New flavonoid glycosides from seeds of Baccharoides anthelmintica

Abstract Baccharoides anthelmintica is the most popular traditional Uighur medicines used for vitiligo. The chemical investigation of the seeds of B. anthelmintica led to the isolation of three new flavonoid glycosides (Vernosides A-C). Their structures were determined by comprehensive analysis of spectroscopic data including 1D and 2D NMR and HRMS data. Vernosides A-C were evaluated for their effects on tyrosinase activity, Vernoside B can enhance tyrosinase activity. Graphical Abstract


Introduction
Traditional Uighur Medicine (TUM) has a history of more than 2,500 years, and very popular in Central Asia. TUM in Xinjiang has abundant experience in treatment of skin disease (Ma et al. 2014). Vitiligo is an acquired pigmentary disorder of unknown etiology that is clinically characterized by the development of white macules related to the selective loss of melanocytes (Alikhan et al. 2011). Extract of Baccharoides anthelmintica ( also named Vernonia anthelmintica) injection is the most popular TUM used for vitiligo (Niu et al. 2017). The pharmacological mechanisms of the injection in treatment of vitiligo has been reported Alam et al. 2010), However, the chemical constitutions of B. anthelmintica are rarely reported. Some flavonoids have been isolated from the plant (Tian et al. 2004;Yadava and Bhargava 2010) and they play important roles in this treatment (Cai et al. 2012). In order to find the active compounds from the seeds of B. anthelmintica, we initiated the fully study of the chemical constitution of B. anthelmintica. Herein, the isolation of three preciously undescribed flavonoid glycosides Vernosides A-C (1-3) ( Figure 1) has discussed. Their structures were elucidated by spectroscopic methods.

Results and discussion
Three preciously undescribed flavonoid glycosides Vernosides A-C (1-3) were isolated from the EtOAc-soluble fraction of the 95% alcohol extract of the powdered seeds of B. anthelmintica ( Figure S1). Their structures were identified by spectroscopic analysis including 1D and 2D NMR and HR-ESI-MS data.
Vernosides C (3) was obtained as a white amorphous powder. The molecular formula was established as C 29 H 30 O 11 on the basis of 13 C NMR data and HR-ESI-MS, requiring 15 degrees of unsaturation. Comparison of the 1 H NMR and 13 C NMR data of 2 and 3 revealed 3 was a 4-methyoxyflavan according to the HMBC correlation ( Figure  S2) of H-11 (d H 3.41) to C-4 (d C 73.3). The ring B of the flavan was a 1,2,4-trisubstitued ring according to the 1 H NMR data [d H 6.87 (d, J ¼ 1.9 Hz, H-2 0 ), 6.80 (d, J ¼ 8.1 Hz, H-5 0 ) and 6.75 (d, J ¼ 1.9, 8.1 Hz, H-6 0 )]. Thus, the structure of 3 was identified as trans- According to the traditional use of B. anthelmintica, these three compounds were evaluated for their effects on tyrosinase activity. Compound 2 significantly enhanced the activity of tyrosinase inhibitory activity in a dose-dependent manner. ( Figure S16). However, compounds 1 and 3 were inactive.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectrum was recorded on a Perkin-Elmer 577 spectrometer using KBr disks. One-dimensional and two-dimensional NMR experiments were performed at room temperature using a Bruker Ascend 500 MHz ( 1 H) and 125 MHz ( 13 C) spectrometer. HR-ESI-MS analyses were carried out on an Agilent 6224 TOF mass spectrometer with an ESI interface. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd.), C 18 reversed-phase (RP-18) silica gel (20-45 lm; Fuji Silysia Chemical Ltd.) were used for column chromatography (CC). Pre-coated silica gel GF 254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC. All solvents used for CC were of analytical grade (Shanghai Chemical Reagents Co., Ltd.).

Plant material
The seeds of Baccharoides anthelmintica were collected from Guangxi Province, People's Republic of China in September 2010, and authenticated by Prof. He-Ming Yang. A voucher specimen (SIMM709) was deposited at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, People's Republic of China.

Acid hydrolysis of 1-3
Compound 1-3 (5 mg each) were individually dissolved 1 N H 2 SO 4 (3 mL) and were heated at 95 C for 4 h. Ethyl acetate was used to extract the organic layer from each reaction mixture. Sugar unit was acquired from aqueous phase. The sugar parts from compounds 1-3 were compared to an authentic sugar sample by TLC GF 254 (Ethyl acetate-methanol-water-acetic acid, 13:3:3:4, R f 0.46 for glucose). Identification of D-glucose in each aqueous layer was carried out by comparing the optical rotation of the liberated glucose with that of an authentic sample of D-glucose ([a] 23 D þ52, c 0.02, H 2 O).

Tyrosinase activity assay
Tyrosinase activity was determined by spectrophotometry and was performed in 96-well plates (Quispe et al. 2017). First, 119 lL of 67 mM potassium phosphate buffer (pH 6.6), 1 lL of compounds were dissolved in dimethyl sulfoxide at the concentrations needed, and 40 lL of levodopa (2.5 mM) solutions were mixed. Then, 40 lL of mushroom tyrosinase solution (Sigma, St. Louis, USA) (100 unit/mL in phosphate buffer) was added to each mixture, final concentrations of compounds were 12.5-200 lM. The mixtures were then incubated at 25 C for 20 min before reading on a Thermo Fisher MK3 absorbance with 490 nm.

Conclusions
Three preciously undescribed flavonoid glycosides Vernosides A-C (1-3) was isolated from the seeds of B. anthelmintica, and were evaluated for their effects on tyrosinase activity. Vernoside B can enhance tyrosinase activity.