Bee pollen peptides as potent tyrosinase inhibitors with anti-melanogenesis effects in murine b16f10 melanoma cells and zebrafish embryos

Abstract

One important functional food ingredient today, valued for its health properties and ability to prevent disease, is bee pollen, which comprises a combination of nectar, pollen from plants, and the secretions of bees. In this research, the tyrosinase (TYR) inhibiting abilities of the peptides derived from bee pollen protein hydrolysates are investigated. Various proteases were utilized to generate these peptides, followed by testing at different concentrations. Tyrosinase inhibition activity was detected in all cases, while the hydrolysate drawn from 5.0% w/v neutrase exhibited the best IC50 value and was thus investigated further via ultrafiltration to separate the active fractions. The highest potential for tyrosinase inhibition was recorded for the fractions below 0.65 kDa. Subsequent purification steps via SEC and RP-HPLC led to the identification of the VDGYPAAGY (named VY-9) peptide via LC-Q-TOF-MS/MS in fraction F1–2, known for its non-toxic and hydrophobic characteristics albeit poor water solubility. The synthesized VY-9 peptide demonstrated competitive inhibition, with IC50 values of 0.55 ± 0.03 µM for mono-phenolase and 2.54 ± 0.06 µM for di-phenolase activities, as confirmed by molecular docking analysis revealing dominant hydrogen bond interactions with TYR. Effective concentrations of 0.2–1.6 µM of VY-9 showed negligible cytotoxicity in B16F10 cells. Melanin synthesis suppression was examined via qRT-PCR, and western blot in MITF, TYR, TRP-1, and TRP-2. Cell death in zebrafish embryos was evaluated in vivo using a toxicity assay which revealed no significant influence from VY-9, while anti-melanogenic effects were observed when the concentration was 4 µM, suggesting bee pollen-derived peptides’ potential in cosmetic and pharmaceutical depigmentation applications.

Introduction

Quality of life can be adversely affected by cosmetic skin problems, and one common remedy involves lightening through the use of either synthetic or natural substances which lighten the skin tone, or in some cases even the skin complexion by lowering the melanin levels in the skin. Accordingly, skin lightening can be used to address problems with freckles, discolored skin, or scars resulting from acne. Skin lightening products typically promise perfect glowing skin, and have long been popular with women, and also today with men1,2,3. In Asia there is significant demand for products to whiten the skin. Since Asian skin tends to be naturally more hydrated, problems of hyperpigmentation or hypopigmentation are more frequently observed. As Asian people age, they are less likely to wrinkle but more likely to exhibit an uneven skin tone. Furthermore, many Asians want to look more western, so try to whiten their skin2. In mammals, melanin is the predominant skin pigment, and it is formed from melanocytes as tyrosine undergoes enzymatic oxidation. Melanin offers protection against ultraviolet (UV) irradiation, DNA damage, and oxidative stress, all of which are harmful to the skin. Protection against UV irradiation also helps in preventing skin cancer. Melanin also has the negative effect, however, of causing mottled or sunburned skin, so substances capable of blocking the synthesis of melanin are likely to be effective as cosmetic products to whiten the skin. The synthesis of melanin takes place in melanosomes whereupon it moves to the nearby epidermal keratinocytes. There are a number of melanocyte-specific enzymes such as tyrosinase (TYR), TYR-related protein 1 (TRP-1), and TYR-related protein 2 (TRP-2) which serve to regulate the process of melanogenesis4,5,6.

One role of TYR, which is a binuclear copper enzyme, is to regulate melanogenesis. It acts as a catalyst for the hydroxylation of tyrosine and the oxidation of 3, 4-dihydroxyphenylalanine (L-DOPA) to o-dopaquinone which are the rate limiting reactions which occur during the synthesis of melanin. Skin color is dependent upon the level of melanin synthesis as well as the type of melanin and the way it is distributed through the keratinocytes. Achieving effective inhibition of TYR with minimal adverse side effects has remained a longstanding challenge within the realms of dermatological and cosmetological sciences. Despite the existence of various potent TYR inhibitors, such as sulfite or kojic acid, their utilization is limited. However, these substances are not utilized due to their high degree of toxicity towards cells and instability in the presence of water or oxygen7,8,9. It is vital that inhibitors used in the food or cosmetics sectors meet the highest safety standards, so while various synthetic and natural TYR inhibitors are known, they are largely unsuitable for use as skin whiteners due to their cytotoxicity, poor solubility, or lack of suitability in terms of cutaneous absorption. Some of the TYR inhibitors which have found application in skin whitening products have been shown to be carcinogenic; the WHO has advised that some skin lightening agents can lead to cancer, and notes that mercury, which is a harmful toxin, is sometimes used in skin whitening products in order to inhibit the production of melanin10,11. In this context, natural or organic products are likely to become increasingly popular, so researchers have sought to focus on natural peptides since they are relatively safe and can be very effective. Accordingly, various studies have been carried out to examine TYR inhibitor peptides derived from synthetic peptides as well as proteins and protein hydrolysates to determine their mechanisms and efficacy. Natural compounds have drawn the greatest attention since they are perceived to lack undesirable side effects. In addition, they offer the advantages of peptides with protein mimicking and high biocompatibility, and therefore have potential to serve as active ingredients in newly developed products for the cosmetic and pharmaceutical sectors12,13. Accordingly, it appears that TYR activity can be inhibited by peptides and proteins from common sources including milk14, split gill mushrooms15, honey16, silk17, feather18, spotted babylon19, and the jellyfish20.

