Antityrosinase Activity of Phycocyanin and Cream Formulation for Hyperpigmentation

Free radicals are involved in the catalytic reactions of tyrosinase to give dopaquinone in melanin biosynthesis. Screening of molecules with antioxidant activity from natural sources which inhibit tyrosinase has become important for cosmetic and medicinal topical products. Tyrosinase inhibitors are used in treating hyperpigmentation. Synthetic tyrosinase inhibitors possess side effects such as skin irritation, dermatitis, dryness, and inflammatory reaction. The objective of the study is to evaluate the tyrosinase inhibition potential of phycocyanin, which is a pigment extracted from the blue green algae Spirulina platensis and formulating a cream that can be used to treat hyperpigmentation. Phycocyanin possesses anticancer, antioxidant, antiviral and anti-inflammatory activities and exhibits the regulative ability of tyrosinase expression and thereby modulates melanogenesis. The spectral characterization of phycocyanin is carried out using UV-vis spectroscopy, FTIR and HPTLC. Phycocyanin showed tyrosinase inhibition potential with an IC50 of 30.88-39.87 μg/ml in SK-Mel-28 melanoma cells. In melanogenesis pathway, tyrosinase regulates the production of melanin by the skin cells and by incorporating phycocyanin in cream will reduce the melanin production and treat hyperpigmentation.


Introduction
Phycocyanin, a phycobiliprotein, is an important pigment found in Spirulina platensis that is utilized as dietary supplement in many countries.The commercialization of Spirulina for the valuable constituents it possesses such as several proteins and vitamins are currently underway in many places as it is inexpensive to obtain [1] [5] [8].
Tyrosinase is a vital enzyme which catalyzes the hydroxylation of L-tyrosine to 3,4dihydroxyphenylalanine (DOPA) including the oxidation of DOPA to dopaquinone which polymerize spontaneously to form melanin in the first two consecutive steps of biosynthesis of melanin.The excess expression of tyrosinase promotes excessive melanin production, that paves to hyperpigmentation effects such as cervical poikiloderma, acanthosis nigricans melasma, age spots, and non-melanoma skin cancer.[4] Insufficient or excessive production of melanin is the cause of several pigmentation issues.Pigmentation is a crucial defense mechanism of the human body and protects against oxidative stress and UV radiation.But abnormal hyperpigmentation of the skin is an aesthetic issue.Novel peptides with low molecular weight compounds are under investigation to regulate skin pigmentation.In-silico digestion of Phycocyanin from S. platensis allowed for construction of an internal library of peptides which were then analyzed for anti-melanogenic activity.[7] Phycocyanin is a good antioxidant and light harvesting protein which has been found to possess potential tyrosinase inhibition properties.[12] 2 The process of Monophenol ortho-hydroxylation and conversion to catechol and successive oxidation forming ortho-quinones is catalyzed by tyrosinase.Tyrosinase is the controller for browning hair and skin pigmentation.Alternatively, low tyrosinase can also cause anomalies like vitiligo or freckles.It also plays a huge role in cancer and Parkinson's disease which is a neurodegenerative disease.Since tyrosinase is a prominent target in categories like food, cosmetics, agriculture and medicine, the development and screening of effective tyrosinase inhibitors has great importance.[11] Present tyrosinase inhibitors are hazardous and or sometimes ineffective, and there is enduring research for improved natural source inhibitors, which could possibly be free of negative side effects [3] Inhibition of the tyrosinase enzyme plays a significant role in the melanogenesis pathway helping in reduction of production of melanin in the skin, and thus regulating skin pigmentation.[10] Some of the drawbacks of synthetic tyrosinase inhibitors are that they cause mild skin irritation and sensitization such as burning and stinging.They can also cause dermatitis, redness, and inflammatory reactions.Hence, natural tyrosinase inhibitors like phycocyanin cream are worth investigating to treat hyperpigmentation.
Creams are regarded to be one of the easiest and most convenient ways to use.People in any age group can use the cream since application is topical and on the surface of the skin only resulting in the formation of semi-solid emulsions.Creams are divided into two types: oil-in-water and waterin-oil.Oil-in-water (O/W) creams are made up of small drops of oil spread in a continuous water phase, and water-in-oil (W/O) creams are made up of tiny drops of water dispersed in a continuous oily phase.Oil-in-water lotions are more pleasant and cosmetically acceptable since they are less oily and simpler to wash off with water.Water-in-oil creams are a little more difficult to work with.phycocyanin was included in the cream formulation due to its tyrosinase inhibitory activity.A tyrosinase inhibitor inhibits the production of tyrosinase in the melanogenesis pathway.Phycocyanin, a natural blue pigment obtained from Spirulina platensis, is used as a natural tyrosinase inhibitor for our study.Thereby reducing the melanin production by treating hyperpigmentation and its associated disorders.Hence, our study reveals that phycocyanin based cream is well suited to remove hyperpigmentation and help in further protection of UV and harmful sun radiations.

