Studies on Stability and Moisture Retention of Urea and Its Derivatives

Lv Yingfeng,Lv Wanqi,Zhou Jingmin

Guangdong Yalget Fine Chemical Co.,Ltd.,China

Abstract The effect of triethyl citrate (TEC) on the stability of cream preparations containing urea or hydroxyethyl urea along with their moisturizing effect on skin was investigated.The results showed that creams incorporating urea or hydroxyethyl urea led to increase in pH value after a long-term and high-temperature storage.pH change in the latter one was relatively smaller.With the addition of TEC,the elevated pH of the formula system was suppressed.In the situation of stable formula,the higher the TEC added,the more significant inhibition effect on the pH change was observed.In the moisture retention test in vivo,compared to 27.19% moisture rate in negative control,cream preparations containing 5% urea or 5% hydroxyethyl urea had remarkable moisturizing effect,which were all higher than 52%.The latter one was relatively more prominent.There is no significant effect on moisturizing effect for product with TEC additive.

Key words urea;hydroxyethyl urea;triethyl citrate;moisturizing properties;pH value

Urea is a moisture absorbent molecule found in the epidermis,and is a component of the natural moisturizing factor (NMF),which is essential for the adequate hydration and integrity of the stratum corneum (SC)[1].Urea binds water in the upper layer of the skin,reduces the rate of transdermal water loss,and helps to improve skin elasticity[2].Human body is able to absorb urea through specific urea transporters and water-glycerol transmembrane transport protein channels[3].In healthy SC,urea makes up 7% of NMF,and it would decrease with ageing.Lack of urea can lead to various skin diseases,such as 50% urea deficiency in dry skin and 60% in psoriasis[2].Topical urea preparation is a well-tolerated treatment,but has certain side effects at high concentrations.[4]Many clinical studies have used urea creams containing 10% or less,but no adverse events have been reported,which can verify the safety of topical urea preparations[5].

However,urea may release unpleasant odor under long-term storage or high temperature conditions,which is caused by volatile amines along with the pH increasing of products.Eventually,it would affect the sensory acceptability and stability of products[6].Usually,ammonia can be smelled when the pH is above 8.

In this paper,the stability and moisturizing effect of the formulae containing urea or its derivative hydroxyethyl urea with the addition of triethyl citrate,a low molecular weight ester,were evaluated.

1 Experimental sector

1.1 Main reagents and apparatus

Urea,Xilong Science Co.,Ltd.;Hydroxyethyl Urea,Akzonobel Corporation;Triethyl citrate (TEC),Shanghai Guanying Biotechnology Development Co.,LTD.;Deionized water,laboratory made;Other reagents used for auxiliary molding and preservative are cosmetic grade.Mixer,Guangzhou Yiren Analytical Instrument Co.,Ltd.Homogenizer,Shanghai Weiyu Electromechanical Manufacturing Co.,Ltd.pH meter,Shanghai Sanxin Instrument Factory;Corneometry,Courage+Khazaka,Germany.

1.2 Preparation of samples

The formulas used in this experiment were composed of two mass concentrations of urea (5%,10%) and two mass concentrations of hydroxyethyl urea (5%,10%),which were pair with five mass concentrations of triethyl citrate (0%,0.25%,0.5%,1%,2%),independently.The other auxiliary ingredients in each formula were added with the same portion,respectively.There were 20 formulae in total.The preparation process of the formula was as follows: the oil-soluble material and water-soluble materials were mixed evenly,respectively,and heated to 80 ℃ with a water bath.The oil phase material was added to the water phase in the agitated state.Keep the temperature not lower than 75 ℃ for 2 min.After stirring and cooling to less than 45 ℃,add preservatives and stir evenly to obtain experimental samples.

1.3 Performance test

Heat resistance stability: 50 g samples were put into the oven at constant temperature (40±2) ℃.At the 0,1,2,4,8,12,16 and 20 weeks,samples were taken out and restored to room temperature to observe the different from the samples stored at room temperature.The pH value of aqueous solution containing 10% samples was measured.

1.4 Moisture test

The corneometry was used to record the skin surface capacitance,which is based on the significant difference between the dielectric constant of water and other substances.According to the different moisture content of the skin SC,the measured skin capacitance value is different.Measurements are given in arbitrary unit (a.u.) ranging from 0 to 120 a.u.Higher capacitance values indicate a higher moisturizing effect of the sample on the skin.This experiment recorded the skin water content before and at 1,2,3 and 4 h after the use of samples.

