Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines

Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. “Radiant”, and their chemical structures were elucidated by UV, IR, ESI-MS, 1H-NMR and 13C-NMR analyses. These compounds, which include trilobatin (A1), phloretin (A2), 3-hydroxyphloretin (A3), phloretin rutinoside (A4), phlorizin (A5), 6′′-O-coumaroyl-4′-O-glucopyranosylphloretin (A6), and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin (A7), all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity.

The dihydrochalcone compound A5 was isolated as a yellow powder, and the 1 H-NMR analysis yielded the following data:  Table 1. Similar results were reported for a compound isolated from strawberries [21], and this compound was thus identified as phlorizin.

In Vitro Cytotoxicity of the Seven Dihydrochalcone Monomer Compounds
The survival rates of tumor cells are shown in Figure 3. When the monomeric compounds A1-A7 were administered at 100 µmol/mL, only A2, A3, A6 and A7 had any effect on A549 cells; A1 and A7 had effects on Bel 7402 cells; A1, A2 and A5 had some effects but A7 dramatically inhibited the growth of HepG2 cells; and A3 had effects on HT-29 cells ( Figure 3A). When the concentration of monomeric compounds A1-A7 was increased to 200 µmol/mL, A2, A3, A6 and A7 significantly inhibited the growth of A549 cells; A1-A4, A6 and A7 had effects on Bel 7402 cells; A2, A3 and A7 had significant effects on HepG2 cells; and A2, A3 and A7 had effects on HT-29 cells ( Figure 3B). Collectively, except for compound A5, the other compounds showed significant cytotoxic activities against one or several of the four cancer cell lines. The two new, rare compounds A6 and A7 were more effective in targeting A549, Bel 7402, HepG2, and HT-29 cells. Methoxyl groups improve the antioxidant activity [25], and A7 had significant effects on all tested cancer cell lines, perhaps due to its methoxyl group bonded to the phloretin structure that dramatically increases the anticancer activity of phloretin. Because A4 and A5 had weaker anti-tumor effects than the other compounds, even under the highest concentration of 200 µmol/mL, we only used A1-A3 and A6-A7 to detect whether their anti-tumor effects depend on the concentration response ( Figure 4). Notably, the results show that the concentration of monomeric compounds A1, A2, A3, A6, and A7 had a positive correlation with the four tumor cell lines' response curves. Because A4 and A5 had weaker anti-tumor effects than the other compounds, even under the highest concentration of 200 μmol/mL, we only used A1-A3 and A6-A7 to detect whether their anti-tumor effects depend on the concentration response ( Figure 4). Notably, the results show that the concentration of monomeric compounds A1, A2, A3, A6, and A7 had a positive correlation with the four tumor cell lines' response curves. These data indicate that the cytotoxicity of dihydrochalcone monomer compounds (DMCs) on the four tumor cell lines are correlated with their bioactivity and was dose-dependent. Moreover, different tumor cells show different sensitivity to the inhibitory effects of the DMCs.

