Cudrania cochinchinensis has shown remarkable medicinal values    . Because leaf and root extracts of C. cochinchinensis have shown good biofunctionality, it has been used as a folk medicine in oriental countries   . Although flavonoids, prenylated xanthones, and other active compounds have been isolated from C. cochinchinensis    , studies on C. cochinchinensis have focused primarily on its roots. To date, no study has been reported on active compounds found in the xylem and pith portions of C. cochinchinensis stem.
Extracts of C. cochinchinensis stem have shown good tyrosinase inhibitory ability  . This inhibitory activity of tyrosinase (EC 22.214.171.124) has been extensively studied in the past      . Tyrosinase catalyzes the oxidation of L-tyrosine to 3,4-dihydroxyphenylalanine (DOPA), which forms DOPAchrome  . These catalyzed reactions result in the formation of melanin, which is responsible for the pigmentation of skin  . Natural medical plants are considered to be a good source of tyrosinase inhibitors  . Zheng et al. (2011) used 95% ethanol to extract C. cochinchinensis, and the extracted solutions showed good tyrosinase inhibitory ability  .
Although C. cochinchinensis extracts have shown the ability to inhibit tyrosinase activity, its inhibitory mechanism has not been studied. Generally, enzyme inhibitors are classified into competitive or noncompetitive inhibitors  . A Lineweaver-Burk plot (Equation (1)), obtained by plotting the inverse values of reaction rate (V) and substrate concentration [S], can be used to determine the inhibitory activity.
The linear regression model applied to the double-reciprocal plot can be used to determine the Michaelis constant (Km) and maximum velocity (Vmax). The x- intercept represents −1/Km, the y-intercept represents 1/Vmax, and the slope of the straight line represents Km/Vmax. Based on the Lineweaver-Burk plot, the inhibitory activity can be determined to be competitive or noncompetitive.
The aim of this study is to analyze the antioxidant activities and tyrosinase inhibitory activity of bark, xylem, and pith extracts of C. cochinchinensis. In addition, the kinetic study on the inhibitory activity of C. cochinchinensis extracts was determined based on the Lineweaver-Burk plot.
2. Materials and Methods
C. cochinchinensis samples were collected from Taiwanese mountain areas, air dried, and then kept in a cool place for further use. Liquiritin was purchased from ChromaDex (
2.2. Preparation of C. cochinchinensis Extracts
A sample of pulverized C. cochinchinensis (1.0 g) was sonicated in an ultrasonic bath (Chrom Tech, Taipei, Taiwan) for 20 min with 7 mL of 70% methanol (methanol/H2O = 7/3, v/v). The suspension was centrifuged at 6000 rpm (HERMLE Z206A, Germany) for 10 min. The supernatant was collected and run through a 0.45 μm filter. The residual solids were extracted with fresh 70% methanol. After the C. cochinchinensis sample was extracted three times, all the collected supernatants were mixed together. Subsequently, 70% methanol was used to make up the total volume to 20 mL. Moreover, 1.0 mg liquiritin was dissolved in 10 mL of 70% methanol and used as the internal standard (IS) solution. Before performing the HPLC analysis, 100 μL of the extracted solution was mixed with 100 μL of the IS solution.
Tetrahydroxyflavanonol (THF) was isolated from C. cochinchinensis stem by Chen et al.  , based on the modified method described by Kobayashi et al.  . Dried C. cochinchinensis samples were extracted four times by methanol under reflux. The extract was partitioned using a mixture of EtOAc and water (1:1, v/v). The EtOAc extract was run through a silica gel column, and then eluted with a mixture of n-hexane and EtOAc/MeOH. Eight fractions were collected during elution. Fraction number 3 was further separated by high performance liquid chromatography (HPLC) using a Cosmosil 5C18-AR column (Nacalai Tesque, Tokyo, Japan) to obtain THF.
