t i l Identification of Ten New N -acetyldopamine Dimers from Periostracum Cicadae

Periostracum Cicadae is the cast-off shell of the cicada Cryptotympana pustulata Fabricius, it is a widely used animal based traditional folk medicine, it is found to have many effects including antipyretic, antiallergic and antioxidant activities. In this study, ten N - acetyldopamine dimers, named Cicadamide C1–C10 (compounds 1–10 ), were isolated from Periostracum Cicadae. One-dimensional NMR, two-dimensional NMR, mass spectrometry, CD spectroscopy, and chemical evidence were performed to further determine their structures. In the results, ten N -acetyldopamine dimers were isolated and their structures were elucidated. This study provides a basic reference for further biological effects study on Periostracum Cicadae.


INTRODUCTION
P eriostracum Cicadae is a well-known animal based traditional folk medicine. It is the cast-off shell of Cryptotympana pustulata Fabricius, commonly known as the black cicada, which is mainly distributed in Shandong, Henan, Hubei, and Sichuan Provinces of China. In traditional Chinese medicinal practice, Periostracum Cicadae, is considered to be cold-natured and have a sweet flavor, and is used for its therapeutic effect against vitiligo (Zhang and Che, 2004), anti-type IV allergic activity (Lin et al., 2001), an inhibitory effect on diabetic retinopathy (Xing, 2010), and anticonvulsant activity (An, 2008).
The clinical efficacy of Periostracum Cicadae is a consequence of its chemical constituents. Its crude extract has been reported to exhibit a variety of biological activities when it was administered by various routes. Hsieh et al. (1991) demonstrated that the water extract of Periostracum Cicadae had anticonvulsitant, sedative and hypothermic effects through pharmacological research. Shin et al. (1999) summarized the effects of oriental medicines including one from Cryptotympana on the systemic anaphylactic reactions induced by compound 48/80, and demonstrated that Cryptotympana atrata could significantly inhibit the rate of mast cells degranulation and systemic anaphylactic reaction, indicating that it may be beneficial to treat nonspecific anaphylaxis. Yang et al. (2013) explored the method of analyzing trace elements from Periostracum Cicadae. Liu et al. (2004) researched the effects of Periostracum Cicadae water extract on hemorheology in hyperlipidemic rats. The results showed that Periostracum Cicadae could significantly improve its hemorheology, which was reflected in the significant reduction of whole blood and plasma viscosity, thrombosis in vitro, erythrocyte aggregation index, serum triglyceride and total cholesterol levels. Wang et al. (2010) used

O n l i n e F i r s t A r t i c l e
different extraction methods and solvents to preliminarily isolate and study the antibacterial activity of the active ingredients of Periostracum Cicadae. It was found that the extracts obtained by different extraction methods had obvious antibacterial effects, but the differences between them were not significant, which showed that the active ingredients of Periostracum Cicadae had strong antibacterial activities. Previous reports on its biological components have revealed that it is rich in dopamine (Noda et al., 2000;Yang et al., 2016;Liu et al., 2019). Oxenkrug and Requintina (2005) studied the effect of N-acetyldopamine on lipopolysaccharide (LPS) induced lipid peroxidation in the form of malondialdehyde (MDA) by measuring the thiobarbituric acid (TBA) reactive substances in rat brain homogenates in vitro, and found that N-acetyldopamine inhibited the formation of MDA in a concentration dependent manner and its effect was stronger than that of melatonin. Xu et al. (2006) isolated two N-acetyldopamine dimers from the methanolic extracts of Periostracum Cicadae and showed that they both exhibited antioxidant and anti-inflammatory activities in LPS induced RAW264.7 cells. Lu et al. (2015) identified three new N-acetyldopamine dimers from Dung Beetle Catharsius molossus, a similar traditional Chinese Medicine from insects.
In this study, we further investigated the phytochemistry of Periostracum Cicadae, with the aim of identifying the previously unknown phthalides with biologically activity from this folk medicine, the structures of new identified compounds were established using spectroscopic methods. In the result, 10 new compounds (1-10) were isolated from Periostracum Cicadae (Fig. 1). Herein, we describe the isolation and structural elucidation of compounds 1-10.

Materials
The dried Periostracum Cicadae in this study was purchased from a traditional medicine market in Urumuqi, Xinjiang, China. It was identified by Prof. Jincai Lu from School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. The sample of Periostracum Cicadae was further deposited at Research Department of Natural Medicine of Shenyang Pharmaceutical University, with a voucher specimen (No. 20081001). The others relevant chemical reagents were analytical pure.

General experimental procedures
HR-ESI-MS was performed on a waters LCT Premier KE399 mass spectrometer (Waters Corp., Milford, MA, USA). A model MOS-450 Chiral Detector (Bio-Logic SAS, Caix, France) was used for CD analysis. The one and two-dimensional NMR spectra were recorded in CD3OD on a Bruker AV-600 spectrometer (1H, 600 MHz; 13C, 150 MHz) (Bruker Corp., Billerica, MA, USA) using tetramethylsilane (TMS) as the internal standard. Preparative HPLC was carried out using a waters 2998 photodiode array detector at 220 nm with a waters 2695 separation module (Waters Corp., Billerica, MA, USA) and a Shim-pack CLC-ODS reversed-phase column (No. 61514407B; Shimadzu Corp., Kyoto, Japan). Silica gel for chromatography was obtained from Oceanview Chemical Group Co. Ltd. (Qingdao, China).

Extraction and isolation
The powder of dried Periostracum Cicadae (5 kg) was extracted with EtOH (50 L) for 3 times under reflux conditions, each time for 3 h. The combined EtOH extracts (76 g) were concentrated in vacuo. A part of the EtOH fraction (70 g) was subjected to silica gel column chromatography (250 g) with a gradient of CHCl 3 /MeOH to afford 14 fractions (100:0-0:100) that were designated A-O.

Ten isolated compounds
As a result of our investigation, 10 new compounds (1-10) were isolated from Periostracum Cicadae (Fig. 1). NMR and CD spectra (Figs. 2 and 3) were used to further identify the structures of Compounds 1-10.

O n l i n e F i r s t A r t i c l e
Ten N-acetyldopamine dimers from Periostracum Cicadae Fig. 3. CD spectral data of compounds 1-10. Note: After spectrum analysis based on 13 C-NMR, 1 H-NMR and HMQC data, compound 5 and compound 3 were found to be isomers, so the CT scan images of compound 5 is not given here.

Structure elucidation of the compounds
Compound 1 was obtained as a white powder. The molecular formula of 1 was established as C 30 H 31 N 3 O 9 on the basis of its HR-ESI-MS data (m/z 578.2160 [M+H] + ; calcd. 578.2139 for C 30 H 32 N 3 O 9 ). The UV spectrum of 1 exhibited λ max at 280.6 nm (MeOH).

CONCLUSION
In this study, the previously unknown phthalides with biologically activity from Periostracum Cicadae were identified. The structures of new identified compounds were established using spectroscopic methods. In the result, 10 new compounds (1-10) were isolated, the structural elucidation of compounds 1-10 were described. This study aims to provide a reference for the further functional research and the development and utilization of Periostracum Cicadae.

Funding
This work was supported by the National Natural Science Foundation of China (No. 81860725).

Supplementary material
There is supplementary material associated with this article. Access the material online at: https://dx.doi. org/10.17582/journal.pjz/20210513050500