Prospective of Monascus Pigments as an Additive to Commercial Sunscreens

Abstract

Red and yellow pigments from Monascus purpureus (NMCC-PF01) were evaluated to enhance sun protection factor (SPF) of commercial sunscreens and Aloe vera extract. The extracted Monascus pigments contain rubropunctamine (red pigment) and the mixture of monascin and ankaflavin (yellow pigment) as major components. Antioxidant activity and in-vitro safety of the pigments were assessed by ferric reduction potential and DPPH radical scavenging assays, human keratinocytes (HaCaT), and erythrocytes (RBCs) cytotoxicity assay, respectively. In results, SPF of commercial sunscreens showed an increase of 36.5% with red pigment compared to the 13% increase by yellow pigment. The in-vitro studies showed 67.6% ferric reducing potential and 27% DPPH radical scavenging activity, neither cytotoxic effect against human keratinocytes nor haemolytic activity. These results confirmed the safe nature of the Monascus pigments; however, in-vivo studies merit further research. In conclusion, screened pigments from Monascus purpureus may act as potential candidates to increase SPF of commercial sunscreen naturally.

Ultraviolet (UV) radiation can damage the skin by multiple modes like mutations in cell DNA, the formation of reactive oxygen species, changes in histochemistry of cell, increased expression levels of the p53 gene, and/or influencing the immune system.¹ Increased consciousness about skin exposure to ultraviolet radiation has tempted the use of commercial sunscreen products. The commercial sunscreen is generally composed of organic, inorganic UV absorbers like aminobenzoates, cinnamates, avobenzone, oxybenzone, and oxides of zinc, titanium, iron. However, increasing side effects of some sunscreen components has made it indispensable to search for natural photoprotectants.² The most commonly utilizing sunscreen ingredients such as oxybenzone linked to sun exposure triggered allergic reactions, generating free radicals, which may be associated with cell damages.^3,4 While the nanoscale TiO₂ and ZnO are responsible for the generation of a substantial amount of reactive oxygen species, which upon UV illumination causes modifications in nucleic acid bases and eventually cell death.^5,6 Consequently, it is the need of an hour to look for other options to replace harmful components of sunscreens.

Nature is an abundant source of metabolites, considering the fact that UV damage is not only limited to humans but also to microbes. In continuation of our research on natural UV protectants,⁷ we targeted pigments from Monascus purpureus, which are well known for their multifaceted use in food coloration and range of other bioactivities. The selection of these pigments was rationalized on their variable bioactivity after linking with different amino acids.⁸ This amino acid-based derivatization of Monascus pigments may change their absorption maxima in the UV region. Here we sought to study the ability of food-grade pigments from Monascus purpureus (NMCC-PF01) (i) to increase the SPF of commercially available sunscreens, (ii) evaluation of their antioxidant potential, and (iii) cytotoxicity testing on human keratinocytes and erythrocytes cells.

Experimental

Commercial Sunscreens, Chemicals, and Plant Extract

Two different commercial sunscreens with labeled sun protection factors (SPFs) of 15 and 24 were brought from local market Jalgaon, India. Ethanol was purchased from Sd-fine chemicals (India). For plant extract, 10 g of Aloe vera leaf was macerated and filtrated through muslin cloth; the resultant extract was used for further studies.

Microorganisms and Cultural Conditions

Monascus purpureus (NMCC-PF01) was procured from North Maharashtra Microbial Culture Collection Centre (NMCC), Jalgaon and used for pigment production. The potato dextrose agar (PDA) (Hi-Media, India) slope was used for the maintenance of fungal culture at 4°C and subcultured periodically.

Microbial Pigments Production

The inoculum preparation and production of Monascus pigments were performed in aliquots as per our previous report.⁹

Extraction and Purification of Pigments

For extraction of extracellular Monascus pigments, fungal mycelium was separated by filtering fermentation broth using Whatman filter paper. The dark red colored filtrate was concentrated under vacuum and extracted by 95% (v/v) ethanol. It was kept rotating for 1 hour at 200 rpm, followed by filtration through Whatman paper to derive crude extract of pigments.¹⁰ The crude Monascus pigments were further purified by chromatographic methods.

