Lignin as a Novel Tyrosinase Inhibitor: Effects of Sources and Isolation

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Lignin as a novel tyrosinase inhibitor: effects of sources and isolation processes Guanhua Wang, Yue Xia, Wenjie Sui, and Chuanling Si ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02234 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Figure 1. Schematic processes for lignin isolation from lignocellulosic biomass (pine, poplar and corn stalk): (a) alkali process; (b) ethanol organosolv process. 124x81mm (300 x 300 DPI)

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Lignin as a novel tyrosinase inhibitor: effects of sources and isolation processes Guanhua Wang †*, Yue Xia †, Wenjie Sui ‡, Chuanling Si †* †

Tianjin Key Laboratory of Pulp and Paper, College of Paper Making Science and

Technology, Tianjin University of Science and Technology, Tianjin 300457, China ‡

Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, China * Corresponding author. Tel.: +86 02260601313 Address: No.29 at 13th Avenue, TEDA, Tianjin 300457, China E-mail address: [email protected] (Guanhua Wang) [email protected] (Chuanling Si)


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ABSTRACT

Lignin is the most abundant aromatic biopolymer in nature and its value-added application has attracted great attention. In this work, the inhibitory effect and mechanism of lignin on tyrosinase activity were investigated to develop lignin as a novel tyrosinase inhibitor. Six lignin samples isolated by alkali and ethanol organosolv processes from three typical lignocellulosic feedstocks were used to evaluate the effects of the lignin sources and isolation processes on the anti-tyrosinase activity. The lignin heterogeneity including purity, molecule weight and chemical structure was characterized detailedly by component determination, GPC, FTIR, 2D NMR, and Py-GCMS analyses. The enzyme studies showed that inhibitory activities of ethanol organosolv lignins were obviously stronger than those of alkali lignins. For lignins from different sources, corn stalk lignin presented highest inhibitory effect with an IC50 value of 0.276 mg/ml, which was comparable to that of positive control p-hydroxy benzaldehyde (0.233 mg/ml). The inhibitory kinetics suggested that the ethanol organosolv lignin from corn stalk was a reversible mixed-type inhibitor. The fluorescence quenching studies demonstrated that the interaction of GOL with the enzyme was a significant molecular mechanism to inhibit the enzymatic activity. Consequently, these results suggest that lignin possesses anti-tyrosinase activity and can be potentially used as an enzyme inhibitor in over-tyrosinase activity control fields. Keywords::

Lignin;

Isolation

process;

Characterization;

Inhibitor; Inhibition mode; Fluorescence quenching


Tyrosinase


INTRODUCTION Lignin, as one of the three main components of lignocellulosic biomass, is the most abundant aromatic polymer in nature. Lignin is deposited in the space between cellulose microfilaments during the progress of plant lignification, thereby to increase the thickness of the cell wall and enhance cellular mechanical holding force and compressive strength. Additionally, the lignin synthesis is also associated with the plant resistance to microbes, insects, and extreme climate.1-2 For example, after pathogens infection, the activity of phenylalanine ammonia-lyase (PAL) in plant cell increases and the accumulation of lignin is accompanied.3 Unlike the versatile functions to plants, lignin is typically underused as a waste stream in most lignocellulosic biomass utilization processes. Currently, the majority of lignin is burned as a fuel and only a small amount (less 2%) is used in industrial applications.4 However, with an increasing demand for a cost-effective biorefinery process, the development of lignin valorization, in particular for high-value products, is becoming an important research focus.4-7 Due to the poly-phenol structure of lignin, many efforts have been dedicated to exploring the value-added applications of lignin as natural products with poly-phenol relevant bioactivities.8-9 In the past decades, the studies of lignin biological activities mostly focused on the evaluation of antioxidant and antimicrobial activities.10-12 Currently, some other benign effects of lignin and its derivatives on human health are also reported, such as antiviral activity, anti-coagulation, anti-emphysema, UV-protection and obesity control.13 Since lignin is found to show potential


