Hinokitiol Inhibits Polyphenol Oxidase and Enzymatic Browning

Food Sci. Technol. Res., 17 (3), 251–256, 2011

1 1 2 1* Saya Okumura , Midori Hoshino , Keiko Joshita1, Takashi Nishinomiya and Masatsune Murata

1 Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan 2 Taisho Technos, 1157-16 Yufune, Oyama-cho, Sunto-gun, Shizuoka 410-1305,Japan

Received December 1, 2010; Accepted February 21, 2011

We screened for inhibitors of enzymatic browning among fermented broths of lactic acid bacteria (58 strains), extracts of spices (7 samples), and food additives (34 samples) from the standpoint of practical use. We found that hinokitiol, a preservative, definitely inhibited the browning of apple sections. An apple section sprayed with a 0.02% hinokitiol solution did not turn brown for 2 h at room temperature. Hinoki- tiol competitively inhibited apple polyphenol oxidase (PPO). The Ki value of hinokitiol against apple PPO was 5.5 μM, while the Km value of chlorogenic acid was 2.5 mM. Furthermore hinokitiol non-competitively inhibited tyrosinase and the discoloration of shrimp caused by tyrosinase during cold storage.

Keywords: hinokitiol, enzymatic browning, polyphenol oxidase

Introduction nols synthesized during storage are successively oxidized by When fruits such as apple and banana are homogenized PPO. Therefore, it is essential for regulation of this type of or crushed, the juices turn brown immediately. When these browning to inhibit polyphenol biosynthesis. It is reported fruits are cut, their surfaces also turn brown within an hour that enzymatic browning of cut lettuce is definitely inhibited or several tens of minutes. These are typical examples of en- by regulating polyphenol biosynthesis by mild heat treatment zymatic browning (Mathew and Parpia, 1971). These fruits (Loaiza-Velarde et al., 1997; Murata et al. 2004) and cinna- contain polyphenols such as chlorogenic acid (CQA) and maldehyde (Fujita et al., 2006). catechins in vacuoles. On the other hand, polyphenol oxi- In both types of enzymatic browning, PPO is an impor- dase (PPO; EC 1.10.3.1) exists in plastids (Mayer and Harel, tant point for the control of browning. Several studies show 1979). When this compartmentation is broken, polyphenols that a molecular biological method is useful to repress the and PPO contact, and polyphenols are oxidized to the cor- expression of PPO (Martinez and Whitaker, 1995). Our responding quinones by the action of PPO (Vaughn and group also cloned the apple polyphenol oxidase gene (Ha- Duke, 1984; Murata et al., 1997) in the presence of oxygen. ruta et al., 1998) and showed that it was possible to inhibit The formed quinones are automatically polymerized to form enzymatic browning of apple by repressing the expression of brown polymers. We call this the immediate type of enzy- PPO by antisense technology (Murata et al., 2000; Murata matic browning. et al., 2001). However, a practical problem exists with the There is another type of enzymatic browning. When molecular biological method in that certain consumers do not lettuce is crushed, the juice does not turn brown. How- willingly accept this technology. ever, when lettuce is cut and stored, the surface of sections Several inhibitors of PPO are known and practically used gradually turns brown over the course of several days. We for regulation of enzymatic browning. However, they are not call this the delayed type of enzymatic browning, because sufficient to regulate various kinds of enzymatic browning. the browning does not happen immediately after cutting or In this study, we screened for inhibitors of enzymatic brown- crushing. Although lettuce does not contain enough poly- ing of apple using fermented broths of lactic acid bacteria, phenols for browning, the biosynthesis of polyphenols in food additives, and extracts of spices from the standpoint lettuce is induced in response to cutting or injury. Polyphe- of practical use, and found that hinokitiol, another name β-thujaplicin, definitely inhibited apple PPO and the enzy- *To whom correspondence should be addressed. matic browning of apple and shrimp. E-mail: [email protected] 252 S. Okumura et al.