CHARCTERISATION OF PHYCOCYANIN
The molecule structure of phycocyanin was measured by FTIR using spectra IR 4000-1000 cm-1.FT-IR spectroscopy was performed to identify characteristic functional groups present in phycocyanin.Phycocyanin was subjected to UV visible spectroscopy characterisation to observe its peak absorption by using the UV visible spectrophotometer.Thin Layer Chromatography was done to validate the quality of Phycocyanin by optimising the solvents n-butanol, acetic acid and distilled water in the ratio of 10:6:7.Further it was characterised by HPTLC technique.It was performed in HPTLC sample applicator (Make: Aspire; Model: AE-05).Two bands were created each 10µl and 20µl was spotted in 10×10cm size precoated silica gel 60 aluminium sheet.The mobile phase solvent is n-butanol: Acetic acid: Distilled water in the ratio of 10:6:7.The sheet was then kept in the chamber for the mobile phase to react with the stationary phase and was then visualised in visible light, UV wavelength of short 265nm and long 364nm.The ingredients are chosen in such a way that they play a significant role in maintaining the homogeneity of the cream and also being skin friendly.Stearic acid is the base of the cream and gives consistency to the cream and a pearlescent appearance to the formulation.Sodium benzoate is used as a preservative so that the cream can be used for a long time.Coconut oil acts as a moisturizer that helps in maintaining the smoothness and hydration of the skin.The oil in water emulsion was prepared for the cream formulation.The oil phase components stearic acid and coconut oil were first combined in the above-mentioned quantity and was then heated to 75ºC in water bath.Simultaneously, the aqueous phase components Glycerine, Cetostearyl alcohol, Sodium benzoate were mixed and heated to 75ºC in water bath.Then the aqueous phase was added slowly to the oil phase and combined well with continuous stirring.After cooling of the product, Phycocyanin was added and mixed well.

TYROSINASE INHIBITION ASSAY
SK-Mel-28 (Human Skin melanoma) Cells were maintained in Dulbecco's Modified Eagles Medium (DMEM-Hi media) -with 10% heat inactivated Fetal Bovine Serum (FBS) + 1% antibiotic cocktail containing Penicillin (100U/ml), Streptomycin (100µg/ml), and Amphotericin B (2.5µg/ml).The naturally present tyrosinase enzyme in SK-Mel-28cells is significantly higher than normal melanin cells.SK-Mel-28 cells were used for this \ assay TC flasks (25cm 2 ) of SK-Mel-28 cells were incubated at 37°C at 5% CO2 environment with humidity in a cell culture incubator.L-DOPA of about 40 L was combined with 80 L of phosphate buffer with pH of 6.8 in the 96 well microtiter plate.The resulting mixture is kept in the incubator at 37 ∘ C for 10 minutes.40 L of varying concentrations Phycocyanin and 40 L of SK-Mel-28 cells (250 U/mL, in PBS) were added to each of the wells on the plate.The absorbance characteristics of the mixtures were analysed using ELISA reader at 475 nm.The results were interpreted at 60-second intervals for a period of 120 minutes.The experiment was done with triplicate (n=3).IC50 value indicates the sample concentration or ascorbic acid concentration in need for inhibiting 50% of the enzyme activity.IC50 value was calculated using Graphpad prism with dose response curves.

Thin Layer Chromatography
The Slides were visualised in the visible and in Ultraviolet light at 254 and 365 nm and it confirmed the movement of the sample in the stationary phase.The formulated cream was analysed for its physicochemical properties by analysing using FTIR spectroscopy.It gave an insight into the functional groups indicating peak values and validated the presence and efficacy of phycocyanin in the formulated cream.

TYROSINASE INHIBITION -COMPARATIVE RESULTS (60 MINS)
Tyrosinase inhibition assay was performed for phycocyanin and ascorbic acid at 60-minute time interval and the results were compared using GraphPad prism software.From the results of tyrosinase inhibition assay, the concentration (µg/ml) and % inhibition was plotted for phycocyanin and ascorbic acid (control) for 60 minutes time interval.GraphPad prism software was used to find out the IC50 values for phycocyanin and ascorbic acid which was found to be 30.88µg/ml and 34.06 µg/ml respectively.Avenanthramide from oats show concentration dependent inhibition of tyrosinase activity in α-MSH-induced SK-MEL-2 cells [14].Flavan Kazinol U isolated from ethyl acetate extract of B. kazinoki showed tyrosinase inhibition in B16F10 mouse melanoma cells at the lower concentration of 20 μM [15].