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applications in some biomedical areas, the compatibility of lignin with human cells is also studied. Ugartondo et al. investigated the cytotoxic effects of industrial lignins and demonstrated that over a range of concentrations the tested lignins were not harmful to normal human cells, indicating the uses for lignins in cosmetic and topical medical formulations.14 In order to confirm the safe use of lignins in cosmetic formulations, Vinardell and his co-workers further studied the irritation effect of lignins on eye and skin and their results suggested that the lignins were all not harmful to eye and skin.15 Tyrosinase (EC 1.14.18.1), as a copper-containing multifunctional polyphenol oxidase, is widely distributed in microorganisms, plants and animals.16 The enzyme catalyzes the hydroxylation of monophenol to o-diphenol and the subsequent oxidation to o-quinones, which undergoes further non-enzymatic polymerization leading to the formation of melanin.17-18 For humans, tyrosinase plays an important role in the production of melanin pigment, which is of importance in the prevention of UV-induced skin injuries. However, excessive accumulation of melanin leads to hyperpigmentation disorders like melanoma and age spots.17-19 Besides, over tyrosinase activity involves in the browning of fruits and vegetables, black spotting in shrimp and lobster, which causes undesirable changes in the color, flavor and nutritive value of the food products.19-20 Therefore, tyrosinase inhibitors are of great interest owing to the effective control of tyrosinase activities and have become increasingly important in agriculture, food and medicinal industries.21 In particular, due to the suppression on melanin hyper-formation, tyrosinase inhibitors is widely applied in cosmetic products for whitening and depigmentation.21-22



Due to the similar structure to the substrate of tyrosinase, many tyrosinase inhibitors are plant-based polyphenols and their derivatives.23-24 Since lignin also has polyphenol structure with considerable phenolic hydroxyl functional groups, this work attempts to evaluate the tyrosinase inhibitory effect of lignin. However, it is well known that extracted lignin is a heterogeneous material having a complex chemical structure. The heterogeneity of lignin structure has an important impact on the molecular weight distribution and functional group content, which will further result in the differences of lignin biological activities, such as antioxidant performance.8, 14 In this work, six various lignins obtained by alkali and ethanol organosolv processes from three typical lignocellulosic biomass (pine, poplar and corn stalk) were used for evaluation of tyrosinase inhibitory capacity. The reasons for choosing the two specific methods are that alkali process that alkali process is a common method used for lignin extraction and ethanol organosolv process can isolate lignin with reserved reactivity.25-26 The lignin heterogeneity caused by the different origins and isolation processes was investigated in detail by purity determination, GPC, FTIR, 2D-NMR, and Pyrolysis GC-MS (Py-GC-MS). The tyrosinase inhibitory activities of six lignins were compared and the inhibition mode was elucidated by kinetics study. Intrinsic fluorescence quenching analysis was conducted to confirm the interaction between lignin and tyrosinase. To the best of our knowledge, it is the first attempt to study the feasibility of lignin as a tyrosinase inhibitor. The aim of this present work is, therefore, to explore the tyrosinase inhibition performance of lignin and open new perspectives in the potential of lignin use as tyrosinase inhibitors in agriculture, food, medicinal and cosmetic industries.


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EXPERIMENTAL SECTION Materials Masson pine chips from the stem of Pinus massoniana were collected in Hunan province, China. Hardwood chips of Populus L. were provided by Huatai Paper Co. Ltd. (Dongying, China). Corn stalk was harvested in the suburb area of Tangshan, China. Tyrosinase isolated from mushroom, was purchased from the Sigma-Aldrich. Tyrosinase stock solution (12.5 kU/mL) was prepared using phosphate buffered saline (PBS, pH 6.8, 0.05 M). The stock solution was stored at 4 oC and was diluted to the required concentrations just before use. Levodopa (L-DOPA) stock solutions were prepared using PBS. Other reagents were analytic purity grade and were obtained from the Sinopharm Chemical Reagent Co (Tianjin, China). Lignin isolation processes In this work, lignins were obtained from three typical lignocellulose including masson pine (softwood), poplar (hardwood) and corn stalk (gramineae plant). The lignin isolation processes including alkali and ethanol organosolv extraction were carried out in a 1 L pressure-tight reactor equipped with an agitator. The schemes for lignin extraction by alkali and ethanol organosolv processes (acid-free) are given in Figure 1.



