Isolation, Purification and Tyrosinase Inhibitory Activity of Anthocyanins and Their Novel Degradation Compounds from Solanum tuberosum L.

To explore the composition of anthocyanins and expand their biological activities, anthocyanins were systematically isolated and purified from tubers of Solanum tuberosum L., and their tyrosinase inhibitory activity was investigated. In this study, two new anthocyanin degradation compounds, norpetanin (9) and 4-O-(p-coumaryl) rhamnose (10), along with 17 known anthocyanins and their derivatives, were isolated and purified from an acid-ethanolic extract of fresh purple potato tubers. Their structures were elucidated via 1D and 2D NMR and HR-ESI-MS and compared with those reported in the literature. The extracts were evaluated for anthocyanins and their derivatives using a tyrosinase inhibitor screening kit and molecular docking technology, and the results showed that petanin, norpetanin, 4-O-(p-coumaryl) rhamnose, and lyciruthephenylpropanoid D/E possessed tyrosinase inhibitory activity, with 50% inhibiting concentration (IC50) values of 122.37 ± 8.03, 115.53 ± 7.51, 335.03 ± 12.99, and 156.27 ± 11.22 μM (Mean ± SEM, n = 3), respectively. Furthermore, petanin was validated against melanogenesis in zebrafish; it was found that it could significantly inhibit melanin pigmentation (p < 0.001), and the inhibition rate of melanin was 17% compared with the normal group. This finding may provide potential treatments for diseases with abnormal melanin production, and high-quality raw materials for whitening cosmetics.


Introduction
The potato (Solanum tuberosum L.) is the fourth most important staple crop worldwide and originated from the highlands of the equatorial Andes in South America [1,2].The potato is a diverse crop that includes varieties with white, yellow, and colored flesh (red, blue, and purple).These differences in flesh color result from significant differences in the phytochemical composition of crops [3,4].Yellow-flesh potatoes are characterized by high levels of carotenoids, whereas red-, blue-, and purple-flesh potatoes contain large amounts of anthocyanins [5].Therefore, potatoes rich in pigments are a good source of anthocyanins [6].
Colored potatoes are a rich source of anthocyanins, especially the acylated derivatives, which account for more than 98% of the total anthocyanins [7].The red potato contains mainly the acylated side of pelargonidin, while the purple potato contains mainly the acylated side of petunidin and peonidin, whereas delphinidin and malvidin have fewer acylated glycosides.[9].The study of polyphenol composition in colored potato skins found that the total anthocyanin content in purple potatoes was 0.863 ± 0.005-1.39± 0.01 mg g −1 (dry weight, DW), including acylated pelargonidin, peonidin, malvidin, and petunidin anthocyanins, as shown in Table 1 [7,[10][11][12].Thus, the types of anthocyanins contained in a crop are closely related to the variety of potato, and the content varies greatly among different varieties and regions.Anthocyanins are a type of flavonoid and glycosidic water-soluble pigment that not only impart red, purple, and blue colors to many fruits, flowers, and tubers, but also confer physiological benefits, reflected in their antioxidant [13][14][15], anti-inflammatory [16,17], hypoglycaemic [18,19], and whitening properties [20][21][22].In particular, tyrosinase inhibitors are gaining research interest as melanin pigmentation can be blocked by inhibiting tyrosinase, a rate-limiting enzyme in melanin production [23,24].Extracts from the seed coat of black soya beans have been reported to possess anti-human tyrosinase activity, and a good correlation has been found between such anti-human tyrosinase activity and the cyanidin 3-O-glucoside content of coat extracts [25].Radio frequency-assisted enzymatic extraction of anthocyanins from Akebia trifoliata (Thunb.)Koidz.flowers showed tyrosinase inhibitory activity (14.67 kojic acid equivalents/g extract) [26].The IC 50 value of anthocyanins from red rice bran, in which tyrosinase inhibitory activity was observed, was reported to be 4.26 µg mL −1 [27].Anthocyanins purified from Lycium ruthenicum Murr.had inhibitory effects on tyrosinase monophenolase (IC 50 = 1.483 ± 0.058 mg mL −1 ), and the type of inhibition was competitive (K i = 39.83 ± 1.4 mg mL −1 ) [28].Moreover, petunidin 3-O-glucoside may act as a tyrosinase inhibitor to block melanin production, and has inhibitory ratios exceeding 55% of the control value at 50 µM, showing dose-dependent inhibitory activity with an IC 50 value of 10.3 ± 1.0 µM [29].Additionally, anthocyanins from Hibiscus syriacus L. inhibit melanogenesis by activating the extracellular regulated protein kinases signaling pathway [30].Therefore, anthocyanins have good potential for tyrosinase inhibitory activity, and thus anti-melanogenesis.
In this study, anthocyanin components from Solanum tuberosum L. were isolated, purified, and prepared using chromatography, spectroscopy, and NMR techniques.Then, their tyrosinase inhibitory activities were evaluated using tyrosinase inhibitor screening kits, molecular docking, dynamic simulation, and through examination of their toxicity and anti-melanogenic effects in zebrafish.These results are expected to provide a more comprehensive understanding of the composition of anthocyanins in purple potato, their tyrosinase inhibitory activity, and potential for anti-melanogenic effects, and promote the wider use of purple potato in these contexts.

