Determination of total phenol (TP) and PAs
Due to the strong correlation between the biological activity and the content of polyphenols, the determination of polyphenol content by Folin phenol colorimetry can explain the mechanism of biological activity of the samples to be tested. Thus, first, we used the FC method to determine TP content and the HCl-N-butanol method to determine PAs content. The contents of TP and PAs in the leaves of C. camphora were 220.6 ± 18.3 mg/g and 152 ± 9.4 mg/g, respectively, and in the branches of C. camphora, they were 118.04 ±11.9 mg/g and 89.6 ± 7.5 mg/g, respectively. The results showed that the content of TP and PAs in the leaves of C. camphora was 22% and 15%, respectively, and that in the branches of C. camphora was 12% and 9%, respectively. The content of PAs in leaves was more abundant than that in branches, suggesting that leaves may be a good raw material for PAs supply.
Reversed-phase HPLC-ESI-MS
The basic structural units and polymer chain lengths of PAs were predicted by measuring the conjugates of acid-catalyzed PAs with benzylmercaptan. The HPLC-ESI-MS results of PAs from C. camphora leaves and branches are shown in Fig. 1. It can be seen that the PAs from leaves and branches have obvious difference in the peak position and intensity, indicating that there are difference in the composition of two parts in C. camphora, in which catechin benzylthioether (C-thio) and afzelechin/epiafzelechin (AF/EAF) were detected in leaves, while no significant AF/EAF was detected in branches. But for both, the terminal units were catechin (C) and the extension units were mainly catechin/epicatechin benzylthioether (C/EC-thio). The difference of active phenolic hydroxyl groups and dihydroxy phthalic groups may be the main reason for the better anti-tyrosinase and antioxidant activity of leaves PAs.
Effect of C. camphora PAs on the activity of tyrosinase monophenolase and diphenolase
Tyrosinase has monophenolase activity of hydroxylation of monophenol to diphenol and diphenolase activity of oxidation of bisphenol to quinine (26, 3). Therefore, we first measured the monophenolase activity using L-tyrosine (L-Tyr) as the substrate. Fig. 2 a-Ⅰ shows the catalytic reaction of the inhibition of monophenolase activity by the PAs and plots the concentration of PAs with steady state activity and lag time (Fig. 2 a-Ⅱ and Fig. 2 a-Ⅲ). The results showed that the PAs from the leaves and branches of C. camphora could both effectively prolong the delay time of monophenolase activity and decrease its steady-state activity, and the inhibitory effect showed an obvious dose-dependent manner. It was found that when the concentration of the PAs from leaves and branches reached 200 μg/mL and 306.67 μg/mL, the lag time increased from 13.14 s to 120 s and 3.6 s to 33 s, respectively, while the steady-state activity decreased from 100% to 43.23% and 42.86%, respectively. The IC50 of PAs was 166.65 ± 18.1 μg/mL and 268.38 ± 23.9 μg/mL, respectively (Table 1), indicating that PAs extracted from leaves possess better inhibitory effect on tyrosinase monophenolase activity than that from branches.
L-DOPA was used as a substrate to determine the activity of tyrosinase diphenolase, and the reaction curve obtained is shown in Fig. 2 b. The enzyme activity decreased with increasing PAs concentration, and the IC50 values were 70.31 ± 6.62 μg/mL and 90.93 ± 8.15 μg/mL, respectively (Table 1). Both of them had good inhibitory activity on tyrosinase diphenolase, especially the extract of PAs from leaves of C. camphora. Qu et al. (23) reported the effects of puerarin on the monophenolase activity with an IC50 value of 0.537 mg/mL. Cui et al. (9) reported that T. grandis “Xiangyafei” seed oil (XYSO) exhibited the highest activities in the tested seed oils against tyrosinase monophenolase ( IC50 = 817.5 μg/mL). XYSO in T. grandis was tested against diphenolase with IC50 values of 227.01 ± 2.68 μg/mL (9). A. aucheri oil exhibited anti-tyrosinase activity at a 50% concentration (IC50) of 6.43 mg/mL (29). Obviously, PAs extracted from the leaves and branches of C. camphora both showed better inhibitory effect on monophenolase and diphenolase, suggesting that C. camphora may be a good resource for tyrosinase inhibitors. Thus, it is feasible to seek potent tyrosinase inhibitor PAs from C. camphora.
Inhibition mechanism of PAs from the leaves and branches of C. camphora on tyrosinase
The inhibition mechanism of PAs on tyrosinase is shown in Fig. 2 c. The slope of straight lines decreased with increasing PAs concentration, and two sets of straight lines passing through the origin were obtained by mapping the enzyme activity to the PAs concentration. These suggested that instead of reducing the amount of effective enzyme, PAs reduced the catalytic efficiency of enzyme to achieve the inhibitory activity, which can be concluded that the inhibition of tyrosinase by PAs is a reversible process. Reduced catalytic efficiency reversible has been a common inhibition mode of PAs from plants on tyrosinase diphenolase (2, 6) (26). As shown in HPLC-ESI-MS, the basic structural units of C/EC and AF/EAF in PAs, which have very active phenolic hydroxy groups and dihydroxy phthalic groups, may be the structural reason for its reversible inhibition of tyrosinase activity.
