INTRODUCTION:

Vitamin C (Vit C) or L-ascorbic acid is a hydrophilic molecule with low molecular weight, which is fundamental to the proper functioning of the human body^1,2. One of the most recognized properties of Vit C is its antioxidant capacity: in-vitro and in-vivo studies confirm the ability of this molecule to protect the cells against UV-induced damage by scavenging free radicals ^3-6. Besides, Vit C is able to regenerate other antioxidants species, such as alpha-tocopherol (vitamin E), inhibiting indirectly lipid peroxidation^3-9. Another essential role of Vit C is the stimulation of collagen biosynthesis.

Vit C directly activates collagen synthesis by enhancing the transcription of its mRNA, which is required for the optimal functioning of several hydroxylases^10,11. In addition, Vit C can suppress the production of melanin by interfering with the active site of tyrosinase¹².

Due to these advantageous effects, Vit C has been used in personal-care formulations for many years^2,13. However, it is necessary to take into account that Vit C must be kept stable in order to maintain its biological activity ⁵. During the degradation process, Vit C is easily oxidized to dehydroascorbic acid and afterward to diketogluconic acid. In this stage, the oxidation is irreversible, and the physiological activities are compromised¹⁴. Nevertheless, this represents a challenge since Vit C is degraded in the aqueous medium, at alkaline pH (maximum stability is obtained at pH ≤ 4), and in the presence of light, oxygen, and metal ions^{15,16, 17,18}.

In order to increase the stability of Vit C, several strategies have been used. Positive results have been achieved with the addition of other antioxidants in the system, such as vitamin E, ferulic acid, and sodium metabisulfite¹⁹. The rate of chemical oxidation may also be affected by the physicochemical aspects of the preparation. Therefore, a careful study and selection of the formulation ingredients may be helpful to improve Vit C stability. Initially, Sheraz et al. demonstrated that certain emollients and humectants might decrease the degradation of Vit C¹. However, improved stability results were found with the utilization of castor oil and glycerin. It was also observed that these excipients increased the viscosity of the formulation and thus affected the degradation rate of Vit C. The utilization of emulsified/encapsulated systems also seems to be advantageous^19,20.

A number of derivatives of Vit C, such as sodium ascorbyl phosphate, ascorbyl palmitate, ascorbyl glucoside, and 3-O-Ethyl ascorbate, have also been used in the preparation of cosmetics and pharmaceuticals¹⁵. However, despite being more stable, they need to be converted in Vit C in- vivo and generally present a limited permeation through the skin. Therefore, the utilization of Vit C in its original form is preferred^1,16.

Nowadays, there has got a tendency to utilize natural ingredients in cosmetics products²¹. Among these materials, clay minerals stand out due to their healing properties as well as their versatility and global accessibility²². In personal-care formulations, they may be used as an active ingredient—assuring anti-inflammatory, antibacterial and wound-healing properties to the product—or as excipients. In this sense, the peculiarity of their physical and chemical properties allows their utilization as emulsion stabilizers, gelling agents, rheological modifiers, topical delivery agents, among others^23,28.

In the last few decades, considerable attention has been given to the use of kaolin minerals. The predominant mineral in this clay is kaolinite. Its structure is of the 1:1 type and consists of alternating sheets of tetrahedral silica and octahedral alumina. This structure is highly organized and well-balanced, with little or no ionic substitution, thus presenting low cation-exchange capacity. The theoretical structural formula of kaolinite is Al2Si5O5(OH)422,24,28. In thepresent work, O/W emulsion containing or not kaolin was developed for the topical delivery of Vit C. The formulation were submitted to chemical stability assays by HPLC.

MATERIALS AND METHODS:

Cetearyl alcohol (and) polysorbate 60 (and) cetearyl glucoside was purchased from Chemyunion; polyacrylate crosspolymer-6 from Seppic (Fairfield, NJ, USA); caprylhydroxamic acid (and) caprylyl glycol (and) glycerin from Inolex (Philadelphia, PA, USA); ascorbic acid – Vitamin C (Fagron, USA), propylene glycol, hydroxyethylethylenediaminetriacetic acid (HEDTA), and sodium metabisulfite, sodium citrate dehydrate, citric acid were obtained from Sigma Aldrich (St. Louis, MO, USA). In addition, sodium hydroxide, methanol, water and ortho-phosphoric acid 85% HPLC grade were obtained from Fisher Scientific (Pittsburgh, PA, USA). The sample SGY^® is impure kaolin clay provided by Terramater (Itajaí, SC- Brazil); the clay was mined from a sedimentary

Deposit and passed through an industrial grinding process to fulfill the grain-size requirements for cosmetic use.

