HPLC/ESI-MS and NMR Analysis of Chemical Constitutes in Bioactive Extract from the Root Nodule of Vaccinium emarginatum

Vaccinium emarginatum Hayata is a medicinal plant that has been historically used in ethnopharmacy to treat diseases in Taiwan. The objective of this study is to evaluate the anti-cancer and anti-bacterial constitutes from the root nodule extract of V. emarginatum. The chemical composition of V. emarginatum fractions was analyzed by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and the chemical constitutes were isolated and structurally identified by nuclear magnetic resonance (NMR) spectroscopy. Bioassay-guided chromatography showed that the ethyl acetate (EA) fraction was bioactive on the hepatocellular carcinoma (HepG2). By LC-ESI-MS/MS analysis, twenty peaks of EA fraction were partially identified and the phytochemical investigation of the fractions led to the isolation and identification of protocatuchuic acid (1), epicatechin (2), catechin (3), procyanidin B3 (4), procyanidin A1 (5), hyperin (6), isoquercetin (7), quercetin (8), lupeol (9), beta-amyrin (10), and alpha-amyrin (11). Both procyanidin B3 and A1 exhibited anti-proliferative activity against HepG2 and gastric adenocarcinoma (AGS) cells at IC50 values between 38.4 and 41.1 μM and 79.4 and 83.8 μM, respectively. In addition, isoquercetin displayed the strongest anti-proliferative activity against the HepG2, lung carcinoma (A549), and AGS cell at 18.7, 24.6 and 68.5 μM, respectively. Among the triterpenoids, only lupeol showed the inhibitory activity against all tested tumor cell lines at IC50 values between 72.9 and 146.8 μM. Furthermore, procyanidins B3, A1 and isoquercetin displayed moderate anti-bacterial activity against Staphylococcus aureus. In conclusion, this study provides background information on the exploitation of V. emarginatum as a potential natural anti-cancer and anti-bacterial agent in pharmaceutical research.

Vaccinium emarginatum Hayata (Ericaceae) is an endemic species distributed across central mountain regions in Taiwan at an elevation of 1500-3000 m [16]. Unlike other Vaccinium species, it is an epiphytic shrub growing on the branches of trees in the broadleaved forests with enlarged root nodules storing the water and nutrients (Figure 1). In Taiwan, the root nodules are used in traditional medicine, which is supposed to be as effective as Lu Rong (the developing antlers of deer; velvet antler). Its leaves are used to treat urinary tract infections and rheumatic pain, while the fruits are used to treat dysentery and enteritis, respectively [9]. To date, few phytochemical studies were reported for V. emarginatum [5,9]. Previously, one epicatechin derivative and three new flavonoids, emarginin A, B, and C, were isolated from the methanolic extract of the whole plants of V. emarginatum. Emarginin C exhibited moderate anti-inflammatory activity [5]. Recently, the cytotoxic and antiinflammatory activity of terpenoids from the whole plant of V. emarginatum was also investigated [9]. Although those studies indicated the high potential of V. emarginatum in anti-inflammatory and cytotoxicity against prostate cancer cells, the anti-infective activity on human pathogens and anti-proliferative effect on tumor cells are still unknown. The object of this study is to evaluate the anti-proliferative and anti-bacterial activities of different chemical fractions from V. emarginatum methanol extracts and to characterize its phytochemical profile. The chemical composition of bioacitve fraction was further analyzed by LC-ESI-MS/MS, and the chemical constitutes were isolated and structurally identified by NMR. Finally, the anti-proliferative activities of isolated compounds were determined by measuring the IC 50 of hepatocellular carcinoma (HepG2), lung carcinoma (A549) and gastric adenocarcinoma (AGS). The anti-bacterial activities of isolated compounds were also tested against seven bacterial pathogens by minimum inhibitory concentration (MIC) methods. It is suggested that all of these isolated compounds may have the potential in clinical treatment as therapeutic agents in the future.