Figure 1. Schematic processes for lignin isolation from lignocellulosic biomass (pine, poplar and corn stalk): (a) alkali process; (b) ethanol organosolv process.

Biomass powders were obtained by mechanical grinding and screening through a 20 mesh sieve (0.83 mm). Alkali and organosolv extractions of lignin from masson pine and poplar were performed by adding 2.5% aqueous NaOH (w/v) and 65% aqueous ethanol (v/v, acid-free), respectively, with a solid/liquid ratio of 1:8 (w/v).26 Due to the porous property of ground corn stalk, alkali and organosolv extractions were carried out by adding 0.75% NaOH and 65% ethanol with a solid/liquid ratio of 1:20 and 1:15 (w/v), respectively. The lignin extraction processes were conducted at 160 oC for 2 h. After extraction, the resulting mixture was filtered to obtain the liquid fraction. Through adjusting the pH to 2.0 by 4 M HCl, the alkali lignins were precipitated and recovered by centrifugation at 4000 rpm for 10 min. For the organosolv lignins, the liquid fraction was evaporated under vacuum to remove ethanol and precipitate lignin. All the precipitated lignins were washed with distilled water twice, freeze-dried and stored at room temperature. Here, the obtained lignins


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were named as softwood alkali lignin (SAL), softwood organosolv lignin (SOL), hardwood alkali lignin (HAL), hardwood organosolv lignin (HOL), gramineae alkali lignin (GAL) and gramineae organosolv lignin (GOL), respectively. Lignin characterization The purity of the six lignin samples were analyzed by component determination using a modified National Renewable Energy Laboratory (NREL) standard protocol.25 The molecular weight distributions were acquired using a hydrophilic gel column (TSK G3000PWxl column) according to the literature 25, 27. Lignin FTIR was obtained on a FTIR spectrophotometer (FTIR-650, Gangdong Sci. & Tech. Co., Ltd, China) using the KBr pellet technique.

28

The region between 4000 and 400 cm-1 with a

resolution of 4 cm-1 and 20 scans was recorded. 2D NMR spectra (HSQC) of six lignin samples were acquired using a Bruker Avance 400 MHz. 0.1 g lignin was dissolved sufficiently in 0.5 ml DMSO-d6 for determination and Bruker standard pulse sequence ‘hsqcedetgpsisp 2.2’ was used.29 The Py-GC/MS determination was conducted by a Frontier Lab Single-Shot Pyrolyzer (Py-2020iS, Japan) concatenated to a gas chromatography equipped with mass spectrometry according to the reported procedure.30 Approximately 100 µg of lignin samples were pyrolyzed in a quartz tube at 600 °C for 12 s under helium as the carrier gas with a mean linear velocity of 1 mL/min. Anti-tyrosinase activity and kinetics assay The anti-tyrosinase activity assay was performed by Zhang et al. with some slight modifications,20 using L-DOPA as substrate and p-hydroxy benzaldehyde as positive control. In brief, six lignin samples with different amounts (3-18 mg) as well as



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p-hydroxy benzaldehyde, were completely dissolved in 1 mL DMSO firstly and then diluted with PBS to 10 mL. 1 mL lignin solution was added to each tube containing 1 mL 1.5 mM L-DOPA in PBS. After sufficient mixing and pre-incubation at 25 oC for 10 min, 1 mL 50 U/mL mushroom tyrosinase in PBS were loaded, followed by another 10 min incubation at 25 oC. The absorbance of the mixture was measured at 475 nm with a UV-visible spectrophotometer (UV-2550, Shimadzu, Japan). Due to the color of lignin samples, a blank was needed and acquired by the absorbance after adding the lignin solutions at an identical concentration without the addition of tyrosinase. The inhibition rate was calculated using the Eq.1. Inhibition rate % = 1 −

× 10

(1)

