Vitamin C as a probable inhibitor of tyrosinase (Tyr) and tyrosinase-related protein-1 (TRP-1) in human gingiva: An analytical study

Abstract

Background:

Vitamin C is a water-soluble substance naturally occurring in citrus fruits, strawberries, kiwifruit, guava, etc., It is required for formation of collagen, healing of wounds, maintaining the health of blood vessels, ligaments, and bones. Vitamin C inhibits the tyrosinase (Tyr) enzyme and reduces melanin synthesis. Tyrosinase along with tyrosinase related protein- 1 and 2 (TRP-1 & TRP-2) are required for melanin synthesis. Hence this study was conducted to assess the effect of Vitamin C on Tyr and TRP-1 in human gingiva.

Aim:

To assess the effect of Vitamin C on tyrosinase and TRP-1 levels in individuals with moderate to heavy gingival melanin hyperpigmentation.

Materials and Methods:

Individuals with complaint of black gums were included. Informed consent was obtained. Initial treatment of scaling and root planing was conducted and they were recalled after 2 weeks. The gingival depigmentation was performed by scalpel technique. The excised layer of epithelial tissue was sent to the laboratory to assess the Tyr and TRP-1 levels. Subsequently, Vitamin C was administered at monthly interval for 6 months. Tyr and TRP-1 levels were assessed at the end of 1 year follow-up.

Results:

The levels of Tyr and TRP-1 in gingival tissue at baseline and at the end of one year follow-up are shown in the table and graph. Tyr and TRP-1 levels were reduced after vitamin C administration.

Conclusion:

Vitamin C administration resulted in reduction of tyrosinase and TRP-1 levels in human gingiva at 1 year follow-up indicating that Vitamin C is a probable inhibitor of melanin synthesis.

INTRODUCTION

Vitamin C is a water-soluble substance naturally occurring in citrus fruits, strawberries, kiwifruit, guava, etc.[1] It is a highly labile vitamin and is lost during food processing. It is required for formation of collagen, healing of wounds, phagocytic function of leucocytes, maintaining the health of blood vessels, ligaments, and bones.[2] It was first discovered by Albert Szent-Gyorgyi in 1912 and isolated by Walter Norman Haworth in 1928. Dr. Albert Szent-Gyorgyi received the Nobel prize in 1937, for identifying the role of Vitamin C in Scurvy and isolating it from red peppers.[3] James Lind was the first person who conducted a clinical trial and reported that scurvy could be cured by citrus fruits.[4]

Vitamin C is produced in plants and animals.[5] However, it is not produced in humans due to the mutations in the gene of gluconolactone oxidase enzyme,[6] hence minimal daily requirement of 40 mg–60 mg is required for a healthy adult.[7] Vitamin C protects the skin from harmful effects of ultraviolet radiations and has been used in the treatment of hyperpigmented spots on the skin. Due to its potent antioxidant property, it protects the skin against oxidative damage.[8]

Melanin is a non-haemoglobin derived endogenous brown pigment that is produced by melanocytes present in the basal and suprabasal layers of the epithelium. It is present on the oral mucosa, gingiva, skin, hair, and iris of the eye. Melanin protects the skin from the harmful effects of ultraviolet radiations.[9] Melanocytes synthesize melanosome granules that produce, store and transport melanin pigment.[10] In humans, tyrosinase (TYR) is the key enzyme involved in the biosynthesis of melanin.[11]

Tyrosinase along with tyrosinase related protein- 1 and 2 (TRP-1 and TRP-2) are required for melanin synthesis.[11] It is dependent on copper and catalyses the initial process of conversion of tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) and subsequently oxidizes it to DOPA quinone.[12] It is the critical rate-limiting enzyme catalysing the hydroxylation and oxidation of tyrosine.[13] Vitamin C reduces melanin synthesis by binding with the copper ions and inhibiting the tyrosinase enzyme.[1]

Gingival tissue is the most pigmented tissue in the oral cavity. The colour of the gingiva is greatly influenced by the presence of melanin pigmentation.[14] Gingival melanin hyperpigmentation results in black gums with an ugly smile especially in individuals with a gummy smile and/or short upper. Hence gingival depigmentation is performed to eliminate the melanin pigmented gingival epithelium either by surgical or non-surgical techniques.[15] Vitamin C has been used as a non-surgical method for treatment of gingival melanin hyperpigmentation.

