3.8. Molecular Interaction Changes after Long-Term Storage

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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In addition, cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells. Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche. Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products. In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.

The interaction at the molecular level between a drug and a polymer is essential to explain the stability in solid dosage forms [46]. FTIR is a useful technique for the determination of molecular interactions between drugs and polymers. Figure 7 shows the FTIR spectra of CP-F before and after storage in different conditions, obtained within the range of 4000 cm−1 to 600 cm−1. The FTIR spectrum of CP showed the band at 1670 cm−1 referred to as C=O stretching. The bands at 1627, 3448, and 3356 cm−1 corresponded to the N–H stretching of CP. The FTIR spectrum of blank nanofibrous film represented absorption peaks at 3290 cm−1 that referred to the O–H stretching vibration of the hydroxyl group of the base polymer. The peaks at 1444 and 2944 cm−1 referred to –CH2 bending and C–H stretching of PVA, respectively [47,48]. The absorption peaks at 1696 cm−1 are referred to as C=O from the amide group of PVP [49]. The peak around 1044 cm−1 was Si–O stretching [50]. The FTIR spectral pattern of CP-F was similar to that of blank nanofibrous film. The absorption peaks at around 1446–1440 cm−1 referred to the CH2 bending of PVA. The weak broad band of the hydroxyl group at the 3500–3200 cm−1 spectral region was assigned to the O–H stretching vibration of the hydroxyl group of PVA. A low-frequency peak of the C=O stretching vibrations spectrum of PVP from 1696 to 1650 cm−1 was observed and a strong absorption peak at 1092 cm−1 was presented.

It was noted that the low frequency of the C=O stretching vibration at 1696 cm−1 of PVP in the blank nanofibrous film was shifted to 1650 cm−1 after loading CP to the nanofibrous film. This might be due to the interaction of the peroxide and PVP [51]. In addition, the strong absorption peak at 1044 cm−1 was due to the siloxane bridge (Si–O–Si) of the formulations. However, after loading CP to the nanofibrous film, this peak was shifted to 1092 cm−1, indicating a molecular interaction with the siloxane bridge. It has been reported that hydrogen peroxide could form a strong hydrogen bond with the oxygen of the siloxane bridge [52]. The spectral shifted peak at 1092 cm−1 represented the interactions of hydrogen peroxide from the molecules of CP, that adsorbed on the silica surface to the siloxane bridge of the silica gel. 

The FTIR spectrum of CP-F after storage at 25 °C/75% RH showed an increase in the intensity of the peak at 3700–3200 cm−1. As previously mentioned, the water content of CP-F could be increased due to the water sorption of CP-F during storage in high humidity, therefore the band in the region of 3700–3200 cm−1 corresponded to the –OH stretching vibration of the hydrogen bonds of the water molecules [53]. However, the FTIR spectrum of CP-F after storage at 45 °C/30% RH exhibited very low intensity at the region of 3700–3200 cm−1, and the peak at 1092 cm−1 was absent. Only the stretching vibration of N–H at 1635 cm−1 was found. These results suggested that high temperatures could lead to a decrease in the water content and hydroxyl groups [54]. Therefore, many peaks were missing due to damage from heat. Interestingly, the FTIR spectrum of CP-F after storage at 25 ◦C/30% RH for 12 months showed no change in the molecular interaction during the storage period. This result suggested the condition of 25 ◦C/30% RH was suitable for keeping CP-F.

3.9. Mechanical Properties Changes after Long-Term Storage

The effect of storage conditions on the mechanical properties of CP-F is of interest. The results as shown in Table 5 indicate that there was no statistically signifificant difference in tensile strength, elongation at break, and Young’s modulus values between initial measurements and after storage at 25 ◦C/30% RH. However, changes in the mechanical properties were detected in CP-F stored at 25 ◦C/75% RH and 45 ◦C/30% RH. The higher-humidity storage led to a decrease in tensile strength and Young’s modulus value of CP-F, while the percentage of elongation at break was increased compared to the initial value. This was likely related to the water molecules in CP-F, which decrease the original interactions in the polymer matrix of the nanofibrous film [55]. Water molecules can restructure the chain networks through inter- and intramolecular hydrogen bonds [56], resulting in an increase in elongation at break and a decrease in tensile strength and Young’s modulus values. In the case of the high temperature of 45 ◦C/30% RH storage, the decrease in tensile strength, elongation at break, and Young’s modulus values was found. It could be noted that the higher temperature affected the strength and flflexibility of the nanofibrous film, resulting in a more brittle film. This result corresponds to the FTIR pattern showing the negative effect of the storage conditions on the molecular interaction of CP-F, thus, changes in mechanical properties also occurred. 