Material and Methods 1/16 sections. A piece was cut into two pieces, which were Materials Food additives used here were supplied by used for a test and a control. A test solution (about 0.20 ml) Taisho Technos (Tokyo, Japan). Each food additive shown was sprayed onto an apple section. A solvent (water, 1% eth- in Table 1 was dissolved in or diluted with water, 1% etha- anol, or 10% ethanol) was sprayed onto another apple section nol, or 10% ethanol at the concentrations of 0.1% and 1.0%. as a control. Apple sections were left at room temperature for Lactic acid bacteria (58 strains; 36 Lactobacillus spp., 13 2 h and the browning was observed. Streptococcus spp., and 9 Leuconostoc spp.) stored in our PPO preparation PPO was prepared as described be- laboratory were incubated in 3 ml of MRS broth for 1-2 d at fore (Tsurutani et al., 2002) with some modifications. An ap- 37℃ with shaking. The supernatant and cells were separated ple was sliced, cored, and homogenized with a mixer for 30 s by centrifugation at 3,000 × g for 15 min. Ethanol (1.0 ml) in a 0.1M Na/K phosphate buffer (pH 7.2) containing 0.4 M was added to the cells, which were mixed well with a vortex sucrose and 0.01 M ascorbic acid. The homogenate was fil- mixer. After centrifugation at 3,000 × g for 15 min, the cell tered through 4 sheets of gauze. The filtrate was centrifuged extract was diluted ten times with water (final 10% ethanol) at 500 × g for 30 min. The precipitate was suspended in a 10- and used as a sample as well as the broth supernatant. Spices fold volume of a 0.01 M phosphate buffer (pH 7.2) contain- (galangal, cinnamon, saffron, nutmeg, black pepper, red pep- ing 3% Triton X-100, and sonicated for 40 s. The supernatant per, and thyme) were purchased at a local market (Tokyo, obtained by centrifugation at 18,000 × g for 10 min was ap- Japan). Each spice (1 g) was extracted with 10 ml of ethanol plied to a column of DEAE-Toyopearl 650M (Tosoh Co., To- for 1 h and diluted ten times by water (final 10% ethanol), or kyo, Japan) equilibrated with 0.01 M phosphate buffer. PPO extracted by boiling water for 1 h. protein was eluted with 0.2 M phosphate buffer, which was Screening for inhibitors of enzymatic browning Mature further fractionated by ammonium sulfate. The precipitate apples (Malus × domestica cv. Fuji) were purchased at a lo- obtained by 55% to 85% saturated ammonium sulfate was cal market (Tokyo, Japan). An apple was vertically cut into dialyzed against 0.02 M phosphate buffer. PPO activity was assayed by the spectrophotometric method at each step of Table 1. Inhibitory effect of food additives against browning of preparation. The enzyme solution (about 3 ml from an apple) apple section. was frozen and stored at −20℃ PPO assay PPO activity was measured by the HPLC Food additive Inhibition Food additive Inhibition method or the spectrophotometric method (Murata et al., Coffee bean extract − Quillaja extract − 1995) at 320 nm to detect the decrease in CQA. A reaction Enzymatically − Sunflower seed − decomposed rice bran extract mixture that consisted of 1.6 ml of McIlvaine buffer (pH 4.0), Eucalyptus leaf extract − Tea extract − 0.2 ml of an enzyme solution, and 0.2 ml of 0.5 mM CQA (E. citriodora) solution was incubated at 30℃. In the HPLC method, 1.0 ml Eucalyptus leaf extract − Yucca foam − (E. polybractea) extract of 8% metaphosphoric acid solution was added to stop the Eucalyptus leaf extract − Betaine − enzymatic reaction at 0 and 0.5 min, and the decreased CQA (E. smithii) was determined by HPLC. The spectrophotometric method Eucalyptus leaf extract − Enju saponin − (E. staigeriana) was used for the preparation of PPO. Grape seed extract − Ferulic acid +++ Tyrosinase assay Tyrosinase activity was measured ac- Hokosshi extract − Gallic acid ++ cording to the method of Matmaroh et al. (2006) with some Horseradish extract − Hinokitiol +++ modifications. Tyrosinase (3.3 mg) from mushroom was Licorice extract − Itaconic acid + purchased from Sigma-Aldrich (St. Louise, MO, U.S.A.) Licorice oil extract − Phytic acid + and dissolved in 6.0 mL of 50 mM phosphate buffer (pH Mannentake extract − ε-Polylysine + 6.5). l-Dopa (0.5, 1.0, 1.5, 2.0, and 3.0 mM) was dissolved Milt protein +++ Trehalose − in 50 mM phosphate buffer (pH 6.5) and used as a substrate. Mousouchiku dry distillate − Eucalyptol − A reaction mixture consisted of 0.6 ml of 50 mM phosphate Mousouchiku Extract − Thyme oil − Mustard extract − Rosemary oil − buffer (pH 6.5), 0.2 mL of 50 mM phosphate buffer (pH 6.5) containing hinokitiol (0, 10, and 20 μM), 0.2 mL of an en- Powdered stevia − White P (SO2) +++ Quassia extract − Kojic acid +++ zyme solution, and 1.0 ml of l-Dopa solution. Each reaction mixture was incubated at 30°C and the absorbance at 475 nm −, no inhibition at 1.0% solution; +, slight inhibition