Extraction optimization, antioxidant activity, and tyrosinase inhibitory capacity of polyphenols from Lonicera japonica

Abstract The objective of this research was twofold: first, to optimize the extraction process of Lonicera japonica polyphenols using a response surface methodology, and second, to study the antioxidant activity and tyrosinase inhibitory capacity of the polyphenols of different purities. High‐speed shearing homogenization extraction was used to extract the polyphenols from L. japonica. The antioxidant activity and the effect of polyphenols on tyrosinase activity were studied using free radical scavenging assay and the tyrosinase method, respectively. The optimal extraction conditions with an extraction yield of 6.96% for polyphenols were determined as follows: ethanol volume fraction 57%, shearing time 3.30 min, and solid–liquid ratio 1:58. Lonicera japonica polyphenols exhibited potent scavenging activity on 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2, 2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS), and inhibitory capacity on tyrosinase. The results suggested that L. japonica polyphenols could be explored as a natural antioxidant and tyrosinase inhibitor.

microwave extraction (Nayak et al., 2015), and ultrasonic-assisted extraction (Sousa et al., 2016). These methods often have an extensive process time, which results in partial destruction of the polyphenols structure and reduced extraction yield. High-speed shearing homogenization extraction (high shear) is an emerging extraction technology, which has less process time, lower energy consumption, and lower extraction temperature with a highly efficient result. High shear is often used to extract polysaccharides, pectin, and other biologically active ingredients (Fan, Lin, Wang, & Wang, 2014;Guo et al., 2017). Some studies have demonstrated the role of polyphenols in inhibiting tyrosinase activity, but not the potential relationships between polyphenols purity and their inhibitory effect. In the present study, the extraction of polyphenols from the L. japonica flowers through high shear technology was optimized using response surface methodology together with a Box-Behnken design. The antioxidant activity and the effect of the extracted polyphenols on tyrosinase activity were studied.

| MATERIAL S AND ME THODS
For this study, the L. japonica was harvested in Quzhou, Anhui province. They were then crushed through a 40-mesh sieve. All chemicals, solvents, and analytical reagents such as deionized water, tyrosinase, L-Dopa, and kojic acid were purchased from Baoman Biotechnology Co., Ltd. (Shanghai, China).

| Total polyphenol yield
The total polyphenol yield was slightly modified based on Evstatieva, Todorova, Antonova, and Staneva (2010) method. A volume of 1.0 ml of Folin-Ciocalteu reagent was mixed with 1.0 ml of sample solution, 5.0 ml of distilled water, and 3 ml of sodium carbonate (15%).
The mixture was then left to stand for 2 hr. Absorbance was then measured at a wavelength of 765 nm, and a standard curve linear regression equation was used to calculate the total polyphenol concentration and yield . The extraction yield was calculated using the following formulation.
In the formula: C-Total polyphenol concentration in the extract sample, mg/ml; V-Fixed volume of extraction, ml.

| Response surface assay
The extraction condition of the polyphenols was optimized by the response surface methodology, with the extraction yield of polyphenols as the response value. Ethanol volume fraction (A), shearing time (B), and solid-liquid ratio (C) were chosen as the independent variables.

| DPPH free radical scavenging assay
The DPPH scavenging activity assay was conducted based on the Zheng, Lin, Su, Zhao, and Zhao (2015) method. A 2 ml sample solution with different contents was mixed with 2 ml of a DPPH ethanol solution (0.2 mM). The reaction mixture was then shaken and allowed to stand for 30 min in a dark environment. The absorbance was measured at a wavelength of 517 nm (A i ). 70% ethanol solution was used as a reference and the absorbance was measured (A j ), which followed the completed reaction of a 2 ml sample solution mixed with a 2 ml of 70% ethanol solution. The absorbance was measured as equal amount of 70% ethanol instead of sample as the blank (A 0 ). With Trolox as a positive control, the DPPH radical scavenging rate was determined.

| ABTS radical scavenging assay
The ABTS free radical scavenging activity was assayed using a method previously described by Marecek et al. (2017) with a slight modification. ABTS reaction solution was prepared by mixing 7 mM of ABTS aqueous solution with 2.45 mM of K 2 S 2 O 8 solution in a dark room for 24 hr. Then, the ABTS stock solution was diluted with absolute ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. Then, 0.4 ml sample solution with different contents or Trolox standard was mixed with 3 ml of the ABTS solution. The mixture was then shaken and allowed to stand for 8 min in a dark environment. The absorbance, set at a 734 nm wavelength, was then measured (A i ).
For the control, absolute ethanol was used instead of ABTS. Then, the absorbance was measured as 0.4 ml distilled water instead of the sample as the blank (A 0 ).

