Experimental Calculations Of Metals Content in Skin-Whitening Creams And Theoretical Investigation For Their Biological Effect Against Tyrosinase Enzyme

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

Tanzeela Ashraf1 & Mehwish Taneez1 & Saima Kalsoom1 & Tahira Irfan2 & Munib Ahmed Shafique3

Abstract

The demand for skin-whitening creams (SWCs) has increased rapidly worldwide due to the sharp rise in product advertisements in the media and the growing awareness. Metals are present either as impurities or added intentionally in creams and may have toxic effects on users. The present study was carried out to determine the content of metals such as mercury (Hg), cadmium (Cd), lead (Pb), arsenic (As), chromium (Cr), nickel (Ni), cobalt (Co), copper (Cu), zinc (Zn), and iron (Fe) in fifteen skin-whitening creams marketed at local shops in Islamabad, Pakistan. The concentrations of metals were analyzed by an inductive coupled plasma-optical emission spectrometer (ICP-OES) after digestion with a mixture of HNO3, HCl, and H2O2. The skin-whitening creams were found to have metal concentrations in parts per million (ppm) in the following range: Hg (1.0–18,210 ppm), Co (0.1992– 1.9931 ppm), Cr (1.0453–2.7455 ppm), Cu (0.6987–0.1997 ppm), Fe (8.8868–28.6213 ppm), Ni (0.7487–1.5958 ppm), Pb (0.2997–4.7287 ppm), and Zn (7819.2–39,696.7 ppm). As and Cd were not detected in any of the fifteen skin-whitening creams. Only one cream (L’Oréal Paris White Perfect) was found in safe limits defined by the Food and Drug Administration for cosmetics. In order to elucidate the mechanism of lower production of melanin in presence of heavy metals, a molecular docking study was carried out by using Molecular Operating Environment (MOE) software. A good correlation was observed between experimental findings and molecular docking studies.

Keywords: Skin-whitening creams. Metals. Molecular docking. Toxicity

cistanche is able to improve skin whitening

Introduction

Fairness is branded as beauty, grace, and high social status across the globe in most of the communities. This perception encourages most women to engage in skin-whitening. Skinwhitening creams, ointments, solutions, and gels are used by both men and women in order to improve the appearance of the skin or as an anti-freckle [1]. During the last few decades, these products have largely been used for beautification [2]. Metals are added intentionally or unintentionally in skinwhitening products and the consumers are mostly unaware of their presence. Human exposure to metals in skin-whitening creams occurs mainly through the skin. These products are applied either to the entire surface of the body or to restricted areas. Some SWCs remain in contact with the skin for several hours or days while others are washed away shortly after application [3]. Continuous use of whitening cream causes accumulation of metals in the body over time and these metals are known to cause a variety of chronic health effects like cancer; reproductive, developmental, and neurological disorders; contact dermatitis; brittle hair; and hair loss [4]. Some metals are strong endocrine disruptors and respiratory toxins. Metals such as Cr, Ni, and Co are well-known skin sensitizers, while Cd, As, Pb, and Hg pose toxicity [4–6]. As, Cd, Co, Cr, Ni, Pb, and Hg and their compounds are among the 1000 chemicals listed as prohibited intentional ingredients of cosmetics in Annex II of the European Council Directive 76/768/EEC because they are considered unsafe due to their toxicological properties [4]. Various forms of cosmetics including powder, skin foundations, skin-whitening creams, lipstick and lip gloss, and lotions are used by women around the world to enhance the appearance of skin. Different concentrations of metals (such as As, Cd, Pb, Co, Ni, Cr, Cu) have been found in many products including eyeliner, henna, tattoos, smears and hair sprays [7], lipsticks, lip moisturizers, and lip balms [7, 8]. Cd, Cr, Cu, Pb, and Ni were present in mascara, eye pencil, body and face creams, sunblock, vaseline, and traditional cosmetics such as eye kohl. Among tested samples, khol was indicated to have the highest content of most of the metals [9]. Moreover, high content of metals was also detected in 91 samples of face paints, and the Zn content was extremely high [10]. In a recent study, five metals (Cd, Cr, Fe, Ni, and Pb) were quantified in different brands of lotions, foundations, whitening creams, lipsticks, hair dyes, and sunblock creams, and lifetime cancer risk (LCR) value was found to be higher than the permissible limit in all cosmetic products except lipsticks [11]. Therefore, regular monitoring of cosmetics and precise information about the presence of metals in the skin-whitening creams are necessary for the safety evaluation of the use of these products due to public health concerns worldwide [4, 12].

