INTRODUCTION:

Vit C(L-ascorbic acid) is widely used for its multiple functions inthe growth and repair of all parts in the body¹ including the skin. It isadministered as antiaging agent due to its important role in collagen synthesis^2,3. by promoting the transcription of mRNA³. It is also a depigmentation agent^4,5,6. Vit C has powerful antioxidant activity^4-10 because it can donate hydrogen atoms to scavenge free radicals^6,12 generated on exposure to sunlight, it protects thereby the skin by scavenging these radicals¹³.

Because of these advantages, Vit C has been used in both cosmetic and pharmaceutical industry¹⁴. However, Vitamin C is easily oxidized in aqueous solutions, in alkaline pH, upon exposure to light, heat, oxygen, metal ions.(Figure 1). The process of oxidation is accompanied by a yellow color¹⁵. Ascorbic acid undergoes degradation upon the absorption of ultraviolet radiation in the wavelength range of 229–330 nm¹⁶. The formation of UV-induced free radicals may acceleratethe degradation of ascorbic acid³.

After eutralizing, ascorbic acid converts into dehydroascorbic acid, which can regain antioxidant activity after accepting hydrogen atoms. Further oxidation of dehydroascorbic acid into diketogulonic acid results in complete loss of bioactivity¹⁷.

Figure 1: Oxidation of vitamin C

Therefore, the main challenge in vitamin C formulation is the maintain of its chemical bioactivity and stable ascorbic acid should be accurately delivered to the desired site for obtaining excellent bioavailability of ascorbic acid. Therefore, several researches have been focused on enhancing vitamin C stability by different strategies³.

Ascorbic acid has been combined with chelating agents (eg. EDTA) and antioxidants (eg. Cysteine, glutathione, metabisulfite Na, hydroquinone¹⁸, alpha-tocopherol^18,19,20. Moreover, the role of different formulation factors has been investigated on vit C stability (increasing viscosity, reducing water activity, pH adjustment)²¹. Formulations containing derivatives of ascorbic acid (such as ascorbyl 2-glucoside (AA2G), 3-o-ethyl-Lascorbic acid (AAE), and ascorbyl 6-palmitate (AA6P) have been found to be more stable^22,23,24, but they do not produce the same effect as that of the parent compound²⁵.

The objective of this research is to assess vitamin C stability mathematically by accelerated stability test. The determination of rate constants of vitamin C degradation will allow the calculation of the shelf lives of vitamin C in different pure aqueous solutions or mixtures: water: alcohol or water: glycerinand to show the effect of the use of HPMC or chitosan as thickening agents.

MATERIAL AND METHODS:

Material:

Vitamin C was purchased fromLobachemie, India. 2,6-dichorophenol:Indophenol Sodium was obtained fromTmmedia, India. HPMCK 4M and chitosan were obtained from Sigma-Aldrich. HCl, alcohol and glycerin were of analytical grade.

Methods:

Preparation of Vit C solutions:

Vit C solutions were prepared according the method cited in Handbook of pharmaceutical excipients²⁶. HPMC was dispersed in 20% of the required amount of waterthen, the remaining water containing Vit C was added to required volume. When glycerin and alcohol were used, HPMC was dispersed into the organic solvent then water containing Vit C was added to final volume. The compositions of formulated solutionsunder different variables were presented in Table 1.

Table 1: Composition of aqueous Vit C solutions

	A	B	C	D	E	F	G
HPMC (g)	1	2	2	2	2	2	-
Chitosan (g)	-	-	-	-	-	-	1
Vit C(g)	10	10	10	10	10	10	20
Glycerin (ml)	-	-	20	40	-	-	-
Alcohol (ml)	-	-	-	-	20	40	-
Water up to	100 ml	100 ml	100 ml	100 ml	100 ml	100 ml	100ml

Vit C solutions characterization:

Visual inspection:

The solutions were inspected for color²⁷. A number from 0 to 5 was used to quantify the color of Vit C solutions from colorless (0) to dark yellow (5) (Table 2).

pH determination:

The pH of solutions were determined by pH meter )Inolab, pH 7110, Germany) in triplicate. The pH of the prepared solutions must be suitable for Vit C stability.