Here, A0, A1, B0 and B1 are the absorbance of: the solution containing lignin without tyrosinase, the solution containing both lignin and tyrosinase, the solution without either lignin or tyrosinase, and the solution with tyrosinase but without lignin. The inhibition type of lignin against tyrosinase was assayed by a Lineweaver–Burk plot.20 The inhibition constants were determined by plots of the apparent 1/Vm or Km/Vm versus the inhibitor concentration, as described by Hridya et al.17 Fluorescence analysis Fluorescence quenching studies were performed according to the protocol from previous references17, 19 using a fluorospectro photometer (F-7000, HITACHI, Japan). The lignin solutions with different concentrations were prepared by dissolving lignin with different amount in 1mL DMSO and then diluting with PBS to 10 mL. The total 3 mL mixture included 1 ml PBS, 1 mL tyrosinase solution and 1 mL lignin solution with


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different concentrations. Since lignin also presents fluorescence emission after exposure to a 290 nm excitation wave (see supporting information Figure S1), the emission spectrum of tyrosinase in the presence of lignin was obtained by subtracting the emission spectrum of lignin at the same concentration. All the tests were performed at room temperature. RESULTS AND DISCUSSION Purity, molecular weight, and structural characterization of lignins Yield and purity In this work, three typical lignocellulosic feedstocks involving masson pine, poplar and corn stalk, from softwood, hardwood and gramineous kingdom respectively, were employed as raw materials for lignin extraction. Two different lignin isolation methods including alkali and ethanol extraction processes were employed, since alkali cooking is the most commonly used for biomass delignification and ethanol organosolv process is thought to be a promising method to extract lignin with reserved reactivity.25-26 The photos of six lignins are shown in Figure 2 (inset) and the lignin colours changed with the variation of sources and isolation processes. Ethanol organosolv lignins had a brighter color compared to alkali lignins. The yield and purity of six lignin samples are determined and tabulated in Table 1. The results indicated that more lignin was dissolved during the alkali process since over 75% lignin was extracted by the alkali process while less than 60% lignin was extracted by the ethanol organosolv process. This result is presumably caused by the higher solubility of lignin in alkali solution than that in ethanol.



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Although less amount of lignin was extracted by the ethanol cooking process, the purity of organosolv lignin was obviously higher than that of alkali lignin (Table.1). This is potentially attributed to the insoluble property of carbohydrates and ash in ethanol.31 The lower content of carbohydrates and ash in organosolv lignin also confirms this suggestion. For a purity comparison from different sources, lignins from corn stalk had the lowest purity due to obviously high xylan and araban content, which indicates that firm covalent bonds between lignin and hemicellulose may exist.32 However, the total lignin content in all lignin samples were all above 85%, suggesting the isolation processes are feasible to obtain high purity lignins that are suitable for further structural and anti-tyrosinase analyses. Table 1. Yield, purity and molecular weight distributions of six lignin samples isolated from three sources via alkali and organosolv methods Lignin samples

Lignin/% Yield/%

a

M

w

M

n

PDI

Carbohydrate/%

Acid-insoluble

Acid-soluble

lignin

lignin

Glucosan

Xylan/mannan

Ash/%

+araban

SAL

75.81

7877

3681

2.14

86.47±0.53

6.73±0.45

0.75±0.39

2.54±0.72

1.34±0.44

SOL

58.25

5378

1775

3.03

90.85±1.16

4.07±0.05

0.36±0.13

1.96±0.01

0.55±0.11

HAL

78.36

4213

1659

2.54

79.31±0.17

8.63±0.38

0.30±0.13

4.12±0.27

1.10±0.13

HOL

56.24

3205

1533

2.09

90.47±0.67

4.44±0.31

0.56±0.30

1.55±0.08

0.09±0.01

GAL

79.29

6456

2281

2.83

79.46±2.99

5.63±0.08

1.04±0.01

6.12±0.02

1.19±0.01

GOL

52.96

4660

2035

2.29

84.94±0.65

4.61±0.40

0.94±0.10

3.78±0.37

0.31±0.01

a

The mass of extracted lignin to the total lignin in raw materials.