Six O/W cosmetic emulsion (Table 1) were prepared. The aqueous phase (Phase B) was pre- mixed using a magnetic bar for 10 minutes to achieve complete dispersion of the kaolin. Then, the oil phase (Phase A) and the aqueous phase (Phase B) were heated to 85 ± 2^oC separately. The aqueous phase was poured into the oily phase with agitation speed of 1500 RPM, using a mechanical stirrer until cooling to below 40^oC. The ascorbic acid, at 5.0 %, was solubilized separately in the citrate buffer (Phase C) and the pH of this solution was corrected into the range of 5.5 and 6.0 with sodium hydroxide aqueous solution at 10% according to Nagase America LLC-New York USA recommendation. This precedent was used in all formulations. The aqueous sodium hydroxide solution and preservative (Phase D) were then poured into the fresh emulsion below 35^oC and homogenized using a Dremel Moto-Toll model 395 homogenizer at ~ 24000 RPM for three minutes, which was considered the ideal homogenization time for the emulsions to achieve complete homogeneity.

Table 1: Qualitative and quantitative (%w/w) composition of emulsions developed

	Formulations
Component (INCI name)	FA	FB	FC	FD	FE	FF
Phase A
Cetearyl alcohol (and) Polysorbate 60 (and) cetearyl glucoside	8.0	8.0	8.0	8.0	8.0	8.0
Polyacrylate crosspolymer-6	0.2	0.2	0.2	0.2	0.2	0.2
Propylene glycol	4.0	4.0	4.0	4.0	4.0	4.0
Phase B
Kaolin SP	-	3.0	3.0	3.0	3.0	3.0
Sodium metabisulfite	-	0.2	0.2	0.2	0.2	0.3
Hydroxyethylethylenediaminet riacetic acid	-	0.2	0.2	-	-	0.3
Disodium EDTA	-	-	-	0.2	0.2	-
Distilled water	q.s 100	q.s 100	q.s 100	q.s 100	q.s 100	q.s 100
Phase C
Citrate buffer (pH = 4.00)	20.0	20.0	-	20.0	-	20.0
Ascorbic acid (Vitamin C)	5.0	5.0	5.0	5.0	5.0	5.0
Sodium hydroxide aqueous solution at 10 %	pH 5.5 -6.0	pH 5.5 -6.0	pH 5.5 -6.0	pH 5.5 -6.0	pH 5.5 -6.0	pH 5.5 -6.0
Phase D
Caprylhydroxamic acid (and) caprylyl glycol (and) glycerin	0.5	0.5	0.5	0.5	0.5	0.5

The blank formulation correspondent named active-free without ascorbic acid (vitamin C). Ascorbyl glycoside samples were quantified using an Agilent 1100 series HPLC (Agilent Technologies, CA, USA) coupled with UV (254nm) and a diode array detector (DAD). A mobile phase of 85% orthophosphoric acid HPLC grade at 0.1% (pH 3.8) was pumped at a flow rate of 1.0 mL/min through a Vydac® TM Reverse Phase C18 / 250 mm x 4.6 mm (ambient temperature) and the volume for each injection was 20 μL. A HP Chemstation software V. 32 was used for data acquisition^25,26,27. The ascorbic acid (vitamin C) was purchased from Fagron, USA. The water and 85% orthophosphoric acid HPLC grade from Fisher Scientific were used to prepare the mobile phase. All chemicals and reagents were used without any further purification.

Table 2: Linear regression for the determination of ascorbic acid in the cosmetic formulations

Compound	Concentration (µg mL^-1)	Regression equation*	r²*
Ascorbic Acid	500, 250, 125, 62.5, 31.25,	y = 60.812x –	0.999
	15.63, 7.81, 3.91	72.569

*n=3

One-way analysis of variance (ANOVA) was used to check for overall differences in variance, and a paired t-test was used to assess differences between formulation A-B, C-D-E, and F.. P- values <0.05 were considered statistically significant. The residual concentration was calculated by dividing the concentration at different time points by the concentration at time zero.

RESULT:

The influence of different components on the stability of vitamin C was evaluated. In Figure 1, the residual percentage of ascorbic acid was obtained for formulation containing clay mineral, chelators ethylene diaminetetraacetic acid, disodium salt (Na2EDTA) and (HEDTA) and antioxidant (sodium metabisulfite) and the emulsionwithout these ingredients (FA) at different point time.

Figure 1: Changes of vitamin C concentration in different formulation with time.

