Antioxidant and tyrosinase inhibitory activities of different parts of oriental cherry (Prunus...

Food Sci. Biotechnol. 21(2): 339-343 (2012)

DOI 10.1007/s10068-012-0045-x

Antioxidant and Tyrosinase Inhibitory Activities of Different Parts of

Oriental Cherry (Prunus serrulata var. spontanea)

Ji Won Park, Hyun Gyun Yuk, and Seung Cheol Lee

Received: 28 June 2011 / Revised: 12 October 2011 / Accepted: 14 October 2011 / Published Online: 30 April 2012

© KoSFoST and Springer 2012

Abstract Six different parts (branch, flesh, flower, fruit,

leaf, and seed) of oriental cherry (Prunus serrulata var.

spontanea) were extracted with ethanol or water, then total

phenol content (TPC), antioxidant activity, and tyrosinase

inhibitory activity of the extracts were evaluated. The

ethanol extracts showed higher TPC and antioxidant activity

than the water extracts regardless of parts. The ethanol

extracts of leaf as well as branch possessed superior TPC

and antioxidant activity. The highest tyrosinase inhibitory

activity was found in ethanol extract of leaf. There was no

dramatic difference of tyrosinase inhibitory activities

according to parts of cherry. The results suggest that leaf

and branch of oriental cherry could be a candidate for

antioxidant and anti-whitening materials in food or cosmetic

industries.

Keywords: oriental cherry (Prunus serrulata var. spontanea),

antioxidant activity, tyrosinase inhibitory activity

Introduction

Reactive oxygen species (ROS) and free radicals, such as

superoxide anion, hydrogen peroxide, and hydroxyl radical,

are constantly formed in the human body by normal

metabolic action. Their action is opposed by a balanced

system of antioxidant defenses, including antioxidant

compounds and enzymes. Upsetting this balance causes

oxidative stress, which can lead to cell injury and death (1).

Synthetic antioxidants such as butylated hydroxyanisole

(BHA) and butylated hydroxytoluene (BHT) are known to

induce carcinogenesis by mutagenicity and toxicity against

human enzymes and lipids (2). Therefore, studies of natural

antioxidants are attractive to investigators for use in foods

or medicinal materials instead of synthetic antioxidants.

Particularly, much research has been focused on natural

products including plant and vegetables sources (3-5).

Tyrosinase is a key enzyme for melanin production in

plants and animals, which is fashioned in melanocytes

located within the basal epidermis (6). It has been also

reported that tyrosinase is associated with neurodegenerative

diseases (7). Recently, tyrosinase inhibitors are becoming

increasingly important in the cosmetic industry, due to their

skin-whitening effects (8).

The Prunus serrulata var. spontanea (commonly called

as oriental cherry, East Asian cherry, or Japanese cherry),

which belongs to the Rosaceae family, is found throughout

Korean peninsula. Cherry been used as a medicinal plant

for a long time in Asia. Especially, red cherry fruits have

been used as a traditional herbal remedy for various

diseases such as heart failure, beriberi, dropsy, and mastitis.

Bark and stems of cherry have been also used for relaxation

and detoxification (9-11). Some researchers have examined

oriental cherry to determine the source and the level of

antioxidant and anticancer activities (12,13). However, the

physiological activity of oriental cherry according to parts

has little been investigated. Therefore, the aim of this study

was to evaluate the antioxidant and tyrosinase inhibitory

activities of 6 different parts (branch, flesh, flower, fruit,

leaf, and seed) of oriental cherry.

Ji Won Park, Seung Cheol Lee ( )Department of Food Science and Biotechnology, Kyungnam University,Changwon, Gyeongnam 631-701, KoreaTel: +82-55-2492684; Fax: +82-505-9992171E-mail: [email protected].