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Molecular weight

SAL Detecter response

SOL

10

11

12

13

14

15

16

17

18

Detecter response

HAL HOL

10

11

12

13

14

15

16

17

18

GAL

Detecter response

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GOL

10

11

12

13

14

15

16

17

18

Elution Time/min

Figure 2. GPC of lignins isolated from three sources via alkali and organosolv methods (inset: lignin photos)

The molecular weight distributions of the six lignins are given in Figure 2. All six lignin samples showed broad molecular weight distributions (elution time from about 11.5 to 17.5 min). The wide molecular weight distributions of lignin are possibly caused by the molecular weight heterogeneity of pristine lignin and the depolymerization/repolymerization during the isolation process.21 Interestingly, the molecular weight of lignins obtained by organosolv process was lower than that by alkali process for all the three raw materials due to the later elution time of main peaks in the chromatograms. This is possibly caused by the intensified condensation reactions of lignin under alkali conditions at elevated temperature.13 Besides, the condensation inhibition by capping effect of ethanol during the acid-free organosolv



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process also results in the low molecular weight of organosolv lignin.33 Thus, this result also indicates that the lignin obtained by acid-free ethanol organosolv extraction may show better reactivity compared with alkali lignin due to the less condensation during the isolation process. The weight-average molecular weight ( M w), number-average molecular weight ( M n) and polydispersity are calculated and given in Table 1. The molecular weights of poplar lignins from alkali and ethanol organosolv processes ( M

w

4213 and 3205,

respectively) were obviously lower than those from masson pine and corn stalk. This is possibly caused by the higher syringyl unit content in the poplar lignin. The methoxyl at the C5 of syringyl unit prevents the formation of C-C bond that is recalcitrant to be broken during the isolation process. Alkali lignin from masson pine presented the highest molecular weight with M

w

7877 and M n 3681. The massive

G unit content in the masson pine lignin (shown in FTIR, 2D-NMR, and Py-GC-MS analyses) are responsible for this result because G unit is easy to form a condensation structure during the alkali cooking process.34 FTIR The FT-IR spectra of six lignins were recorded in the range of 400-4000 cm−1 wavenumbers (Figure S2) and the band assignments were based on literature data.25, 30, 35

All lignin samples presented a broad band at about 3420 cm-1 attributed to OH

stretching, stretching vibrations corresponding to C-H of methyl (2930 cm-1) and methylene groups (2580 cm-1) and characteristic vibrations of aromatic rings identified approximately at 1600, 1510 and 1425 cm-1. The absorptions at 1322, and 1120 cm-1 were attributed to syringyl structure while the band at 1270 cm-1 was associated with guaiacyl ring breathing. The absence of absorption at 1322 cm-1 and


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tiny peaks at 1120 cm-1 for SAL and SOL suggests that softwood lignin hardly has syringyl unit, which is in agreement with the results found by Pandey.36 Besides, the peak intensities at 1270 cm-1 for SAL and SOL were much higher than those for lignins from poplar and corn stalk, indicating a higher guaiacyl unit content. Interestingly, compared with ethanol lignins, alkali lignins showed a lower absorption at 1270 cm-1 regardless of the lignin sources. The decrease of 1270 cm-1 peak intensity is presumably due to the structure alteration by lignin condensation, which also corresponds to the molecular weight analysis of lignins obtained by the two different ways. 2D-NMR To obtain detailed information on the inter-unit linkages as well as the unit compositions, the six lignins were analyzed by 2D-NMR (shown in Figure 3 and Figure S3). The aliphatic (δC/δH 50−90/3.0−6.0) and aromatic (δC/δH 100−150/6.0−8.0) regions of the HSQC NMR spectra are shown in Figure 3a-f. The main lignin substructures identified according to previous publications 37-40 are presented in Figure 3g. In the aliphatic regions, all lignins shared a characteristic of a relatively higher proportion of methoxyl (δC/δH, 55.40/3.75) and β-O-4 ether linkage (A), and a lower proportion of β-5 carbon-carbon linkage (B). The pinoresinol structure formed by β-β’ linkage (C) was observed from pine and poplar lignin 2D-NMR while a unique dibenzodioxocin structure was detected in the spectra of corn stalk lignin. The minor amounts of C-C linkages detected by 2D NMR suggest that no serious condensation occurs during the ethanol isolation process, which confirms the molecular weight



analysis. In the aromatic region (Figure 3d, e and f), the main cross-signals corresponding to the aromatic rings of syringyl (S) and guaiacyl (G) were clearly observed in the spectra of poplar and corn stalk lignins, while the pine lignin only presented the signals of G unit. This result is exactly in conformity with the FTIR results about the unit compositions among the lignins from the three sources. Besides S and G units, a unique p-hydroxybenzoate substructure was identified obviously in the poplar lignin spectra, which is in agreement with the previous study.41 For the corn stalk lignin, two notable peaks at δC/δH 145.52/7.44 and 114.21/6.31 attributed to phenolic acid signals (Pα and Pβ) were found. In previous literature

