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 (H2
) 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 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.
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
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 2
prove 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.
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.
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.