at 1.0% solution; ++, slight inhibition at 0.1% solution; +++, definite inhibition was monitored. at 0.1% solution. White P (0.1% and 1.0%) and kojic acid (0.1% and Measurement of CQA by HPLC CQA concentration 1.0%) were used as positive controls. was determined by HPLC. HPLC conditions were as follows; Hinokitiol Inhibits Polyphenol Oxidase and Enzymatic Browning 253 pump, JASCO PU-980 (Tokyo, Japan); column, TSKgel by distillation of the trunk of hinoki (a Japanese cypress), is a ODS-100V 3 μm (i.d. 4.3 mm × 150 mm; Tosoh, Tokyo, Ja- preservative and known as an antimicrobial compound (Trust pan); detector, Hitachi L-4200 (Tokyo, Japan); detection, 325 et al., 1973; Arima et al., 2003). This compound is a deriva- nm; flow rate, 1.0 ml/min; eluent, 5% acetic acid : CH3CN tive of tropolone (Fig. 1-B). Although tropolone is known (80:20). as a PPO inhibitor, there is no report showing that hinokitiol Estimation of browning of apple sections and shrimp A inhibits PPO. There is a recent report showing that hinokitiol test solution (about 0.2 mL) was sprayed to an apple section inhibited reddish coloration in wounded scales of Hippeast- as described above. Apple sections were left at room tem- rum bulbs (Saniewski et al., 2007). perature for 2 h. The browning was visually observed, and Inhibitory effect of hinokitiol against apple PPO and inhibition was estimated; 0, no inhibition; 1, slight inhibi- tyrosinase Apple PPO activity is usually measured by the tion (inhibition was partly observed); 2, moderate inhibition spectrophotometrical method because of its ease. However, (inhibition was clearly observed but partly turned brown); 3, when hinokitiol was added to the reaction mixture, the absor- definite inhibition (almost similar to original color). Shrimp bance at 320 nm was disturbed by the absorption of hinoki- (black tiger; Penaeus indicus) was purchased at a local mar- tiol. Therefore, we measured the amount of CQA in the reac- ket (Tokyo, Japan). After shrimp was immersed in 0.05% tion mixture by HPLC. Although this method was not simple and 1.0% hinokitiol solution (1% ethanol) or water for 16 h, and was time-consuming, reproducible results were obtained. the shrimp was put into a Petri dish and stored at 4℃ for 6 d. Apple PPO was first pre-incubated with hinokitiol, and The darkening of shells, legs, and tails of shrimp was visu- the effect of pre-incubation on the inhibitory activity of hi- ally estimated; 1, no darkening; 2, slight darkening; 3, moderate darkening; 4, extreme darkening. Table 2. Secondary screening for inhibitors of enzymatic browning of apple section. Results and Discussion Sample Inhibition Sample Inhibition Screening for inhibitors of enzymatic browning As the purpose of this study was to find a new practical inhibitor for Milt protein Hinokitiol enzymatic browning, we selected fermented broths of lactic 1.0% 2 0.1% (6 mM) 3 acid bacteria (58 strains), extracts of spices (7 samples), and 0.5% 0.5 0.05% (3 mM) 3 food additives (34 samples) as test samples. Apple sections 0.2% 0 0.02% (1.2 mM) 3 were used for the screening. After a sample was sprayed onto 0.1% 0 0.01% (0.6 mM) 3 the surface of an apple section, the browning of the surface 0.005% (0.3 mM) 3 was observed. No fermented broth of lactic acid bacteria Ferulic acid 0.002% (0.12 mM) 3 inhibited the browning of apple sections. The extracts of sea- 0.5% 3 0.001% (0.06 mM) 2 sonings, except galangal, did not inhibit the apple browning. 0.2% 2 0.0005% (0.003 mM) 1 Table 1 shows the results of 1st screening for the inhibitors 0.1% 1 No addition 0 of enzymatic browning of apple sections among food addi- 0.05% 0 White P (SO2) 3 tives. Several food additives such as ferulic acid, hinokitiol, and milt protein as well as kojic acid (a PPO inhibitor; Chen 3, definite inhibition; 2, moderate inhibition; 1, slight inhibition; 0, no inhibition (n = 2). et al., 1991) and white P (a sulfite preparation, 94% NaS2O3; McEvily et al., 1992) inhibited browning. Kojic acid and white P were used as positive controls. Table 2 shows the O inhibition of enzymatic browning of apple sections by these O food additives (2nd screening). The low concentration of milt OH protein (0.01 − 0.1%) did not inhibit the apple browning, and OH the inhibitory effect was not constant. Ferulic acid inhibited the browning at the concentration of 0.1 − 0.2%. However, the inhibitory effect gradually weakened, and apple sections turned brown after 1 h. On the other hand, hinokitiol definitely inhibited browning at the concentration of 0.002%, and A B this inhibitory effect continued for more than 2 h. From these results, we selected hinokitiol as an inhibitor of enzymatic browning of apple. Hinokitiol (Fig. 1-A), which is prepared Fig. 1. Structures of hinokitiol (A) and tropolone (B). 254 S. Okumura et al.