42

and our earlier publication,31 phenolic acids are

also found in lignins extracted from corn stalk. Phenolic acids including p-coumaric acid and ferulic acid are postulated as potential tyrosinase inhibitors in previous studies.43-44 The result suggests that lignins from corn stalk may exhibit preferable anti-tyrosinase activities among the three sources.


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R

α

OH

γ Oα

γ

β O

β

OMe

αO

β

β

α

γ

O

MeO

O

O

OMe

2

6 5

OH

OMe

6

2

6

2

MeO

OMe

O

O

O

HO γ

6

2

5

3

OR

OR

OR

OH

G

S

H

PB

OR γ

β

α

(MeO) O(R)

A

B

C

D

Figure 3. Aliphatic (a, b and c) and aromatic (d, e and f) regions of 2D NMR spectra of ethanol organosolv lignins from Masson pine (a and d), poplar (b and e) and corn stalk (c and f). Main substructures (g) identified by 2D NMR: (A) β-O-4’ aryl ether linkage; (B) phenyl-coumaran structure formed by β-5’ and α-O-4’ linkages; (C) pinoresinol structure formed by β-β’, α-O-γ’ and γ-O-α’ linkages; (D) dibenzodioxocin structure; (G) guaiacyl unit; (S) syringyl units; (H) p-hydroxyphenyl unit; (PB) p-hydroxybenzoate structure; (P) phenolic acid.


P


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Py-GC/MS

O

OH

O

OH OH

O

SOL

HO

O

OH

OH

OH

OH 0

10

20

30

40

50

60

70

80

90

O

100

OH

O

OH

OH

O

O

OH

HOL 0

10

20

30

40

50

60

70

80

90

100

50

60

70

80

90

100

GOL 0

0

10

20

30

40

5

10

15

20

25

Retention time(min)

Figure 4. Pyrograms (total-ion chromatograms) of ethanol organosolv lignins from different sources. The identities and relative abundances of compounds released by Py-GC/MS are listed in bar charts.

The lignins from different sources were further analyzed by Py-GC/MS and their chromatograms are shown in Figure 4, including the main identities and relative abundances of the released compounds as well as structural formula. The main diagnostic compounds from the pyrolysis of lignin were classified into six types, including guaiacol, syringol, phenol, phenyl, heterocycle and fatty hydrocarbon.30 It could be found that for the softwood lignin, guaiacyl type compounds were the dominant products while not any syringol type compounds were detected. Syringol type compounds including syringol and 3-methoxy-benzenediol formed by


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demethylation of syringol were observed in the pyrolysis products of hardwood lignin and corn stalk lignin. These results are in accordance with the finding from the FTIR and 2D-NMR analyses. According to the relative percentage of guaiacol, syringol and phenol types from lignin pyrolysis, the G:S:H ratio could be further calculated.45 For the three lignins from different sources, these values are determined to be: G83.05S0H16.95 for pine lignin, G48.12S38.67H13.21 for poplar lignin and G40.16S22.00H37.94 for corn stalk lignin. According to del Río, lignin originated from hardwoods were composed mainly by S and G units in varying ratios, while softwood lignin was made of primarily G units and a small amount of H units.46 In our previous study, we demonstrated that the unit composition of lignin from corn stalk lignin were about G42.82-54.72S34.40-27.45H22.78-17.83 calculated form H1-NMR analysis.31 The higher H unit amount in this work is possibly derived from the demethoxylation reactions of G/S units during the pyrolysis process.45 Tyrosinase inhibition by lignins Inhibition activity To evaluate the anti-tyrosinase activity, the assay of inhibition of L-DOPA oxidized by diphenolase to produce dopaquinone was performed. In this assay, results were expressed as the ratio percentage of the absorbance decrease of dopaquinone in the presence of lignin. Since the solubility of most lignin samples tested was low in the phosphate buffer (pH 6.8), 10% DMSO (v/v) was used to assist the dissolution of lignin into the buffer solution (see experimental section). It was found that there was no obvious effect on the tyrosinase activity after adding 10% DMSO (Figure S4), indicating the reliability of this assay.