Spreadability test:

Spreadability is a measure of lubricity²⁸. It depends on viscosity of the formulation and on physical properties of the thickening polymers²⁹. Higher spreadability values increase surface area available for drug permeation³⁰. Hence, therapeutic efficacy may be enhanced³¹.

This test was carried out according to previous study³²with little modifications. Briefly, 0.5 g of each solution was placed between two horizontal plates (20 cm × 20 cm) and the weight of the upper plate was standardized at 60g. The mean spreading diameter "d" (in vertical and horizontal axes) was determined after one minute and the areas of circles "S" (S=d²π/4) were recorded as spreadability values. Spreadability studies were performed on solutions at room temperature and 37˚ C in the 14^th and 21^th days after preparation for monitoring possible changes in viscosities.

Vit C determination:

The bioactive Vit C was determined using 2,6-dichlorophenol:indophenol (DCPIP) method according to AOAC official method (1984)³³ with little modification in which the metaphosphoric acid was substituted by hydrochloride acid HCl (0.2%). Diluted Vit C solutions from different solutions were titrated by standardized solution DCPIP.

Vit C Stability Analysis Using Accelerated Stability test:

Seven closed tubes containing 10 or 20% ascorbic acid were placed in two ovens (A & E Lab, UK) at two temperatures: 37˚ and 45˚ C for accelerated stability test. Vit C degradation rate increases with the temperature³⁴. These temperatures were chosen based on prior studies of Vit C stability^35,36. Preparations were stored in the dark and samples were taken periodically at 0 (100 % ascorbic acid), 7, 14, 21 and 28days from the starting point of the experiment. An aqueous solution of Vit C (10%) without additives was used as a control for determining its stability.

Kinetic modeling:

A zero-order model: C=C₀-Kt (1) and first order model: ln C=lnC₀-Kt (2) were fitted to the kinetic of Vit C degradation under the investigated conditions of this study³⁷.

Shelf life determination:

Based on the obtained data from the kinetics of degradation reaction rate, the following were calculated: the degradation reaction rate constants of Vit C (k) at the temperature of 37 and 45 °C (K₃₇ and K₄₅). Using Arrhenius equation (K=A.e^-Ea/RT)and the rate constants: K₃₇ and K₄₅,the activation energies (Ea) were calculated³⁸.By using Arrhenius equation again and substituting Ea and either K₃₇ or K₄₅, the rate constant at room temperature (K₂₀) was then calculated and the shelf lives of Vit C(t₉₀) were determined. The shelf life (t₉₀) is the time after which Vit C concentration decreases by 10 % in the solution³⁹.

RESULTS AND DISCUSSION:

Vit Csolutionswere yellowish and translucent. As the time passes the color becomes more yellow du to Vit C decomposition. Table 2 shows the numbers coding the intensity of color as the time goes on.

pH values ranged between 2.45 and 3.23. These values are acidic and probably due to high concentration of Vit C that makes the pH falls down⁴⁰. Under these conditions, vit C is at its unionized form (pKa=4.2)⁴¹.

It has been reported that the Vit C oxidation is pH dependent with a minimum at pH 2.5 to 3.0⁴²and its further decomposition is probably due to others factors rather than the pH.

Spreadability test:

Table 3 shows spredability values of Vit C solutions at room temperature and at 37⁰C. when HPMC concentration is increased, spreadability value decreased and this is explained by the fact that the viscosity of HPMC solutions is concentration related (handbook). From the Table 3, it is evident that spreadabilities of formulations with glycerin or alcoholdecrease when compared with formulation non-containing these solvents. Glycerin increases water viscosity so the two formulations: C and D are more resistant to spread in comparison with B non-containing glycerin and the spreadabilty decreases with increasing glycerin content.On the other hand, alcohol does not increase water viscositybut it is not the solvent of first choice for HPMC and HPMC solutions of organic solvents tend to be more viscous²⁶. Therefore, HPMC channels might be more coiled in alcoholic aqueous solutions when compared with purely aqueous solutions (E versaB) and this effect is more important whenalcohol content is increased (E and F).The influence of other factors is not clear under the investigated conditions.