Column Chromatography

The chromatography column (300 × 18 mm sintered disc glass column) with silica mesh (60 × 120) was used with a solvent gradient system of 100: 0, 100: 10, 50: 50 chloroform: methanol gradient 4 mL/min.⁹

Thin-Layer Chromatography

The pigment fractions collected after column chromatography were concentrated and spotted on TLC (silica gel 60 Merck Darmstadt, Germany) plate, and pigments were separated with a solvent system of chloroform : methanol : acetic acid (285: 24: 9). The two separated (yellow and red color) bands on TLC plate were scrapped and redissolved in ethanol and centrifuge at 3000 rpm for 15 minutes to settle down the silica. The supernatant was collected and concentrated for further study.^11,12

Pigments Characterization

Two dominant colored pigments (yellow and red) were obtained and characterized using UV-Visible absorption (Shimadzu spectrophotometer), Fourier transform infrared spectroscopic analysis (Shimadzu FTIR 8400, range 400 cm⁻¹ to 4000 cm⁻¹), and mass spectrophotometry analysis (WATER Q-TOF MICROMASS).

Determination of Sunscreen Protection Factors

The sunscreen protection factors (SPFs) of commercial sunscreens (SPF-15 & 24) and sunscreens enrich with Monascus pigments were determined by the in vitro spectrophotometric method with slight modification.^7,13,14 The commercial sunscreens with labeled SPF of 15 and 24 and Aloe vera extract were constituted as control while, in the test, Monascus pigments at a concentration of 4% and 8% w/w were added to the above commercial sunscreens. Multiple samples (0.1 g) each from control and test sunscreens formulations were taken and separately added to volumetric flasks containing 10 mL ethanol. All samples (ie, controls and pigment-supplemented samples) were ultrasonicated for 5 minutes and then filtrated through a muslin cloth. The 0.5 mL aliquots of filtrate were collected in tube and made up to 2.5 mL by addition of ethanol.

The optical density of each of these solutions was measured in the UV range from 290 to 320 nm at 5 nm intervals using ethanol as blank. Three independent analyses were performed, and SPFs were calculated, according to Mansur et al. (1986)¹⁵ using the following formula:

S P F = C F \times \sum_{290}^{320} E E (λ) \times I (λ) \times A b s (λ)

where CF (correction factor) = 10; EE (λ) = erythmogenic effect of radiation with wavelength λ; Abs. (λ) = absorbance value of a solution; and I = solar intensity spectrum. EE (λ) $\times I$ is constant and determined by Sayre et al. (1979),¹⁶ as shown in Supplemental table S1.

Ferric Reducing Potential

Antioxidant potential of the pigment samples was determined by evaluating ferric reducing activity as per our previous reports.^7,14 In brief, individual test samples, including red and yellow pigments (10 mg each) were added to 2.5 mL phosphate buffer (pH 6.6) and 2.5 mL of 1 % w/v potassium ferricyanide and incubated for 20 minutes. Further, the mixture was added with 10 % w/v trichloroacetic acid (2.5 mL) and centrifuged at 3000 rpm for 10 minutes (Remi C-24 BL). Then, 2.5 mL of the mixture from the upper layer was mixed with distilled water (2.5 mL), and freshly prepared 0.5 mL of ferric chloride solution (0.1% w/v) was added, and the volume was made up to 100 mL with distilled water. The optical density was recorded at 700 nm. Percent reduction in the ferric chloride was determined by comparing with the ascorbic acid as a positive control. The experiment was carried out in triplicates.

DPPH Radical Scavenging Assay

The radical scavenging activity was determined by DPPH (1,1-Diphenyl-2-picrylhydrazyl) assay with a slight modification of Chang et al¹⁷ method. In brief, 40 µl of Monascus pigments (10 mg/mL in DMSO) was mixed with 2.96 mL DPPH (0.1 mM) solution. The reaction mixture was vigorously shaken and incubated in dark condition at room temperature for 30 minutes. The absorbance was measured at 517 nm with DPPH as control. Ascorbic acid was used as positive control. The % radical scavenging activity was calculated using the following formula,

% I n h i b i t i o n = \frac{A b s C o n t r o l - A b s S a m p l e}{A b s C o n t r o l} x 100

where Abs control is the absorbance of DPPH solution and Abs sample is the DPPH +pigments.