Bee pollen is produced in hives from nectar, the saliva of the bees, and flower pollen grains. It can be safely harvested from the hive by beekeepers. It is rich in nutrition, providing lipids, carbs, proteins, and dietary fibers along with minerals, vitamins, and both phenolic and volatile compounds. Around 200 different substances have been found in the pollen from various plant species, but bee pollen remains notable due to its biologically active components. From a nutritional perspective it can be classified as a functional food, which has invited studies to examine its potential in both the medical and food industries21,22. In recent years, studies have investigated the possibility of improving upon the capabilities of bee pollen via the process of enzymatic hydrolysis. The bee pollen protein hydrolysate (BPPH) can undergo hydrolysis to create natural bioactive peptides, as reported by Saisavoey et al.23. who showed that the enzymatic hydrolysates derived from bee pollen are effective in NO scavenging, while the neutrase hydrolysate can produce peptides which have anti-inflammatory properties. The production of NO is inhibited by the smallest peptide fraction (< 0.65 kDa), which can also counteract the LPS-induced expression of IL-6, COX-2, iNOS, and TNF-α transcripts in RAW264.7 macrophage cells, confirming that enzymatic hydrolysis can lead to better anti-inflammatory activity. Saisavoey et al.24. reported that bee pollen treated with alcalase could serve as an antioxidant, while antiproliferation activity was exhibited towards lung cancer cells (ChaGo-K1) by the BPPH, thus promoting apoptosis. The production of TIPs (tyrosinase inhibitory peptides) from BPPH has not, however, been widely reported. It is claimed that these bioactivities result from the greater variety and overall numbers of bioactive peptides which can be generated through enzymatic hydrolysis. The role of bioactive peptides in human nutrition has attracted widespread interest, but this study focuses on bee pollen bioactive peptides derived from enzymatic hydrolysis and investigates their ability to inhibit melanogenesis. We took the view that bee pollen might be a potential source of TIPs with the capacity to inhibit the expression of TYR in B16F10 mouse melanoma cells through disruption of the melanogenesis signaling cascades. In addition, in vivo testing of the ability of peptides to inhibit melanin generation was performed in zebrafish embryos. It can thus be concluded that bee pollen may be a source of valuable bioactive peptides which can find useful applications in the cosmetic sector as an important ingredient.

Results and discussion

Enrichment of bee pollen peptides treated with different proteases

TIPs are now widely prepared via enzymatic hydrolysis, due to the ease of controlling the process and the absence of extreme reaction conditions. There are many commercially produced enzymes which are available to support TIP production, among which are trypsin, chymotrypsin, flavourzyme, alkaline protease, neutral protease, papain, and so forth. It is understood that different enzymes can differ in their hydrolytic impact upon a given material because the binding specificity between substrate and enzymes will vary. However, in the case of the enzymolytic effect of an enzyme, the same enzyme will produce different results on different materials12. In this study, the enzymes under investigation included neutrase, alcalase, flavourzyme, papain, and pepsin-pancresin. Hydrolysis of the bee pollen powder was conducted with various proteases at three specific concentrations before comparisons were drawn to consider the TYR inhibition of the bee pollen hydrolysates, with findings shown in Table 1. The lowest IC50 values for the mono-phenolase activity (50.01 ± 1.15 µg/mL) and di-phenolase activity (39.93 ± 0.60 µg/mL) were recorded for the protein hydrolysate which was prepared using 5% (w/v) neutrase. The hydrolysates comprise a number of peptides, and thus differ from kojic acid, which is a single molecule and a very potent standard TYR inhibitor. For this reason, the true concentration of the active compound is not as high as that stated for the IC50 value. This study therefore places emphasis upon those hydrolysates which offer the greatest enzyme inhibition7. A similar example can be found in the work of Zhao et al.25. whose hydrolysis of the “Fengdan” peony (Paeonia ostii) seed meal protein with neutrase (at 1 mg/mL) revealed hydrolysates achieving 59.7% TYR inhibition. Before carrying out the process of enzymatic hydrolysis it is essential to select the appropriate proteases and raw materials, because the protein amino acid compositions vary and this leads to different sized peptides being released, which offer different levels of bioactivity.