Studies have been conducted assessing the efficacy of tyrosinase inhibitors like Vitamin C in dermatology. However, no such study has been conducted in the gingiva. Further, no study has been conducted to assess the effect of vitamin C administration on tyrosinase and TRP-1 in human gingiva. Hence this study was conducted to assess the effect of Vitamin C on Tyr and TRP-1 in human gingiva.

MATERIALS AND METHODS

Systemically healthy individuals who reported with the complaint of black gums and ugly smile were considered for the study. Before the start of the study, Ethical clearance was obtained from the Institutional Ethics Committee (Registration File no: EC/NEW/INST/2019/329). A thorough intraoral examination was done and gingival melanin hyperpigmentation was graded using Dummett Gupta Oral Pigmentation Index 1964.

Systemically healthy individuals in the age range of 20–45 years of either sex with DOPI Score 2 (moderate pigmentation) and DOPI Score 3 (heavy pigmentation) were included for the study.[16] Individuals with history of medications causing gingival melanin hyperpigmentation, smokers, drug addicts, alcoholics, and those with autoimmune or endocrine disorders were excluded.

In initial phase of therapy, scaling and root planing was done. Participants were explained about home care oral hygiene measures to be taken and were recalled after 2 weeks. On receiving normal blood report values, surgery was scheduled. Before the start of the surgery, the subjects were explained in detail about the surgery and signed consent for surgery was taken.

2% Lignocaine hydrochloride with 1:80,000 dilution adrenaline was used to anesthetize the surgical area. Preoperative baseline intraoral clinical photograph of gingival melanin hyperpigmentation was taken [Figure 1]. Gingival depigmentation was performed by scalpel technique using Bard parker blade no: 11 mounted on BP handle no: 3. Then about 2 mm of the excised layer of epithelial tissue along with thin layer of underlying connective tissue was sent to the laboratory to assess the tyrosinase and TRP-1 levels by real-time polymerase chain reaction (RT-PCR) technique as per the manufacturer’s guidelines (Thermofisher). RT-PCR analysis was performed using an Applied Biosystems Quant Studio 5 (QS5) RT-PCR system using Blue-Ray TurboCycler Lite Unit [Figure 2].

Figure 1.

Intraoral clinical photograph of gingival melanin hyperpigmentation at baseline

Figure 2.

Blue-ray turbocycler lite unit used for real time-polymerase chain reaction

The surgical area was irrigated with saline. Any left-over pigmented epithelial tissue was removed. Periodontal pack was placed and instructions for postoperative care were given. After 1 week the periodontal pack was removed and the area was examined to assess the healing. Subsequently, the participants were recalled after 1 month for administration of Vitamin C (250 mg/ml) with the help of an insulin syringe. 0.1 ml–0.2 ml of Vitamin C[17,18,19] was administered into the mesiobuccal, midbuccal and distobuccal area of the attached gingiva and interdental papilla in relation to each tooth [Figures 3 and 4]. It was repeated at monthly intervals for a period of 6 months. At the end of one year follow-up, there was minimal gingival repigmentation seen [Figure 5]. About 1 mm–2 mm of gingival tissue was excised from the distobuccal gingiva in relation to the premolar region from the eight selected sites and sent to the laboratory for assessment of tyrosinase and TRP-1 levels.

Figure 3.

Administration of Vitamin C using Insulin syringe at 6 months postoperatively into the attached gingiva

Figure 4.

Administration of Vitamin C using Insulin syringe at 6 months postoperatively into the interdental gingiva

Figure 5.

One-year postoperative intraoral clinical photograph showing minimal gingival repigmentation

RESULTS

The tyrosinase and TRP-1 levels in the gingival tissues were assessed at baseline and at the end of 1 year follow-up in terms of relative gene expression. The computed tomography (CT) values are usually obtained in the semi-quantitative method, where gel images are observed. In the present study, the RT-PCR was performed by fully quantitative method, which gave the values of the relative gene expression as digital read out. Hence there were no CT values and conversion required.

