Biofunctionality Studies of Cudrania cochinchinensis Extracts

Cudrania cochinchinensis has been found to show remarkable medicinal values. The total phenolic and flavonoid contents of C. cochinchinensis extracts were analyzed, and the antioxidant activity and reducing ability of C. cochinchinensis extracts were also evaluated. Tetrahydroxyflavanonol (THF) was isolated from the xylem and pith portions of C. cochinchinensis stem; however, the bark portion of C. cochinchinensis stem was found to contain no THF. Consequently, solutions extracted from the xylem and pith portions of C. cochinchinensis showed good antioxidant activity. The IC50 values of pith, xylem, and bark extracts were 0.779, 3.020, and 3.507 mg/mL, respectively. As the pith portion of C. cochinchinensis stem contained more THF and had a higher flavonoid content, it exhibited better antioxidant activity and reducing ability. In addition, C. cochinchinensis pith extracts reduced tyrosinase activity in a dose-dependent manner with IC50 = 16.1 μg/mL. The inhibitory activity was determined to be noncompetitive with Km = 0.23 mM.


Introduction
Cudrania cochinchinensis has shown remarkable medicinal values [1] [2] [3].Because leaf and root extracts of C. cochinchinensis have shown good biofunctionality, it has been used as a folk medicine in oriental countries [4] [5].Al-DOI: 10.4236/ajac.2018.91001 2 American Journal of Analytical Chemistry though flavonoids, prenylated xanthones, and other active compounds have been isolated from C. cochinchinensis [4] [6] [7], 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.
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 [17].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 (K m ) and maximum velocity (V max ).The xintercept represents −1/K m , the y-intercept represents 1/V max , and the slope of the straight line represents K m /V max .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.

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/H 2 O = 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. [18], based on the modified method described by Kobayashi et al. [19].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.

Analysis of Total Phenolic and Flavonoid Contents
The total phenolic content was measured following the method described by Singleton et al. [20].An amount of 200 µL of different concentrations of samples was mixed with 200 µL of 0.5 N Folin-Ciocalteu reagent, to which 200 µL of 10% (w/v) Na 2 CO 3 and 40 µL of distilled water were added.The mixture was incubated at room temperature for 1 h in the dark.After incubation, the mixture was centrifuged at 5000 rpm for 10 min.An amount of 200 µL of the supernatant was transferred to a 96-well plate and the absorbance of each well was measured using an ELISA reader at a wavelength of 700 nm.Gallic acid was used as a posi-American Journal of Analytical Chemistry tive control.Each measurement was performed at least in duplicate.
The flavonoid content was measured according to the method described by Chandra et al. [21].Different concentrations of samples (50 µL) were mixed with 100 µL of 10% (w/v) AlCl 3 .The mixture was incubated at room temperature for 10 min in the dark.The absorbance of the mixture at 430 nm wavelength was measured using an ELISA reader.Quercetin was used as a positive control.Each measurement was performed at least in duplicate.

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. [22] [23] [24].The radical scavenging activity of ascorbic acid, used as a positive control, was also measured.The sample (50 µL) was mixed with 50 µL of freshly prepared 160 µM 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 (TECAN R , Austria).Each measurement was performed at least in duplicate.The radical scavenging activity was calculated as follows: where A Sample and A Blank 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. [25].Samples of different concentrations (100 µL each) were individually mixed with 100 µL of 1% (w/v) K 3 Fe(CN) 6 and 100 µL of 2 mM phosphate buffer (pH 6.6).The mixture was incubated at 50˚C for 20 min.After incubation, 100 µL of 10% (w/v) trichloroacetic acid was added to it, and the mixture was centrifuged at 3000 rpm for 2 min.An amount of 100 µL of the supernatant was transferred to a 96-well plate.Each well contained 100 µL of distilled water and 20 µL of 0.1% (w/v) FeCl 3 solution.BHA was used as a positive control.The absorbance of each well was measured using an ELISA reader at 700 nm wavelength.Each measurement was performed at least in duplicate.

Analysis of Tyrosinase Inhibition Activity
An amount of 20 μL of extracted C. cochinchinensis pith solution (500 μg/mL, in ( ) The absorbance of sample (OD sample ) and control (OD control ) was measured at 475 nm.The IC 50 value was determined by regression of a constructing doseresponse curve at which 50% target activity was lost. In

Statistical Analysis
Statistical evaluation was performed by running one-way analysis of variance (ANOVA) with SAS R software (version 6.08, SAS Institute Inc., Cary, NC, USA).
All data were presented as mean ± standard deviation (SD).Differences were considered to be statistically significant when the p-value was less than 0.05.

Total Phenolic and Flavonoid Content Analyses of C. cochinchinensis Extracts
The total phenolic content of C. cochinchinensis extracts is shown in Figure 1

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  Derivatives of both flavonol and flavanone have been approved as good antioxidants [27].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 [28].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 [28].Because the pith portion of C. cochinchinensis extracts contained more THF, pith extracts had a better reducing abil- ity than other extracts.Likewise, the bark portion contained the least THF and had the worst reducing ability.

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 0.1 mM of L-DOPA was used as the substrate, tyrosinase activity was increased with the addition of more tyrosinase.A linear relationship of the first-order was observed between the tyrosinase activity and tyrosine concentration (Figure 4).However, tyrosinase activity reduced with the addition of  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 IC 50 value (36.3 μg/mL) of ethanol extracted C. cochinchinensis stem solution was reported by Zheng et al. [8].In this study, the pith portion of C. cochinchinensis stem was further extracted with methanol.In this study, it was required to determine whether the inhibitory activity of C. cochinchinensis pith extracts was competitive or noncompetitive.

Conclusion
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 IC 50 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 whi- tening 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.
(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.

Figure 1 (Figure 1 .
Figure 1(c).The DPPH radical scavenging activity of the THF standard is shown in Figure 1(d).The IC 50 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

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 IC 50 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.
/mL) American Journal of Analytical Chemistry

Figure 5 .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).
[12]solved 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[12][26] 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: 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 American Journal of Analytical Chemistry