, while the dead cells are incapable of converting it,
which allows
for the differentiation between the viable and dead cells. RWS, RWB and RWM showed a cell viability
higher than 50%, while SDS showed 8.6% and the medium 100%. Hence, all rice waters can be
considered as non-irritant. However, the HaCaT cell lines are more sensitive than skin, since skin has
a functional barrier that prevents the total absorption of these compounds when applied to the skin.
The rate-limiting barrier to the absorption of topical cosmetics is the stratum corneum, due to its length,
which comprehends the number of cell layers, the thickness, the cell size and the difficulty of this
pathway that the substances have to cross.
These assays were determinant for the selection of rice water to be further incorporated into
a hydrogel
. Considering the results, RWB was chosen to be the main ingredient of the gel composition.
The amount of carbohydrates, proteins and lipids were determined for RWB and the results
obtained were 0.20%, 0.14% and 0.013%, respectively. These results may reflect the fact that we have
used paddy rice for the rice water preparation as the rice pericarp prevents the release of the rice grain
(white rice) components into the water. In addition, the content of insoluble phenolic compounds is
significantly higher in brown rice than in white rice, a fact which also can prevent the release of rice
components to the water [23].
We evaluated also the antimicrobial activity of rice waters prepared. None of them presented
antibacterial or antifungal activity. However, according to other research studies, boiled rice water
and soaked rice water showed antimicrobial and antifungal activity, due to zinc and selenium content
present in these types of rice waters [24].
Cosmetics 2018,5, 26 9 of 12
3.3. Preparation and Characterization of Rice Water Hydrogel
Semisolid dosage forms, such as a hydrogel, promote the consistency and the adhesiveness and
could be more suitable for topical application than the liquid ones. The rice water presenting the best
antioxidant and anti-elastase activity (RWB) was incorporated into a hydrogel formulation with very
few ingredients in order to incorporate almost 96% of rice water into the composition.
After preparing the RWB hydrogel and the control hydrogel, both odour and transparency were
consistent with the ingredients used. The hydrogel with rice water had a yellowish colour compared
to the control hydrogel, whitish in colour.
The pH values of both formulations prepared ranged between 5.4 and 5.9, meaning they are all
pH compatible with skin.
The hydrogel containing rice water also appeared to be more fluid than the control hydrogel.
This might
be due to the presence of phospholipids which results in low viscosity pastes and with
lower transmittance power [
25
]. The hydrogel also had a homogeneous appearance and an appropriate
consistency, but was easy to spread over a surface.
The rheology of the rice water hydrogel was studied since it is an important parameter in the
development of a semisolid formulation for topical application as it determines the spreadability.
The rice
water hydrogel appeared to have pseudoplastic and thixotropic flow, as shown in Figure 2.
In addition, the results presented in Figure 2prove that the hydrogel containing rice water is less
viscous than the control hydrogel, which is in agreement with that previously described. The results
also indicate that the rice water hydrogel has a rheological profile adequate for topical application.
Figure 2. Rheograms of the rice water hydrogel (RWG) and the control hydrogel (HG).
3.4. Biological Effects and Cosmetic Properties of Rice Water Hydrogel
During the HRIPT study, in the initial 3 weeks contact and even after the final challenge contact,
no reactions or skin sensitization/irritation were observed. Therefore, the repeated application of the
product did not induce any sensitization on the volunteers’ skin. The formulation presented very good
skin compatibility and absence of allergenic potential.
The skin is often exposed to physical and chemical agents which may affect the skin barrier.
Corneometry is a technology that is used to measure the hydration of the outer layer of the epidermis
(stratum corneum). Volunteers applied the Rice Water Gel (Test) formulation on the forearm during
28 days and the results were compared with a defined control area. A placebo formulation (HG)
was applied for the same period on the other forearm of the volunteers. Results (Figure 3) showed,
at day 28
, a 10% increase of hydration in the area where the rice water hydrogel was applied, relative
to day 14; however, there is no statistically significant difference between day 14 and day 28.
Cosmetics 2018,5, 26 10 of 12
Figure 3.
Skin hydration values (Arbitrary Units, AU) in terms of capacitance during 28 days. Control area:
forearm without any formulation application; Placebo: hydrogel; Test: Rice Water Gel (RWG).
Sebometry is a method employed for quantifying the fat content of the skin. Sebometry was measured
in the same tested area and the results were zero at day 1, 14 and 28 for all volunteers, which means that
rice water hydrogel did not significantly increase skin surface lipids compared to the control.
The cosmetic properties of the rice water hydrogel were assessed using a simple sensory survey,
in which 12 volunteers participated. The questions focused on the basic characteristics evaluated by
volunteers, during and after application (texture, skin feel, among others). Results showed that rice
water hydrogel met consumer appeal and acceptance requirements (Figure 4). Rice water gel presents
the highest score for spreadability, ease of application and stickiness, with a low score for the fragrance
and oiliness. Hydration and freshness have also high scores. These observations are in agreement with
the results obtained so far in corneometry and sebometry.
Figure 4. Sensory analysis of the rice water hydrogel.
4. Conclusions
In this work, we produced rice water by different methods. The rice water obtained after boiling
the rice presented
in vitro
biological antioxidant activity comparable to that of ascorbic acid and
remarkable elastase inhibition activity. Its incorporation into a hydrogel formulation has led to the
development of a semisolid dosage form suitable for topical application and with adequate cosmetic
properties. It would be very important to identify the component or the class of compounds responsible
for the antioxidant activity. Another important evaluation would be how such antioxidant activity
Cosmetics 2018,5, 26 11 of 12
could be used to supplement skin antioxidant capacity. Rice water should be thus considered as
an anti-aging ingredient to be used in skincare products.
Acknowledgments:
We thank Ana Neves and Casa Agricola Quinta das Barracas da Rainha for providing the
paddy rice samples.
Author Contributions:
Joana Marto and Sandra Simões conceived and designed the experiments; Joana Marto
was responsible for the evaluation of antioxidant activity; Ângela Neves prepared rice water and made the sensory
analysis evaluation; Lídia Maria Gonçalves evaluated elastase activity; Pedro Pinto was responsible for biological
evaluation (HRIPT); Cristina Almeida performed physico-chemical characterization of rice water; Sandra Simões
prepared rice water and rice water gel and was responsible for rice water characterization. All authors contributed
to the manuscript preparation.
Conflicts of Interest: The authors declare that they have no conflicts of interest with the content of this article.
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