3.10. Adhesive Property Changes after Long-Term Storage 

The adhesion of the nanofibrous film is important as it affects the intended function of tooth whitening. The freshly prepared CP-F could adhere to the surface of the mucosa and the measured adhesive force was found to be 0.79 ± 0.07 N. After storage at 25 ◦C/30% RH for 12 months, the formulation did not show a signifificant difference in the adhesive properties of the film from its initial value. The adhesive force of the stored film was 0.75 ± 0.06 N. The adhesive force of CP-F after storage at 25 ◦C/75% RH and 45 ◦C/30% RH for 12 months was decreased to 0.54 ± 0.03 N and 0.31 ± 0.05 N, respectively. It was therefore suggested that the humidity and temperature influenced the adhesive properties of CP-F.

3.11. CP Remaining after Long-Term Storage 

The stability of CP during long-term storage under different conditions is presented as degradation profiles, as shown in Figure 8. After storage for 12 months at 25 ◦C/75% RH and 45 ◦C/30% RH, CP content was significantly decreased from the initial value (p < 0.05). However, CP in CP-F kept at 25 ◦C/30% RH showed significantly higher stability than that kept at the other storage conditions. A slight reduction in CP was observed, without a signifificant difference in CP content between time intervals. At the end of the test period of 12 months, the remaining CP content in this condition was found to be up to 96.23 ± 3.05%, followed by that kept at 25 ◦C/75% RH (68.37 ± 4.17%). Stored at 45 ◦C/30% RH, CP could not be found after 6 months had passed, suggesting that all CP might have been completely degraded. The results also indicate that temperature had a higher effect on CP degradation than humidity. 

According to the short-term stability under stress conditions of 60, 70, and 80◦C as mentioned above, the calculated shelf-life of CP in CP-F, obtained from the predicted degradation rate of Arrhenius plots at 25◦C, is approximately 1 year. This result is in agreement with the actual measured value of CP in CP-F stored at 25◦C/30% RH. However, at 25 ◦C/75% RH, the results show that CP degradation occurred after 3 months. This result indicates that the presence of humidity in the environment can increase the CP degradation rate.

4. Conclusions 

Supplementary Materials: The followings are available online, Figure S1: HPLC chromatogram of (a) triphenylphosphine oxide and residual of triphenylphosphine after oxidation by CP and (b) HPLC chromatogram of triphenylphosphine.

Author Contributions: Conceptualization, S.O., P.C., and A.K.; methodology, S.O., P.C., and A.K.; validation, S.O.; formal analysis, S.O., and A.K.; investigation, A.K.; writing—original draft preparation, A.K.; writing—review and editing, S.O. and A.K.; supervision, S.O.; project administration, S.O.; funding acquisition, S.O. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Thailand Research Fund through the Research and Researcher for Industry (Grant No. PHD58I0012), the Agricultural Research Development Agency, and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission.

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable. 

Data Availability Statement: Data are available upon request to the corresponding author. 

Acknowledgments: The authors are grateful to the Research Center of Pharmaceutical Nanotechnology, Chiang Mai University, Thailand, for equipment and facility support. 

Conflicts of Interest: The authors declare no conflict of interest. 

References 

1. Joiner, A.; Luo, W. Tooth color and whiteness: A review. J. Dent. 2017, 67, S3–S10. [CrossRef] 

2. Gold, S.I. Early Origins of hydrogen peroxide used in oral hygiene: A historical note. J. Periodontol. 1983, 54, 247. [CrossRef]

3. Farrell, G.; McNichols, W. Efficacy of various medicaments in the treatment of Vincent’s stomatitis. J. Am. Med. Assoc. 1937, 108, 630–633. [CrossRef]

4. Bonesi, C.D.M.; Ulian, L.S.; Balem, P.; Angeli, V.W. Carbamide peroxide gel stability under different temperature conditions: Is manipulated formulation an option? Braz. J. Pharm. Sci. 2011, 47, 719–724. [CrossRef] 

5. Joiner, A. The bleaching of teeth: A review of the literature. J. Dent. 2006, 34, 412–419. [CrossRef] 

6. Dahl, J.E.; Pallesen, U. Tooth bleaching-A critical review of the biological aspects. Crit. Rev. Oral Biol. Med. 2003, 14, 292–304. [CrossRef] 

7. Kawamoto, K.; Tsujimoto, Y. Effects of the hydroxyl radical and hydrogen peroxide on tooth bleaching. J. Endod. 2004, 30, 45–50. [CrossRef] [PubMed] 

8. Christensen, G.J. Are snow-white teeth so desirable? J. Am. Dent. Assoc. 2005, 136, 933–935. [CrossRef] 

9. Putt, M.S.; Proskin, H.M. Custom tray application of peroxide gel as an adjunct to scaling and root planing in the treatment of periodontitis: Results of a randomized controlled trial after six months. J. Clin. Dent. 2013, 24, 100–107. 

