Natural Antioxidants, Tyrosinase and Acetylcholinesterase Inhibitors from Cercis glabra Leaves

Cercis glabra is a plant belonging to the legume family, whose flowers and barks are commonly used as food and traditional Chinese medicines. However, its leaves are usually disposed of as wastes. This research comprehensively investigated the bioactive constituents of C. glabra leaves, and two new phenolic, ceroffesters A-B (1–2) and thirteen known compounds (3–15) were isolated. Their structures were elucidated by spectroscopic methods such as nuclear magnetic resonance (1D NMR and 2D NMR), high-resolution electrospray ionization mass spectra (HR-ESI-MS), optical rotatory dispersion (ORD) and electronic circular dichroism (ECD). All of them were assessed for their antioxidant activities through ABTS, DPPH and PTIO methodologies, and evaluated for inhibitory activities against two enzymes (mushroom tyrosinase and acetylcholinesterase). As a result, compounds 3–6, 10 and 13 exhibited evident antioxidant activities. Meanwhile, compounds 5, 10 and 13 showed the most potent tyrosinase inhibitory activities, with IC50 of 0.64, 0.65 and 0.59 mM, and compared with the positive control of 0.63 mM (kojic acid). In the initial concentration of 1 mg/mL, compounds 3, 5 and 6 demonstrated moderate inhibitory activities against acetylcholinesterase with 85.27 ± 0.06%, 83.65 ± 0.48% and 82.21 ± 0.09%, respectively, compared with the positive control of 91.17 ± 0.23% (donepezil). These bioactive components could be promising antioxidants, tyrosinase and acetylcholinesterase inhibitors.


Antioxidant, Tyrosinase and Acetylcholinesterase Inhibitory Activities
The ABTS, DPPH and PTIO radicals have been widely used to evaluate the antioxidant capacity of natural products or extracts. In this study, fifteen isolates (at initial concentration of 1 mg/mL) from C. glabra leaves were explored with L-ascorbic acid as the positive control (Table 1 and Figure 4).
Of these bioactive components, all except gallic acid (13) are flavonoids. Therefore, the phenolic hydroxyl groups may be important for the radical scavenging activity. The ABTS, DPPH and PTIO methods are commonly used to evaluate the scavenging ability of free radicals. Thus, their reaction may be regarded as a direct antioxidant process. However, their scavenging mechanisms are in fact rather different. For example, DPPH and ABTS are nitrogen-centered radicals, while PTIO is an oxygen-centered radical. The ABTS radical is scavenged mainly involving one-electron transfer (ET), while DPPH and PTIO scavenging have been demonstrated to be involved in H + transfer (HAT) [35]. As can been see from Table 1, compounds 3, 5, 6 and 10 could effectively scavenge three types of free radicals through ET and HAT pathways, indicating that they could be used as novel effective antioxidants.
In Table 2, most of the isolated compounds showed moderate-to-strong inhibitory activities against mushroom tyrosinase at 1 mg/mL. In particular, the new compounds 1-2 showed moderate tyrosinase inhibitory activities, while compounds 5, 10 and 13 showed the most potent tyrosinase inhibitory activities, with IC 50 of 0.64, 0.65 and 0.59 mM, respectively, while the positive control was 0.63 mM (kojic acid). Tyrosinase inhibitors, characterized by reducing melanin production and improving skin elasticity, are widely used in pharmaceutical and skincare industries [36]. However, current tyrosinase inhibitors may induce unwanted adverse reactions such as unequal pigmentation, skin irritation and even cancer [37]. Therefore, it is still necessary to explore safe, stable and effective tyrosinase inhibitors. As natural compounds extracted from medicinal plants, compounds 5, 10 and 13 are of great pharmaceutical value both in cosmetics and pharmaceuticals, due to their potent biological properties. [a] At initial concentration of 1 mg/mL. Results were expressed as means ± SEMs.
The initial concentration was 1 mg/mL, and compounds 3, 5 and 6 exhibited moderate acetylcholinesterase inhibitory activities, with a percentage inhibition value of 85.27 ± 0.06%, 83.65 ± 0.48% and 82.21 ± 0.09%, respectively, with donepezil used as the positive control (91.17 ± 0.23%). The inhibition of AChE serves as a strategy for the treatment of neurologi-cal disorders, including myasthenia gravis, glaucoma, Parkinson's disease, senile dementia and ataxia [38]. The findings of this study reveal that ethanol extract from the C. glabra leaves may be a potential therapeutic agent for the treatment of Alzheimer's disease, due to its phytochemical components.
Myricetin (3) and kaempferol (6) are two flavonoids widely distributed in natural plants [39,40] and have been previously reported to possess strong antioxidant and acetylcholinesterase inhibitory abilities [41][42][43][44]. Therefore, the results for 3 and 6 were generally consistent with the previous research. Quercetin (5) spread widely in fruits and vegetables [45], and was found to show antioxidant, tyrosinase and acetylcholinesterase inhibitory abilities [46,47], which fits well with the results of this research. Compound 10 has been tested to be a potential natural antioxidant [28]; however, the present study demonstrated for the first time that it is a promising tyrosinase inhibitor. Compound 13 was found to show antioxidant and tyrosinase inhibition activities [48,49], which was further confirmed.