Hyun Gyun YukFood Science and Technology Programme, Department of Chemistry,National University of Singapore, Science Drive, Singapore 117543,Singapore

RESEARCH ARTICLE

340 Park et al.

Materials and Methods

Materials Cherry (Prunus serrulata var. spontanea) was

collected in May to August 2010 at Changwon-si area,

Korea, and was divided into 6 different parts; branch, flesh

without seed, flower, fruit with seed, leaf, and seed.

Arbutin, ABTS, dimethyl sulfoxide (DMSO), and DPPH

were purchased from Sigma-Aldrich (St. Louis, MO, USA).

L-Ascorbic acid, hydrogen peroxide, potassium chloride,

potassium phosphate, sodium chloride, sodium hydroxide,

tyrosinase (from mushroom), and L-tyrosine were also

purchased from Sigma-Aldrich. Folin-Ciocalteu reagents

were from Wako Pure Chemical Industries, Ltd. (Osaka,

Japan). All the other organic solvents and chemicals used

in this study were of analytical grade.

Preparation of extracts from cherry Each 10 g of 6

different parts of the cherry was extracted with 200 mL of

70%(v/v) ethanol or distilled water in a shaking incubator

(100 rpm) overnight at room temperature, then the extracts

were filtered through a Whatman No. 1 filter paper (Advantec,

Tokyo, Japan). Solvent was removed by evaporation in

vacuo, and the dried extract was then obtained. The dried

extracts were dissolved in DMSO at concentration of

50 mg/mL for experiments. The dissolved extracts were

diluted with DMSO when needed.

Total phenolic content (TPC) TPC was determined by

a modified method of Gutfinger (14). Each extract

(1.0 mL) was mixed with 1.0 mL of 2% Na2CO3 and then

the mixture was allowed to stand at room temperature for

3 min. After addition 0.2 mL of 50% Folin-Ciocalteu

reagent, the reaction was carried out for 30 min. The

mixture was centrifuged at 13,400×g for 5 min, and the

absorbance of supernatant was measured with a

spectrophotometer (UV-1601; Shimadzu, Tokyo, Japan) at

750 nm. TPC was expressed as gallic acid equivalents

(GAE).

DPPH radical scavenging activity DPPH radical

scavenging activity was determined according to the

method of Lee et al. (11). After 0.1 mL of extracts in

DMSO was mixed with 0.9 mL of 0.041 mM DPPH in

ethanol for 10 min, the absorbance of the mixture was

measured at 517 nm by a spectrophotometer. The mixture

of DMSO (0.1 mL) and DPPH (0.9 mL) was used as a

control. Radical scavenging activity was expressed as a %

inhibition and it was calculated by using the following

formula:

DPPH radical scavenging activity (%)

=[1−(Abssample/Abscontrol)]×100

ABTS radical scavenging activity The scavenging

activity for ABTS radical was measured according to the

method of Muller (15) with slight modification. Each

extract (0.1 mL), potassium phosphate buffer (0.1 mL,

0.1 M, pH 5.0), and hydrogen peroxide (20 µL, 10 mM)

were mixed and the mixture was incubated at 37oC for

5 min. ABTS (30 µL, 1.25 mM, in 0.05 M phosphate-

citrate buffer, pH 5.0) and peroxidase (30 µL, 1 unit/mL)

were added to the mixture and then it was also incubated

at 37oC for 10 min. DMSO instead of the extract was used

in control. The absorbance level was obtained with a

multiplate reader (Sunrise RC/TS/TS Color-TC/TW/BC/

6Filter; Tecan Austria GmbH, Grödig, Austria) at 405 nm,

and the ABTS radical scavenging activity was calculated

by the following formula:

ABTS radical scavenging activity (%)


Reducing power (RP) RP was determined according to

the method of Oyaizu (16). Extracts in DMSO (1.0 mL),

sodium phosphate buffer (1 mL, 0.2 M, pH 6.6), and

potassium ferricyanide (1.0 mL, 10 mg/mL) were mixed

and incubated at 50oC for 20 min. Trichloroacetic acid

(1.0 mL, 100 mg/mL) was added to the mixture followed

by centrifuged at 12,000×g for 5 min. The supernatant

(1.0 mL) was mixed with distilled water (1.0 mL) and

ferric chloride (0.1 mL, 1.0 mg/mL), and then its absorbance

was measured at 700 nm.