Human Keratinocytes Cytotoxicity Assay

The cytotoxicity of the pigments was analyzed using human keratinocytes (HaCaT) cell line (obtained from NCCS, Pune, India). Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and the 1X antibiotic solution was used for cells maintenance at 37°C in a CO₂ incubator. The cells were harvested and seeded into a 96-well plate at a concentration of 1 × 10⁴ cells/well. After 24 hour incubation, the samples were added at different concentrations (0.25-2 mg/mL), and the cells were incubated for further 24 hour. The test samples were prepared in 10% DMSO. The cytotoxicity of the samples was evaluated using the standard method of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.¹⁸ Twenty microliter (stock-5 mg/mL) MTT reagent was added in each well and kept for incubation in the CO₂ incubator at 37°C for 4 hour. Then 100 µL of DMSO was added to each well and kept in the dark for 1 hour. The absorbance was measured at 595 nm wavelength. The cells without treatment were taken as a control (added 10% DMSO) for the study. Experiments were done in triplicate, and the percentage viability was calculated by using the following equation:

% C e l l v i a b i l i t y = (O D_{595} (T e s t) / O D_{595} (C o n t r o l)) \times 100

Human Erythrocytes Cytotoxicity Assay

The cytotoxicity testing of the pigment molecules was performed by hemolysis assay according to the method of Borase et al¹⁴ with appropriate modifications. Five-milliliter blood was collected from a healthy volunteer, and it was added with ethylene diamine tetra-acetic acid (EDTA) to inhibit the coagulation of blood cells. The RBCs (red blood cells) were separated from blood, 1 mL of blood was mixed into 5 mL of phosphate buffer saline (PBS) (pH-7.4) and the mixture centrifuged to 6000 rpm for 10 minutes at 37°C (Remi C-24 BL, India) to remove the supernatant containing platelets and plasma to obtain pellets of RBCs. The pellet of RBCs was resuspended into 10 mL of PBS and (a process of washing was repeated four times) again centrifuged them.

One milliliter aliquots of uniformed cells suspension (RBCs) were taken in eppendorf tubes, and 1 mg/mL of yellow and red Monascus pigments were added in test vial and mixed by gentle shaking. One milliliter of sterile water and phosphate buffer saline was added in the positive and negative control, respectively. The mixtures in all tubes were incubated in the dark for 4 hour and centrifuged for 10 minutes at 1000g. Supernatants from all the tubes were further subjected to spectroscopic absorption at 541 nm, and the optical densities were used to calculate percent hemolysis by the following formula:

% H e m o l y s i s = \frac{S a m p l e a b s o r b a n c e - N e g a t i v e c o n t r o l a b s o r b a n c e}{P o s i t i v e c o n t r o l a b s o r b a n c e - N e g a t i v e c o n t r o l a b s o r b a n c e} \times 100

The hemolysis of the RBCs for different treatment and positive and negative control were also confirmed by using Foldscope (Foldscope Instrument Inc., CA).

The partially purified pigments from M. purpureus were first characterized by UV visible (Supplemental figure S1) and mass spectroscopic analysis (Supplemental figure S2). The pigment components were identified as red pigment fraction representing rubropunctamine (m/z 354.16 MH⁺), while yellow representing a mixture of monascin (m/z 359.23 MH⁺) and ankaflavin (m/z 387.28 MH⁺).

The SPF was calculated by measuring the absorbance within the range of 290-320 nm.¹⁵ Two commercial sunscreens with claimed SPF values of 15 and 24 empirically showed SPF values of 16.26 ± 0.75 and 24.42 ± 1.06, respectively, whereas SPF of Aloe vera extract was 0.1 ± 0.01. In combinational studies, red pigment (4% w/w) combined with sunscreen having 15 and 24 labeled SPF showed 17.0% and 13.26% increase, respectively, while the yellow pigment (4% w/w) with same sunscreens showed a marginal increase of 1.6% and 3.48%, respectively. The same pigments with 8% (w/w) concentration increased the SPF by 36.53% and 30.67% (rubropunctamine) and 13.16% and 12.40% (monascin and ankaflavin), respectively (Table 1). Similarly, with the rubropunctamine (4% and 8%) combined with Aloe vera extract, the SPF was 2.55 ± 0.38 (2550%) and 3.87 ± 0.37 (3870%), respectively (Table 1). A mixture of monascin and ankaflavin increased the SPF of Aloe vera to 1.74 ± 0.11 (1740%) and 2.13 ± 0.124 (2130%) with respective concentrations of 4% and 8% (Table 1).