2.3. HPLC Method
HPLC analysis was performed on an Agilent 1200 system with a reverse phase column (Cosmosil 5C18-AR II, 5 μm, 25 cm × 4.6 mm I.D.; Nacalai Tesque, Kyoto, Japan). The detection wavelength was set at 254 nm. The flow rate was 0.8 mL/min with a linear solvent gradient of A-B (A = 10 mM KH2PO4, pH 4.6; B = CH3CN/CH3OH/H2O, 1.5/2.5/1, v/v/v). as follows: 0 min, 40% B; 10 min, 40% B; 20 min, 60% B; 30 min, 70% B; and 50 min, 100% B.
2.4. Analysis of Total Phenolic and Flavonoid Contents
The total phenolic content was measured following the method described by Singleton et al.  . An amount of 200 mL of different concentrations of samples was mixed with
The flavonoid content was measured according to the method described by Chandra et al.  . Different concentrations of samples (50 mL) were mixed with
2.5. Analysis of Antioxidant Activity
Radical scavenging activities of THF marker standards and C. cochinchinensis extracts were measured respectively using the methods of Singh and Rajini and Chan et al. and Azman et al.    . The radical scavenging activity of ascorbic acid, used as a positive control, was also measured. The sample (50 mL) was mixed with 50 mL of freshly prepared 160 mM DPPH in ethanol. The mixture was kept in the dark for 30 min. The absorbance of the mixture at 517 nm wavelength was measured using an ELISA reader (TECANR,
where ASample and ABlank represent the absorbance of sample and blank solution, respectively.
The reducing ability of the samples was measured following the method described by Canabady-Rochelle et al.  . Samples of different concentrations (100 mL each) were individually mixed with 100 mL of 1% (w/v) K3Fe(CN)6 and 100 mL of
2.6. Analysis of Tyrosinase Inhibition Activity
An amount of 20 μL of extracted C. cochinchinensis pith solution (500 μg/mL, in 3.3% of DMSO) was placed in a 96-well plate. Then, 40 μL of tyrosinase solutions of various concentrations (0.277, 0.554, 1.662, 3.324, and 6.648 μg/mL) and 0.1 mM of L-DOPA solution (dissolved in a sodium phosphate buffer at pH 6.8) were added to it.
Another 20 μL of extracted C. cochinchinensis pith solution (31.25, 62.5, 125, 250, and 500 μg/mL, in 3.3% of DMSO) was placed in a 96-well plate, to which 40 μL of tyrosinase solution (6.648 μg/mL) and 0.1 mM of L-DOPA solution (dissolved in a sodium phosphate buffer at pH 6.8) were added. These mixed solutions were kept at room temperature (25˚C) for 25 min. The absorbance was measured at 475 nm   using the Microplate-Reader (Sunrise Basic, Grödig, Austria). Kojic acid was used as a positive control. The tyrosinase inhibition rate (%) was calculated from the following equation:
The absorbance of sample (ODsample) and control (ODcontrol) was measured at 475 nm. The IC50 value was determined by regression of a constructing dose- response curve at which 50% target activity was lost.
In a 96-well plate, 20 μL of extracted C. cochinchinensis pith solution (7.8125, 15.625, 31.25, and 62.5 μg/mL, in 3.3% of DMSO) was placed, to which 40 μL of tyrosinase solutions (6.648 μg/mL) was added. The substrate was L-DOPA solution, which was prepared by dissolving L-DOPA (0.1, 0.3, 0.5, 0.7, and 1.0 mM) in sodium phosphate buffer at pH 6.8. The Line weaver-Burk plot was obtained by plotting the inverse values of reaction rate (V) and concentration of L-DOPA (Equation (1)).
2.7. Statistical Analysis
Statistical evaluation was performed by running one-way analysis of variance (ANOVA) with SASR software (version 6.08, SAS Institute Inc.,
3. Results and Discussion
3.1. Total Phenolic and Flavonoid Content Analyses of C. cochinchinensis Extracts
The total phenolic content of C. cochinchinensis extracts is shown in Figure 1(a). The pith extract contained more phenolic components than the xylem and bark extracts. No significant difference was observed in the total phenolic content between the bark and xylem extracts. Flavonoid contents of C. cochinchinensis extracts are shown in Figure 1(b), which were in the order pith > xylem > bark. Thus, the pith portion of C. cochinchinensis stem was expected to have better antioxidant activity.