Tyrosinase inhibitory activity Tyrosinase inhibitory

activity was determined using the method described by

Vanni et al. (17). All extracts were dissolved in DMSO at

50 mg/mL concentration and then diluted to 100 µg/mL

concentrations with DMSO. Each diluted extract (100 µL)

was mixed with 140 µL of 0.05 mM sodium phosphate

buffer (pH 6.8) in a 96-well plate and then 40 µL of

1.5 mM L-tyrosine solution and 20 µL of mushroom

tyrosinase (1,500 units/mL) were added into a 96-well

plate. The test mixture (300 µL) was mixed well and

incubated at 37ºC for 30 min. DMSO instead of the extract

was used in control. The absorbance level was obtained

with a multiplate reader at 492 nm, and the % inhibition of

tyrosinase activity was calculated by the following

formula:

Inhibition (%)


Statistical analysis All measurements were done in

triplicate, and analysis of variance (ANOVA) was conducted

according to the procedure of the general linear model

Antioxidant Activity of Cherry 341

using SAS software (SAS Institute, NC, USA). Student-

Newman-Keul’s multiple range tests were used to compare

the significant differences of the mean value among

treatments (p<0.05).

Results and Discussion

Extraction yield and TPC of cherry extracts The

extraction yields of ethanol and water extract according to

parts of cherry were shown in Table 1. Flesh part showed

high yield regardless of extracting solvent, followed by

fruit, leaf, and flower parts. Extraction yields of branch and

seed parts were relatively low.

Phenolic compounds are very important components in

plants because of their radical scavenging ability due to

their hydroxyl groups (18). TPC of each extract (at

concentration of 1 mg/mL) was expressed in Table 2.

Overall, in same cherry parts, the ethanol extracts possessed

significantly (p<0.05) higher TPC than the water extracts.

It’s noticeable that ethanol extracts of branch and leaf

(110.48±0.48 and 121.41±0.53 mg GAE/g, respectively)

showed 3.8 and 11.3 times of TPC than water extracts of

branch and leaf (29.05±0.23 and 10.75±0.55 mg GAE/g,

respectively). In our previous study (11), ethanol extract of

cherry blossom also showed higher TPC than water extract.

Branch, flower, and leaf parts showed higher TPC than

flesh, fruit, and seed parts. Especially, the highest TPC was

detected in ethanol extract of leaf (121.41±0.53 mg GAE/

g). The results indicated that ethanol soluble phenolic

compounds are abundant in leaf, branch, and flower parts

of cherry. Phenolic acids such as chlorogenic, neochlorogenic,

p-hydroxybenzoic, p-coumaric, and caffeic acids were

identified in cherry wines (19).

DPPH radical scavenging activity Antioxidant activity

of the cherry extracts was analyzed by measuring DPPH

radical scavenging activity. DPPH radical scavenging

activity was determined at 50, 100, 500, and 1,000 µg/mL

concentration of each extract, and IC50 values, the

concentration required for scavenging 50% of DPPH

radicals, of the extracts was compared with that of L-

ascorbic acid (IC50=26.90±0.15 µg/mL) used as the

positive control in this study (Table 3). DPPH radical

scavenging activity of the extracts showed similar pattern

with TPC, which means the extract possessed higher TPC

has lower IC50 value, which is stronger DPPH radical

scavenging activity. For example, the ethanol extract of

cherry leaf, which has the highest TPC, showed the lowest

IC50 (78.87±10.73 µg/mL) for DPPH radical scavenging

activity. IC50 of ethanol extract of cherry leaf was lower

than that of ascorbic acid, however, it might be significant

antioxidant activity because L-ascorbic acid was a purified

single compound while the extracts was mixture of crude

various compounds. In same cherry parts, DPPH radical

scavenging activities of the ethanol extracts were higher

than those of water extracts. Sun and Ho (20) reported a

significant correlation between DPPH radical scavenging

activity and TPC of buckwheat extracts. Linear regression

analyses between IC50 values for DPPH radical scavenging

activity and TPC showed a good correlation (r2=0.896;