Compounds Structure Identification
Anthocyanins and their novel degradation compounds (9 and 10) were extracted from fresh slices of potato tubers using acidic water-ethanol, enriched with macroporous resin, and purified via semi-preparative chromatography, as shown in Figure 1.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 18 inhibitory activity with an IC50 value of 10.3 ± 1.0 μM [29].Additionally, anthocyanins from Hibiscus syriacus L. inhibit melanogenesis by activating the extracellular regulated protein kinases signaling pathway [30].Therefore, anthocyanins have good potential for tyrosinase inhibitory activity, and thus anti-melanogenesis.In this study, anthocyanin components from Solanum tuberosum L. were isolated, purified, and prepared using chromatography, spectroscopy, and NMR techniques.Then, their tyrosinase inhibitory activities were evaluated using tyrosinase inhibitor screening kits, molecular docking, dynamic simulation, and through examination of their toxicity and anti-melanogenic effects in zebrafish.These results are expected to provide a more comprehensive understanding of the composition of anthocyanins in purple potato, their tyrosinase inhibitory activity, and potential for anti-melanogenic effects, and promote the wider use of purple potato in these contexts.
Although lyciruthephenylpropanoid D/E was first identified in Lycium ruthenicum Murr., lyciruthephenylpropanoid D/E were first discovered and obtained in potato [39].Interestingly, Solanum tuberosum L. and Lycium ruthenicum Murr.Belong to the Solanaceae Juss. in which the major anthocyanins are all petanin, and norpetanin also was purified from Lycium ruthenicum (Unpublished work from our research group).Consequently, these results may imply novel degradation or metabolic pathways of anthocyanins, such as from petanin to norpetanin to lyciruthephenylpropanoid D/E to 4-O-(p-coumaryl) rhamnose, and need to be supported by more direct evidence.
Anthocyanins purified from Lycium ruthenicum Murr.had inhibitory effect on tyrosinase monophenolase (IC50 = 1.483 ± 0.058 mg mL −1 ), and the maximum inhibitory activity of the purified anthocyanins (3.00 mg mL −1 ) on tyrosinase diphenolase was 42.16% ± 0.77% [28].Petunidin 3-O-glucoside may act as a tyrosinase inhibitor to block melanin production, and its IC50 value was 10.3 ± 1.0 μM [29].As shown in Table 4 and Figure 3A, regarding the tyrosinase activity of petanin and its degradation compounds, petanin and 4-O-(pcoumaryl) rhamnose were shown to be significantly different (p < 0.01).However, there was no significant difference between petanin, norpetanin, and lyciruthephenylpropanoid D/E (p > 0.05).Therefore, the acyl group containing two sugar groups may be the 'key' to the 'lock' for tyrosinase, whereas 4-O-(p-coumaryl) rhamnose may not have the same effect because the molecule is too small.In fact, according to the results of the evaluation of toxicity and anti-melanogenic effects in zebrafish, petanin not only had a very safe tolerated dose (MTC = 0.15%), but also showed a significant anti-melanogenic effect (17%, p < 0.05) at a concentration of 0.1% compared with the normal group, as shown in Petanin, a major compound in purple potato, was tested for its safety and antimelanogenic effects in zebrafish, as shown in Table S3 and Figure 3B,C.
Anthocyanins purified from Lycium ruthenicum Murr.had inhibitory effect on tyrosinase monophenolase (IC 50 = 1.483 ± 0.058 mg mL −1 ), and the maximum inhibitory activity of the purified anthocyanins (3.00 mg mL −1 ) on tyrosinase diphenolase was 42.16% ± 0.77% [28].Petunidin 3-O-glucoside may act as a tyrosinase inhibitor to block melanin production, and its IC 50 value was 10.3 ± 1.0 µM [29].As shown in Table 4 and Figure 3A, regarding the tyrosinase activity of petanin and its degradation compounds, petanin and 4-O-(p-coumaryl) rhamnose were shown to be significantly different (p < 0.01).However, there was no significant difference between petanin, norpetanin, and lyciruthephenylpropanoid D/E (p > 0.05).Therefore, the acyl group containing two sugar groups may be the 'key' to the 'lock' for tyrosinase, whereas 4-O-(p-coumaryl) rhamnose may not have the same effect because the molecule is too small.In fact, according to the results of the evaluation of toxicity and anti-melanogenic effects in zebrafish, petanin not only had a very safe tolerated dose (MTC = 0.15%), but also showed a significant anti-melanogenic effect (17%, p < 0.05) at a concentration of 0.1% compared with the normal group, as shown in Figure 3B,C.Ultimately, petanin and its analogues inhibited tyrosinase activity, and its parent nucleus and acyl group may be important active groups.