Determination of Vit C shelf life under different conditions:

Table 4 shows R² values of degradation rates and shelf lives of Vit Cin different formulations at both temperatures: 37⁰C and 45⁰C.

Table 2: Color and pH values of different vit C solutions

		A		B		C		D		E		F		G
		Color	pH	Color	pH	Color	pH	Color	pH	Color	pH	Color	pH	Color	pH
0	25˚C	0	2.63	0	2.81	0	2.76	0	2.73	0	2.9	0	3	0	2.75
21^th day	37˚C	3	2.56	3	2.64	2	2.68	1	2.67	2	2.92	3	2.69	3	2.69
21^th day	45˚C	3	2.59	3	2.63	2	2.7	1	2.64	3	2.8	3	2.97	3	2.71
28^th day	37˚C	3	2.61	3	2.57	3	2.45	2	2.63	3	2.58	3	2.62	5	2.63
28^th day	45˚C	4	3.02	4	3.23	3	3.18	2	2.71	4	2.61	4	2.83	5	2.69

Table 3: Spreadability values (mm²) of different vit C solutions

Day	Temperature	A	B	C	D	E	F	G
7^th day	25˚C	122.66	113.04	86.55	46.54	90.72	53.43	76.16
7^th day	37˚C	125.62	113.04	56.72	41.26	90.72	50.24	103.82
14^th day	25˚C	99.35	82.47	60.10	35.77	60.10	35.77	99.35
14^th day	37˚C	86.55	67.17	41.26	25.95	74.62	38.47	86.55

Table 4: The determination coefficients: R²₁ (first order)and R²o (zero order) of Vit C degradation kinetics at 37˚ and 45˚ C

		A	B	C	D	E	F	G
37˚ C	R²₁	0.96	0.9917	0.97	0.99	0.98	0.95	0.97
37˚ C	R²o	0.94	0.9912	0.93	0.94	0.97	0.92	0.96
45˚ C	R²₁	0.99	0.97	0.99	0.999	0.99	0.98	0.999
45˚ C	R²o	0.98	0.96	0.98	0.99	0.98	0.97	0.99

Figure 2 illustrates the profiles of Vit C degradation at 37C according to first order.

From Table 4, it is obvious that the Vit C’ decomposition follow the equation for first-order reactions and this is in accordance with previous studies^35,43,44.

The calculated shelf lives of Vit C are gathered in Table 5. They range between 13 and 40 days.

Table 5: The shelf lives (t₉₀) of Vit C in different formulations

	A	B	C	D	E	F	G
t₉₀ (day)	21.7	20.11	26.9	30.3	22.6	13.9	40.5

Vit C degradation depends on manyfactors such as oxygen, temperature, light, pH, metal ions and storage conditions^45-50. Therefore, any factor that decrease the transfer or diffusion of oxygen may be favorable for Vit C stabilization.

The impact of oxygen mass transfer to the reaction site was studied by Mohr⁵¹. He stated that the transfer of oxygen (governing by diffusion) might be limiting for Vit C degradation. It has been noticed earlier that an increase in the viscosity would lead to lower rate of Vit C degradation^52,53. Viscous systems prevent diffusion of molecules and enhancements in Vit C stabilities are therefore proven⁵⁴. In this sense, both formulations C and D formulated with glycerin showedenhancedVit C stabilities as shown in Table 5 (26.9 and 30.3 days respectively). Previous works reported the role of glycerin as viscosity-increasing agent and stability enhancer^47,55. In addition, glycerin is humectant and can attract water molecules via hydrogen bonds reducing therebyits activity⁵⁶. Reduced water activity is related to low free water enhancing thereby Vit C stability.