A 100 )

% 80 100 ( y ) t i

% 60 v i ( 80 t c a 40 n O o

i 60 P t i

P 20 b i

h 40 n

I 0 20 0 mM 0.2mM 2 mM 10 mM 0 Concentration of Cu (II) acetate

0 10 20 30 Fig. 3. Effect of Cu(II) acetate on the inhibitory effect of hinokitiol Incubation time (min) against apple PPO. Black bar, no addition; white bar, addition of Cu(II) acetate. B 1/v (min) -1 Added hinokitiol Added hinokitiol 1/v (min ) 800 Km 2.5 mM 20 µM 5 2.0 µM Ki 5.5 µM 600 Km 0.30 mM 4 Ki 0.15 µM 10 µM 1.0 µM 400 3

200 0 µM 2 0 µM 1 0 -50 0 50 100 150 0 1/S (mM-1) -4 -3 -2 -1 0 1 2 3 4 -1 Fig. 2. Inhibitory effect of hinokitiol on apple PPO. 1/[L-Dopa] (mM ) A, Effect of pre-incubation on inhibition by hinokitiol against apple Fig. 4. Inhibitory effect of hinokitiol on tyrosinase. PPO. B, Lineweaver-Burk plot. nokitiol against apple PPO was examined. As shown in Fig. and a substrate, respectively. As shown in Fig. 4, hinokitiol 2-A, the inhibitory effect was not affected by pre-incubation. non-competitively inhibited tyrosinase. The Km values of l- The inhibitory effect of hinokitiol was then examined. As Dopa and Ki value of hinokitiol were 0.30 mM and 0.15 μM, shown in Fig. 2-B, hinokitiol seemed to inhibit apple PPO respectively. competitively. The Ki value of hinokitiol was 5.5 μM, while Effect of hinokitiol on the discoloration of apple sec- the Km value of CQA was 2.5 mM. Under this condition, the tions and shrimp The inhibitory effect of hinokitiol on the Ki value of tropolone was 3.9 μM. Hinokitiol thus inhibited browning of apple sections was compared with that of tropo- apple PPO at a similar level as tropolone. As it was reported lone. As the visually evaluated score for browning of cut let- that the inhibition of tropolone was restored by the addition tuce corresponded well to a-value by a colorimeter (Hisami- of copper (Valero et al., 1991; Espin and Wichers, 1999), the nato et al., 2001), the degree of browning of apple sections effect of copper on the inhibition was examined. As shown in was visually estimated after 2 h. As shown in Table 3, the Fig. 3, little restoration by copper was observed. inhibitory effect of tropolone and hinokitiol was identical. In some enzymatic browning, l-tyrosine and l-Dopa be- Both completely inhibited the browning of apple sections at come the major substrate. In this case, PPO is called tyrosi- the concentration of 0.1 mM for 2 h. In a preliminary experi- nase. Melanin formation in animals, fungi, and crustaceans is ment, an apple section sprayed with a 0.1 mM hinokitiol attributed to tyrosinase. Inhibitory effect of hinokitiol against solution showed a slight aroma of hinokitiol and resulted in a tyrosinase was then examined. Commercial tyrosinase origi- good sensory evaluation. Next, the effect of the combination nated from mushroom and l-Dopa was used as an enzyme of hinokitiol and other chemicals such as NaCl and ascorbic Hinokitiol Inhibits Polyphenol Oxidase and Enzymatic Browning 255

Table 3. Inhibitory effect of hinokitiol and tropolone on enzy- Table 5. Inhibitory effect of hinokitiol on the discoloration matic browning of apple section. of shrimp.