As observed, the addition of chelators and antioxidants to clay emulsion improved vitamin C stability since the residual percentage of formulation FB-FF remained similar to the initial value after 10 hours. In contrast, in the base emulsion (FA), the residual percentage reduced to 40% before this time point.

There were statistically significant differences between formulation A and all other formulations with a (p-value 0.04), and static differences between formulation B and C (P-value 0.03), formulation C and D (P-value 0.04), and formulation D and E (P-value 0.04).

Clay minerals have been used in cosmetic preparations due to their high ion-exchange capacity, thermal stability, softness, the small size of their particles, and their attractive adsorptive properties^22,30. Kaolinite is a 1:1 type dioctahedral layered structure whose interlayer space is composed of different surfaces (tetrahedral silica sheet and octahedral alumina sheet) linked through hydrogen bounds^31,32. This clay mineral group can form intercalation complexes with polar substances and, thus, act as a potential drug carrier and prevent the speedy decomposition of drugs^32-35.

DISCUSSION:

Intercalation is a process in which molecules are incorporated into the interlayer space and stabilized through electrostatic interactions. Formation of clay-drug complexes depends on the polarity, binding ability, and size of the guest substances^28,31,36. It is proposed that these molecules ideally have one of the following characteristics: (i) acceptor and donor sites, (ii) a high dipole moment and (iii) large cations of short-chain fatty acids ²⁸. Thus, compounds such as salt, ionic liquids, and dipolar organic molecules can covalently link to hydroxyl groups on the alumina sheet and spontaneously align due to dipolar interlayer environment induction^34,35,37.

Intercalation and adsorptive properties of clay minerals have been applied in the pharmaceutical and cometic fields to improve the solubility, photo and thermal stability of various classes of drugs such as antibiotics, anti-inflammatories, and antioxidants^{30,31,33,38,39,41,42.}

Among the sensitive bioactive molecules, vitamin C represents a challenge to the cosmetic industry due to its chemical instability in semisolid formulation. Different strategies such as the incorporation of this antioxidant molecule in carrier systems, exclusion of the oxygen, protection against light and temperature, and utilization of antioxidant system are applied to prevent degradation of the compound. In addition, formulation excipients with antioxidant and chelating Functions can help prevent vitamin C degradation^2,40,43,44.

Thus, it was observed in this study that the use of sodium metabisulfite (antioxidant), disodium EDTA and HEDTA (chelators) associated with the application of clay as a potential carrier system contributed to the increase in vitamin C stability compared to base emulsion (F1).

Several strategies can be used to enhance ascorbic acid stability in cosmetic formulations. The application of kaolinite as a potential drug-delivery system, associated with the use of antioxidant and chelating ingredients, resulted in a degradation reduction of the active molecule compared to the formulation without these ingredients.

REFERENCES:

1. Rincón-Fontán M et al. Novel Multifunctional Biosurfactant Obtained from Corn as a Stabilizing Agent for Antidandruff Formulations Based on Zn Pyrithione Powder. ACS Omega. 2020 Mar 16;5(11):5704-5712. doi: 10.1021/acsomega.9b03679.

2. Caritá AC et al. Vitamin C: One compound, several uses. Advances for delivery, efficiency and stability. Nanomedicine. 2020 Feb;24:102117. doi: 10.1016/j.nano.2019.102117. Epub 2019 Oct 30.

3. Santhoshakumari Tmi et al. Role of vitamin c and vitamin E on hypertension. Asian J Pharm Clin Res. 2019; 12. doi: 10.22159/ajpcr.2019.v12i9.32634.

4. Colven RM, Pinnell SR. Topical vitamin C in aging. Clin Dermatol. 1996 Mar-Apr;14(2):227-34. doi: 10.1016/0738-081x(95)00158-c.

5. Humbert PG et al. Topical ascorbic acid on photoaged skin. Clinical, topographical and ultrastructural evaluation: double-blind study vs. placebo. Exp Dermatol. 2003 Jun;12(3):237-44. doi: 10.1034/j.1600-0625.2003.00008.x.

6. Elhabak M et al. Topical delivery of l-ascorbic acid spanlastics for stability enhancement and treatment of UVB induced damaged skin. Drug Deliv. 2021 Dec;28(1):445-453. doi: 10.1080/10717544.2021.1886377.

7. Sunil Kumar BV et al. Anticancer potential of dietary vitamin D and ascorbic acid: A review. Crit Rev Food Sci Nutr. 2017 Aug 13;57(12):2623-2635. doi: 10.1080/10408398.2015.1064086.