The values of tyrosinase and TRP-1 were calculated in units of relative gene expression. Statistical analysis was not conducted, as the gene expression at two-time intervals (baseline and after 1 year) were measured and compared. Table 1 and Graph 1 shows the tyrosinase and TRP-1 levels in the gingival tissue.

Table 1.

Tyrosinase and tyrosinase related protein 1 gene expression levels

Sites	Tyrosinase gene expression		Tyrosinase related protein 1 gene expression
	At baseline	At the end of 1 year	At baseline	At the end of 1 year
Site 1	0.649	0.438	0.569	0.284
Site 2	0.616	0.445	0.459	0.184
Site 3	0.728	0.364	0.543	0.311
Site 4	0.693	0.347	0.593	0.339
Site 5	0.813	0.436	0.478	0.298
Site 6	0.684	0.342	0.498	0.291
Site 7	0.618	0.309	0.512	0.255
Site 8	0.668	0.334	0.465	0.234

Graph 1.

Tyrosinase and tyrosinase related protein 1 gene expression levels

It was observed that, the sites with severe melanin pigmentation showed higher levels of tyrosinase and TRP-1 at baseline. Although there was a decrease in the tyrosinase and TRP-1 levels after vitamin C administration at the end of 1 year follow-up, the decrease in the tyrosinase and TRP-1 levels was lesser at the sites with severe melanin pigmentation as compared to the sites with moderate pigmentation.

DISCUSSION

Vitamin C is a water-soluble vitamin that is required for antiaging, antioxidation, dermal collagen formation in the skin of humans. It is a weak acid, barely stronger than vinegar. The melanocytes undergo acidification in the presence of Vitamin C. This could inhibit the activity of tyrosinase, which is the rate-limiting enzyme required for melanin biosynthesis.[18,19]

The inhibition of tyrosinase activity and melanin synthesis by Vitamin C occurs in a dose-dependent manner. Copper ions activate the tyrosinase enzyme by binding at the active sites. Activation of tyrosinase is an important step in the production of melanin. Vitamin C binds with the copper ion and inactivates the tyrosinase.[1] The level of tyrosinase enzyme expression has been corelated with the degree of melanin pigmentation of skin.[20]

In dermatology, skin whitening/lightening agents have been developed for management of melanin hyperpigmentation. These agents contain tyrosinase inhibitors, which may specifically inhibit the melanogenesis in cells without side effects on other cells and tissues. Many tyrosinase inhibitors such as hydroquinone, arbutin, kojic acid, azelaic acid, L-ascorbic acid, ellagic acid, tranexamic acid has been used as skin-whitening agents.[21]

Studies have been conducted assessing the efficacy of tyrosinase inhibitors in dermatology. There is lack of evidence of such studies been conducted to assess efficacy of tyrosinase inhibitor namely Vitamin C on the human gingiva. The present study was conducted to assess the effect of Vitamin C on Tyr and TRP-1 in human gingiva. Further the role of Vitamin C as a probable inhibitor of melanin synthesis was studied.

Reoccurrence of gingival melanin pigmentation after surgical depigmentation[22] is a common complaint of the patients, which may require repeat surgical depigmentation. Administration of Vitamin C may help prevent reoccurrence of gingival melanin pigmentation and thereby help to avoid a second surgical depigmentation procedure. This study emphasizes on the role of TRP-1 in inhibiting melanin synthesis along with tyrosinase. Administration of Vitamin C inhibits tyrosinase, as well as tyrosinase related protein-1, thereby reducing melanin synthesis.

The observation of the present study suggests a positive correlation between the levels of Tyrosinase and TRP-1 levels and degree of gingival melanin hyperpigmentation. There was a decrease in the tyrosinase and TRP-1 levels after administration of Vitamin C. These observations show that Vitamin C is a probable inhibitor of tyrosinase and TRP-1 in human gingiva.

CONCLUSION

From the observations of the present study, it can be concluded that, there exists a positive correlation between levels of tyrosinase and TRP-1 and the degree of gingival melanin hyperpigmentation. Administration of Vitamin C reduced the levels of tyrosinase and TRP-1. Hence the Vitamin C can serve as a probable inhibitor of tyrosinase and TRP-1 in human gingiva. Administration of Vitamin C may help maintain the results of surgical depigmentation for longer duration.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.