10. Bentley, C.D.; Leonard, R.H.; Crawford, J.J. Effect of whitening agents containing carbamide peroxide on cariogenic bacteria. J. Esthet. Dent 2000, 12, 33–37. [CrossRef] 

11. Yao, C.S.; Waterfifield, J.D.; Shen, Y.; Haapasalo, M.; MacEntee, M.I. In vitro antibacterial effect of carbamide peroxide on oral biofilm. J. Oral Microbiol. 2013, 5, 1–6. 

12. Polydorou, O.; Hellwig, E.; Auschill, T.M. The effect of different bleaching agents on the surface texture of restorative materials. Oper. Dent. 2006, 31, 473–480. [CrossRef] 

13. Buchalla, W.; Attin, T. External bleaching therapy with activation by heat, light or laser-A systematic review. Dent. Mater. 2007, 23, 586–596. [CrossRef] [PubMed] 

14. Matis, B.A.; Matis, J.I.; Wang, Y.; Monteiro, S.; Al-Qunaian, T.A.; Millard, R. Labeled vs. actual concentration of bleaching agents. Oper. Dent. 2013, 38, 334–343. [CrossRef] 

15. Blanco, M.; Coello, J.; Sánchez, M.J. Experimental design for optimization of peroxide formulation stability and cost. J. Surfactants Deterg. 2006, 9, 341–347. [CrossRef] 

16. Francine, K.V.M.; Celso Afonso, K.J.; Eduardo, G.R.; Rubem Beraldo, D.S.; Fernando Freitas, P.; Keiichi, H. Storage temperature influences the carbamide peroxide concentration of at-home bleaching agents. Biomed. J. Sci. Tech. Res. 2018, 9, 6898–6902. 

17. Kurthy, R. The science of whitening gel refrigeration. A KöR Whitening Sci. Pap. 2016, 10, 9–15.

18. Shetab Boushehri, M.A.; Dietrich, D.; Lamprecht, A. Nanotechnology as a platform for the development of injectable parenteral formulations: A comprehensive review of the know-how and state of the art. Pharmaceutics 2020, 12, 510. [CrossRef] 

19. Kriegel, C.; Arrechi, A.; Kit, K.; McClements, D.J.; Weiss, J. Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Crit. Rev. Food Sci. Nutr. 2008, 48, 775–797. [CrossRef] 

20. Persano, L.; Camposeo, A.; Tekmen, C.; Pisignano, D. Industrial upscaling of electrospinning and applications of polymer nanofibers: A review. Macromol. Mater. Eng. 2013, 298, 504–520. [CrossRef]

21. Tian, Y.; Orlu, M.; Woerdenbag, H.J.; Scarpa, M.; Kiefer, O.; Kottke, D.; Sjöholm, E.; Öblom, H.; Sandler, N.; Hinrichs, W.L.J.; et al. Oromucosal films: From patient centricity to production by printing techniques. Expert Opin. Drug Deliv. 2019, 16, 981–993. [CrossRef] 

22. Okonogi, S.; Kaewpinta, A.; Rades, T.; Müllertz, A.; Yang, M.; Khongkhunthian, S.; Chaijareenont, P. Enhancing stability and tooth bleaching activity of carbamide peroxide by the electrospun nanofibrous film. Pharmaceuticals 2020, 13, 381. [CrossRef] [PubMed]

23. World Health Organization. Guidelines for stability testing of pharmaceutical products containing well-established drug substances in conventional dosage forms (Annex 5). In WHO Technical Report Series; World Health Organization: Geneva, Switzerland, 1996; pp. 65–80. 