Determination of the inhibition type and inhibition constant of PAs from C. camphora on tyrosinase
In the diphenolase detection system, by measuring the effect of L-DOPA on tyrosinase activity, a group of straight lines with different slope intersected in the second quadrant were obtained, as shown in Fig. 3 a and Fig. 3 b, in which the Km increased and Vm decreased with the increase of substrate concentration, showed mixed competitive inhibition. The inhibition constant KI and KIS of leaves PAs can be calculated as 33.36 μg/mL and 344.44 μg/mL, respectively (Table 1). For PAs from branches of C. camphora, the inhibition constants KI and KIS were 17.97 μg/mL and 349.89 μg/mL, respectively. For PAs from the leaves and branches of C. camphora, KIS was 10 and 19 times that of KI, respectively. KIS were much larger than KI further indicated that the binding ability of PAs extracted from the two parts of C. camphora to free enzyme were much stronger than that to the enzyme substrate complex. In conclusion, the results demonstrated for the first time that the leaves and branches of C. camphora might be good sources for further development of tyrosinase inhibitors, which indicated the possible application of these compounds, especially PAs from leaves, in food, agricultural, cosmetic and medical industries.
Scanning study
To further analyzed the inhibition mechanism of PAs on tyrosinase catalytic activity, UV-Vis spectroscopy was used to assess the L-Tyr oxidation and L-DOPA oxidation of PAs. Fig. 4 showed the time-dependent UV spectra of PAs acting on tyrosinase catalyzed oxidation of tyrosine. The absorption value in 475 nm increased with the increase of catalytic time, suggesting that 475 nm is the characteristic peak of the oxidation of L-Tyr by tyrosinase. Compared with Fig. 4a, the absorption values of absorption peaks at 475 nm caused by PAs decreased with time(Fig. 4 b-c), indicating that PAs could effectively inhibit the the oxidation of L-Tyr by tyrosinase.
L-DOPA oxidation
The product of L-DOPA catalyzed by tyrosinase can also be obtained from the non enzymatic catalysis,such as oxidized by NaIO4. Using spectral analysis to assess the effect of PAs on the oxidation of L-DOPA, and Fig. 5 showed that the characteristic peaks of L-DOPA oxidized by NaIO4 are located at 475 nm after adding PAs, and the absorbance value obviously decreased. It is suggested that PAs could decline the L-DOPA oxidation products. Therefore, PAs in C. camphora could inhibit the oxidation of L-Tyr by tyrosinase, and the effect of PAs on the spectra of oxidation products of L-DOPA indicated that PAs could depress the oxidation products of L-DOPA and prevent the formation of L-DOPA pigment, which leads to the reduction of characteristic absorption peak of the product.
Determination of antioxidant capacity
Due to the rich hydroxyl structure, PAs can prevent the chain reaction of free radicals by releasing H+ in the structure to compete with free radicals, eliminate free radicals and protect lipids from oxidation (34, 31, 1, 32, 13). Thus, DPPH, ABTS and FRAP methods were used to investigate the antioxidant capacity of PAs in leaves and branches of C. camphora. The relationship between the scavenging capacity of PAs from two plant parts on DPPH free radicals were shown in Fig. 6 a. In the range of experimental concentrations, with increasing concentration, the scavenging effect of each part on DPPH free radicals also showed an increasing trend. The antioxidant activities of different extracts were significantly different, and the DPPH scavenging ability of PAs extracted from these two parts was relatively weak compared to that of VC. The IC50 of DPPH radical scavenging activity of leaves and branches were 77.51 ± 12.6 μg/mL and 273.53 ± 28.3 μg/mL, respectively (Table 2). The DPPH scavenging ability of leaves was higher than that of branches. Fig. 6 b shows that the ABTS radical scavenging rate of PAs and VC were positively correlated with the concentration, and the change trend was similar to the scavenging rate of DPPH radicals. The IC50 values of leaves and branches in Table 2 are greater than those of VC. The results of the FRAP assay were expressed in the form of VC equivalent, that is, the ability of a 1 g sample to scavenge free radicals is equivalent to the capacity in mmol of VC. It can be seen from the results in Table 2 that leaves PAs (4.74 ± 0.46 mmol AAE/g) > branches PAs (3.58 ± 0.35 mmol AAE/g). Wang et al. reported that the Fuwan 8, Dongliang and FD97 varieties of Dimocarpus longan Lour. had the strongest DPPH scavenging activity, with an IC50 of 1.03 g/mL (31). The anthocyanins isolated from Lycium ruthenicum Murr posessed ABTS radical scavenging capacity ,with IC50 of 0.5023 ± 0.011 mg/mL (17). Because the total phenol content (TPC) is the greatest antioxidant contributor in the DPPH and FRAP assays, the radical scavenging ability of PAs in leaves was better than that of PAs in branches, which may be due to the relatively high TPC. In short, the results demonstrated for the first time that PAs from the leaves and branches of C. camphora might be good sources for the further development of antioxidants.