Three new phenylacetamide glycosides from Dracocephalum tanguticum Maxim and their anti-hyperglycemic activity

Abstract Three new phenylacetamide glycosides (1–3) together with one known phenylacetamide glycoside (4) and two known flavonoid glycosides (5–6) were isolated from whole plants of Dracocephalum tanguticum. The structure of all compounds were elucidated based on spectroscopic data analysis and comparison with data reported in related literature. Compounds (1–3) were evaluated for their anti-hyperglycemic and anti-fungal (Candida albicans) activities, the results revealed that all of them showed moderate activity with 3T3-L1 adipocytes glucose consumption rate of 20.80 ± 1.47%, 21.48 ± 2.44%, and 21.57 ± 1.35%, respectively at the final concentration of 25 μM. However, none of them showed obvious Candida albicans inhibitory activity. Graphical Abstract


Introduction
The genus Dracocephalum (Labiatae) includes about 60 species and is distributed all over the world. About 32 species and 7 varieties are found in China, which are distributed in the northeast, north, northwest and southwest (Wu and Li 1977) of China. The chemical constituents of this genus are complex which mainly contain flavonoids (Guo and Liu 1980), glycosides (Zeng et al. 2011), triterpenoids (Zheng et al. 2007), caffeic acid tetramer (Zhang et al. 2018), flavonoid glycoside (Yang et al. 2013), and diterpenoids (Uchiyama et al. 2003). Studies have shown that many obtained chemical constituents exhibited interesting biological properties, such as anti-oxidant, anti-hypoxic, and anti-HBV (Dastmalchi et al. 2007;Hai 2005;Shen et al. 2009).
Dracocephalum tanguticum, a perennial herb, is one of the commonly used herbs for Tibetan medicine. The aerial parts of the plant can be used for treatment of liver heat, hemostasis, and hemorrhoids (China Pharmacopoeia Committee 1995). In order to find compounds with diverse structures and high biological activity, three new phenylacetamide glycosides (1-3) along with one known phenylacetamide glycoside (4) and two known flavonoid glycosides (5-6) have been isolated from Dracocephalum tanguticum. Compounds (1-3) were evaluated for their anti-hyperglycemic activity. Herein, the isolation, structure elucidation, and biological activities of these compounds are described.
Compound 1 was obtained as an amorphous powder with a molecular formula of C 40 H 49 NO 17 , as determined by 13 C NMR data and an HRESIMS ion at m/z 838.2893 [M þ Na] þ (calcd 838.2893) with 17 indices of hydrogen deficiency. Its IR absorption peaks indicated the presence of the hydroxyl group (3420 cm À1 ), the conjugated carbonyl group (1712 cm À1 ), and the aromatic rings (1638,1509,1489, and 1450 cm À1 ). Its 13 C NMR and DEPT data (Table S1) 106.4, 105.7, and 98.3) suggested the presence of three sugar residues. Acid hydrolysis of compound 1 afforded D-glucose and L-rhamnose based on HPLC analysis of their chiral derivatives. The two D-glucose units were determined to have b-configurations from the 3 J H1,H2 coupling constants (7.6 and 7.7 Hz), and the a-configuration of L-rhamnose was established from the chemical shift of C-5 00 (d C 70. 1) (Lv et al. 2014). In addition, the molecule also includes a 1,4-disubstituted benzene ring and a monosubstituted benzene ring structural unit [d C 155.9, 135.8, 134.3, 132.6, 130.8 (2C), 129.6 (2C), 128.2 (2C), 117.5 (2C)]. Based on the above data and comparsion with reported literatures, it was confirmed that the compound was a phenylacetamide glycoside.