8. Rupali Kirtawade, Pallavi Salve, Anita Kulkarni, Pandurang Dhabale. Herbal antioxidant: Vitamin C. Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 58-61.

9. Shrada. B. Kumar,. Dhanraj. M. Role of Vitamin C in Body Health. Research J. Pharm. and Tech 2018; 11(4): 1378-1380.

10. Nusgens BV et al. Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis. J Invest Dermatol. 2001 Jun;116(6):853-9. doi: 10.1046/j.0022-202x.2001.01362.x.

11. Zhou W et al. Storage stability and skin permeation of vitamin C liposomes improved by pectin coating. Colloids Surf B Biointerfaces. 2014 May 1;117:330-7. doi: 10.1016/j.colsurfb.2014.02.036. Epub 2014 Mar 2.

12. Pullar JM et al. The Roles of Vitamin C in Skin Health. Nutrients. 2017 Aug 12;9(8):866. doi: 10.3390/nu9080866.

13. Maia Campos PM et al. Application of tetra-isopalmitoyl ascorbic acid in cosmetic formulations: stability studies and in vivo efficacy. Eur J Pharm Biopharm. 2012 Nov;82(3):580-6. doi: 10.1016/j.ejpb.2012.08.009.

14. Maia AM et al. Validation of HPLC stability-indicating method for Vitamin C in semisolid pharmaceutical/cosmetic preparations with glutathione and sodium metabisulfite, as antioxidants. Talanta. 2007 Feb 15;71(2):639-43. doi: 10.1016/j.talanta.2006.05.006. Epub 2006 Jun 12.

15. Stamford NP. Stability, transdermal penetration, and cutaneous effects of ascorbic acid and its derivatives. J Cosmet Dermatol. 2012 Dec;11(4):310-7. doi: 10.1111/jocd.12006.

16. Sheraz MA et al. Stability and Stabilization of Ascorbic Acid. A Rev. Household Pers. Care Today 2015;10(3):22-26.

17. Akansha Bhandarkar, et al. Formulation and Evaluation of Ascorbic acid Lozenges for the treatment of Oral Ulcer.Research J. Pharm. and Tech 2018; 11(4):1307-1312.doi: 10.5958/0974-360X.2018.00243.3

18. Wardhani DH et al. Performance of glucomannan-alginate combination as a pH sensitive excipient of vitamin C encapsulation using gelation method. Int J Appl Pharm, 11, 185-92.

19. Lin FH et al. Ferulic acid stabilizes a solution of vitamins C and E and doubles its photoprotection of skin. J Invest Dermatol. 2005 Oct;125(4):826-32. doi: 10.1111/j.0022-202X.2005.23768.x.

20. Kumari P et al. (2021) An recent advancement in topical dosage forms: a review. IJCPR, 13(1); 01-08.

21. Caritá AC et al . Stabilization of vitamin C in emulsions of liquid crystalline structures. Int J Pharm. 2021 Jan 5;592:120092. doi: 10.1016/j.ijpharm.2020.120092.

22. Mota AH et al. Design and evaluation of novel topical formulation with olive oil as natural functional active. Pharm Dev Technol. 2018 Oct;23(8):794-805. doi: 10.1080/10837450.2017.1340951.

23. Moraes JDD et al. Clay minerals: Properties and applications to dermocosmetic products and perspectives of natural raw materials for therapeutic purposes-A review. Int J Pharm. 2017 Dec 20;534(1-2):213-219. doi: 10.1016/j.ijpharm.2017.10.031.

24. Viseras, C., Carazo, E., Borrego-Sánchez, A., García-Villén, F., Sánchez-Espejo, R., Cerezo, P., & Aguzzi, C. (2019). Clay minerals in skin drug delivery. Clays and Clay Minerals, 67(1), 59–71. https://doi.org/10.1007/s42860-018-0003-7

25. Sharmin Reza Chowdhury, Mahfuza Maleque, Mahbubul Hoque Shihan. Development and Validation of a Simple RP-HPLC Method for Determination of Caffeine in Pharmaceutical Dosage Forms. Asian J. Pharm. Ana. 2(1): Jan.-Mar. 2012; Page 01-04.

26. Sharmin Reza Chowdhury, Mahfuza Maleque, Mahbubul Hoque Shihan. Development and Validation of a Simple RP-HPLC Method for Determination of Caffeine in Pharmaceutical Dosage Forms. Asian J. Pharm. Ana. 2(1): Jan.-Mar. 2012; Page 01-04.