Table 1

Sunscreen Protection Factors (SPFs) for Commercial Sunscreen Preparations and Natural Plant Extracts Before and After Supplementation with Pigments From Monascus purpureus

Commercial sunscreens	Ingredients	SPF	Calculated SPF	SPF pigment + sunscreens
				Red pigment		Yellow pigment
				4%	8%	4%	8%
Sunscreen 1	Aqua, Paraffinum, Liquidum, Emulsefying Wax, Titanium Dioxide, Steric Acid, Borex, Almond Oil, Vit-e, Isopropyl Myristate,Bhenzophenone-3, Octylmethoxy Cinnamate, Propylene Glycol, Allantoin, Phenoxyethanol, Methyl Paraben, Myristate, Ethylene Diamine Tetra Acetate, Imidazolidinyl Urea, Allantoin, Propyl Paraben, Isoproply, Fragnance	15	16.26 ± 0.75	19.03 ± 0.74	22.2 ± 1.2	16.95 ± 0.62	18.4 ± 0.52
Sunscreen 2	Aqua, Ethylhexyl, Salicylate, Avobenzone, Glycerine, Steric Acid, Palmitic acid, Phenylbenzimidazol Sulphonic Acid, Glycol Stearate, Steramide AMP, PEG-100 Stearate, Dimethicone, Glyceryl Stearate, Xanthan Gum, Potassium Hydroxide, Niacinamide, Disodium EDTA, Propylene Glycol, Carbomer, Cetyl Alcohol, Sodium Hydroxide, Cymbopogon, Schoenanthus (Lemom Grass) Extract, CucumisSativis (Cucumber) Fruit Juice, Lactic Acid, Phenoxyethanol, Methyl and Propylparaben	24	24.42 ± 1.06	27.66 ± 0.81	31.91 ± 1.63	25.27 ± 0.33	27.45 ± 1.54
Aloe vera plant extract		NA	0.1 ± 0.01	2.55 ± 0.38	3.87 ± 0.37	1.74 ± 0.11	2.13 ± 0.124

NA-, not applicable.

The rationale behind this SPF increment probably lies in a combination of pigment with sunscreen components that resulted in significant changes in the infra-red spectrum (Supplemental figure S3c). For yellow pigment and sunscreen combination, the changes were indicated by the broadening of the peaks at 3460.41 cm⁻¹ for the alcoholic group and 2967.58 cm⁻¹ for C-H. The frequency at 1652.03 cm^-1 indicated α-β unsaturated ketone, while the other two peaks at 1922.13 and 1229.48 cm⁻¹ were newly detected. The infra-red results indicate that the Monascus pigments may chemically interact with commercial sunscreen and undergo derivatization by making some changes in functional groups of chemical constituents present in sunscreen. The outcome of the study specifies the use of Monascus pigments as an additive to sunscreen, which may provide more protection from UV-B radiation. These results are in agreement with Suryawanshi et al and Borase et al.^7,14

The genesis of free radicals may occur when the skin is exposed to UV light. Antioxidants are well known to protect the skin from free radicals. Thus, a variety of antioxidant compounds are incorporated in cosmetic products, which are protecting the skin from oxidative damage. In the present study, whether, the pigments have the potential to protect the skin from free radicals were determined by antioxidant assay. The Monascus fermented products and extracted compounds are known to use in cosmetic preparation, for their antioxidant and nutricosmetic potential.¹⁹ During the study, red pigment (rubropunctamine) showed 67.6% ferric reduction potential as compared to 100% for standard antioxidant, ascorbic acid, and 3.2% for yellow pigments. Similarly, the DPPH assay revealed that 27% and 14.51% radical scavenging activity for red and yellow pigments, respectively. These signify that red pigments have high antioxidant potential as compared to yellow pigments. Similarly, the antioxidant activity of Monascus fermented products and/or extract such as rice, corn, and sorghum have been reported and correlated with the high amount of red pigments present.²⁰

The extracted Monascus fermented soybean in methanol and 80% ethanol were shown the highest Trolox equivalent antioxidant activity (3.13 ± 0.06 mM TE/g) and oxygen radical scavenging activity (2.79 ± 0.09 mM TE/g), respectively. Besides, pigments showed significant inhibition of enzymes, tyrosinase, hyaluronidase, and elastase, which are related to skin aging.²¹ The results of the present study have also following these previous reports, which ascertain the antioxidant potential of Monascus pigments.