For formulation B containing 2% HPMC in comparison with A containing 1%, the effect of viscosity on stability was not noticed (although, Table 3 shows lower spreadability of B versus A). The shelf life of vitamin C in B was slightly lower than that of A (20 vs 22 days respectively) and the color of formulation B at room temperature was more intense than that of A. This might be explained by the impurities in HPMC (eg. Propylene oxide) which are sensitive to oxidizing agents²⁶. In presence of Vit C, propylene oxide could be protected from these oxidizing agents viaVit C, consumption of more Vit C will be thereby shown.

When alcohol is introduced in formulation E, an enhancement in Vit C stability is noticed (shelf life of Vit C was 27 days vs 20.11 days in B). This is attributed to reduction of water activity and thereby reduction of vitamin degradation⁵⁷.

It has been reported in other works that anincreasein the solvent dielectric constant leads to an increase in the rate of photolysis⁴². For this reason, formulation C containing glycerin (dielectric constant=40) would haveprobably a smaller shelf life than that of E containing alcohol (dielectric constant=24).However, the impact of viscosity and lowering water activity induced by glycerin has a more significant effect than its polarity on the shelf life of Vit C (26.9 in C containing glycerin vs 22.6 days in E containing alcohol). At high concentrations of glycerin and alcohol (D and F respectively) the positive effects of glycerin may overcome the effects of alcohol very significantly and the shelf life in formulation D is more important than that of formulation F (30.3 vs 13.9 days respectively).

However, when alcohol content increased from 20 to 40% (E and F) the stability of Vit C reduced. These results agree with those of Hsu et al.(2012) and Barril et al.(2011) who reported that ascorbic acid (as an antioxidant) undergoes oxidative degradation more rapidly in aqueous ethanol solutions compared with that in pure aqueous solutions^44,58. Chuang et al. 2016 explained the impact of ethanol on the degradation of ascorbic by the fact of lowering of water activity, thereby allowing easier dehydration of xylosone (one particular product of dehydroascorbic acid). This would lead to an accelerated decrease in dehydroascorbic acid which would in turn induce the direction of the reaction towards the products. In our results, the effect of ethanol on lowering Vit Cstability has been noticed in formulation F containing 40% and not in formulation E with 20% ethanol and the shelf life of Vit C in F is smaller than that of B (pure aqueous solution).

Chitosan is a cationic polysaccharide with strong chelating properties³ which may reduce the effect catalyzer of metal ions. Vit C can interact with the amino groups in the backbone of chitosan to form strong hydrogen bonds, which can retain Vit C on the polysaccharide^59,60 and thus keeps the high antioxidant capacity of Vit C³. The highest shelf life was reported with chitosan (40.5 days)although the number 5 has been given to the color of its solution(Figure 3). This is possibly due to the high concentration of Vit Cin formulation G, therefore the degraded amount of Vit C is more important than that in other formulations (eg. A).

Figure 3: Change of color in formulation G containing chitosan

Chitosan may be very good choice for dermal preparations because it is a biodegradable polymer and it has shown excellent biocompatibility characteristics⁶¹. Chitosan can disturb keratin fluidity by enhancing water content so chitosan can be used as permeation enhancer⁶².

The studied parameters (HPMC, Chitosan, Alcohol and Glycerin) have been shown improvement in vit C stability relatively to aqueous solutions without any additives (degradation of Vit C to 38%, 25% and 17% after one week at room temperature, 37˚C and 45˚C respectively).

CONCLUSION:

Aqueous solutions containing Vit C were formulated and the effects of different factors were studies. Both viscosity of the vehicle and polarity of solvents played an important role in Vit C stability. When alcohol was increased to 40% the shelf life decreased and long shelf life (30.3 days) wasnoticed in formulation containing high concentration of glycerin. Chitosan also extended Vit C shelf life to 40.5 days. Further studies with these optimal conditions would be promising.

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Received on 15.12.2023 Modified on 16.01.2024

Research J. Pharm. And Tech 2024; 17(8):3874-3880.

DOI: 10.52711/0974-360X.2024.00601