Cytotoxic Effects of Dihydrochalcone Monomeric Compounds on Four Human Cancer Cell Lines
After exposure to A1-A7, the IC 50 values of the four cancer cell lines was determined and are shown in Table 3. Our data shows that different cells had different sensitivity to the inhibition effect of the dihydrochalcone monomers. The IC 50 value reflects the intrinsic sensitivity of the acetylcholinesterase molecule to the inhibitor in rats [26]. In our experiments, the IC 50 value can be understood as the concentration required to achieve 50% DMC-induced apoptosis of tumor cells. This concentration is known as the 50% inhibitory concentration. Namely, when the ratio between the apoptotic cells and all of the cells is greater than or equal to 50%, the IC 50 is satisfied for every DMC concentration. Moreover, IC 50 values can be used to measure the ability of DMC-induced apoptosis, and a stronger induction ability is associated with a lower IC 50 value. The four tested cancer cell lines showed different sensitivities to the seven DMCs. For the A549 cells, the DMC induction ability order was A2 > A7 > A3 > A6 > A1 « A4 « A5; for Bel 7402 cells, the DMC induction ability was A2 > A3 > A7 > A1 > A6 > A4 « A5; for HepG2 cells, the DMC induction ability was A2 > A3 > A7 > A6 > A1 « A4 « A5; and for HT-29 cells, the DMC induction ability was A2 > A3 > A7 > A6 > A1 « A4 « A5. The values of A2, A3, A6 and A7 indicate strong action against the four tumor cells. Although A1 had strong anticancer activity for Bel 7402, its induction ability against A549, HepG2 and HT-29 cells was relatively weaker, and both A4 and A5 had weak induction ability against the four types of tumor cells.
Havsteen reported that the number and position of hydroxyl groups attached to the A-ring and the nature and position of the carbohydrate units in the glycosides could influence flavonoids' medicinal uses [27]. Davide reported that the presence of a glycosyl moiety bound to the chalcone structure decreased the antimicrobial activity of phloretin [2]. These finding may explain why phloretin and its derivatives exhibited different induction abilities. In addition, phloretin (A2) showed the strongest induction ability among the derivatives: one explanation is its small molecular mass; the other is that some glycoside moiety is bound to the phloretin structure and decreases the anticancer activity of phloretin, such as is trilobatin (A1). Phlorizin (A5) has a glucoside, phloretin-rutinoside (A4) has a rutinoside, and 6 11 -O-coumaroyl-4 1 -O-glucopyranosylphloretin (A6) and 3 111 -methoxy-6 11 -O-feruloy-4 1 -O-glucopyranosylphloretin (A7) have a glucopyranosyl moiety.

Materials and Instruments
The Malus crabapples "Radiant" leaves used in the experiments were collected from 6-year-old Malus crabapple trees. Trees with similar growth vigor were planted in the Crabapple Germplasm Resource Garden of BUA (Beijing, China). When the leaves matured in mid-June, we collected 30 kg leaves for our experiments. Column chromatography with silica gel (#200-300 mesh), thin layer chromatography with silica gel GF254 (Qingdao Marine Chemical Plant, Qingdao, China); reverse phase silica gel C18 (including 50 m, YMC, Kyoto, Japan), Sephadex LH-20 (GE, Uppsala, Sweden); and MCI-gel CHP-20 p (70-150 µm, Mitsubishi Chemical Corporation, Tokyo, Japan). Analytically pure solvents were used. AB 8 macroporous resin (Hebei Bao An Resin Products Limited Liability Company, Baoan, China), Sephadex LH-20 from GE were used. The flavonoid composition of the crabapple leaves was detected by HPLC using an analytical column (Agilent Extend C18 4.6ˆ250 mm) for HPLC analysis and a preparative chromatographic column (Agilent Extend, C18 9.4ˆ250 mm) was used for isolating the monomeric compounds. NMR was performed with a Bruker DRX-500 Nuclear Magnetic Resonance Instrument (Zug, Switzerland); Mass spectrometry analysis was performed with a Finnigan LCQ DECA XP PLUS Type Ion Trap Mass Spectrometer (San Jose, CA, USA) coupled to an Agilent 1260 HPLC (Palo Alto, CA, USA).

Purification of Dihydrochalcones from Crabapple "Radiant" Leaves
The air-dried crabapple leaves were powdered and extracted three times for 2 h with 70% MeOH at 60˝C. After the solvent evaporated, the pooled residues were suspended in water, and extracted sequentially with 30% MeOH, 60% MeOH on AB-8 macroporous resin.

