Isoprenylated Flavonoids and 2-Arylbenzofurans from the Root Bark of Morus alba L. and Their Cytotoxic Activity against HGC27 Cancer Cells

Three new compounds (1, 11, and 12), together with 32 known ones, were isolated from the root bark of Morus alba L. using various chromatographic methods. The structures of the undescribed compounds were elucidated based on 1D, 2D NMR, and HRESIMS dataanalysis, while the known ones were identified by comparison of their spectroscopic data with those reported in the literature. All the isolates were evaluated for their cytotoxic activities against human gastric cancer HGC27 cells by CCK-8 assay. Among them, compounds 5, 8, 10, and 30 exhibited cytotoxic activities on HGC27 cells with IC50 values of 33.76 ± 2.64 μM, 28.94 ± 0.72 μM, 6.08 ± 0.34 μM, and 10.24 ± 0.89 μM, respectively. Furthermore, compound 10 was confirmed to reduce proliferation ability, increase apoptosis rate, and inhibit cell migration pathway by annexin V/PI double staining experiment, transwell experiment, and Western blot analysis.


Introduction
Morus alba L., the root bark of Morus mulberry, is a well-known traditional Chinese medicine (TCM) documented in Chinese Pharmacopoeia [1].The root bark of M. alba has been widely used in Chinese herbal medicinal prescriptions for the treatment of pneumonia, hypertension, diabetes, and arthritis [2,3].Previous phytochemical studies revealed that M. alba contained a variety of polyphenolic constituents such as prenylated flavonoids, 2-arylbenzofurans, stilbene, coumarin, and mulberry Diels-Alder-type adducts.In addition, pharmacological studies exhibited that some polyphenols from M. alba had a broad spectrum of biological activities, such as hepatoprotective activity, whitening ability, and antiplatelet effects [4][5][6][7][8][9].Previous studies also showed that diverse individual compounds or extracts from different parts of various mulberry plants (M.alba, M. australis, M. wittiorum, and M. yunanensis) exhibited anti-tumor activity against different tumor cell lines, such as lung cancer, liver cancer, colon cancer, breast cancer, rectal adenocarcinoma, and renal adenocarcinoma [10][11][12][13][14].Our preliminary pharmacological experiment showed that the 75% ethanol extract of M. alba exhibited certain anti-tumor activity against gastric cancer cells, which had rarely been reported.Thus, the present paper focuses on detailed phytochemical investigation of the 75% ethanol extract of M. alba and evaluation of antitumor activity against gastric cancer cells of the isolated individual compounds.As a result, thirty-five compounds including three new ones were obtained.Among them, compound 10 (Albanol B) shows the most significant cytotoxicity with an IC 50 value of 6.08 ± 0.34 µM.Moreover, the potential mechanism was preliminarily explored.

Biological Activities of Compounds against HGC27 Cancer Cells
The effect of compounds (1-35) on the cell viability of HCG27 cells was evaluated by CCK-8 assay.As shown in Table 3, four compounds 5, 8, 10, and 30 had a certain inhibitory effect on the viability of HCG27 cells compared with the model group (control).In contrast, other compounds had no significant inhibitory effects on the viability of HCG27 cells; therefore, compounds 5, 8, 10, and 30 were screened to evaluate the semi-inhibition concentration (IC 50 ) values (Table 4).Among the four compounds, compound 10 has showed the best cytotoxicity against HCG27 cells, with an IC 50 value of 6.08 ± 0.34 µM, and compounds 5, 8, and 30 have showed certain cytotoxicity, with IC 50 values of 33.76 ± 2.64 µM, 28.94 ± 0.72 µM, and 10.24 ± 0.89 µM, respectively.Compound 10 effectively inhibited the clone formation of HGC27 cells (Figure 3a).When cells were treated with 6 µM compound 10, the number of cell clones formed in the administered group was significantly reduced and cell growth was significantly inhibited compared to the control group (Figure 3b).We used Annexin V-FITC (Annexin V-Fluorescein Isothiocyanate) double staining assay to detect apoptotic cells.As shown in Figure 3c, the apoptosis rate of HGC27 cells treated with compound 10 significantly increased compared to the control group.Quantification of apoptotic cells by flow cytometry revealed that the percentage of HGC27 apoptotic cells increased in a dose-dependent manner (Figure 3d).Cell migration is an essential process in tumor metastasis.The effect of compound 10 on migration was examined by the transwell method.The effect of compound 10 on the migration of HGC27 tumor cells was investigated in the premise of HGC27 cell death.As shown in Figure 3e, in vitro migration studies showed that compound 10 significantly inhibited the migratory ability of HGC27 cells in a dose-dependent manner.The number of migrating HGC27 cells was reduced from 647 to 357 compared to the control group (Figure 3f).Caspase-3 is an important protein in the regulation of cell apoptosis [44].After treating HGC27 cells with 10 µM and 20 µM of compound 10 for 48 h, Western blot analysis showed the expression of apoptosis-related protein Caspase-3 was significantly increased compared with the control (Figure 4).Thus, compound 10 had a substantial cytotoxic effect on HGC27 cells (Figure 4).We also analyzed the expression levels of proteins Bax and Bcl-2 related to cell apoptosis [45].Bax was upregulated in HGC27 cells, whereas Bcl-2 was downregulated.These results indicated that compound 10 has the capacity to induce apoptosis (Figure 4).Cell migration is an essential process in tumor metastasis.The effect of compound 10 on migration was examined by the transwell method.The effect of compound 10 on the migration of HGC27 tumor cells was investigated in the premise of HGC27 cell death.As shown in Figure 3e, in vitro migration studies showed that compound 10 significantly inhibited the migratory ability of HGC27 cells in a dose-dependent manner.The number of migrating HGC27 cells was reduced from 647 to 357 compared to the control group (Figure 3f).Caspase-3 is an important protein in the regulation of cell apoptosis [44].After treating HGC27 cells with 10 µM and 20 µM of compound 10 for 48 h, Western blot analysis showed the expression of apoptosis-related protein Caspase-3 was significantly increased compared with the control (Figure 4).Thus, compound 10 had a substantial cytotoxic effect on HGC27 cells (Figure 4).We also analyzed the expression levels of proteins Bax and Bcl-2 related to cell apoptosis [45].Bax was upregulated in HGC27 cells, whereas Bcl-2 was downregulated.These results indicated that compound 10 has the capacity to induce apoptosis (Figure 4).