In the present study, 15 samples of skin-whitening creams (SWCs) were analyzed for Hg, Pb, Ni, Cr, Co, Cu, Zn, Fe, As, and Cd contents, and their binding pattern with skin target was determined by computational docking studies. Molecular docking is defined as an optimization problem, which determines the “best-fit” orientation of a ligand that binds the particular target of interest and is used to predict the structure of the intermolecular complex formed between two or more molecules. There are several possible mutual conformations in which binding occurs, commonly called binding modes [13]. Molecular docking is routinely used for understanding drug-receptor interaction. It provides useful information about drug-receptor interactions and is frequently used to predict the binding orientation of small molecule drug candidates with their protein targets to predict the affinity and activity of the small molecule [14].

natural ingredients Echinacoside of Cistanche

Materials and Methods

Sample Collection and Metal Extraction

Fifteen skin-whitening creams including one imported cream for comparison were purchased from local stores of 1-10, Islamabad, Pakistan, based on the popularity of the brand, namely L’Oreal Paris (LP), Stillman’s Skin (SS), White Face (WF), Face Fresh (FF), Faiza Beauty (FB), Due Beauty (DB), Golden Pearl (GP), Fair & Lovely (FL), White Look (WL), Bfresh (BF), Goree Beauty (GB), Sara Whitening (SW), Silk face (SF), Evergreen (EG), and Nisa Extra Glowing (NS). Prices of the SWCs ranged between 250 and 1500 PKR. Details of each product such as color, manufacturing date, expiry date, details of manufacturers, net weight, and sampling city/location were provided in Table S1 (Supplementary Information). All the reagents used were of analytical grade nitric acid (HNO3 69%; BDH, England), hydrochloric acid (HCl 37%; Sigma-Aldrich, Germany), and hydrogen peroxide (H2O2 35%; Sigma-Aldrich, Germany). All the apparatus used in the extraction of metals was prewashed with 10% HNO3 and distilled water. One gram of each cream sample was placed in a 250-mL flask and 20 mL of HNO3 and H2O2 mixture was added. The samples were covered with a condenser tube and left to stand overnight at room temperature. The mixture was then heated with continuous refluxing for 4–5 h on a hot plate at 70–100 °C. Aliquots of HCl and HNO3 were added until no brown fumes appeared. The samples were evaporated to dryness and allowed to cool [15, 16]. All samples along with blanks were digested in triplicate, diluted to 10 ml with distilled water, and filtered using 0.45-μm syringe filters before analysis on ICP-OES (iCAP 6500, Thermo Scientific, UK). The detailed procedure of metal analyses and the most sensitive line for analyzed metals are presented in Table. S2 of supplementary information.