Results
2.1. Biological Activity of V. emarginatum Extract 2.1.1. Anti-Proliferation Activity of the V. emarginatum Extract on HepG2 Cell Line The cytotoxic effect of different fractions of V. emarginatum extract was examined on the HepG2 cell line over 72 h. Aliquots of DCM (dichloromethane), EA (ethyl acetate), BuOH (n-butanol), and water fractions (for a concentration range from 2.5 to 40 µg/mL) were evaluated for the anti-proliferative activity. The DCM and BuOH fractions displayed moderate cytotoxicity, with a relative cell viability of 91.6% to 93.9%, and 86.3% to 96.9% on HepG2 cells, respectively, in comparison with the control (Figure 2). It was revealed that the EA fractions exhibited dose-dependent inhibitory activities on the HepG2 cell, with a relative cell viability of 65.9% to 85.6%. The results show that the HepG2 cell was potentially inhibited by the EA fraction of V. emarginatum extract. Figure 2. The anti-proliferative activity of different fractions from V. emarginatum on human hepatocellular carcinoma cells (HepG2 cell line). The HepG2 cells were treated with a different concentration from 0 to 40 ug/mL of DCM (dichloromethane), EA (ethyl acetate), BuOH (n-butanol), and water fractions of V. emarginatum. * p < 0.05; ** p < 0.01; *** p < 0.001.

Anti-Bacterial Activity of the V. emarginatum Extract by the Disc Diffusion Method
Seven strains of pathogens, including Staphylococcus aureus (ATCC 43300), Enterococcus faecalis (ATCC 29212), Listeria monocytogenes (ATCC 7644), Bacillus cereus (ATCC 9139), Escherichia coli (ATCC 35150), Salmonella enterica (ATCC 13311), and Pseudomonas aeruginosa (ATCC 27853), were selected to evaluate the anti-bacterial activity. Among all of the partitioned fractions, ethyl acetate (EA) fractions and the butanol (BuOH) displayed higher anti-bacterial activity against all tested pathogens. The BuOH fraction presented a slightly lower anti-bacterial activity than the EA fraction. As shown in Table 1, the EA fraction of V. emarginatum extract showed anti-bacterial activity against the test strains of S. aureus, E. faecalis, L. monocytogens and B. cereus with average inhibition zones of 14, 7, 9 and 10 mm by using the disc diffusion assay. The EA-15 fraction revealed the most significant and broad anti-bacterial spectrum against the strains of S. aureus, E. faecalis, L. monocytogens and B. cereus, with average inhibition zones of 18, 9, 12 and 14 mm, respectively.

LC-ESI-MS/MS Analysis of EA Fraction of V. emarginatum Extract
The chromatographic profile EA fraction of V. emarginatum extract was determined through LC-ESI-MS/MS analysis ( Figure 3). The data of both positive and negative MS and MS 2 spectra were obtained and the component of the EA fraction was identified partially according to the literature.
As shown in Table 2, twenty-two peaks can be characterized and twenty peaks can be identified by comparing the spectrum data to references reported previously. For example, loss of CO 2 was observed for dihydroxyisophthalic acid and protocatechuic acids, giving the [M−H-44]as a characteristic ion [17]. As described by Sanchez-Rabaneda F. et al., catechin and epicatechin generated a loss of a −CH 2 CHOH− group (m/z 245) [17]. Peak 11 showed free (epi)catechin ions at m/z 289, indicating condensed tannin dimers consisting of (Epi)afzelechin-(Epi)catechin at m/z 561 [18].  Table 2) [19]. Those peaks were identified as procyanidin B1, B2 and B3 according to the literature and isolated chemicals [19]. For Peak 6 and 13 in Figure 3 [19]. Furthermore, Peak 13 was identified as procyanidin A1 according to the literature and isolated chemicals.
Fragmentation pattern of Peaks 9, 11, 15 and 16 displayed the diagnostic ions at m/z 301, suggesting that these compounds were quercetin and quercetin derivatives [20]. For the hyperin and isoquercitin, the spectra showed the ion related to the deprotonated aglycone [A − H] − , which is formed by loss of the galactose or glucose moiety from the flavonol glycosides [17]. In addition, the peaks were identified as lupeol, β-amyrin, α-amyrin and friedelin according to the retention time, the literature and the isolated chemicals in this study [21][22][23].