2 Results and discussion

2.1 Mechanism of urea hydrolysis

The hydrolysis rate of urea is fast when the temperature is higher than 80 ℃,and the higher the temperature is,the faster the hydrolysis rate is.When the temperature is lower than 80 ℃,the hydrolysis rate of urea is relatively slow.The hydrolysis process can be divided into two steps:[7]

Equation (2-1) is the process of hydrolysis of urea to ammonium carbamate,which is rapid and exothermic;Equation (2-2) is the reaction of ammonium carbamate decomposed into ammonia gas and carbon dioxide,which requires heat and the reaction is violent.In terms of the total reaction(Equation (2-1)),the hydrolysis reaction of urea needs to absorb heat,and high temperature promotes the decomposition of urea.This also explains that formulas containing urea,when tested for heat resistance,give off a more pronounced ammonia smell than those at room temperature.Water,as a solvent,is usually the largest component in O/W cream preparations.Therefore,the large amount of water in the O/W preparation will also promote the urea hydrolysis equilibrium reaction to the right.The solubility of CO2produced by the reaction is very low in water,and its solubility can be ignored.NH3is present in the solution,but a small amount may escape.Urea as the only source of ammonia nitrogen in the solution,measuring the content of NH3in the solution can be an indication of the decomposition degree of urea to a certain degree.

2.2 Stability test

The pH value of each formula with different concentration of urea or hydroxyethyl urea and TEC were measured at different storage time span.As a result,the pH of each formula changed with time can be obtained,as shown in Figure 1.

Figure.1 pH change with respect to time for different formula

As depicted in Figure 1a and 1b,during the 20-week storage period,the pH of each formula generally increases with the extension of storage time.In addition,the pH range of the material in the first week was particularly sharp.This is because NH3 produced by urea hydrolysis reaction,under acidic conditions,will react with H+to form NH4+,increasing the consumption of NH3and thus shifting the hydrolysis balance of urea to the right.When pH reaches neutral,the hydrolysis rate slows down.The change in pH at a high concentration of urea was more pronounced than that at a low concentration,as increasing the concentration of urea caused the reaction shift to the right,and the final pH was higher.In addition,the range of pH change in the formula with TEC was smaller than that in the formula without TEC.The higher the amount of TEC added,the smaller the pH changed.However,at 2%TEC concentration,pH value eventually fluctuated at around 6.8.Except for experimental error,this might be due to the reversible reaction of ammonia in water and the effect of slow hydrolysis of TEC,which formed a buffer effect on the system.In addition,demulsification and delamination occurred in the high/low concentration urea cream containing 1.0%and 2.0%TEC at the 16th week due to the limited emulsification and suspension ability of the system,as well as urea degradation.

Consistent with the urea cream,the change of pH in the formulation containing hydroxyethyl urea also decreased with the increase of TEC.The difference was that the pH increase in week 1 was much more moderate than that of the urea cream,and the final pH increment was smaller.As illustrated in Figure 1c and 1d,with the increase of pH,the hydrolysis rate of hydroxyethyl urea generally shows a slowing trend when the system tended to be neutral and alkaline.After 20 weeks of stability testing for TEC free urea cream,the pH raised to about 3.5.In hydroxyethyl urea cream,the maximum pH change is about 2.5.Therefore,the stability of hydroxyethyl urea in the formulation was better than urea.Structurally,the hydroxyethyl urea derives from urea by means of replacing one of the hydrogen atoms in the amino group with a hydroxyethyl group,thus there is a less tendency to be degraded,and it is better retain its activity.According to the final results of the experiment,the pH change value of the formulae with high or low hydroxyethyl urea content could be stabilized at about 1 by 2%TEC,and no stability problem appeared.Thus,it suggests that the stability of TEC and hydroxyethyl urea compound formula is much better than the combination of TEC and urea.

2.3 Stabilizing mechanism of TEC

On the one hand,TEC can slowly hydrolyze into citric acid in water,which can partially neutralize the increase in pH caused by urea hydrolysis.On the other hand,TEC is a low molecular weight ester compound,which can be structurally combined with the polymer by a mean of weak force and without changing the macromolecular structure.TEC can fill the gap of polymer matrix and extend the distance between polymer molecules,thus increasing the thickness of the film and making the molecular structure more stable[8].In addition,the hydrophilic properties of urea enable it to function as a hygroscopic agent and form hydrogen bonds with TEC and other molecules,which is also conducive to improving the thickness of the membrane during the swelling process of the polymer[9].This consolidate its structure in the formula system,thus greatly slowing the tendency of urea hydrolysis in long-term storage and high temperature.The capability of TEC to increase membrane flexibility is widely implemented in drug sustained-release and food packaging[8,9].