Those proteins offering strong TYR inhibition typically contain high levels of hydrophobic amino acids, such as Trp, Phe, Gly, Val, Leu, Ile, Ala, Pro, and Met, and also aromatic amino acids including Tyr, Trp, and Phe. Neutrase, which can be described as a type of neutral protease has a tendency to hydrolyze proteins to generate peptides which have hydrophobic amino acids including Tyr, Trp, or Phe as C-terminals26. Accordingly, neutrase could serve as an appropriate protease for the preparation of high-activity TIPs. Reactions take place between hydrogen donor hydrophobic amino acids and the various residues, free radicals, or metal ions, while the aromatic amino acids possess conjugated planar rings, which are able to absorb ultraviolet rays while also engaging in π-π interactions with the copper ions of TYR. The oxidative activity of TYR tends to be disrupted by the conjugation with Cu2+, which in turn limits melanin synthesis12. To summarize, the current approach to obtain TIPs involves the screening of enzyme species followed by optimization of the enzymolytic process along with efficacious purification. This study employed neutrase in sequence since this was the approach which provided the highest levels of TYR inhibition. These findings were then taken forward to guide the ultrafiltration (UF) stage.

UF of the prepared BPPH

Having obtained the enzymatic hydrolysates from protein fractions and peptides, it is necessary to perform further filtration in order to separate these peptides prior to carrying out additional experimentation. The peptides must be purified isolated from other substances before it is possible to determine their abilities in melanogenesis inhibition in cell culture or to assess their anti-TYR properties through in vitro assay. This is because impurities could affect the outcomes of these analyses. Most research into bioactive peptides makes use of membrane fractionation techniques to commence the analysis. The purification stage begins by filtering the protein hydrolysates and peptides using membranes which are able to separate the fractions by molecular size. The food sector normally requires UF in which the sub-fractions fall within a range of 1 to 100 kDa for molecular size27. The initial screening for bioactivity can then be carried out by comparing the activity levels of the different sub-fractions. The most promising protein hydrolysate can be separated by membranes to form different peptide sizes, typically of MW > 10 kDa, MW 5–10 kDa, MW 3–5 kDa, MW 3-0.65, and MW < 0.65 kDa. Assays can then be conducted for in vitro TYR inhibition to determine the capacity to serve as TYR inhibitors in mono-phenolase and di-phenolase contexts, as explained in the section describing the experimental procedures. Table 2 presents the findings. The lowest IC50 values for both mono-phenolase activity (1.08 ± 0.50 µg/mL) and di-phenolase activity (1.82 ± 0.80 µg/mL) were obtained for protein hydrolysates which were prepared with 5% (w/v) of neutrase at a molecular weight < 0.65 kDa. Meanwhile, Prakot et al.19. reported the highest level of TYR inhibition from peptides derived from the protein hydrolysates of the spotted babylon snail, for which the molecular weight was lower than 3,000 Da. Deng et al.28. reported similar findings, noting that greater TYR inhibition was exhibited by the smaller molecular weight fractions of the protein hydrolysates from Chinese quince seeds following UF. In line with this trend, the 0–3 kDa fraction was chosen to undergo additional purification. Pongkai et al.18. confirmed the benefits of using the UF membrane to enhance the activity of a peptide fraction in terms of TYR inhibition, since the inhibitory activity of a peptide is closely related to its molecular weight. Accordingly, the protein hydrolysates prepared using 5% (w/v) neutrase which has a molecular weight < 0.65 kDa were shown to offer strong TYR inhibition, since they were able to exert their inhibitory influence at low concentrations, thus exhibiting strong dependence upon molecular weight. The differences observed might be attributable to the susceptible bonds in the amino acid sequences, which might have been dependent on the mechanism for catalysis of the immobilized enzyme, the breaking point preference in the protein sequence, and protein isolates composition. The fraction with molecular weight < 0.65 kDa which offered strong mono-phenolase and di-phenolase inhibition was selected to be purified further using size exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP-HPLC).