cistanche bodybuilding

Molecular Docking Studies

In order to better understand the effective binding patterns of ligands (heavy metals) with the target (tyrosinase enzyme), molecular docking studies were performed. The software used to study the binding pattern of ligands was Molecular Operating Environment (MOE) version 2016.08 by Chemical Computing Group Inc. Three standard inhibitors hydroxyquinone, kojic acid, and niacinamide along with ten metals were used as ligands for docking studies. The 2D structures of these ligands were built by the builder tool of MOE and saved in MDB file format. For the binding pattern mode of standard drugs and heavy metals, a suitable target protein, tyrosinase 1 (human TYRP1, PDB code: 5M8N), was selected. The crystal structure of the protein was downloaded from Protein Data Bank PDB ID: 5M8N and was imported into MOE as shown in Fig. S1 (Supplementary info.). These structures were 3D protonated and energy minimized after eliminating water molecules, and all hydrogen atoms with their standard geometry were added to the structure. Site Finder was used to search systematic confirmation of the resulting model at default parameters with an RMS gradient of 0.0001 kcal/mol. Protein comprises 4 chains, A, B, C, and D. To identify the active site of the protein, the Site Finder tool of MOE was used. The longest chain was selected as per protocol and applied. Alpha centers were created followed by the insertion of dummies; the docking was performed along with the dummy atoms. Ten conformations were generated for each ligand. The lowest binding energy conformation of each ligand was selected for binding pattern analysis [17].

Results and Discussion

Metal Content in SWCs

The average concentrations (ppm) of Hg, Pb, Cr, Ni, Co, Cu, Zn, As, Cd, and Fe in SWCs are shown in Table 1. As and Cd were not detected in any of the samples while other metals were present in all samples with varying concentrations. Mercury was detected in thirteen SWCs except in Stillman’s Skin (SS) and Goree Beauty (GB) creams. Levels of Hg in different SWCs ranged from 1.0 ± 0.09 to 18,210 ± 479 ppm. We found exceptionally high concentrations of Hg in SWCs. The mean concentrations of Hg in SWCs were above the permissible limit, i.e., 1 ppm by the World Health Organization (WHO) and US Food and Drugs Administration (FDA) [18]. Though the detected concentrations of metals in SWCs are too high and pose toxicity in spite of this, they are added in cosmetics. Based on the mean Hg concentration levels, the samples were arranged in the following decreasing order: WF > NS > SF > SW > FF > GP > DB > FB > BF > EG > FL > WL > LP. In the present study, Hg content was lower than that in SWCs (i.e., 2.46 to 23,222 ppm) marketed in Saudi Arabia [19] but greater than that reported by another study (i.e., 1.18 to 5650 ppm) [20]. Similarly, facial creams in Thailand, Lebanon, and England showed also a high level of Hg ranging from 1281 to 5650 ppm [21]. Hg levels in all these studies exceeded the permissible levels in whitening creams set by the FDA in cosmetic products [4, 18]. Exposure to a high level of Hg can induce changes in the central nervous system resulting in irritability, sleeping disorders, tremors, reduced memory and intelligence, headaches, hallucinations, and even death in severe cases. Moreover, chronic exposure to Hg can cause damage to kidneys and developing fetuses due to its accumulation in the cells [19]. Skin is more liable to skin cancer by inhibiting the production of melanin. Application of inorganic mercury salts causes nephrotoxic effects. Hg exposure to placental cells causes damage to the developing fetus [22, 23].

Pb was detected in fourteen SWCs except for BF. The level of Pb differed significantly among the SWCs ranging from 0.1997 ± 0.19 to 4.7287 ± 0.0428 ppm. The order of Pb in SWCs was NS > SS > SF > DB > GP > FF > SW > EG > LP > WL > WF > FB > GB > FL > BF. SWCs in Nigeria were found to have similar concentrations of Pb (i.e., 0.8 to 4.50 ppm) [24], while in Saudi Arabia, 76% of the SWCs were found to have Pb content up to 10 ppm [25]. Therefore, cosmetic products containing Pb whether applied once or a number of times per day can lead to human exposure to Pb. To limit human exposure to Pb, the FDA has drafted guidelines for the cosmetic industry, i.e., Pb concentration in cosmetics should not exceed 10 ppm as an impurity. This guidance applies to cosmetic lip products (such as lipsticks, lip glosses, and lip liners) and externally applied cosmetics (such as eye shadows, blushes, shampoos, and body lotions) sold in the USA [18, 26].