Anti-Proliferative Activity of Isolated Compounds
Anti-proliferative activities of Compounds 1-11 were determined by measuring the IC 50 of hepatocellular carcinoma (HepG2), lung carcinoma (A549) and gastric adenocarcinoma (AGS). As shown in Table 3 In addition, Compound 7 displayed the strongest anti-proliferative activity against the HepG2, A549, and AGS cell proliferation at 18.7, 24.6 and 68.5 µM, respectively. Among the triterpenoids (Compound 9-11), only Compound 9 showed the inhibitory activity against all the tested tumor cell lines at IC 50 values between 72.9 and 146.8 µM.

Anti-Bacterial Activity of Isolated Compounds
The minimum inhibitory concentration (MIC) method was applied for an anti-bacterial activity exploration of isolated compounds. As shown in Table 4, only procyanidins dimer (Compound 4 and 5) and one flavonoid glycoside (Compound 7) displayed moderate antibacterial activity against S. aureus. Most of the triterpenoids displayed non-anti-bacterial activities against all tested pathogens, expect for Compound 7, which showed moderate activity against E. faecalis at a concentration of 64 µg/mL. In addition, only Compound 5 showed moderate antimicrobial activities against B. cereus and L. monocytogenes. None of the isolated compounds had anti-bacterial activities against S. enterica in this study.

Discussion
In this study, the biological activities of different chemical fractions from V. emarginatum methanol extracts were evaluated. The results indicate that the EA fraction showed pronounced anti-cancer and anti-bacterial activity, and its phytochemical profile was fur-ther analyzed by LC-ESI-MS/MS. Twenty-two peaks were identified as five flavonoids, three flavonol glycosides, six procyanidins, one phenolic acid, five triterpenoids and two unknown chemicals. LC-ESI-MS/MS with negative ion detection had been dramatically used for tentative identification of a variety of phenolic compounds in fruits [3,17,19], flowers [20], and traditional herb medicine [18,22]. LC-ESI-MS/MS is a great analytical tool because of its high selectivity for analyzing complex components in plants. Moreover, negative ESI-MS provided a qualitative analysis useful in the screening of phenolics, flavonoids, and procyanidins. It provides simple MS/MS spectra in which it was possible to observe the loss of units more easily in contrast to positive ESI-MS fragmentation with Na + adducts [18,33].
Triterpenoids have been analyzed for a long period and are still widely detected and identified using gas chromatography mass spectrometry (GC-MS) [34,35]. Most triterpenoids are lacking a chromophore group, for example, neutral triterpenes have only one hydroxyl group on their structure, such as lupeol and amyrin, which are not easily ionized by ESI. Although liquid chromatography is more suitable for non-volatile chemicals, the triterpenoids founded in this study can be identified by both negative and positive LC-ESI-MS/MS and structurally identified by NMR.
Previously, the new flavonoid emarginin C, which was isolated from the whole plants of V. emarginatum, exhibited anti-inflammatory activity with an IC 50 value of 27.99 µM [5]. In addition, the triterpenoid coumaroyl and feruloyl esters exhibited strong cytotoxicity against PC-3 prostate cancer cells, with 85.6-90.2% inhibition at 10.0 µg/mL [9]. In this study, the anti-proliferative activities of isolated compounds were determined by measuring the IC 50 of hepatocellular carcinoma (HepG2), lung carcinoma (A549) and gastric adenocarcinoma (AGS). The results indicate that isolated procyanidin B3 and A1 are the promising lead compounds of V. emarginatum for anti-proliferative activities. Other procyanidins, such as procyanidin A2, showed an IC 50 value of 62.19 µg/mL on human hepatoma HepG2 cells [36]. All of these findings suggest that procyanidins may act as anti-proliferative agents in human hepatoma cells.
Vaccinium species are perennial and low-growing herbs found in the temperate climate regions. The berries of Vaccinium contain abundant flavonoids, including flavonol glycosides, anthocyanins, flavan3-ols, and procyanidins [37]. Among flavonoids, procyanins are the greatest contributors to the antioxidant capacity in the genus Vaccinium [38]. Most of the wild and cultivated cranberries contain both A-and B-type procyanidins. In this study, the EA fraction of V. emarginatum methanol extract also contained both dimeric and trimeric procyanidins. Peak 2, 4 and 10 were identified as B-type procyanidins B1, B2 an B3 according to the data of retention time, mass spectrum and MS/MS ion [19,24]. In addition, A-type procyanidins, including dimer A1 and three unknown trimers, were also found in this study. The procyanidin trimer with more OH groups in one benzene unit may increase the antioxidant activity significantly [26]. Flavonoids with a dihydroxy structure on the B ring have been shown to be effective in biological function [39]. In the group of flavonoids, this catechol unit is found in catechins, epicatechin, quercetin, and procyanidins. All of these flavonoids were also found in the root nodule of V. emarginatum, indicating that not only the berries but also the root nodule of V. emarginatum can be a great source of procyanidins.
In this study, the anti-bacterial activities of isolated compounds were also tested against seven bacterial pathogens by minimum inhibitory concentration (MIC) methods. The determination of MIC against Gram-positive bacteria gave a value of 64 µg/mL for procyanidin B3 (4) and A1 (5). Previous reports revealed a moderate for certain procyanidins against Bacillus cereus, Klebsiella pneumoniae, Proteus vulgaris and Streptococcus pyogenes at concentrations below 100 µg/mL [40]. In addition, procyanidin B2, which was also found in the EA fraction by LC-ESI-MS/MS analysis in this study, generated anti-bacterial activity against S. aureus at an MIC value of 100 µg/mL [41]. All of these results indicate that procyanidin dimers displayed moderate antimicrobial activity against certain pathogens. As a potential anti-cancer agent, procyanidin has been shown to inhibit the proliferation of various cancer cells [26,42]. Moreover, several assays demonstrate potential biological functions of procyanidins, including antioxidant, radical-scavenging properties [26], and enzyme inhibiting [43] has also been reported. It is suggested that procyanidin might be the valuable group of V. emarginatum as a source of promising compounds for the development of future pharmacological applications. The NMR spectra data were measured on an Avance NEO 400 spectrometer (Bruker Daltonics Inc., Billerica, MA, USA) and an Agilent Technologies DD2 600 spectrometer (Agilent, Santa Clara, CA, USA). ESI-MS spectra were performed on a Bruker Daltonics Esquire HCT spectrometer (Bruker Daltonics Inc., Billerica, MA, USA).

Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry (LC-ESI-MS/MS)
LC mass analysis for the chemical components in the EA fraction was applied to LC-ESI-MS/MS) analysis (Esquire HCT, Bruker Daltonics, Billerica MA, USA). At a flow rate of 0.25 mL/min, the Atlantis T3 RP-18 column (150 × 2.1 mm; 3 µm; Waters, Milford, MA, USA) was eluted with Buffer A (distilled water/acetonitrile/formic acid; 95/5/0.1, v/v/v) and Buffer B (acetonitrile/formic acid; 100/0.1, v/v) at 25 • C. Elution started initially with 100% Buffer A, followed by a linear increase in Buffer B to 10% from 0 to 5 min, and maintained in 10% Buffer B for another 5 min. A secondary linear increase in Buffer B to 20% was carried out from 10 to 20 min and maintained in 20% Buffer B for another 10 min. From 30 to 35 min, the column was further eluted with a third linear increase in Buffer B to 60% and maintained for another 5 min. A final linear increasing of Buffer B was carried out from 60% to 95% and maintained for 5 min from 40 to 45 min. The column was finally equilibrated with Buffer A for another 10 min. The ion source temperature is maintained at 100 • C and the dry temperature is 365 • C. The flow rate of N 2 desolvation gas is sustained at 12 L/min. Product ion scans for mass were accomplished by low-energy collision (20 eV) using argon as the collision gas. MS spectrum data of both positive and negative ionization mode were collected. All data were analyzed by Bruker Daltonics Data software (version 4.0).

