Iridoid glycosides from Callicarpa nudiflora Hook

Abstract Four new iridoid glycoside derivatives (1–4), along with ten known iridoid glycosides (5–14), were isolated from Callicarpa nudiflora Hook et Arn. The structure of the new iridoid glycosides was elucidated as 5″-methoxy-ampicoside (1), 6″-O-trans-caffeoylcatalpol (2), 6″-O-trans-feruloylcatalpol (3) and 3″-methoxy-agnucastoside C (4) on the basis of spectroscopic analysis. Compounds 1–11 were reported from this plant for the first time. The cytotoxic activity of the isolated compounds against human cervical carcinoma Hela cells and ovarian carcinoma HeyA8 cells was evaluated using the microculture tetrazolium assay. Compounds 4, 5, 8, 12 and 13 showed cytotoxic activity against the Hela cell line with IC50 values of 25.3, 48.1, 17.3, 38.3 and 28.2 μM, respectively. While only compound 8 showed cytotoxicity against the HeyA8 cell line, with an IC50 of 35.5 μM


Introduction
Callicarpa nudiflora Hook et Arn., belonging to the family of Verbenaceae, is widely distributed from southern China to Malaysia (Dong et al. 2014). This plant has been used as folk medicine in China for eliminating stasis to subdue swelling, treating respiratory tract infections, hepatitis, antiphlogosis haemostasis and memory promote function. Phytochemical investigations of this plant led to the isolation of diterpenoids, triterpenoids, flavonoids, phenylpropanoid glycosides and phenolic acids (Liang et al. 2011;Luo et al. 2014;Hjørnevik et al. 2015). However, cytotoxicity studies on iridoids have rarely been reported.
Moreover, the 1 H nMR spectrum displayed signals for two aromatic protons at δ H 7.25 (2H, s, H-2″,6″), indicated the presence of a 1,3,4,5-tetrasubstituted benzene ring. And the two methoxyl groups at δ H 3.83 (6H, s, 3″ and 5″-OCH 3 ) corresponding to δ C 56.6 was also observed on the bases of HSQC spectrum. In the HMBC spectrum (Figure 2), the correlations from OCH 3 to C-3″ and C-5″ suggested that the two methoxyl groups were linked to C-3″ and C-5″, respectively. Besides, a signal for carbonyl at δ C 166.1 (C-7″) was presented in the 13 C nMR. The further correlation signals from H-2″ and H-6″ to C-7″ observed in HMBC spectrum suggested the carbonyl is located at C-1″. Meanwhile, in the HMBC spectrum, the correlation from H-6 to C-7″ confirmed that carbonyl connects with the C-6 position of catalpol through ester linkage. Thus, the structure of 1 was similar to the iridoid glycoside ampicoside except for the presence of methoxyl group at C-5″ (Wang et al. 1993). Concluded all the information above, the structure of 1 was established as 5″-methoxy-ampicoside.
Compound 2  in the eSI-MS spectrum. The 1 H and 13 C nMR spectroscopic data of 2 and comparison with spectra of known compounds showed that 2 was again an ester of catalpol. The chemical shifts for the 6′-CH 2 group at δ H 4.51 (1H, dd, Figure 2. the hMBc correlations of compounds 1-4. J = 12.0 and 1.6 Hz, H-6′a) and 4.44 (1H, dd, J = 12.0 and 5.6 Hz, H-6′b) showed that this was the position of esterification. The remaining signals in the spectrum were typical for a caffeoyl group, and the connection to the catalpol moiety was confirmed by the correlation from H-6′b to C-9″ in the HMBC spectrum. Therefore, the structure of 2 was deduced as 6″-O-trans-caffeoyl-catalpol.
Compound 3, a white amorphous powder, was assigned as C 25 H 30 O 13 on the basis of its HR-eSI-MS data at m/z 561.1596 [M + na] + (Calcd for C 25 H 30 naO 13 : 561.1579) and its eSI-MS data m/z 561.2 [M + na] + and m/z 1075.3 [2M − H] − . It was apparent from the nMR spectroscopic data ( 1 H, 13 C, COSY, HSQC, HMBC and nOeSY) that this compound differed from 2 only for an additional methoxyl group. In the 13 C nMR spectrum, the C-atom signal at δ C 149.4 (C-3″) significantly shift to low field compared with 2 (δ C 145.9) and the C-atom signal at δ C 56.5 was presented, which indicated that the methoxyl group instead of the hydroxyl group in compound 3. In addition, the correlation signal between CH 3 O-3″ at δ H 3.90 and C-3″ was observed in HMBC spectrum, which suggested the methoxy group is located at C-3″. So, the structure of 3 was deduced as 6″-O-trans-feruloylcatalpol.
Compound 4  Analysis of the 1 H and 13 C nMR data indicated compound 4 consisted of an 8-epiloganic acid skeleton, a caffeoyl group, a cinnamyl group and a glucose moiety fragment. Combined the 1 H nMR, 13 C nMR and HMBC spectra, the correlations from H-1′ at δ H 4.72 (1H, d, J = 8.0 Hz) to C-1 at δ C 96.2 and H-7 at δ H 4.91 to C-9′′′ at δ C 169.0 deduced the β-d glucopyranosyl unit and the cinnamyl ester group are located to C-1 and C-6 of the aglycone, respectively. The correlation from H-6' at δ H 4.54 and 4.42 to C-9″ at δ C 169.1 indicated that the caffeoyl ester group is attached to C-6″ of the glucoside. The above information together constructed an agnucastoside C structure of 4. Meanwhile, detailed nMR analysis showed an additional methoxyl group in 4 at δ H 3.83 (3H, s, OCH 3 ) and δ C 56.5. The location of the methoxyl group was at C-3″, confirming from the significant correlation peaks from the methoxyl proton to C-3″ in the HMBC spectrum [ Figure 2]. The relative configuration of 4 was established by a combination of the 1 H nMR spectrum, nOeSY experiment and the literature report (Liang et al. 2011). The presence of nOe interactions of H-1, H-7 and H-10 indicated that the hydroxyl group at C-7 should be β-orientation. Thus, the structure of 4 was elucidated as 3″-methoxy-agnucastoside C.
Besides the four new iridoid glycosides, ten known iridoid glycosides were also isolated from this plant. Their structure was identified by comparing the nMR properties with the reported data.
The cytotoxicity on Hela and HeyA8 cell lines in vitro of compounds 1-14 was determined using the microculture tetrazolium (MTT) assay, with taxtol as positive control (Table 1) 1-3, 6, 7, 9-11 and 13), demonstrated that the agnucastoside C skeleton with an COOH substitution at C-4 plays an important role in the cytotoxicity against Hela cancer cell line, and the presence of caffeoyl group and cinnamyl group promotes the cytotoxicity of the iridoid glycosides.