Figure 5a illustrates the tyrosinase inhibitory effects of the six tested lignins as well as the lignin-derived unit p-hydroxy benzaldehyde which is demonstrated to be an effective tyrosinase inhibitor.47 As indicated in Figure 5a, all the tested lignins showed anti-tyrosinase activity. However, there were significant differences in inhibitory activities among the six lignins. It was observed that the inhibitory activities of organosolv lignins were stronger than these of alkali lignins. One possibility to this result is the high purity of ethanol organosolv lignin. Besides, by comparing the tyrosinase inhibitory effect of lignins from different sources, it was concluded that the corn stalk lignins exhibited the highest inhibition activity followed by the lignins from softwood, and the hardwood lignins had lowest inhibition rate to the tyrosinase. The high anti-tyrosinase activity of corn stalk lignins is possibly because of the phenolic acids (Figure 3) which present strong affinity to the active copper ions in the tyrosinase and inhibit the tyrosinase activity.43 Therefore, the GOL obtained from corn stalk by organosolv process is the most potent tyrosinase inhibitor among the six lignins. In order to acquire the IC50 value of GOL, the inhibition rates were determined at different lignin concentrations and the results are given in Figure 5b. A dose dependent inhibitory effect of lignin against tyrosinase activity was observed and the IC50 was about 0.276 mg/ml. It was noteworthy that the IC50 of GOL was only 18.45% higher than that of positive control p-hydroxy benzaldehyde (0.233 mg/ml), suggesting the effective inhibition against tyrosinase activity. Since lignin has been proved to be not harmful to normal human cells,14-15 our results suggest that GOL can potentially contribute as a novel tyrosinase inhibitor in food, cosmetic and pharmaceutical industries.


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(b)

(a) 0.4mg/ml 0.5mg/ml

80

PHB GOL

80

60

Inhibition rate (%)

Inhibition rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40

20

0

60

40

20

0 SAL

HAL

GAL

SOL

HOL

GOL

PHB

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Lignin concentration (mg/mL)

Figure 5. Inhibition activity of lignins isolated from three sources via alkali and organosolv methods as well as p-hydroxy benzaldehyde (PHB)

Inhibition mechanism and type The inhibition mechanism and type on tyrosinase by lignin were further investigated. Figure 6a shows the relationship between enzyme activity and concentration in presence of different concentrations of GOL. As shown in Figure 6a, the plots of the remaining enzyme activity versus the concentrations of enzyme gave a family of straight lines which all passed through the origin. A linear slope decrease with increasing concentration of GOL indicated that the inhibition mechanism of lignin on tyrosinase was reversible.19-20 Therefore, the inhibitor does not reduce the amount of effective enzyme, but reversibly combines with the enzyme in the form of non-covalent bonds to decrease the enzyme activity for the oxidation of DOPA.



140

Tyrosinase 0.01mg/mLGOL 0.02mg/mLGOL 0.03mg/mLGOL 0.04mg/mLGOL 0.05mg/mLGOL

2500

100

Fluorescence intensity

[I]=0 [I]=0.1mg/mL [I]=0.3mg/mL [I]=0.5mg/mL

120

Activity (mM/min)

80 60 40

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20

0 0 0

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[E](U/ml)

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Wavelength/nm 0.035

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0.030

i=0 mg/mL i=0.1 mg/mL i=0.5 mg/mL i=1.0 mg/mL

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Slope

0.10

0.06 1/[V] (min/mM) -2.0

0.020 0.015 0.010 0.005

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-0.04 1/[S] (mM-1)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.014 0.012 0.010 0.008 0.0

0.2

0.4

0.6

0.8

1.0

[I] (mg/mL)

Figure 6. a: Effects of tyrosinase concentrations ([E]) on its activity different GOL concentrations ([I]); b: Inhibition kinetics of GOL on tyrosinase by Lineweaver–Burk plots. The two figures in the right represent the secondary slope and the intercept of the straight lines versus concentration of GOL, respectively; c: fluorescence spectra of tyrosinase in the presence of GOL at different concentrations.