The HMBC correlations of H-2 (d H 7.16) with C-7 (d C 35.7), H-8 (d H 3.59) with C-7 0 (d C 170.3), and H-2 0 (d H 7.77) with C-7 0 (d C 170.3) determined the main structure of the compound. According to the H-1 00 (d H 5.60) correlation with C-4 (d C 155.9) in HMBC spectrum, it can be presumed that the C-1 00 substitute at the C-4 position; H-6 00 (d H 1.19) correlation with C-5 00 (d C 70.1) and H-1 00 correlation with C-5 00 revealed that the sugar attached to the benzene ring is a-L-rhamn-Pyranosyl group. H-2 00000 (d C 7.96) correlation with C-7 00000 (d C 167.8); H-6 0000 (d H 4.70, 4.40) correlation with C-7 00000 (d C 167.8) indicating that C-7 00000 was substituted at the C-1 00000 position of the phenyl ring, so that the benzoyl group is substituted at the C-6 0000 of the glucopyranosyl group. H-5 0000 (d H 3.76) correlates with C-6 0000 (d C 66.1) and C-1 0000 (d C 106.4), H-2 00 (d H 4.00) correlates with C-1 0000 , these correlations suggested that C-1 0000 was located at C-2 00 . The HMBC correlations of H-3 00 (d H 4.41) with C-1 000 (d C 105.7) suggested that C-1 000 was located at C-3 00 . Therefore, compound 1 was assigned as shown. Compound 1, named dratanguticumide A, was determined to be N- Compound 2 was obtained as an amorphous powder. The HRESIMS spectrum gave an [M þ Na] þ ion at m/z 640.2361(calcd 640.2364), which together with the 13 C NMR data indicated a molecular formula of C 31 H 39 NO 12 with 13 indices of hydrogen deficiency. Its IR absorption peaks indicated the presence of the hydroxyl group (3416 cm À1 ), the conjugated carbonyl group (1734 cm À1 ), and the aromatic rings (1655, 1616, 1509, and 1449 cm À1 ). Its 13 C NMR and DEPT data (Table S1) exhibited 31 carbon resonances including two methyl group (d C 21.2 and 17.9), three methylene groups (d C 62.3, 42.3, and 35.7), and 21 methines (11 of which are olefinic methines), five quaternary carbons (including two conjugated carbonyl carbons d C 172.6 and d C 168.6), The 1 H NMR spectrum (Table S1) displayed from 3.22 to 5.43 ppm (including two anomeric proton resonances d H 5.43 and d H 4.47) suggested the presence of two sugar residues. The sugar unit of compound 2 was composed of one a-L-rhamnosyl and one b-D-glucosyl, as determined from the acid hydrolysis experiment, the chemical shift of C-5 00 of rhamnose, and the coupling constants of the anomeric protons. The above data confirmed that the compound was a phenylacetamide glycoside.
The HMBC correlations of H-2 (d H 7.19) with C-7 (d C 35.7), of H-8 (d H 3.51) with C-9 0 (d C 168.6), of H-7 0 (d H 7.50) with C-9 0 , and of H-2 0 (d H 7.53) with C-7 0 (d C 141.7) determined the main structure of the compound. According to the HMBC correlations of H-4 00 (d H 5.14) with C-2 0000 (d C 172.6) and C-6 00 (d C 17.9) suggested that C-4 00 position was replaced by an acetyl group. Correlations of H-1 000 (d H 4.47) with C-2 00 (d C 79.7), it can be presumed that the glucose group was located at C-2 00 position. The H-7 0 and H-8 0 coupling constants as 15.8 Hz, it shows the double bond between the C-7 0 and C-8 0 was trans. Therefore, the structure of compound 2 was elucidated. Compound 2, named dratanguticumide B, was determined to be ( Compound 3 was obtained as an amorphous powder, Its molecular formula was established as C 29 H 39 NO 12 by HRESIMS (m/z 616.2364[M þ Na] þ ) (calcd 616.2364) with 11 indices of hydrogen deficiency. Comparisons of the 1 H and 13 C NMR data of 3 with those of 2 (Table S1) indicated that both compounds have identical skeletons, the difference is that double bond between the C-7 0 and C-8 0 in compound 2 was hydrogenated in compound 3, a hydroxyl group is substituted at the C-4 0 position, and the acetoxy group substituted at C-4 00 position disappeared. These conclusions were verified by two methylene groups in compound 3 replaced two methine groups in compound 2, and by the HMBC correlation H-3 0 (d H 6.70) with C-4 0 (d C 156.8). The structure of compound 3 was thus elucidated. Compound 3, named dratanguticumide C, was determined to be 3-(4-hydroxyphenyl)-N-phenethylpropanamide-4-O-[b-D-glucopyranosyl-(1!3)]-a-L-rhamnopyranoside.