MS System and Conditions
A Xevo G2-S QTof (Waters MS Technologies, Milford, MA, USA), a quadrupole and orthogonal acceleration time-of-flight tandem mass spectrometer, was used with an ESI source. Both positive and negative ion modes were used for compound ionization. The MSE data collection mode was used. At one sample injection, the mode could collect the exact mass data of the quasi-molecular ions and fragment ions by quickly alternating between low and high collision energies. The detecting conditions used were: capillary voltage of 0.45 kv, cone voltage of 40 v, source temperature of 120˝C, desolvation temperature of 500˝C, cone gas flow of 50 L/h, desolvation gas flow of 700 L/h, low energy of 6 V, and high energy ramp of 20 to 40 V. The Tof-MS mass range was m/z 100 to m/z 1200.
The scan time was 0.2 s. All analyses were obtained using the Lockspray feature to ensure accuracy and reproducibility. The mass-to-charge ratio of leucine-enkephalin was used as the lockmass at a concentration of 200 ng/mL and a flow rate of 10 µL/min. The data were acquired by real time collection (scan time of 0.5 s, interval of 15 s). The UPLC-QTof-MS data of the samples were acquired and analyzed by the Waters UNIFI 1.7 software.

Cancer Cell Growth Inhibition
A MTT [3-(4,5)-dimethylthiazolyl)-3,5-diphenyltetrazolium bromide] cell viability assay was used. Succinate dehydrogenase in the mitochondria of live cells can convert the insoluble violet crystalline formazan produced from MTT and deposit it in the cells, but dead cells cannot not. Dimethyl sulfoxide (DMSO) could dissolve the formazan in cells, and its light absorption value was measured by ELISA at 490 nm. The number of living cells can be determined from the measured absorbance value (OD value).
Human lung adenocarcinoma cell line A549, human hepatoma cell line Bel7402, human cancer colorectal adenomas cell line HepG2 and colon cancer cell HT29 (ATCC, Manassas, VA, USA) were obtained from Dr. Wen Xu (College of Pharmacy, Fujian University of Traditional Chinese Medicine). These four tumor cell lines were picked in their logarithmic growth phase with trypsin digestion and cultured supplemented with 10% fetal bovine serum (GIBCO BRL, Carlsbad, CA, USA) in RPMI1640 Dulbecco's modified Eagle medium (DMEM) and reached a 150,00/mL cell suspension. Then, these cells were seeded into 96-well plates at 190 µL/plate at 37˝C for 24 h under 5% CO 2 . An aliquot of 10µL of the test compounds was added to cells and cultured at 37˝C for 3 days under 5% CO 2 . The cells were measured by a modified MTT assay [28,29]. The A549, Bel7402, HepG2 cells were treated with MTT solution (final concentration of 0.5 mg/mL in DMEM) for 4 h at 37˝C in a 96-well plate, the supernatant was carefully removed, and DMSO (150 µL) was added to each well to dissolve the precipitate. The absorbance at 570 nm was measured using a Model 680 microplate reader (BIO-RAD, Hercules, CA, USA).

Statistical Analysis
Data are presented as the mean˘standard deviation (SD). Analysis of variance (ANOVA, SPSS 17.0 software, SPSS Inc., Chicago, IL, USA) of all values was used to assess differences in the means among different samples (p < 0.05). Duncan's multiple analysis and Student's t-test were used to identify significant differences among groups (p < 0.05, p < 0.01). Graphs were prepared in Origin Pro 8.0 SR4 (Origin Lab, Northampton, MA, USA) and Adobe Photoshop CS6.

Conclusions
Seven dihydrochalcone compounds separated and purified from Malus crabapple var. "Radiant" leaves displayed remarkable biological activities. Compounds A2, A3, A6 and A7 had a strong protective effect against the four tested tumor cell lines A549, Bel 7402, HepG2 and HT-29. Therefore, we propose that the dihydrochalcones may have beneficial effects on human health and can be considered as possible therapeutic agents against cancer. This article shows that Malus crabapples may be a valuable resource of anticancer effects, and the two new rare compounds A6 (6 11 -O-coumaroyl-4 1 -O-glucopyranosylphloretin) and A7 (3 111 -methoxy-6 11 -Oferuloyl-4 1 -O-gluco-pyranosylphloretin) could be developed into promising anticancer agents.