| Determination of tyrosinase inhibitory capacity
Tyrosinase inhibition rate was determined using L-Dopa as substrate (Paun, Neagu, Albu, & Radu, 2015). Briefly, 200 μl of sample solution and 200 μl of mushroom tyrosinase (0.5 mg/ml in 0.2 mM PBS %Extraction yield = C × V × Dilution times 1.00 × 1,000 × 100. %Purity = Total polyphenols mass in the sample Total sample mass × 100. buffer, pH 7.0) were mixed with 3,700 μl of PBS and pre-incubated at 30°C for 15 min. Finally, the L-Dopa (100 μl, 10 mM) was added to the mixture and the absorbance (A i ) was measured at 475 nm after 3 min. The sample solution was replaced by an equal amount of PBS, and the absorbance (A 0 ) was measured under the same conditions.
Kojic acid was used as a positive control.

| Statistical analysis
All measurements were done in triplicate and the results were expressed as mean value ± standard deviation (n = 3). The data between the control and experimental groups were analyzed by pvalues. p-values <0.05 were considered as significant. To obtain the best extraction process, ANOVA and 3D surface maps of the model (Table 1) were used to analyze the experimental and predictive data to determine the accuracy of the model. Figure 1a shows the effect of solid-liquid ratio on the total polyphenol yield. The extraction yield increased gradually with increasing solidliquid ratio with the maximum value being obtained at 1:50 (g/ml) ratio.

| Single factor assay: Effect of solid-liquid ratio on polyphenol extraction yield
TA B L E 1 Box-Behnken design matrix for optimization of parameters and the response values for the extraction yield of polyphenols. Ethanol volume fraction (A), shearing time (B), and solid-liquid ratio (C)  The extraction yield remained low at ratios 1:10 to 1:30, but beyond 1:30 ratio, the extraction yield increased rapidly. The extraction yield decreased with the solid-liquid ratio decreasing from 1:50 to 1:60.

| Response surface assay
Design-expert software (Design Expert 8.0.6; Stat-Ease Inc.) was used for data statistical analysis. Regression analysis second-order polynomial equation was developed to study the relationships between input process variables and respective responses: Extraction yield = 6.83 As illustrated in Table 2, the highly dominant factors that influenced the extraction of the total polyphenols were C, AC, A 2 , and C 2 , and the dominant factor was A. The coefficient of variation was 1.93, which indicated that the test was reliable. The signalto-noise ratio (11.688, larger than 4) signified that the model had later. Figure 2c shows that extraction yield initially increased with the increase in ethanol volume fraction but decreased later with further increase in ethanol volume fraction, but shearing time had the same effect on the extraction yield. When taking into account the convenience of practical operation, and after further adjustment, the highest yield was achieved using 57% ethanol.
Consequently, the extraction time became 3.30 min, with a 1:58 solid-liquid ratio.

| DPPH free radical scavenging assay
Historically, DPPH free radical scavenging assays are used to evaluate the activity of antioxidants, as it is a very stable free radical that can be stored for a long period. The DPPH free radical is an aromatic radical which has three aromatic ring structures (Guo et al., 2004).
As shown in Figure 3a, the scavenging rate of DPPH increased as

| D ISCUSS I ON AND CON CLUS I ON S
Our results showed that an increase in contact area of the solvent with L. japonica powder caused a decrease in material viscosity and an enhancement in the osmotic pressure and intermolecular driving force. As a result, polyphenols dissolved easily, which is consistent with the conclusions of Wong, Li, Li, Razmovski-Naumovski, and Chan (2017)  In contrast, we also concluded that the extraction yield might be affected by the structure of polyphenols. Previous studies have classified polyphenols as flavonoids and nonflavonoid compounds; two main forms are as follows: glycosides and aglycone (Santhakumar, Battino, & Alvarez-Suarez, 2018). In keeping with the principle of similar dissolution, overall polarity will enhance with the decrease of the ethanol mass fraction in a solution, which favors the solubility of polar substances. As the mass fraction of ethanol increases, the nonpolarity enhances and the amount of dissolved polar species decreases. Aglycone is a polar substance, so we speculate that L. japonica polyphenols might be polar isoflavones, which is similar to the result found by Wong et al. (2017). The yield of polyphenol compounds in less polar solvents is affected, thus reducing the solubility of polyphenolic compounds. Lonicera japonica polyphenols  (Wong et al., 2017). Secondly, long-term shear may cause damage to the cell wall and modify the polyphenols microstructure. Thirdly, excessively long shearing process generates heat, which increases oxidation.
There are many kinds of phenolic substances in L. japonica (Seo et al., 2012), including chlorogenic acid, caffeic acid, ferulic acid, coumaric acid, cinnamic acid, rutin, and luteolin. Its biological function is mainly attributed to the specific chemical structure of polyphenols. Due to the aromatic nature and highly conjugated systems with multiple hydroxyl groups, these compounds have beneficial electron or hydrogen atom donors, which neutralize free radicals and other reactive oxygen species. Each phenolic compound exhibits an antioxidant effect (Zhang et al., 2018), achieved by hydrogen supply and chelation of metal ions; an anticancer effect (Lee et al., 2016), primarily through antioxidant functions; the ability to prevent cardiovascular disease (Croft et al., 2018), which regulates blood lipid density in the blood, inhibits the oxidation of low-density lipoprotein, and promotes vasodilation, thereby achieving cardiovascular protection. For other phenolic compounds, their ability to scavenge ABTS free radicals is primarily affected by the number and location of free hydroxyl groups in the structure. This indicates that the polyphenolic compound contained in A in Figure 3b has more phenolic hydroxyl groups than B and C.
As shown in the Figure 3a, L. japonica polyphenols have DPPH free radical scavenging activity in different purity. When the concentration was 10 μg/ml, the purity of the three different elution did not reach 50%, the clearance of 75% elution purity was 38.10%, and the purity of 35% elution only reached 13.90%. At this time, Trolox's clearance rate reached 44.65%. When the concentration reached 25 μg/ml, the clearance rate reached more than 50%, and the 75% elution purity clearance rate was the highest. At a concentration of 40 μg/ml, there was no significant difference in the ability to remove free radicals with 35% elution purity and 50% elution purity, but all were below 75% elution purity, at which point 75% elution purity removes free radicals. The ability is close to Trolox. The above results indicate that the different elution purity of L. japonica polyphenols has the ability to scavenge DPPH free radicals and is a good donor of hydrogen protons. However, the ability of different purity of L. japonica polyphenols to remove DPPH free radicals is significantly less than that of Trolox.