General experimental procedures
1D NMR spectra were recorded on a Brucker AVANCE III 400 MHz spectrometer with TMS as the internal standard. 2D NMR spectra were recorded on a Brucker AVANCE III 500 MHz spectrometer with TMS as the internal standard, all chemical shifts (d) are expressed in ppm relative to the solvent signals. IR spectra were obtained on a Tenor 27 FT-IR spectrometer using KBr disks. HR-ESI-MS was performed on a Agilent 1290 UPLC/6540 Q-TOF spectrometer. UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer. Optical rotation were acquired with Jasco P-1020 polarimeter.
3.4. Acid hydrolysis and HPLC analysis of 1 À 3 Compounds 1 2 3 (2.0 mg each) were dissolved in 5.0 mL 2 M HCl(aq) and refluxed at 100 C for 4 h. The residue was extracted 3 times with EtOAc/H 2 O after dried in vacunnm. The H 2 O layer was dried under vacuum and redisolved in pyridine (1 mL) containing 1 mg/mL L-cysteine methyl ester hydrochloride. After stirred reaction at 60 C for 1 h, 2 lL o-Tolylisothiocyanate was added, and reaction was continued for another hour. After removal of the solvent, the residue was dissolved in MeOH and analyzed by RP-HPLC at 25 C, with isocratic elution of CH 3 CN:H 2 O:HCOOH ¼ 25:75:0.1(v/v) for 40 min and subsequent washing of the column with 100% CH 3 CN at a flow rate of 1 mL/min (Lv et al. 2014). The retention time of the acid hydrolysates derivative products of compounds 1 2 3 were compared with D-glucose and L-rhamnose (t R 15.680 min and t R 26.996 min) which were derivatizated in the same manner. It was confirmed that both the monosaccharide configuration of compounds 1 2 3 were D-glucose and L-rhamnose.

Anti-hyperglycemia and anti-fungal (Candida albicans) assay
In order to evaluate the anti-hyperglycemic activity of the new compounds, glucose consumption in 3T3-L1 adipocytes with glucose oxidase-peroxidase (GOD-POD) procedure was determined followed by the GOD-POD method previously described (Zhu et al. 2010). The experiment procedures were as follows: The compounds (1-3) were dissolved in DMSO and diluted with DMEM medium to 10 mmol/L. 3T3-L1 preadipocytes were cultured and passaged in DMEM medium containing 25 mmol/L glucose and 10% fetal bovine serum (FBS) at 37 C, a volume fraction of 5% CO 2 and saturated humidity. 2-day postconfluent cells were placed in 10% FBS-DMEM containing 250 nmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine and 1 lg/mL insulin. After 2 days, the medium was changed to 10% FBS-DMEM containing only 1 lg/ mL of insulin, and then replaced with 10% FBS-DMEM after 2 days (Reed and Lane 1980). Thereafter, the medium was changed every 2 days until induction of differentiation into mature adipocytes. After differentiation of 3T3-L1 cells into adipocytes, the cells were digested and inoculated into 96-well plates and cultured overnight. After washing the cells once with a low-sugar medium (glucose concentration: 1800 mg/L), and then 200 lL low sugar medium of containing different drugs was added. Blank control group (DMSO group), positive control group (0.1 lM insulin), and the sample group to be tested were incubated with the final concentration of 25 lM. Three repeating holes were set for each group sample. After incubation for 24 hours, 10 lL of supernatant medium was aspirated, and the concentration of glucose in the medium was measured by glucose oxidase-peroxidase (GOD-POD) method. The glucose consumption rate of the active compound was calculated and compared.
The compounds (1-3) were dissolved in DMSO and diluted into 20 mg/mL, the Candida albicans broth was added to a 96-well culture plate at a final concentration of 1 Â 105 CFU/mL, cultured at 37 C for 24 h. The OD value at 625 nm was measured by microplate reader. At the same time, a medium blank control and an amphotericin B positive drug control were set. The inhibition rate of compounds was calculated against Candida albicans.