The assessment of the toxicological properties of the ingredients of cosmetics and pharmaceutical formulations is an essential regulatory requirement.²² Various in vitro and in vivo toxicological methods were executed for the evaluation of toxicity. In the case of cosmetic products, considering the specific route of topical application, the in vitro cytotoxicity testing on skin cell lines (in particular keratinocytes and fibroblasts) can be used for primary toxicological evaluation of new ingredients such as surfactants, colorants, preservatives, and additives.^2,23
-25 In this study, the human keratinocyte (HaCaT cell line) was used for evaluation of the Monascus pigments skin toxicity by using in vitro MTT assay.

The results of the cytotoxicity of Monascus pigments against normal human keratinocytes were shown in Table 2 which indicated that Monascus pigments are nontoxic toward the normal skin cell in the in vitro study. Since the cells even were treated with a higher concentration (2 mg/mL) of pigments, no significant cell mortality (<10% mortality) was recorded. Likewise, the yellow Monascus pigment, ankaflavin showed selective cytotoxicity to human hepatocellular carcinoma cells (Hep G2), but low toxicity to normal fibroblast, while, no cytotoxicity of monascin against the normal human lung fibroblast (WI-38 and MCR-5 fibroblast cells). Also, the red Monascus pigments are not reported for toxicity against the normal human cells. In recent year, Yuliana et al studied the toxicity of Monascus pigments by using computerized structure-activity relationships, against fish, daphnid, and green algae and the toxicity were determined by ecological structure-activity relationships (ECOSAR).²⁶ Among the 57 tested Monascus pigments, almost all red pigments were shown no toxicity toward the tested organism. This suggesting that the pigments derived from Monascus are nontoxic nature and safe to be used in commercial sunscreen formulations, which are applied on the skin.

Table 2

In Vitro Toxicity Testing of Red Monascus Pigments in Human Keratinocytes (HaCaT) Cell Line

Cytotoxicity against human keratinocytes
Pigments concentrations (mg/mL)	% Viability of cells
Pigments concentrations (mg/mL)	Red pigment	Yellow pigment
0.00	100 ± 0.24	100 ± 1.39
0.25	97.43 ± 1.02	97.72 ± 1.33
0.50	96.44 ± 0.95	94.49 ± 1.12
1.00	95.25 ± 0.57	92.76 ± 0.89
2.00	91.72 ± 1.42	90.64 ± 0.96

In cytotoxicity analysis, the potential of pigments to damage the RBC was measured by the amount of hemoglobin released from treated RBCs. RBCs test enables the quantification and evaluation of the in vitro irritant effects of the surfactants (added into a range of cosmetics products) and sunscreens formulations.^27,28 In the present investigation, both pigment molecules were responsible for a negligible percentage of hemolysis, that is, 0.11 ± 0.01% by rubropunctamine and 0.36 ± 0.04% by a mixture of monascin and ankaflavin. Sterile water served as a positive control, which was considered to show 100% hemolysis (Figure 1). In microscopic observation, RBCs in test samples were healthy and well ordered compared with positive control showing disorganized and faint cells.

Figure 1

Toxicity Testing of Monascus Pigments by RBCs Hemolysis: (a) 1 - PBS-Negative Control, 2-Red Pigment +RBCs Test, 3-Sterile Water Positive Control, 4- Red Pigment, and (b) 1 Positive Control, 2-Yellow Pigment +RBCs Test.

Red and yellow Monascus pigments, in combination with commercial sunscreens, may form different functional groups that consequently responsible for absorption of light in UVB region (290-320 nm). The potential to increase SPF, antioxidant activity, no cytotoxicity against the healthy skin cells as well as earlier reported antimicrobial activities of these pigments might help to put more emphasis on the use of microbial pigments in commercial sunscreens. Though Monascus is food grade pigments, their use on skin rest on in vivo safety studies and consumers’ choice.

Supplemental Material

Supplementary material - Supplemental material for Prospective of Monascus Pigments as an Additive to Commercial Sunscreens

Supplemental material, Supplementary material, for Prospective of Monascus Pigments as an Additive to Commercial Sunscreens by Sunil H. Koli, Rahul K. Suryawanshi, Bhavana V. Mohite and Satish V. Patil in Natural Product Communications

Footnotes

Acknowledgments

SVP acknowledges the Department of Biotechnology, New Delhi, for the Indo-US Foldscope Major Research Project. The authors are thankful to UGC-SAP and DST-FIST for providing financial support to the SOLS department.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Mr Sunil Koli thankful to UGC-BSR for providing fellowship to research work (Fie No. NMU/SLS/491/2015(UGC BSR, Dated 11 Aug.2015).

ORCID ID

Satish V. Patil

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