HPLC-ESI-MSn Identification and NMR Characterization of Glucosyloxybenzyl 2R-Benzylmalate Deriva-Tives from Arundina Graminifolia and Their Anti-Liver Fibrotic Effects In Vitro

Four new glucosyloxybenzyl 2R-benzylmalate derivatives, named Arundinoside H (2), I (5), J (6), K (8) as well as four known compounds Arundinoside D (1), G (3), F (4), E (7) were isolated and characterized by a combination of chemical and spectroscopic methods, including HR-ESI-MS, 1D and 2D NMR experiments. Besides, 24 unreported compounds were inferred from ESI-MSn data. The anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of lipopolysaccharide (LPS)-induced activation of rat hepatic stellate cells (HSC-T6). The result suggested Arundinosides D, H, F, I and K showed moderate inhibitory effects in vitro.


Introduction
Arundina graminifolia (D. Don) Hochr., a species widely distributed in subtropical Asia and known as bai-yang-jie in Chinese, has a long history of use as one of the major drugs in a formula "BaoGan Capusle" with the efficacy of heat clearing and detoxifying, dispersing blood and relieving pain, reducing inflammation and promoting urination and so on [1]. Previous phytochemical investigation focusing on the chloroform and ethyl acetate exacts of A. graminifolia had resulted in the separation of stilbenoids [2][3][4], phenols [5][6][7], flavonoids [8,9] and other ketones [3,10,11]. However, the works on the polar parts of the plant are few.
In the course of our studies on pharmacology, it was proved that the formula "BaoGan Capusle" was effective in the treatment of hepatic fibrosis and liver injury of model rat [12][13][14][15]. As a continuing study on bioactive constituents of A. graminifolia, a series of phytochemical and biological experiments of the n-butanol (n-BuOH) extract was thus performed to yield the isolation of four new and four known glucosyloxybenzyl 2R-benzylmalates. In this paper, we described the isolation and structural elucidation of these derivatives, as well as their anti-liver fibrotic activities in vitro. Furthermore, the fragmentation pathways of eight isolates were studied in positive ESI-MS n , and then 24 unreported glucosyloxybenzyl 2R-benzylmalate derivatives were predicted by HPLC-ESI-MS n .
In 1 H and 13   The molecular formula of compound 5 was determined to be C49H60O25 based on the HR-ESI-MS ion [M + NH4] + at m/z 1066.3766. 1 H and 13 C NMR data of compound 5 indicated that it was a glucosyloxybenzyl 2R-benzylmalate derivative with three acetyl groups as the same as compound 6, but one group substituted position was different. The structure of compound 5 was further confirmed by HSQC and HMBC experiments. The substituent positions of three acetyl groups were determined at C-2′′′′′′, C-4′′′′′′ and C-6′′′′′′ according to HMBC correlations from  The molecular formula of compound 5 was determined to be C 49 H 60 O 25 based on the HR-ESI-MS ion [M + NH 4 ] + at m/z 1066.3766. 1 H and 13 C NMR data of compound 5 indicated that it was a glucosyloxybenzyl 2R-benzylmalate derivative with three acetyl groups as the same as compound 6, but one group substituted position was different. The structure of compound 5 was further confirmed by HSQC and HMBC experiments. The substituent positions of three acetyl groups were determined at C-2 , C-4 and C- ) to 170.1 suggested that four acetyl groups of compound 8 substituted at C-6 , C-2 , C-3 , C-6 , respectively. Therefore, compound 8 was identified as 1-(β-D-glucopyranosyloxybenzyl-6 -acetyl)-2-(β-D-glucopyranosyl-2 ,3 ,6 -triacetyl)-4-(β-D-glucopyranosyloxybenzyl)-2R-benzylmalate, and named Arundinoside K.

MS Fragmentation Pattern
HPLC-ESI-MS n experiments were carried out to analysis structural characterization and discuss the fragmentation behaviors of glucosyloxybenzyl 2R-benzylmalates 1-8 from A. graminifolia. The target glucosyloxybenzyl 2R-benzylmalates recorded at retention times were designed as A1-A6, B1-B6, C1-C3, D1-D6. The positive ion mode was performed on each of these components, and ESI-MS n data were summarized in Tables 3 and 4.     Figure 4 showed the proposed fragmentation pathway of compound 6 [20]. The same rules were found in the MS n analysis of other isolates listed in Table 3.
The  (Figure 5b,c). Then, the ion at m/z 329 obtained by loss of C11H10O4 (206 Da) from m/z 535, combining the ion at m/z 247 (Figure 5d), indicated the presence of 2-benzyl-malic acid moiety in A8. Based on its fragmentation behaviors and previous studies, A8 was inferred to be the structure shown in Figure  6. Moreover, the mass spectra of A5 showed the same molecular formula and similar fragmentation ions with A8, but the retention time on HPLC with the same conditions were different, which indicated A5 was an isomer of A8. The succession of neutral loss of 310 Da and 268 Da obtained from the ion at m/z 845 produced by precursor ion at m/z 1113 suggested one of Ac groups in A5 was located at G2, and not at G1. The same experimental procedures were applied to analyze other