3.2. Antioxidant Activity Analysis of C. cochinchinensis Extracts
The DPPH radical scavenging activities of the extracts were measured as the decrease of absorbance at a wavelength of 517 nm, and the results are shown in Figure 1(c). The DPPH radical scavenging activity of the THF standard is shown in Figure 1(d). The IC50 value of the THF standard was 0.122 mg/mL and those of the pith, xylem, and bark extracts were 0.769, 2.809, and 3.34 mg/mL, respectively. The pith portion of C. cochinchinensis stem showed better DPPH
(a) (b) (c) (d)
Figure 1. (a) Total phenolic content of C. cochinchinensis stem extracts (■: bark; □: xylem; ▲: pith); (b) Flavonoid content of C. cochinchinensis stem extracts (■: bark; □: xylem; ▲: pith); (c) DPPH radical scavenging activity of C. cochinchinensis stem extracts (◆: ascorbic acid; ■: bark; □: xylem; ▲: pith); (d) DPPH radical scavenging activity of the standard (◆: ascorbic acid; ●: THF); (e) Reducing ability of C. cochinchinensis stem extracts (■: bark; □: xylem; ▲: pith); (f) Reducing ability of the standard (◆: BHA; ●: THF).
radical scavenging activity. The reducing ability was measured as the change in absorbance at a wavelength of 700 nm. Figure 1(e) and Figure 1(f) show the reducing ability of C. cochinchinensis extracts and the THF standard, respectively. Generally, higher flavonoid contents result in better reducing ability. On the other hand, better reducing ability represents stronger antioxidant activity.
Figures 2(a)-(c) show the representative HPLC chromatograms of C. cochinchinensis stem extracts from the bark, xylem, and pith portions respectively. THF was well separated by HPLC with a retention time of 15 min. Based on
Figure 2. Representative HPLC (high performance liquid chromatography) chroma- tograms of C. cochinchinensis stem extracts: (a) bark; (b) xylem; (c) pith.
the chromatographic analysis results, THF was found only in xylem and pith extracts of C. cochinchinensis (691.5 ± 3.2 and 1,489.7 ± 5.5 μg/g, respectively). The THF content of bark extract was not detectable. The pith portion of C. cochinchinensis stem contained more THF than the xylem portion. Therefore, the pith extracts showed better radical scavenging activity and reducing ability. Although xylem and bark extracts had similar antioxidant results, only xylem extracts were found to contain THF. Bark extracts may contain some other ingredients which may also provide antioxidant activity. More studies are required to explain why bark extracts had similar antioxidant activity without containing THF.
Derivatives of both flavonol and flavanone have been approved as good antioxidants  . Flavonol derivatives such as THF and quercetin have similar chemical structures (Figure 3). Multiple hydroxyl groups, especially on the B-ring, improve the antioxidant activity of flavonoids  . Likewise, flavanone derivatives such as tetrahydroxyflavanone and luteolin also have similar chemical structures (Figure 3). The difference between THF and tetrahydroxyflavanone is that THF has one extra hydroxyl group on the B-ring. Consequently, THF has better antioxidant activity  . Because the pith portion of C. cochinchinensis extracts contained more THF, pith extracts had a better reducing ability than other extracts. Likewise, the bark portion contained the least THF and had the worst reducing ability.
3.3. Tyrosinase Inhibitory Ability of C. cochinchinensis Pith Extracts
Pith extracts of C. cochinchinensis showed the ability to inhibit the formation of DOPA chrome, which can be detected with a spectrophotometer at a wavelength of 475 nm. When
(a) (b) (c) (d)
Figure 3. Chemical structures of some flavonoids: (a) THF; (b) quercetin; (c) tetrahydroxyflavanone; (d) luteolin.
Figure 4. Influence of C. cochinchinensis pith extracts on tyrosinase activity when
more C. cochinchinensis pith extracts. This confirmed that the extracted C. cochinchinensis pith solution could inhibit tyrosinase activity. In addition, the slope of the linear lines decreased with the addition of more C. cochinchinensis pith extracts (Figure 4).