In the presence of GOL, the inhibition kinetics of GOL on the enzyme was studied using the double-reciprocal Lineweaver-Burk plots. The results (Figure 6b) showed that the plots of 1/v versus 1/[S] presented a set of straight lines with different slopes that intersected one another in the second quadrant, which suggested that GOL was a mixed-type inhibitor for the enzyme.20, 48 This behavior indicates that lignin can bind not only with free tyrosinase, but also with the tyrosinase-substrate complex 20.


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The equilibrium constants for its binding with free enzyme (KI) and with enzyme-substrate complex (KIS), were obtained from the second plots of the Km/Vm and 1/Vm versus lignin concentration, respectively. According to Ma et al.

49

, the

inhibition constants of KI and KIS were calculated to be 0.495 mg/mL and 0.802 mg/mL. The higher value of KIS indicates that the affinity of lignin for the free enzyme is stronger than that for the enzyme-substrate complex.17, 20 Intrinsic fluorescence quenching analysis Since the inhibition kinetics study suggests that the binding of lignin to free tyrosinase is the main reason for lignin as an effective inhibitor, an intrinsic fluorescence quenching analysis of tyrosinase in the presence of lignin was conducted to confirm the interaction between lignin and tyrosinase. As shown in Figure 6c, the fluorescence emission spectrum of tyrosinase presented a strong peak at 340 nm, due to the presence of fluorophore aromatic amino acid residue (e.g. tyrosine, tryptophan and phenylalanine).17, 48 After the addition of GOL in increasing concentration, the intrinsic fluorescence of tyrosinase was decreased gradually because of the quenching effect in a dose dependent manner. Besides, a slight red shift of the spectra occurred with the increase of lignin concentration. These results reasonably suggest that GOL has a binding affinity towards the tertiary structure of tyrosinase inducing the inhibition of enzyme activity. CONCLUSIONS Six lignins obtained by alkali and ethanol extraction from three typical lignocellulosic feedstocks (pine, poplar and corn stalk) were used for evaluation of tyrosinase inhibitory



capacity. Lignins obtained by ethanol organosolv process present higher purity but lower yield and molecular weight. There is mainly G unit in pine lignin while the three basic units are all observed in poplar and corn stalk lignin. The lignin sources and isolation processes have a significant effect on the tyrosinase inhibition activity. GOL isolated from corn stalk by acid-free ethanol organosolv process presents highest anti-tyrosinase activity. The enzyme activity analysis indicates that GOL inhibits tyrosinase activity in a dose dependent manner and kinetics study reveals GOL reversibly inhibits tyrosinase in a mixed type manner. The interaction of GOL with the enzyme is a significant molecular mechanism to explain its efficient inhibition. Therefore, this study confirms the efficient tyrosinase inhibitory effect of lignin and opens a new perspective on the potential usage of lignin as a tyrosinase inhibitor in over-tyrosinase activity control fields. ASSOCIAED CONTENT Supporting information: Fluorescence spectra of GOL (Figure S1). FTIR spectra of lignins (Figure S2). 2D-NMR of three alkali lignins (Figure S3). Time scanning curves of L-DOPA catalyzed by tyrosinase in PBS with and without the addition of 10% DMSO (Figure S4). ACKNOWLEDGMENT Financial supports for this study were kindly provided by the National Key Research and Development Program of China (2017YFB0307903), Natural Science Foundation of China (31700515), Natural Science Foundation of Tianjin City (16JCQNJC05900) and Tianjin Municipal Education Commission (2017KDYB23).


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For Table of Contents Use Only Abstract Graphic

Synopsis: Renewable lignin is proved to be an effective tyrosinase inhibitor which can be potentially used in over-tyrosinase activity control fields.


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Lignin as a Novel Tyrosinase Inhibitor: Effects of Sources and Isolation

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