Isolation and Identification of Chemical Constitutes from V. emarginatum Extract
Six kilograms of V. emarginatum root nodules was air-dried (630 g) and extracted with methanol thrice following the standard extraction procedures. The methanolic extract was concentrated to gain 37.3 g dry residue and then partitioned by dichloromethane, ethyl acetate, butanol with H 2 O to the obtained portion of DCM (5.90 g), EA (8.59 g), BuOH (7.28 g), and an aqueous layer (14.86 g), sequentially.

Anti-Bacterial Assay
The disc diffusion method was applied to anti-bacterial-guided fractionation. The chemical fractions were dissolved in DMSO at a concentration of 100 µg/µL. According to the standard protocol (Clinical and Laboratory Standards Institute, CLSI) [44], 10 µL of dried extracts was applied to paper discs (6 mm in diameter) and placed on Mueller Hinton agar plates, which were inoculated with overnight-cultured pathogens. The plates were incubated at 37 • C for 18 h. The diameters of the inhibition zones were measured within 24 h, and discs containing DMSO and antibiotics were served as a negative and positive control. At least three independent determinations were repeated.
The anti-bacterial activity of purified compounds was conducted by the minimum inhibitory concentration (MIC) method. According to the guidelines of the CLSI [45], the MIC values were determined by the broth micro-dilution method. Briefly, all bacterial strains were cultured on nutrient agar and incubated at 37 • C for 24 h. Bacteria were suspended in 0.85% NaCl solution and diluted to give an inoculum with a final density of 5 × 10 5 cfu/mL. Isolated compounds were dissolved in DMSO at a final concentration of 2560 µg/mL. Two-fold dilutions were made serially in a concentration range from 0.5 to 128 µg/mL. In each 96-well plate, all of the wells were calculated for bacterial growth by spectrophotometer at 590 nm. The lowest concentration that inhibited the growth of the respective pathogen was defined as the MIC value. The antibiotics (ampicillin and tetracycline) were used as a positive control. All tests were carried out in triplicate.

Anti-Proliferative Assay
Anti-proliferative assay was determined against HepG2 2.2.15 (human hepatocellular carcinoma cells) and A549 cells (human lung adenocarcinoma cell line) using the MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay (Promega, USA). Briefly, HepG2 2.2.15 and A549 cells were treated with 2-fold serial dilutions of the tested compounds ranging from 0.19 to 200 µM for 3 days. After incubation, the medium from the wells was removed carefully. To each well, 50 µL of serium-free medium and 50 µL of MTT solution (5 mg/mL in PBS) were added. The plate was incubated for 3 hours in 37 • C incubator with 5% CO 2 . After incubation, 150 µL MTT solution was added into each well and the plates were shacked on a shaker for 15 min. Mixing well by micropipette may be required to fully dissolve the MTT formazan. The presence of viable cells was visualized by the formation of formazan crystals in purple color. Plates were read at 570 nm for cytotoxicity assay within one hour.
The human gastric adenocarcinoma (AGS cell line) was cultured in RPMI-1640 medium supplemented with antibiotics (100 µg/mL of streptomycin and 100 U/mL of penicillin) and 10% FBS (fetal bovine serum). The AGS cell was treated with compounds ranging from 0.19 to 200 µM for 2 days. The trypan blue exclusion protocol was used for in vitro cytotoxicity assay. In brief, 40 µL of trypan blue was mixed with 10 µL of cell suspension and the numbers of dead cells (stained) and live cells (unstained) were counted using a hemocytometer. After treatment, the cell viability as the percentage of cell survival was recorded and the value of IC 50 was also calculated. All measurements were performed in triplicate, and epigallocatechin gallate (EGCG), 5-fluorouracil, and doxorubicin were used as the positive control.

Conclusions
In this study, twenty peaks of EA fraction from V. emarginatum extract were identified by LC-ESI-MS/MS analysis, and eleven of them were isolated and identified by NMR analysis. The ability of procyanidin B3, A1, isoquercetrin, quercetin and lupeol to inhibit the tumor proliferative activity means that they have the potential to constitute a valuable group of therapeutic agents in the future.