Hinokitiol (mM) Inhibition Tropolone (mM) Inhibition Storage days after treatment

0.50 3.0 0.50 3.0 1 2 3 5 0.40 3.0 0.40 3.0 Control 0.20 3.0 0.20 3.0 Shell 1 1 1 2 0.10 3.0 0.10 3.0 Leg 0 0 1 1 0.08 3.0 0.08 3.0 Tail 1 1 2 3 0.06 2.3 0.06 2.3 0.025% Hinokitiol 0.04 1.3 0.04 1.3 Shell 0 0 0 1 0.00 0 0.00 0 Leg 0 0 0 0 A solution of hinokitiol and tropolone was sprayed to an apple sec- Tail 0 1 1 2 tion, which was left for 2 h at room temperature. The browning of was visually estimated (n = 3). 3, definite inhibition; 2, moderate 0.05% Hinokitiol inhibition; 1, slight inhibition; 0, no inhibition. Shell 0 0 0 1 Leg 0 0 0 0 Table 4. Inhibitory effect of combination of chemicals on the Tail 0 0 0 1 enzymatic browning of apple section. 0.1% White P Chemical Inhibition Shell 0 0 0 1 0.2% NaCl 2.0 Leg 0 0 0 0 0.1% NaCl 1.0 Tail 0 0 0 0

0.2% AsA 2.0 Shrimp (Black tiger) was dipped into water (control), hinokitiol so-

0.1% AsA 0.0 lution, and white P (NaS2O3 solution (positive control) for 16 h and 0.0017% (0.10mM) Hinokitiol 3.0 stored at 4℃. 0, Discoloration was visually estimated (n=2). 1, no discoloration; 2, slight discoloration; 3, moderate discoloration; 4, 0.001%(0.06 mM) Hinokitiol 2.3 extreme discoloration. 0.00067% (0.04 mM) Hinokitiol 1.0 0.1% NaCl, 0.1% AsA 1.0 0.1% NaCl, 0.001% hinokitiol 2.7 and 0.05%) inhibited the darkening of shrimp during cold 0.1% AsA, 0.001% hinokitiol 2.7 storage. 0.1% NaCl, 0.1% AsA, 0.001% hinokitiol 3.0 In conclusion, hinokitiol, a preservative, inhibited PPOs and the browning of apple sections and shrimp. Hinokitiol is Each solution was sprayed to an apple section, which was left for 2 h considered to be a useful inhibitor for enzymatic browning. at room temperature. The browning of was visually estimated (n = 3). 3, definite inhibition; 2, moderate inhibition; 1, slight inhibition; 0, no inhibition. References Arima, Y., Nakai, Y., Hayakawa, R. and Nishino, T. (2003). Anti- bacterial effect of β-thujaplicin on staphylococci isolated from acid (AsA) on the browning of apple sections was examined atopic dermatitis: Relationship between changes in the number of (Table 4). Although 0.10% AsA and 0.10% NaCl, slightly viable bacterial cells and clinical improvement in an eczematous inhibited the browning, the combinations of 0.0001% (0.06 lesion of atopic dermatitis. J. Antimicrob. Chemother., 51, 113- mM) hinokitiol and 0.10% AsA and of 0.0001% hinokitiol 122. and 0.10% NaCl definitely inhibited the browning. The color Chen, J.S., Wei, C.-I., Rolle, R.S., Otwell, S., Balaban, M.O. and of the sections treated with 0.001% hinokitiol, 0.10% AsA, Marshall, M.R. (1991). Inhibitory effect of kojic acid on some and 0.10% NaCl was almost similar to just after cutting. plant and crustacean polyphenol oxidases. J. Agric. Food Chem., The effect of hinokitiol on the darkening of shrimp dur- 39, 1396-1401. ing cold storage was further examined. The darkening or dis- Espín, J.C. and Wichers, H.J. (1999). Slow-binding inhibition of coloration of shrimp is considered to be mainly attributed to mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone. tyrosinase. As shown in Table 5, the tail and shell of shrimp J. Agric. Food Chem., 47, 2638-2644. tended to turn dark during cold storage. Hinokitiol (0.025% Fujita, N., Tanaka, E. and Murata, M. (2006). Cinnamaldehyde in- 256 S. Okumura et al.

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