TYROSINASE INHIBITION -COMPARATIVE RESULTS (120 MINS)
Tyrosinase inhibition assay was performed for phycocyanin and ascorbic acid at 120-minute time interval and the results were compared using GraphPad prism software.From the results of tyrosinase inhibition assay, the concentration (µg/ml) and % inhibition was plotted for phycocyanin and ascorbic acid (control) for 120 minutes time interval.GraphPad prism software was used to find out the IC50 values for phycocyanin and ascorbic acid which was found to be 39.87 µg/ml and 17.84 µg/ml respectively.

CREAM FORMULATION
Phycocyanin incorporated cream was prepared in oil-in-water formulation, which resulted in a significant blue colour cream due to the presence of phycocyanin a naturally blue pigmented compound.

pH of the cream
The pH meter was used to examine the pH of the formulated cream.It was calibrated with Standard buffer.0.5 g of the cream was dissolved in about 50 ml of distilled water and tested.The results revealed the pH to be 5.9 and indicate an appropriate match to the skin.

Homogeneity
The homogeneity of the cream was examined by visualising the consistency of the cream by touch and feel.It exhibited uniform distribution upon application to the skin.

After Application Feel
The cream was applied topically on the skin and spread in circular motions.It left no granules or other particles and applied and absorbed into the skin very smoothly.The emollience and slipperiness of the cream that was left after application was found to be good.

CONCLUSION
Phycocyanin showed concentration dependent tyrosinase inhibition in SK-Mel-28 cells against positive control ascorbic acid.Phycocyanin incorporated cream was formulated by taking appropriate ingredients in the oil and aqueous phase mixed in the ratio of 2:1 which resulted in a homogeneous mixture The cream had a significant blue colour due to the presence of phycocyanobilin in phycocyanin.This cream can be applied topically to the affected areas that are infected by over exposure to harmful sun and UV radiation.Thus, our study revealed that Phycocyanin, a naturally occurring blue colour pigment extracted from Spirulina platensis potentially inhibited the tyrosinase enzyme in the melanogenesis pathway.Tyrosinase inhibition will reduce the melanin production to a

Fig. 1 .
Fig. 1.1 represents the role of phycocyanin in melanogenesis.When the skin cells are over exposed to sunlight, UV radiation or chemotherapy, it attacks the normal melanocyte cells which results in abnormal melanocytes due to the production of increased ROS and tyrosinase.This favors hyperpigmentation disorders such as non-melanoma skin cancer, acanthosis nigricans melasma, cervical poikiloderma, lentigines and periorbital hyperpigmentation.

Fig. 2 .
Fig. 2.2.1 Flow chart representing the process of cream formulation.

Fig. 2 . 3 . 1 1 FTIR
Fig. 2.3.1 Microscopic image of SKMEL cells at 10X (inverted phase contract)3.RESULTS AND DISCUSSIONS3.1 FTIRPhycocyanin was characterised using Fourier Transform Infrared Spectroscopy from wavenumber range 500 to 4000 cm -1 .From the above figure of the infrared spectrum the evident peak at 3600.77519 cm -1 wavenumber denotes the O-H Stretch of free hydroxyl group.The peak at 3232.55814 cm -1 corresponds to C-H Stretch.Similarly peak at 1740.31008 cm -1 corresponds to C-O Stretch.The peaks at 1642.44106 and 1533.83539cm -1 of the spectra corresponds to the amino N-H bend[2] [6][9].

Figure 3 .
3.1(c) is captured under visible light, (a) is the image captured at 365 nm of UV light and (b) is at 254 nm.The three captures confirmed the movement of the sample.

Fig 3 . 4
Fig 3.4.1 (a) Fig 3.4.1(b) HPTLC scan at (a) UV 254 nm and at (b) 365 nmThe lane 1 is result of the spot loaded with 10 l of Phycocyanin and lane 2 is with 20l.

Fig 3 . 4 . 2
Fig 3.4.2Phycocyanin lane 1 band 2 in UV 365 nm The above figure is the UV long 365 nm scan of the sample and the band here indicates the Phycocyanin being illuminated by the UV light.Below is the chromatogram of the Lane 1 and Lane 2-band 2. Further the Rf value of 0.68 was obtained and it validated the compound in accordance with the literature review.

Table 2 .
2.1 Components and quantity of oil and aqueous phase in cream formulation

Table 2 .
2.2 Role of ingredients of oil and aqueous phase