p<0.05). This correlation suggests that phenolic compounds

in the cherry extracts may contribute to DPPH radical

Table 1. Extraction yield of extracts from several parts oforiental cherry (unit: %1))

Parts of cherryExtraction solvent

Ethanol Water

Branch 05.5 02.5

Flesh 72.5 80.0

Flower 27.0 16.0

Fruit 51.0 47.4

Leaf 27.6 22.5

Seed 06.7 13.3

1)Yield (%)=(extract weight/dry weight)×100

Table 2. Total phenolic contents of extracts from several partsof oriental cherry (unit: mg GAE/g)


Ethanol Water

Branch 110.48±0.48b1) 29.05±0.23b

Flesh 020.96±0.90d 08.13±0.08d

Flower 084.66±1.01c 55.85±1.15a

Fruit 008.63±0.41e 06.72±0.12e

Leaf 121.41±0.53a 10.75±0.55c

Seed 008.00±0.88e 05.43±0.27f

1)All measurements were done in triplicate, and all values aremean±SD; a-fDifferent letters within a column are significantlydifferent (p<0.05).

Table 3. DPPH radical scavenging activity of ethanol andwater extracts from several parts of oriental cherry

(unit: IC501))


Ethanol Water

Branch 0,222.00±12.66b2) 1,376.00±39.83c

Flesh 1,194.00±4.38d 3,208.53±52.75d

Flower 0,406.45±10.73c 0,494.97±60.01a

Fruit 2,376.00±16.90e 3,797.22±72.77e

Leaf 00,78.87±10.73a 0,941.07±46.92b

Seed 4,995.00±343.50f 9,260.00±397.60f

Ascorbic acid 26.90±0.15

1)IC50 (µg/mL), concentration for scavenging 50% of DPPH radicals2)All measurements were done in triplicate, and all values aremean±SD; a-fDifferent letters within a column are significantlydifferent (p<0.05).

342 Park et al.

scavenging activity. Yook et al. (21) have reported that

cherry fruits showed strong antioxidant activity, however,

they did not compare the activity between parts of cherry.

ABTS radical scavenging activity Another stable free

radical cation, ABTS, was used to evaluate antioxidant

activity of the cherry extracts. The ABTS radical scavenging

activity of the cherry extracts was shown as IC50 values

(Table 4). As like as DPPH radical scavenging activity, the

ethanol and water extracts of branch part showed lower

IC50 values (202.50±15.14 and 401.29±12.66 µg/mL,

respectively), whereas IC50 of L-ascorbic acid was 18.81±

0.57 µg/mL. Regardless of extraction solvents, cherry fruit

showed higher IC50 values. In the case of guava, the

extracts of branch and leaf also showed relatively higher

antioxidant activity that those of fruit and seed (22),

however, it has still not been elucidated how and why

branch and leaf contains high amounts of phenolics and

high antioxidant activity.

Reducing power (RP) The power of certain antioxidants

is associated with their RP (23). The reducing capacity of

a compound may serve as a significant indictor of its

potential antioxidant activity (24). The reducing properties

are generally associated with the presence of reductones

(25). It is reported that the antioxidant action of reductones

is based on the breaking of the free radical chain by

donating a hydrogen atom, or reacting with certain precursors

of peroxide to prevent peroxide formation (26). RP was

expressed as IC50 (the values indicate 0.5 increase of

optical density) values (Table 5). Except seed part, the

other parts showed stronger RP in ethanol that in water

extracts. The highest RP was found in ethanol extracts of

leaf (IC50=210.75±13.54 µg/mL) followed by branch (IC50

=222.56±10.17 µg/mL). A significant linear correlation

was observed between RP and TPC (r2=0.852; p<0.05).