Molecular Docking and Dynamic Simulation of Tyrosinase Inhibitors
Evaluation of petanin and its degradation compounds were performed against tyrosinase using AutoDock Vina.They depicted considerable docking energy and formation of intermolecular interactions with the essential residues of tyrosinase enzyme in the respective docked complexes (Figure 4 and Table 4).In addition, the ADMET of petanin was similar to that of norpetanin, as shown in Figure S18.
To further demonstrate the degree and stability of the binding between the compound and the protein, molecular dynamics simulations of 100 ns were performed for each set of docking results.The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), number of hydrogen bonds, and Gibbs free energy plots were analyzed in each set of molecular dynamics simulation trajectories (Figure 5).To further demonstrate the degree and stability of the binding between the compound and the protein, molecular dynamics simulations of 100 ns were performed for each set of docking results.The root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), number of hydrogen bonds, and Gibbs free energy plots were analyzed in each set of molecular dynamics simulation trajectories (Figure 5).Molecular docking results showed that petanin and its degradation compounds had a huge binding energy with tyrosinase, but 4-O-(p-coumaryl) rhamnose had smaller binding energies and fewer hydrogen bonds than the other compounds, as shown in Table 4 and Figure 4.However, hydrogen bonding and hydrophobic interactions are well established as essential factors in the stability of ligands at the active pocket of receptors [41].Molecular docking results showed that petanin and its degradation compounds had a huge binding energy with tyrosinase, but 4-O-(p-coumaryl) rhamnose had smaller binding energies and fewer hydrogen bonds than the other compounds, as shown in Table 4 and Figure 4.However, hydrogen bonding and hydrophobic interactions are well established as essential factors in the stability of ligands at the active pocket of receptors [41].Thus, the differences observed in the tyrosinase inhibitory activity of petanin and its degradation compounds may be related to the number and position of hydrogen bonds they form with tyrosinase.
Results of root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), number of hydrogen bonds, and Gibbs free energy plots in the molecular dynamics simulation trajectories of the complex are shown in Figure 5.The RMSD fluctuation curve of molecular dynamics simulation reached a stable state without large fluctuations within 100 ns, and the fluctuation range was within 0-0.3 nm.This indicates that these compounds can form stable complexes with tyrosinase [42].The RMSF showed that the amino acid residues in the complex all fluctuated around the amino acid residues at positions 80 and 250.This may be a normal fluctuation caused by the binding of the component small molecules to the protein [43].The Rg curves all fluctuated in the range of 0.1-0.2nm, indicating that each complex formed a tight and stable complex structure [44].Calculation of the number of hydrogen bonds between the compound and the protein showed that petanin and norpetanin had four hydrogen bonds on average, lyciruthephenylpropanoid D/E had three hydrogen bonds, and 4-O-(p-coumaryl) rhamnose had only two.The difference in the number of hydrogen bonds formed may be the main factor affecting the stability of the complex.Gibbs free energy plots showed that petanin formed the most stable complex with tyrosinase compared to its other compounds [45].In conclusion, petanin and its degradation compounds formed stable complexes with tyrosinase, and this stability benefited from conformational fluctuations of the protein, fluctuations in the level of protein residues, protein folding, and the number of hydrogen bonds.

Safety Evaluation and Verification of Anti-Melanogenic Effect
Wild-type AB strain zebrafish were used for safety evaluation in the experimental system.The age of zebrafish was 6 h after fertilization (6 hpf).The sample size of each group was 30 tails (n = 30).The adult fish were raised and bred according to the laboratory standards and in accordance with the requirements of the International AAALAC certification (Certification number: 001458).The experimental protocol was as follows: 1.The zebrafish were randomly selected in six-well plates, with 30 fish in each well.2. Samples were dissolved in water, and the normal group and petanin 0.1% group were set up at the same time.The volume of each well was 3 mL.3. The cells were incubated at 28 • C for 45 h in the dark.4. The maximum tolerated concentration (MTC) of samples was determined for normal zebrafish based on zebrafish death count, death rate, and toxicity.
The anti-melanogenesis effect of petanin was also tested in zebrafish.The experimental protocol was as follows: 1.The zebrafish were randomly selected in six-well plates, with 30 fish in each well.2. Samples were dissolved in water, and the normal group and petanin 0.1% group were set up at the same time.The volume of each well was 3 mL.3. The cells were incubated at 28 • C for 45 h in the dark.4. Ten zebrafish in each experimental group (n = 10) were randomly selected and photographed under a microscope, and the data were analyzed and collected by ImageJ.According to the Formula (1), the melanin inhibition rate of each sample was calculated and judged.) for molecular docking.The ADMET of petanin and its derivatives were predicted by using the SwissADME (http://www.swissadme.ch/).

Figure 1 .
Figure 1.Anthocyanins and their novel degradation compounds from Solanum tuberosum L.

2. 1 . 1 .
Resolution and Identification of New CompoundsCompound 9 was obtained as a yellowish amorphous powder with a molecular formula of C36H42O21, as determined using HR-ESI-MS (m/z 833.2117 [M + Na] + , calcd.

Figure 1 .
Figure 1.Anthocyanins and their novel degradation compounds from Solanum tuberosum L.

Table 1 .
The anthocyanins composition of purple potato skins.

Table 3 .
The 1 H and13C NMR spectral data for 10 in CD 3 OD.