The ability of C. cochinchinensis extracts to inhibit tyrosinase activity could be attributed to the presence of phenolics in the extracts. The IC50 value (36.3 μg/mL) of ethanol extracted C. cochinchinensis stem solution was reported by Zheng et al.  . In this study, the pith portion of C. cochinchinensis stem was further extracted with methanol. Figure 5(a) shows the inhibition rate of tyrosinase activity using C. cochinchinensis pith extracts, which reduced the tyrosinase activity in a dose-dependent manner. The slope and intercept of the linear regression line were 0.5015 and 41.914, respectively. The IC50 value of methanol extracted C. cochinchinensis pith solution was calculated to be 16.1 μg/mL. Comparing this result with the results reported by Zheng et al., C. cochinchinensis pith extracts were found to exhibit a better inhibitory ability. Figure 5(b) shows a comparison of tyrosinase inhibitory rate between C. cochinchinensis pith extracts and kojic acid, which was used as a positive control. Based on Figure 5(b), the inhibition rate of tyrosinase activity was 70.4% when 250 μg/mL of C. cochinchinensis pith extracts were added. This inhibition rate was close to that of kojic acid (70.5%), at a concentration of 62.5 μg/mL. Although the inhibitory ability of C. cochinchinensis pith extracts was approximately 25% of that of kojic acid, C. cochinchinensis extracts are natural ingredients and may possibly be used in cosmetic products.
In this study, it was required to determine whether the inhibitory activity of C. cochinchinensis pith extracts was competitive or noncompetitive. Figure 6
Figure 5. (a) Inhibition rate of tyrosinase activity using C. cochinchinensis pith extracts as the inhibitor (40 μL of tyrosinase solution (6.648 μg/mL) and 0.1 mM of L-DOPA solution were added for each measurement); (b) Inhibition rate of tyrosinase activity using C. cochinchinensis pith extracts as the inhibitor (□: Kojic acid as the positive control; ■: C. cochinchinensis pith extracts).
shows the Line weaver-Burk double reciprocal plot of C. cochinchinensis pith solutions. The substrate was L-DOPA. Based on Figure 6, the x-intercept (−1/Km) remained the same but the y-intercept (1/Vmax) increased with increasing concentrations of C. cochinchinensis pith extracts. As a result, Km remained unchanged (0.23 mM), but Vmax decreased by the introduction of an inhibitor. The binding of C. cochinchinensis pith extracts to tyrosinase had no effect on
Figure 6. Lineweaver-Burk double reciprocal plot of extracted C. cochinchinensis pith solution (○: 0; ■:7.8125; △: 15.625; ●: 31.25; □: 62.5 μg/mL of C. cochinchinensis pith extracts; V: absorbance change rate, △OD475nm/min; [S]: concentration of L-DOPA).
the binding of L-DOPA to tyrosinase. The binding sites of L-DOPA and C. cochinchinensis pith extracts to tyrosinase were different. Based on the Lineweaver-Burk double reciprocal plot shown in Figure 6, the inhibitory activity was determined to be noncompetitive.
The C. cochinchinensis extracts of bark, xylem and pith were shown different antioxidant activities. The pith extracts showed better antioxidant activity and higher reducing ability, which might because of the higher THF content. In addition, C. cochinchinensis pith extracts could reduce tyrosinase activity successfully. The IC50 value of C. cochinchinensis pith extracts was 16.1 μg/mL, and the tyrosinase inhibitory activity was determined to be noncompetitive. C. cochinchinensis pith extracts could be used in cosmetic formulations as a natural whitening agent. Based on our studies, the pith extracts of C. cochinchinensis stem contained THF and showed good whitening ability. The future study suggests using B16F10 murine melanoma cells to perform in vivo tests. Results can be used to verify the whitening ability of the pith extracts in cells.
The authors are grateful for the financial support of this study by the Ministry of Science and Technology, Taiwan China, under contract number MOST 104- 2622-E-241-003-CC3.
Declaration of Interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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