Tyrosinase inhibitory activity Tyrosinase inhibitory

activities of the cherry extracts (at 100 µg/mL) and arbutin

(at 1,000 µg/mL) were shown in Table 6. The highest

tyrosinase inhibitory activity of cherry was found to be

63.39±0.02% for ethanol extract of leaf. There was no

dramatic difference of tyrosinase inhibitory activities

according to parts of cherry as like as antioxidant activities.

When considering result of arbutin (49.88±3.62%), the

positive control, tyrosinase inhibitory activity of cherry

parts was significantly high because the concentration of

extracts used was only 10% of that of arbutin. It is

noteworthy that the water extracts of flesh and fruit, which

possessed relatively lower antioxidant activities, showed

higher tyrosinase inhibitory activity than the other extracts

except ethanol extract of leaf, indicating that different

compounds in the extracts might be responsible for

antioxidant or tyrosinase inhibitory activity.

This study was the first attempt to evaluate antioxidant

and tyrosinase inhibitory activities of oriental cherry

Table 4. ABTS radical scavenging activity of ethanol andwater extracts from several parts of oriental cherry

(unit: IC501))


Ethanol Water

Branch 00,202.50±15.14b2) 0,401.29±12.66b

Flesh 03,772.91±310.32d 2,884.98±29.70d

Flower 01,420.11±122.93c 0,454.41±11.68c

Fruit 12,301.18±859.03e 7,144.91±359.40e

Leaf 01,261.51±352.87a 2,724.42±782.53a

Seed 03,971.90±997.50f 1,745.96±420.10f


1)IC50 (µg/mL), concentration required for inhibiting 50% of activity2)All measurements were done in triplicate, and all values aremean±SD; a-fDifferent letters within a column are significantlydifferent (p<0.05).

Table 5. Reducing power of ethanol and water extracts fromseveral parts of oriental cherry (unit: IC50

1))


Ethanol Water

Branch 0,222.56±10.17b2) 1,413.00±8.11b

Flesh 1,401.00±29.87d 2,167.50±10.52d

Flower 00,399.1±17.36c 0,446.43±26.29c

Fruit 4,371.00±30.41e 4,833.00±80.75e

Leaf 0,210.75±13.54a 2,182.00±10.91a

Seed 4,144.00±16.70f 2,054.00±19.70f


1)IC50 (µg/mL), concentration for increasing 0.5 value in opticaldensity


Table 6. Tyrosinase inhibitory activity of ethanol and waterextracts from several parts of oriental cherry (unit: %)

Parts of cherry1)Extraction solvent

Ethanol Water

Branch 041.33±0.00e2) 40.31±0.02d

Flesh 40.63±0.01f 53.00±0.04b

Flower 50.19±0.01b 50.77±0.01c

Fruit 45.15±0.01c 58.16±0.01a

Leaf 63.39±0.02a 39.03±0.01e

Seed 41.45±0.01d 35.40±0.01f

Arbutin 49.88±3.62

1)Tyrosinase inhibitory activity of the extracts of cherry parts wasdetermined at the concentration of 100 µg/mL, whereas arbutin wasat 1,000 µg/mL.


Antioxidant Activity of Cherry 343

according to parts. For practical application of the results to

food and cosmetic industries, ethanol and water were used

for preparing the extracts. The ethanol extracts of branch as

well as leaf might be a strong candidate with antioxidant

activity. The ethanol extract of leaf also had powerful

tyrosinase inhibitory activity. Further researches on the

identification of valuable compounds of oriental cherry

will be needed.

Acknowledgments This study is supported by a research

grant from Kyungnam University, Korea, in 2012.

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