Cr, Ni, Co, Zn, and Fe were detected in all the fifteen SWC samples. Cr with concentration ranging from 1.0453 ± 0.0503 to 2.7455 ± 0.35 ppm is exceptionally low as compared to the concentrations detected in another study where Cr ranged from 4.25 to 8.0 ppm in creams [27]. The concentration was in order of SS > GP > FF > WL > EG > WF > DB > FB > BF > GB > NS > LP > FL > SF > SW. It is recommended that consumer products should not contain more than 5 ppm of Cr, or for good health protection, levels should not exceed 1 ppm [28]. Therefore, the concentrations of Cr detected in the present study were higher than these permissible limits. The concentrations of Cr in products were in the range that can induce contact dermatitis in sensitive individuals. Exposure to Cr can cause severe redness, skin ulcers, and swelling of skin. There is no regulation to limit the use of chromium in cosmetics, although the listing regulation for the color additive FD & C Blue No. 1 limits chromium as an impurity to 50 ppm [18].

The range of Ni in the SWCs was found to be between 0.7478 ± 0.0492 and 1.5958 ± 0.2969 ppm which is not high and follows the order: GP > SS > WF > FF > DB > FL > WL > BF > EG > NS > FB > GB > LP > SW > SF. Ni concentrations were consistent with literature values found in SWCs (0.03 to 1.65 ppm) while skin moisturizers have up to 10.7 ppm of Ni [29]. Body creams in Nigeria were found to have Ni level of 5.09 ppm and absorption of metals from these creams to skin is facilitated due to the presence of fat-soluble substances [30]. Ni is one the most common contact allergens used in patch test. Exposure to Ni from SWCs can result in sensitization [31]. Once in contact with the skin, metallic Ni oxidizes to form soluble diffusible compounds that may penetrate the intact stratum corneum via the appendageal (hair follicles, sweat glands, and sebaceous glands), transcellular, or intracellular route [32]. The recommended limit of Ni for consumer products is 5 ppm; however, the level should not exceed from 0.5 to 1.0 ppm for better health protection [3, 28]. Ni concentration of about 0.5 ppm is sufficient to cause contact dermatitis in skin [28]. The levels of Ni found in the SWCs could trigger contact dermatitis in people with hypersensitivity. Similarly, Co is assumed to be skin allergen. The Co content in the studied SWCs was slightly higher than the suggested acceptable limits, i.e., 0.1992 ± 0.0003 to 1.9931 ± 0.003 ppm, and followed the order: WL > WF > FF > SS > GP > DB > LP > FB > FL > EG > SF > GB > BF > SW > NS. However, the permissible level of Co impurity has not been regulated so far but suggested that the consumer products should not contain more than 1 ppm of Co [28]. Only, WL was found to have Co concentration above recommended level. It is further evident from the literature that Co powders penetrate the damaged skin more easily than the intact skin. Volunteers exposed cutaneously to Co had higher concentrations of urinary Co [33]. A dose-response study with 72 Coallergic patients identified a stimulation concentration at 50 ppm [31], whereas Co-allergic patients could react to Co test at a concentration of 19 ppm [34]. Although Cu is rarely a skin sensitizer, in some cases, immune reactions occurred due to Cu exposure from intra-uterine devices. The use of prosthetic materials in dentistry has also created a risk of sensitization for Cu [35]. We found very low concentration of Cu (i.e., 0.1997 ± 0.1997 to 0.6987 ± 0.0999 ppm) in all SWCs: FL > GB > SS > FF > NS > WL > BF > EG > GP > WF > SF > LP > SW > DB > FB. Previously, low concentrations of Cu were also reported in SWCs (0.3–10.0 ppm) compared to moisturizing creams (0.5–17.5 ppm) [29].