24. Kaewpinta, A.; Khongkhunthian, S.; Chaijareenont, P.; Okonogi, S. Preparation and characterization of rice gels containing a tooth bleaching agent. Drug Discov. Ther. 2018, 12, 275–282. [CrossRef]

25. Stark, G.; Fawcett, J.P.; Tucker, I.G.; Weatherall, I.L. Instrumental evaluation of the color of solid dosage forms during stability testing. Int. J. Pharm. 1996, 143, 93–100. [CrossRef] 

26. Jantrawut, P.; Boonsermsukcharoen, K.; Thipnan, K.; Chaiwarit, T.; Hwang, K.M.; Park, E.S. Enhancement of antibacterial activity of orange oil in pectin thin film by microemulsion. Nanomaterials 2018, 8, 545. [CrossRef]

27. Kaewpinta, A.; Khongkhunthian, S.; Chaijareenont, P.; Okonogi, S. Tooth whitening efficacy of pigmented rice gels containing carbamide peroxide. Drug Discov. Ther. 2018, 12, 126–132. [CrossRef]

28. Gimeno, P.; Bousquet, C.; Lassu, N.; Maggio, A.F.; Civade, C.; Brenner, C.; Lempereur, L. High-performance liquid chromatography method for the determination of hydrogen peroxide present or released in teeth bleaching kits and hair cosmetic products. J. Pharm. Biomed. Anal. 2015, 107, 386–393. [CrossRef]

29. Yoshioka, S.; Stella, V.J. Stability of Drug and Dosage Forms; Springer: Boston, MA, USA, 2002; pp. 1–270. 

30. Hunt, J.P.; Taube, H. The photochemical decomposition of hydrogen peroxide. J. Phys. Chem. 1952, 74, 5999–6002. 

31. Lima, D.A.N.L.; Aguiar, F.H.B.; Liporoni, P.C.S.; Munin, E.; Ambrosano, G.M.B.; Lovadino, J.R. In vitro evaluation of the effectiveness of bleaching agents activated by different light sources. J. Prosthodont. 2009, 18, 249–254. [CrossRef]

32. World Health Organization. Stability testing of active pharmaceutical ingredients and finished pharmaceutical products (Annex 10). In WHO Technical Report Series, No. 1010; World Health Organization: Geneva, Switzerland, 2018; pp. 310–351. 

33. Huang, L.; Wang, S. Effects of heat treatment on tensile properties of high-strength poly(vinyl alcohol) fibers. J. Appl. Polym. Sci. 2000, 78, 237–242. [CrossRef] 

34. Johnston, W.M.; Kao, E.C. Assessment of appearance match by visual observation and clinical colorimetry. J. Dent. Res. 1989, 68, 819–822. [CrossRef]

35. Wijanarko, T.A.W.; Kusumaatmaja, A.; Chotimah, R.; Triyana, K. Effect of heat treatment on morphology and crystallinity of electrospun poly(vinyl alcohol) nanofibers. Am. Inst. Phys. Conf. Proc. 2016, 1755, 1–4.

36. Moraes, R.R.; Marimon, J.L.M.; Schneider, L.F.J.; Correr Sobrinho, L.; Camacho, G.B.; Bueno, M. Carbamide peroxide bleaching agents: Effects on surface roughness of enamel, composite and porcelain. Clin. Oral Investig. 2006, 10, 23–28. [CrossRef] 

37. Ranganathan, S.; Sieber, V. Recent advances in the direct synthesis of hydrogen peroxide using chemical catalysis—A review. Catalysts 2018, 8, 379. [CrossRef] 

38. Seif, S.; Franzen, L.; Windbergs, M. Overcoming drug crystallization in electrospun fibers—Elucidating key parameters and developing strategies for drug delivery. Int. J. Pharm. 2015, 478, 390–397. [CrossRef] [PubMed] 

39. Feng, X.; Ye, X.; Park, J.B.; Lu, W.; Morott, J.; Beissner, B.; Lian, Z.J.; Pinto, E.; Bi, V.; Porter, S.; et al. Evaluation of the recrystallization kinetics of hot-melt extruded polymeric solid dispersions using an improved Avrami equation. Drug Dev. Ind. Pharm. 2015, 41, 1479–1487. [CrossRef] [PubMed] 

40. Ueda, H.; Kadota, K.; Imono, M.; Ito, T.; Kunita, A.; Tozuka, Y. Co-amorphous Formation Induced by Combination of Tranilast and Diphenhydramine Hydrochloride. J. Pharm. Sci. 2017, 106, 123–128. [CrossRef] [PubMed]

41. Polaskova, M.; Peer, P.; Cermak, R.; Ponizil, P. Effect of thermal treatment on the crystallinity of poly(ethylene oxide) electrospun fibers. Polymers 2019, 11, 1384. [CrossRef] 