Plant Materials
The fresh leaves of C. glabra were collected from Yanling Zhonglin Garden Engineering Co., Ltd., Xuchang, China, in May 2021. The species was identified by Prof. Lin Yang at Lanzhou Technology University. A specimen (No. SPH2021B) was stored in Xuchang University, China.

Computational Section
Monte Carlo conformational searches were carried out by means of the Spartan′s 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g level in gas. The theoretical calculation of ECD was conducted in MeOH using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compound 2 and its isomers. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using  Table 3  Experimental data of compounds 3-15 can be found in Section 2.1.

Computational Section
Monte Carlo conformational searches were carried out by means of the Spartan's 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31+g level in gas. The theoretical calculation of ECD was conducted in MeOH using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compound 2 and its isomers. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

ABTS Radical Scavenging Activity
The ABTS radical scavenging assay was modified according to the method with slight modifications [50]. Briefly, the ABTS radical cation was obtained by mixing ABTS diammonium salt stock solution (7.4 mM) with potassium persulfate (2.6 mM) in equal proportion and reacting it at 37 • C in darkness for 12-16 h. Before used, the absorbance of light green ABTS radical test solution at 745 nm was controlled to be 0.70 ± 0.02 by diluting with methanol [51]. Sample solution and ABTS methanol solution (10 µL:190 µL) were added to 96-well microplate, and L-ascorbic acid was the positive control. After incubation at 37 • C for 10 min, the absorbance was tested at 745 nm using a microplate reader. Scavenging rate was calculated according to Equation (1).

ABTS radical scavenging activity (%) =
where A C and A S are the absorbance of the blank control and the compounds to be tested, respectively.

DPPH Radical Scavenging Activity
The scavenging ability on DPPH radical was conducted based on the method with some modifications [19]. The sample solution and DPPH methanol solution (20 µL:180 µL) were added to a 96-well microplate. L-ascorbic acid was the positive control. The absorbance at 517 nm was measured using a microplate reader after the solution had stood for 30 min at 37 • C under dark conditions. The calculation formula of DPPH radical scavenging activity is consistent with the formula of ABTS radical scavenging activity.

PTIO Radical Scavenging Activity
The PTIO radical scavenging activity was assayed by referring to relevant methods [52]. Briefly, PTIO radical solid (3 mg) was dissolved in 20 mL of methanol, and sample solution and PTIO methanol solution (40 µL:160 µL) were added to a 96-well microplate. The absorbance was determined at 585 nm using a microplate reader after 30 min of incubation and the scavenging rate was calculated on the basis of Equation (2).
where A S is the absorbance of the compounds to be tested and A C is the absorbance of the untreated control.

Tyrosinase Inhibitory Activity
The mushroom tyrosinase inhibitory activity was partially improved on the basis of reports [53,54]. Mushroom tyrosinase (400 U/mL) and L-tyrosine (3 mM) were added separately in 0.05 M potassium phosphate buffer (pH 6.5). A total of 80 µL of potassium phosphate buffer (pH 6.5), 80 µL of L-tyrosine (3 mM) solution, 20 µL of sample solution and 20 µL of mushroom tyrosinase (400 U/mL) were added to a 96-well microplate. The mixture reacted for 1 h at 37 • C. Kojic acid was selected as the positive control. The absorbance was measured at 490 nm using a microplate reader and Equation (3) was used to calculate the inhibition rate.

Tyrosinase inhibition activity (%) =
Equation (3) is the absorbance of the test compound and A C is the absorbance of the untreated control.

Acetylcholinesterase Inhibitory Activity
The experiment of acetylcholinesterase inhibition activity was slightly modified according to the literature [55]. Acetylcholinesterase (AChE) and acetylthiocholine iodide (ATCI) were dissolved in 0.1 M phosphate buffer (pH 8.0). 5,5 -Dithiobis-(2-nitrobenzoic acid) (DTNB) was prepared in 10 mL of 0.1 M phosphate buffer (pH 7.0) with a small amount of NaHCO 3 . A total of 120 µL of 0.1 M phosphate buffer (pH 8.0), 20 µL of 3 mM DTNB solution, 20 µL of sample solution and 20 µL of AChE (0.2 U/mL) were sequentially added to the 96-well microplate, and the mixture reacted for 10 min at 37 • C. The reaction was started by adding 20 µL of 3 mM ATCI and the mixture was incubated at 37 • C for 20 min. Donepezil was chosen as the positive control. The absorbance was tested at 412 nm and the inhibition rate was calculated based on Equation (4).
Acetylcholinesterase inhibition activity (%) = 1 − (A S -Aj) A C × 100 where A s and A j are the absorbance of the compound to be tested and tested compound blanks, respectively, and A C is the absorbance of the untreated control.