General experimental procedures
Optical rotations were measured with a Perkinelmer 341 polarimeter with MeOH as solvent. The UV spectra were performed on a Thermo Scientific evolution 300 UV-visible spectrophotometer in MeOH. The 1 H (500 MHz), 13 C (125 MHz) and 2D nMR spectra were recorded on a Bruker DRX-500 instrument using TMS as internal standard. eSI-MS were collected on a MDS SCIeX API 2000 LC/gC/MS instrument. HR-eSI-MS were carried out on a Bruker Bio-TOF-IIIQ mass spectrometer. Preparative HPLC was run with a Waters 1525 pump and a Waters 2489 ultraviolet-visible detector using an Xterra® prep MS C18 column (10 μm, 7.8 × 150 mm).

Plant material
The aerial part of C. nudiflora was collected from Five Finger Mountain of Hainan Province, People's Republic of China, in April 2013. The plant was identified by BintaoLi of Shenzhen Fairy Lake Botanical garden, Chinese Academy of Sciences. A voucher specimen (no. 20130916) has been deposited in the herbarium of Shenzhen Fairylake Botancial garden, Chinese Academy of Sciences.

GC analysis of the sugar moieties in 1-4
Compounds 1-4 (each 4 mg) in 1 M HCl (5 mL, dioxane-H 2 O 1:1, v/v) were heated at 95 °C for 6 h, respectively. The reaction mixtures were evaporated in vacuo. The residues were dissolved in water and then extracted with CHCl 3 for three times, respectively. After evaporating the aqueous phases to dryness in vacuo, the residues were dissolved in pyridine (5 mL) and 1-(trimethylsilyl)-imidazole (0.5 mL) at room temperature for 30 min. The reaction mixtures were dried with a stream of n 2 . The residues were partitioned between CHCl 3 and H 2 O. The organic layers were subjected to gC analysis using an L-Chirasil-Val column (0.32 mm × 25 m) [14]. Temperature of the injector and detector was 200 °C. A temperature gradient system was used for the oven; the initial temperature was maintained at 100 °C for 1 min and then increased up to 180 °C at a rate of 5 °C/min. Peaks of the hydrolysate of 1-4 were, respectively, detected at 14.56, 14.57, 14.55 and 14.57 min, suggested that all the sugar moieties of 1-4 are d-glucose. Retention times for authentic samples of d-glucose (Sigma-Aldrich, St. Louis, MO, USA) after being treated in the same manner with 1-(trimethylsilyl)-imidazole in pyridine were detected at 14.56 min.

Cytotoxicity assay
Hela and HeyA8 cell lines (purchased from Shanghai Institutes for Biological Science, Chinese Academy of Sciences) were cultured in DMeM (gibco BRL, grand Island, nY, USA) supplemented with 10% foetal bovine serum (FBS) (gibco BRL, grand Island, nY, USA). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 . Cytotoxicity was measured by a MTT assay with minor modification to the reference procedure (Mosmann 1983). Briefly, cells were seeded in 96-well microculture plates and cultured for 24 h and then various concentration of tested drug was added, with taxtol (Sigma) as positive control. After the incubation for another 48 h, 20 μL of MTT (5 mg/mL) was added to each well, and the incubation continued for 4 h at 37 °C. After 4 h, 100 μL of DMSO was added to each well to dissolve the formazan crystals of the viable cells. The plates were read at a wavelength of 590 nm using microplate reader. IC 50 values were calculated using SPSS 17.0 statistical analysis software.

Disclosure statement
no potential conflict of interest was reported by the authors.