2.4 Quantitative analysis

Measuring the pH of the sample can indicate the hydrolysis of urea in the formula to a certain extent,but the factors affecting the pH value are not only the hydrolysis of urea,but also the hydrolysis of TEC.The decomposition rate of urea can be intuitively reflected by quantitative measurement of urea in the material.As urea and hydroxyethyl urea are the only sources of ammonia nitrogen in the formula,the measurement of ammonia content in the material can also be used as an indicator of the degree of hydrolysis.Among the current cosmetic standards,“Determination of Ammonia Content in Cosmetics Titration Method (GB/T40970-2021)” is used to determine the content of free ammonia in the cosmetic products through the principle of acid-base neutralization.In practice,the ammonia content in the above formulations is mostly about 0.2% according to this method,which cannot be used as a good basis for determining the degree of urea hydrolysis.In addition to the accuracy of acid-base titration experiment itself,the reasons may include long-term high-temperature storage,the tightness of PET bottle,and frequent opening of the cap process,which lead to the leakage of a large number of volatile amines,thus affecting the accuracy of experimental results.Therefore,the accelerated aging test needs to be designed more strictly,such as the use of vacuum bottles,so that the results obtained by this method will be more reliable.In addition,“Determination of urea content in cosmetics enzyme catalytic method GB/T 29661-2013” shows the use of urease,which is specificity to urea,can directly measure the urea content.Without cost consideration,the use of liquid-mass spectrometer (LC-MS) allows for better qualitative and quantitative analysis of the substances in the product.The principle is to separate the sample through chromatography.After ionization,substances can be separated according to the molecular weight of ion fragments through the mass analyzer of mass spectrometer.This method can simultaneously detect the final content of urea or hydroxyethyl urea and triethyl citrate.

2.5 Moisture retention

A total of 12 volunteers aged between 20 and 45 were recruited for the study.The test environment was kept at the temperature of 20～ 22 ℃ and the humidity of 40%～60%.None of the subjects experienced adverse reactions during the experiment.The whole experimental test procedure followed the Guidelines for Evaluation of Moisturizing Effect of Cosmetics(QB/T4256-2011).Three 3 cm×3 cm areas were delimited on the subject’s left and right forearms,with a spacing of at least 1cm between each area.Apply an equal amount of the corresponding sample to the test area and measure the water content of the skin every 1 hour.5 formulas,cream containing 5% urea,5%hydroxyethyl urea with three gradient TEC (0%,1%,2%) and the negative control group,were tested in this study.As shown in Figure 2,the negative control,urea cream and three samples containing hydroxyethyl urea were applied on the skin,and the moisture rate results after 4 h were 27.19%,52.29%,64.63%,65.94% and 59.42%,respectively.Among them,hydroxyethyl urea cream containing 1%TEC had the most significant moisturizing effect within 1 h,and the measured result was as high as 73.56%.Urea also had a significant moisturizing effect,but its moisturizing ability was lower than other positive formulas.The results showed that both 5% urea and 5% hydroxyethyl urea had significant moisturizing effect on skin (P<0.005),and the latter one was better than the former.However,the moisturizing effect of high TEC content on skin would be worse than that of no or low TEC supplemental level.This may be because the high percentage of TEC affects the bearing capacity of the emulsion system,which was manifested as excessive softening of the product.

Figure.2 Moisture retention ability with respect to time for different formulae

3 Conclusion

Studies have shown that in formulas containing urea and urea derivatives,there is a certain extent of degradation during prolonged high-temperature storage,which eventually leads to an increase in pH.The addition of TEC can slow down the rising of pH.On the basis of a stable formulation,the higher the addition amount is,the better the reduction effect is.In addition,TEC does not stabilize urea formulation as well as hydroxyethyl urea.Regardless of low content or high content of hydroxyethyl urea,2%TEC can stabilize the pH change of the formula at around 1 increment.In the 4h moisture test,both 5% urea and 5% hydroxyethyl urea have significant moisturizing effect on skin,and the moisture ability of hydroxyethyl urea is better than urea.Among them,1%TEC is the best.The 2%TEC hydroxyethyl urea cream also have significant moisturizing effect,but its performance is slightly worse than that of the 1%TEC cream.In general,compared with urea,hydroxyethyl urea has a better moisturizing effect,skin feeling,and is able to maintain the long-term stability of cosmetics.However,high TEC dosage may increase the cost of the formula,and may also have a negative impact on the emulsifying system.Attention should be paid to adjusting the emulsifying suspension performance while applying TEC.In the field of cosmetics,it remains to be developed whether there are other more efficient,more stable and cheaper methods to stabilize pH change more effectively throughout the life of the product.