The Zn was detected in quite high concentrations, i.e., 6.0231 ± 0.3515 to 39,696.7 ± 174.23 ppm, in the cream samples. The highest concentration was found to be present in the sample SF followed by SS > SW > NS > GP > FF > WL > EG > FB > FL > BF > LP > SW > DB > FB. A high concentration of Zn was reported for moisturizers (17.3 to 372.0 ppm) and SWCs (24.7 to 267.5 ppm). The concentrations of Zn in the present study are far much higher than the previously reported Zn concentrations. The high content of Zn may be due to ZnO which is used as an ultraviolet (UV) radiation filter in creams. In recent years, ZnO nanoparticles are frequently added to sunscreens and the high refractive index of Zn makes the skin look unnaturally white by inhibiting melanin production. Zn used in anti-dandruff shampoos has been shown to cause allergic contact dermatitis [36, 37], while high exposure over time can cause brittle hair and nails, neural abnormalities, gastrointestinal disorders, and convulsions [38]. The mean concentration levels of Fe present in SWCs ranged from 8.8868 ± 0.1043 to 28.6213 ± 0.0926 ppm. The amount of Fe decreased in creams in the following order: NS > SS > GP > EG > WL > SF > GB > BF > WF > SW > FL > FF > LP > FB > DB. The content of Fe in the present study was lower compared to previous studies reported from Pakistan [11, 22], and Nigeria [10] where the body creams and skinlightening creams were found to have Fe up to 2468 ppm and 211.6 ppm, respectively. But exposure to small doses of Fe from consumer products may result in cell death [39] or colorectal cancer due to cumulative effects [40]. Generally, there are no national and international standard limits for Cu, Cd, Fe, and Zn contents in cosmetics and SWCs but the regular use of SWCs causes the accumulation of these elements in the body. Despite the fact that Fe and Zn are essential for regulating some physiological functions in the body, the values obtained in this study raise safety concerns due to the cumulative effect arising from continuous exposure. The elevated levels Table 1 of these metals in some creams are likely due to the use of some natural or inorganic pigments such as clay, mica, and iron oxides, or the use of metallic devices during production.

Molecular Docking

In order to evaluate the molecular docking studies of metals and three standard compounds with tyrosinase enzyme (PDB code: 5M8N), the lowest energy docked conformation of each ligand was used for analysis. Docked binding energies of almost all complexes range from 5.25 to 8.62 kcal/mol. These energy values showed the instability binding mode of these metals with the tyrosinase enzyme. Other docked binding energies also showed high values which could be responsible for allosteric site generation in the tyrosinase enzyme. Binding interactions were predicted by the log plot of MOE software as shown in Figs. 1, 2, and 3. Ligand and active site conformation of target protein obtained from docking were taken as input into the MOE. The interactions were studied between the ligand and the active site of the target by selecting the atoms within 5 Å. In the natural process, Zn binds with Co-crystallized ligand MMS and initiates the process of melanin production. The ligand was lined with amino acid residues, known to play a critical role in the catalytic activity of TYRP1 as shown in Fig. 1. The observed key residues in close proximity to the cavity were Tyr 362, Asn378, Leu382, His215, His377, His404, Phe400, Ser394, Thr391, His381, Gly389, Gln390, and Arg374. Hydrophobic amino acids in the active site were shown by the green sphere while hydrophilic was represented by the purple sphere. co-crystallized ligand showed metal ligation with Zn (gray sphere). An aromatic moiety of MMS had arene-π interactions with Thr391 which also showed H binding with ammonium ion of MMS. Two different protons of Arg374 showed H binding with carbonyl moiety of MMS as shown in Fig. 1. These three different types of interactions (metal ligation, H binding, and arene-π) are responsible for natural melanin production.

All three standard compounds hydroxyquinone, kojic acid, and niacinamide, and most of the metals were gorged into the catalytic amino acid triad as shown in Figs. 2 and 3. The catalytic triad of TYRP1 was located within the 5 Å of the docked ligands.