Structural Prediction of Glucosyloxybenzyl 2R-Benzylmalates Unreported
We also examined unknown glucosyloxybenzyl 2R-benzylmalate derivatives in fractions I~VI of n-BuOH extract with CH 3 CN-H 2 O (32:68, v/v) for mass spectrometry analysis. In the MS n spectra, similar fragmentation pathways as described above were observed, and the possible structures of chemical components A1-A6, B1-B6, C1-C3, D1-D6 were inferred (Table 4). Herein, the analytic procedures were explained by peak A8 and A5.
The  (Figure 5b,c). Then, the ion at m/z 329 obtained by loss of C 11 H 10 O 4 (206 Da) from m/z 535, combining the ion at m/z 247 (Figure 5d), indicated the presence of 2-benzyl-malic acid moiety in A8. Based on its fragmentation behaviors and previous studies, A8 was inferred to be the structure shown in Figure 6. Moreover, the mass spectra of A5 showed the same molecular formula and similar fragmentation ions with A8, but the retention time on HPLC with the same conditions were different, which indicated A5 was an isomer of A8. The succession of neutral loss of 310 Da and 268 Da obtained from the ion at m/z 845 produced by precursor ion at m/z 1113 suggested one of Ac groups in A5 was located at G2, and not at G1. The same experimental procedures were applied to analyze other molecules list in Table 4, and the main fragments observed in MS n spectra of the [M + Na] + precursor ions were summarized.

Anti-hepatic Fibrosis Activity
Emerging studies indicated that HSC in resting state could be induced to activated state by LPS [21], while the inhibition of proliferation of activated HSC, has been considered as an effective target for liver fibrosis [22]. In addition, Considering the bioactive results obtained for the "BaoGan capsule" in our previous work, the anti-hepatic fibrosis activities of the isolates 1-8 were tested on the proliferation of LPS-activated HSC-T6 cells in vitro by MTS method. Legalon (silymarin capsules) was taken as a positive control. As shown in Figure 7, compound 1, 2, 4, 5, 8 were exhibited moderate anti-proliferative activity with significantly different values while the concentration was 100 µg/mL, while positive control showed a significant difference at 50 µg/mL.

Anti-hepatic Fibrosis Activity
Emerging studies indicated that HSC in resting state could be induced to activated state by LPS [21], while the inhibition of proliferation of activated HSC, has been considered as an effective target for liver fibrosis [22]. In addition, Considering the bioactive results obtained for the "BaoGan capsule" in our previous work, the anti-hepatic fibrosis activities of the isolates 1-8 were tested on the proliferation of LPS-activated HSC-T6 cells in vitro by MTS method. Legalon (silymarin capsules) was taken as a positive control. As shown in Figure 7, compound 1, 2, 4, 5, 8 were exhibited moderate anti-proliferative activity with significantly different values while the concentration was 100 μg/mL, while positive control showed a significant difference at 50 μg/mL.

Plant Material
The whole plant of A. graminifolia was bought from Dai hospital of Xishuanbanna Autonomous Prefecture, Yunnan Province, China. A voucher specimen (batch number: 20111128) was collected in the laboratory.

Plant Material
The whole plant of A. graminifolia was bought from Dai hospital of Xishuanbanna Autonomous Prefecture, Yunnan Province, China. A voucher specimen (batch number: 20111128) was collected in the laboratory.

In vitro Evaluation of Anti-Liver Fibrotic Activity
HSC-T6 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator with constant temperature at 37 • C and a humidified atmosphere of 5% CO 2 . The cells were trypsinized and passaged to new plates every two or three days. HSC-T6 cells were seeded in 96-well plates (5 × 10 3 /100 µL) for 24 h to ensure fully adhesion and good condition. The cell medium in the wells was changed into fresh RPMI-1640 medium supplemented with 5% fetal bovine serum for further incubation. Cells incubated with LPS (1 µg/mL) in the different concentration of compounds 1~8 (0, 5, 50, 100, 300 µg/mL) were cultivated for another 24 h. Each group was provided with 6 independent duplicates. Cell viability was determined using MTS/PMS assay. Absorbance values were read at 490 nm on an ELISA reader. 0.1% DMSO was considered as blank control and legalon (silymarin capsules) as positive control. Cell viability was expressed as a percentage of control cells at 100% viability. Statistical analysis was performed using origin Pro 8.0 (OriginLab Corpration, One Roundhouse Plaza, Northampton, MA, USA).

Conclusions
Glucosyloxybenzyl 2R-benzylmalates are a class of naturally occurring substances distributed in Orchidaceae. They were noticed for their novel type of structure and significant activities, while the research of glucosyloxybenzyl 2R-benzylmalates was limited by their higher polarity and less content. In present work, basis on the information acquired from HPLC-ESI-MS n experiment combined with NMR analysis, it was possible not only to identify 8 compounds isolated from A. graminifolia, but also to predict the structures of 24 previously unreported glucosyloxybenzyl 2R-benzylmalates in the extract. The ESI-MS n experiments provide a useful guide for gaining the large structural information of novel compounds, which are important to drug design, although the analytical method cannot confirm the accurate substitution position of Ac groups of these glucosyloxybenzyl 2R-benzylmalates.
In addition, a cell model associated with hepatic fibrosis was established by using LPS to stimulate HSC-T6. The isolates were carried to hepatic fibrosis experiment while compounds 1, 2, 4, 5, 8 showed moderate anti-hepatic fibrosis effects. Nevertheless, the studies on the quantitative structure-antihepatic fibrosis relationship of predicted glucosyloxybenzyl 2R-benzylmalates will be further investigated.

Patents
Two patents resulting from the work about new structures, anti-liver fibrotic activity and MS fragment pathway have been submitted to the Chinese Patent Office.