42. Rumondor, A.C.F.; Stanford, L.A.; Taylor, L.S. Effects of polymer type and storage relative humidity on the kinetics of felodipine crystallization from amorphous solid dispersions. Pharm. Res. 2009, 26, 2599–2606. [CrossRef]

43. Peresin, M.S.; Habibi, Y.; Vesterinen, A.H.; Rojas, O.J.; Pawlak, J.J.; Seppälä, J.V. Effect of moisture on electrospun nanofiber composites of poly(vinyl alcohol) and cellulose nanocrystals. Biomacromolecules 2010, 11, 2471–2477. [CrossRef]

44. Ueda, H.; Aikawa, S.; Kashima, Y.; Kikuchi, J.; Ida, Y.; Tanino, T.; Kadota, K.; Tozuka, Y. Anti-plasticizing effect of amorphous indomethacin-induced by specific intermolecular interactions with PVA copolymer. J. Pharm. Sci. 2014, 103, 2829–2838. [CrossRef] 

45. Prudic, A.; Ji, Y.; Luebbert, C.; Sadowski, G. Influence of humidity on the phase behavior of API/polymer formulations. Eur. J. Pharm. Biopharm. 2015, 94, 352–362. [CrossRef] 

46. Tran, T.T.D.; Tran, P.H.L. Molecular interactions in solid dispersions of poorly water-soluble drugs. Pharmaceutics 2020, 12, 745. [CrossRef] 

47. Alwan, T.J.; Toma, Z.A.; Kudhier, M.A.; Ziadan, K.M. Preparation and characterization of the PVA nanofibers produced by electrospinning. Mar. J. Nanotechnol. Nanosci. 2016, 1, 1–3. [CrossRef] 

48. Subramanian, U.M.; Kumar, S.V.; Nagiah, N.; Sivagnanam, U.T. Fabrication of polyvinyl alcohol-polyvinylpyrrolidone blend scaffolds via electrospinning for tissue engineering applications. Int. J. Polym. Mater. Polym. Biomater. 2014, 63, 462–470. [CrossRef]

49. Huang, S.; Zhou, L.; Li, M.C.; Wu, Q.; Kojima, Y.; Zhou, D. Preparation and properties of electrospun poly (vinyl pyrrolidone)/cellulose nanocrystal/silver nanoparticle composite fibers. Materials 2016, 9, 523. [CrossRef]

50. Wei, Y.; Zhang, W.; Li, S.; Patel, A.C.; Wang, C. Electrospinning of porous silica nanofibers containing silver nanoparticles for catalytic applications. Chem. Mater. 2007, 19, 1231–1238. 

51. Panarin, E.F.; Kalninsh, K.K.; Pestov, D.V. Complexation of hydrogen peroxide with polyvinylpyrrolidone: Ab initio calculations. Eur. Polym. J. 2001, 37, 375–379. [CrossRef] 

52. Zegli ´Ski, J.; Piotrowski, G.P.; Pieko´s, R. A study of the interaction between hydrogen peroxide and silica gel by FTIR spectroscopy and quantum chemistry. J. Mol. Struct. 2006, 794, 83–91. [CrossRef]

53. Ping, Z.H.; Nguyen, Q.T.; Chen, S.M.; Zhou, J.Q.; Ding, Y.D. States of water in different hydrophilic polymers—DSC and FTIR studies. Polymer 2001, 42, 8461–8467. [CrossRef] 

54. Vasudevan, P.; Thomas, S.; Biju, P.R.; Sudarsanakumar, C.; Unnikrishnan, N.V. Synthesis and structural characterization of sol-gel derived titania/poly (vinyl pyrrolidone) nanocomposites. J. Sol-Gel Sci. Technol. 2012, 62, 41–46. [CrossRef]

55. Tian, H.; Yan, J.; Rajulu, A.V.; Xiang, A.; Luo, X. Fabrication and properties of polyvinyl alcohol/starch blend films: Effect of composition and humidity. Int. J. Biol. Macromol. 2017, 96, 518–523. [CrossRef] [PubMed] 

56. Abral, H.; Chairani, M.K.; Rizki, M.D.; Mahardika, M.; Handayani, D.; Sugiarti, E.; Muslimin, A.N.; Sapuan, S.M.; Ilyas, R.A. Characterization of compressed bacterial cellulose nano paper film after exposure to dry and humid conditions. J. Mater. Res. Technol. 2021, 11, 896–904. [CrossRef]

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