Metal forms complexes with MMS and replaces Zn. This causes deformation and change in the energy profile of complexes. These changes in both structure and energies of docked complexes minimize the production of melanin. Most metals bind in the active site of tyrosinase and disturb the energy pattern and conformations of amino acids in the active site which results in suppression of melanin production. Docked poses of metal enzyme complexes were also showed deferent types of H binding of MMS with nearby amino acids as compared to origin tyrosinase enzyme complex. Metallic binding of Zn in the active site with MMS disturbed due to difference in energy pattern. Zn metal showed conformational changes in placement, refinement, electrostatic, and score energies as shown in the docked output file. 3D docked patterns of some metals (Cr, Zn, Ni, and Hg) in the vicinity of tyrosinase are shown in Fig. 2. A Three-dimensional docked representation of the lowest binding energy interactions of the metals Cd, Cu, Fe, Co, Pb, and As is given in Figure S2 (supplementary info.). Docked energy values of these compounds range from 5.25 to 8.62 kcal/ mol. The energy required for melanin production is 11.96 kcal/ mol. Conformation changes in amino acids patterns were also responsible for the suppression of melanin production as shown in 3D docked view of metals in the active site. The toxic behavior of these compounds was also checked by using in silico PKCSM. Dry lab pharmacokinetics behavior of these metals showed that a high concentration of these metals was also responsible for hepatotoxicity. These metals do not metabolize in our body and accumulate in target complex, which results in cytotoxicity.

Docking of Standard Whitening Compounds with TYRP1

In order to compare the whitening effect and toxic behavior produced by these toxic metals, three standard compounds were also used for molecular docking studies. These standard whitening compounds were kojic acid, hydroquinone, and niacinamide. The ligand enzyme complex with the highest binding affinity (kojic acid) was studied and the same exercise was repeated with hydroquinone and niacinamide. Binding energy was found in the range of 9.355 to 12.07 kcal/mol. Their docking pattern showed that they showed metal ligation with Zn inactive site and disturb the melanogenesis. Kojic acid showed both strong H binding and arene-π interactions with Gl388, Ser39, His404, and His381 respectively. These types of interactions may be responsible for the low production of melanin as different amino acids were observed (Fig. 3) in binding with a standard whitening agent (kojic acid, hydroquinone, and niacinamide) as compared with a binding complex of MMS with tyrosinase as shown in Fig. 1.

However, their toxic effect is very less as compared to metal because these compounds are easily metabolized and excreted from the body. 2D and 3D docked patterns of these whitening compounds kojic acid and hydroquinone are shown in Fig. 3 and the niacinamide docked view is presented in figure S2 (supplementary info.).

Conclusion

Metals are added in cosmetics either intentionally for whitening purposes or unintentionally during processing as contamination. Their presence in SWCs may cause health risks to users. Fifteen skin-whitening creams were evaluated for metal (As, Cu, Cd, Ni, Cr, Co, Hg, Zn, Fe, and Pb) concentrations. In SWCs, all the metals analyzed were detected except As and Cd. All the SWCs were found to be contaminated by metals except L’Oréal Paris White Perfect when compared with the permissible limits of metals defined by the US FDA and WHO in cosmetics. Hg level was alarmingly high in most of the cream samples. Metals in SWCs are responsible for whitening by decreasing the melanin production and aging effect in users. However, high concentrations of these metals and regular use can induce toxicity. These factors were also studied using molecular docking studies. In silico studies were performed to check the decrease in the production of melanin by the binding of these metals with the tyrosinase enzyme. Molecular docking studies showed that these metals bind with the MMS and replace the zinc metal which is responsible for the lower production of melanin. The toxic behavior of these metals was also checked using in silico PKCSM. These metals do not metabolize in our body and accumulate in the target complex, which results in cytotoxicity. A good correlation was observed between the experimental findings and computational docking studies. There is a dire need for public awareness-raising (through social, print, and electronic media), regarding the high level of hazardous mercury and other chemical contents in SWCs and their effects both on the skin and on human health. People need to understand that “healthy” skin is beauty, not its “complexion” and people should not hunt for SWCs which lead to unhealthy and ugly skin.

cistanche bodybuilding