27. Hamid Khan. Analytical Method Development in Pharmaceutical Research: Steps involved in HPLC Method Development. Asian J. Pharm. Res. 2017; 7(3): 203-207. Doi: 10.5958/2231-5691.2017.00031.4

28. Viseras, C., Aguzzi, C., Cerezo, P., & Lopezgalindo, A. (2007). Uses of clay minerals in semisolid health care and therapeutic products. Applied Clay Science, 36(1-3), 37–50. https://doi.org/10.1016/j.clay.2006.07.006

29. Lfa AR, J J. Synthesis of bentonite nanoclay and incorporation of cassia fistula leaf extract to form organobentonite: characterization and its biomedical applications. Asian J Pharm Clin Res. 2018;11. doi:http://dx.doi.org/10.22159/ajpcr.2018.v11i9.26717.

30. Awad ME et al. Kaolinite in pharmaceutics and biomedicine. Int J Pharm. 2017 Nov 25;533(1):34-48. doi: 10.1016/j.ijpharm.2017.09.056.

31. Zhou Y et al. Molecular Structure and Decomposition Kinetics of Kaolinite/Alkylamine Intercalation Compounds. Front Chem. 2018 Jul 27;6:310. doi: 10.3389/fchem.2018.00310.

32. Detellier C. Functional Kaolinite. Chem Rec. 2018 Jul;18(7-8):868-877. doi: 10.1002/tcr.201700072.

33. Zheng W et al. Formation of intercalation compound of kaolinite-glycine via displacing guest water by glycine. J Colloid Interface Sci. 2014 Oct 15;432:278-84. doi: 10.1016/j.jcis.2014.06.016..

34. Jafarbeglou M et al. Clay nanocomposites as engineered drug delivery systems. Rsc Adv. 2016;6(55):50002-16. doi: 10.1039/c6ra03942a.

35. Massaro M et al. The Use of Some Clay Minerals as Natural Resources for Drug Carrier Applications. J Funct Biomater. 2018 Oct 19;9(4):58. doi: 10.3390/jfb9040058.

36. Khurana IS et al. Multifaceted role of clay minerals in pharmaceuticals. Future Sci OA. 2015 Nov 1;1(3):FSO6. doi: 10.4155/fso.15.6.

37. Kenne Dedzo G, Detellier C. Characterization and Applications of Kaolinite Robustly Grafted by an Ionic Liquid with Naphthyl Functionality. Materials (Basel). 2017 Aug 29;10(9):1006. doi: 10.3390/ma10091006.

38. Elbokl TA, Detellier C. Intercalation of cyclic imides in kaolinite. J Colloid Interface Sci. 2008 Jul 15;323(2):338-48. doi: 10.1016/j.jcis.2008.04.003.

39. Kim MH et al. Review of Clay-Drug Hybrid Materials for Biomedical Applications: Administration Routes. Clays Clay Miner. 2016;64(2):115-130. doi: 10.1346/CCMN.2016.0640204.

40. Khatoon N et al . Nanoclay-based drug delivery systems and their therapeutic potentials. J Mater Chem B. 2020 Aug 26;8(33):7335-7351. doi: 10.1039/d0tb01031f.

41. Ermi Abriyani, Lia Fikayuniar. Screening Phytochemical, Antioxidant Activity and Vitamin C Assay from Bungo perak-perak (Begonia versicolar Irmsch) leaves. Asian J. Pharm. Res. 2020; 10(3):183-187.

42. Sankalp Goswami, et al.Phytopharmaceuticals as Cosmetic Agents: A Review. Res. J. Topical and Cosmetic Sci. 2(1): Jan. –June 2011 page 11-13

43. Sneha Mali, et,al. Overview of Nutraceuticals. Asian Journal of Pharmaceutical Research. 2022; 12(1):61-0. Doi: 10.52711/2231-5691.2022.00010

44. D. A. Bhagwat, et, al. More. Formulation and Evaluation of Herbal Lipstick using Lycopene Extracted from Solanum lycopersicum L. Research J. Pharm. and Tech. 2017; 10(4): 1060-1064.Doi: 10.5958/0974-360X.2017.00192.5

45. D. A. Bhagwat, et al. More. Formulation and Evaluation of Herbal Lipstick using Lycopene Extracted from Solanum lycopersicum L. Research J. Pharm. and Tech. 2017; 10(4): 1060-1064.Doi: 10.5958/0974-360X.2017.00192.5

Received on 12.04.2022 Accepted on 19.05.2022

Research J. Topical and Cosmetic Sci. 2022; 13(1):9-13.

DOI: 10.52711/2321-5844.2022.00002