F I G U R E 4
The effects of total polyphenols on the inhibitory ability of tyrosinase Past research shows that tyrosinases can catalyze the first two steps of melanin production and are associated with hyperpigmentation disorders such as melasma, solar lentigines, and postinflammatory hyperpigmentation (Fisk, Agbai, Lev-Tov, & Sivamani, 2014;Xue et al., 2018). Plants that contain natural active ingredients can reduce melanin synthesis through inhibiting tyrosinases and antioxidant effects. Therefore, the application of tyrosinases inhibitors can inhibit pigmentation and achieve whitening effect (Rescigno, Sollai, Pisu, Rinaldi, & Sanjust, 2002). Plant cosmetics have become an increasingly popular alternative to decolorizers because they are safer than standard de-colorants (Fisk et al., 2014). Tyrosinases cause browning reactions which are unfavorable in fruits and vegetables (Olmedo et al., 2018), but can be slowed by tyrosinases inhibitors (Chang, 2009).
In addition, tyrosinase inhibitors can be used as an insect control (Balabanidou et al., 2018;De, Adhikari, Nandy, Saha, & Goswami, 2018;Rezaei, Mohammadi, Mahdavi, Shourian, & Ghafouri, 2018) because the physiological responses of insects, such as the thickening of the stratum corneum or changing the cuticle composition, are related to tyrosinase action. Therefore, the development of safe and effective tyrosinases inhibitors is necessary in medicine, agriculture, cosmetics, and food industry.
It can be seen from the Figure 4 that with the increase in polyphenol concentration, the inhibition rate of tyrosinase of different purity of L. japonica polyphenols has a certain upward trend, indicating that the inhibition is continuously enhanced; and at 50-100 μg/ ml, the slope of the line is large, indicating that the inhibitory effect on tyrosinase is obvious when the concentration of polyphenol is low, but the inhibition rate is not strong. When the inhibition rate reached 50%, the purity of 30%, 50%, and 75% polyphenols was (355.270 ± 0.84) μg/ml, (282.229) ±1.12) μg/ml, and (205.826 ± 0.23) μg/ml, indicating that the higher the purity of the polyphenol, the better the inhibition effect. The tyrosinase inhibition of L. japonica polyphenols may be due to a group of multiple hydroxyl groups on the ring.
In conclusion, shearing time of 3.30 min, ethanol volume fraction of 57%, and a solid-liquid ratio of 1:58 were found to be optimal conditions for the extraction L. japonica polyphenols. The polyphenols from L. japonica had the effect of scavenging free radicals, antioxidation, antiaging, and whitening. Results from this study validated L. japonica as an antioxidant and a tyrosinase inhibitor (Jeong, Jeong, Hwang, & Kim, 2015). The purification effect of macroporous resin was not ideal in this experiment as the monomer substance was unable to be separated and extracted. Subsequent experiments can use HPLC to extract effective monomer substance.

ACK N OWLED G M ENTS
This study was supported by the Fundamental Research Funds for the Central Universities (2572016CA06), China Postdoctoral Science Foundation (2016M600239), Heilongjiang Youth Science Fund (QC2016021). All authors express their thanks to Professor Bob Tuck from Australia who edited and refined an early version of the paper. We wish to thank our colleagues and members of our laboratories for useful discussions.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing. Written informed consent was obtained from all study participants.

CO N FLI C T O F I NTE R E S T
The authors declare that we do not have any conflict of interest.