Flavonoids with antioxidant and tyrosinase inhibitory activity from corn silk (Stigma maydis)

Abstract Corn silk (Stigma maydis), being the styles and stigmas of maize, is a famous traditional medicine and functional tea in China. Research into the chemical composition of corn silk led to the identification of an unreported flavone (1, silkone A), accompanying with three known flavonoids (2–4). And their structures were elucidated through comprehensive spectroscopic analysis. Each obtained compound was evaluated for antioxidant capacity by DPPH, ABTS and FRAP assays. As a result, all tested compounds exhibited stronger radicals scavenging activities than Trolox in ABTS radical assay and displayed relatively weak antioxidant capacity in the other two experiments. Tyrosinase inhibitory activities of compounds 1–4 were also investigated, and compounds 3 and 4 demonstrated moderate inhibitory activities to tyrosinase with IC50 values of 0.49 and 0.21 mM, respectively, which was further investigated through molecular docking calculation. These results may contribute to the development of novel antioxidants and tyrosinase inhibitors from corn silk. Graphical Abstract


Introduction
Maize (Zea mays Linn.), which belongs to the Gramineae family, is extensively cultivated around the world and has been a crop of important economic value (Song et al. 2020). Corn silk (Stigma maydis), being the styles and stigmas of maize, is widely consumed as a traditional medicine and functional tea in China, due to its therapeutic effects on edema, hypertension, cystitis and similar ailments (Guo et al. 2018).
Pharmacological study and clinical practice demonstrated that its extract possesses various biological activities, such as antioxidant activity, neuroprotective activity and anti-inflammatory activity (Zhou et al. 2021). Phytochemical research of corn silk revealed the presence of flavonoids, sesquiterpenes and diterpenes (Qi et al. 2018).
Aiming at exploring bioactive components from corn silk, phytochemical investigation on this herbal medicine was carried out, which resulted in the isolation of an unreported flavone (1), along with three known flavonoids (2-4). Considering that flavonoids are important natural antioxidants (Berczy nski et al. 2020), DPPH, ABTS free radical scavenging assays and FRAP assay (Gandia et al. 2009;Sugiyama et al. 2009;Dai et al. 2015) were conducted to comprehensively evaluated the in vitro antioxidant capacity of isolated compounds. Since the elimination of free radicals by antioxidants is a key element to the inhibition of tyrosinase (EC 1.14.18.1, a crucial enzyme involved in the melanin biosynthesis) (Choi et al. 2021), compounds 1-4 were also assessed for their tyrosinase inhibitory activities and molecular docking calculation was performed to probe the interactions between the tested compounds and the residues in the active site of tyrosinase (Ferro et al. 2018).

Results and discussion
Compound 1, being a yellow powder, was determined to possess a formula of C 19  These spectral features manifested that compound 1 might be a flavone. Detailed HMBC analysis ( Figure S6) revealed the key correlations of H-3 with C-2, C-4, C-10 and C-1 0 , of H-8 with C-6, C-7, C-9 and C-10, of H-2 0 with C-1 0 , C-4 0 , C-6 0 and C-2, and of H-6 0 with C-4 0 , which indicated that compound 1 possessed a flavone skeleton. Besides, the correlations from 2 00 -OCH 3 and H-1 00 to C-2 00 delineated the presence of a methyl ester moiety and the position of the moiety was certified to locate at C-6 by the correlations from H-1 00 to C-5, C-6 and C-7. Furthermore, HMBC cross coupling between 3 0 -OCH 3 and C-3 0 indicated the attachment of a methoxyl at C-3 0 , as expected. In the light of these observations, 1 was elucidated to be a new flavone (Figure 1), and it was endowed the trivial name silkone A.
Tyrosinase inhibitory activities of isolated compounds were determined by adopting mushroom tyrosinase as the enzyme and L-tyrosine as the substrate (Dai et al. 2006). During the experiment, compounds 3 and 4 exhibited tyrosinase inhibitory effects with IC 50 values of 0.49 and 0.21 mM, respectively (0.15 mM for arbutin). And the IC 50 values of compounds 1 and 2 were found to be greater than 1 mM. These suggested that compounds 3 and 4 demonstrated moderate tyrosinase inhibitory effects, while compounds 1 and 2 showed weak activities (Table S2).
To study the binding interactions between the active compounds (3 and 4) and the residues in the active site of tyrosinase (PDB ID: 2Y9X), molecular docking calculation was conducted using Schr€ odinger software suite (Ferro et al. 2018;Somaia et al. 2018). As a result ( Figure S10), the positive control arbutin formed six hydrogen bonds with His 85, Glu 322, His 244 and Ala 323. While the less active compound 4 (IC 50 : 0.21 mM) followed by the same skeleton compound 3 (IC 50 : 0.49 mM) were surrounded by more hydrophobic amino acids, and only formed two hydrogen bonds with Cys 83/Asn 81 and His 244/Ala 323, respectively. It is speculated that hydrogen bonding interactions between ligands and the residues in the active site may play a crucial role in inhibiting the tyrosinase activity.

Conclusions
In conclusion, an unreported flavone together with three known flavonoids were obtained from corn silk. The structures of isolated compounds were elucidated by extensive spectroscopic data. All isolates were investigated for their in vitro antioxidant activity by three different methods. In ABTS assay, all compounds demonstrated stronger free radical scavenging capacity than Trolox. In addition, compounds 3 and 4 also showed moderate tyrosinase inhibition effects. A molecular docking study revealed that hydrogen bonding interactions may be important for tyrosinase inhibitory potency. This study proved the potential of isolated flavonoids in antioxidation and tyrosinase inhibition and afford the possibility of developing promising natural antioxidant and anti-melanin products from corn silk.

Disclosure statement
The authors report there are no competing interests to declare.

Funding
This work was supported by Science and Technology Planning Project of Liaoning Province (2021JH1/10400049).