General Experimental Procedures
IR and UV spectra were recorded through a PerkinElmer 1725X-FT spectrometer with KBr disks and a PerkinElmer (Boston, MA, USA) Lambda 35 spectrometer, respectively, and optical rotations were determined by using a Perkin-Elmer 341 polarimeter.1D and 2D NMR spectra were recorded using Bruker Avance 400 and 600 spectrometers.The HR-ESI-MS data were from micrOTOF-QII mass spectrometer.The precoated silica gel GF254 10-40 µm (Qingdao Marine Chemical Group, Qingdao, China) was used for TLC.Analytical HPLC was used with the Prominence LC-20AT with a model SPD-M20A detector and Ultimate ® C18 column (250 mm × 4.60 mm, 5 µm).Preparative HPLC was carried out on P3000 with a UV3000 detector (Chengdu LaiPu Science and Technology, Chengdu, China) and Ultimate ® C18 column (250 mm × 21.2 mm, 5 µm; 250 mm × 50 mm, 10 µm, respectively).Column chromatography (CC) was performed with silica gel (100-200 mesh and 200-300 mesh, Qingdao Marine Chemical Group, China) and RP-C18 (20-40 µm, Fuji, Japan) was used for column chromatography (CC).All chemical solvents were obtained from Chron Chemicals Reagent of Chengdu.The 35 compounds with more than 96% purity were prepared in our own laboratory.

Plant Material
The root bark of M. alba (Sang-Bai-Pi) was purchased from Lotus Pond Chinese Herbal Medicine market, Sichuan, China.It has been identified by Professor Weikai Bao (Chengdu Institute of Biology, Chinese Academy of Sciences).A voucher specimen (CIB-A-431) has been deposited at the Laboratory of Phytochemistry, Chengdu Institute of Biology, Chinese Academy of Sciences.

Extraction and Isolation
The air-dried and powdered roots (30.0 kg) were extracted three times with 75% ethanol (100 L × 3, each 3 days).The resulting solution was filtered, combined, and concentrated under reduced pressure to give 3.0 Kg of brown residue.The 75% ethanol extract (3.0 Kg) was partitioned between H2O and ethyl acetate.The ethyl acetate part (2.2 Kg) was separated by silica gel column (6.0 Kg, 150 × 40 cm) eluted with petroleum ether

General Experimental Procedures
IR and UV spectra were recorded through a PerkinElmer 1725X-FT spectrometer with KBr disks and a PerkinElmer (Boston, MA, USA) Lambda 35 spectrometer, respectively, and optical rotations were determined by using a Perkin-Elmer 341 polarimeter.1D and 2D NMR spectra were recorded using Bruker Avance 400 and 600 spectrometers.The HR-ESI-MS data were from micrOTOF-QII mass spectrometer.The precoated silica gel GF254 10-40 µm (Qingdao Marine Chemical Group, Qingdao, China) was used for TLC.Analytical HPLC was used with the Prominence LC-20AT with a model SPD-M20A detector and Ultimate ® C 18 column (250 mm × 4.60 mm, 5 µm).Preparative HPLC was carried out on P3000 with a UV3000 detector (Chengdu LaiPu Science and Technology, Chengdu, China) and Ultimate ® C 18 column (250 mm × 21.2 mm, 5 µm; 250 mm × 50 mm, 10 µm, respectively).Column chromatography (CC) was performed with silica gel (100-200 mesh and 200-300 mesh, Qingdao Marine Chemical Group, China) and RP-C18 (20-40 µm, Fuji, Japan) was used for column chromatography (CC).All chemical solvents were obtained from Chron Chemicals Reagent of Chengdu.The 35 compounds with more than 96% purity were prepared in our own laboratory.

Plant Material
The root bark of M. alba (Sang-Bai-Pi) was purchased from Lotus Pond Chinese Herbal Medicine market, Sichuan, China.It has been identified by Professor Weikai Bao (Chengdu Institute of Biology, Chinese Academy of Sciences).A voucher specimen (CIB-A-431) has been deposited at the Laboratory of Phytochemistry, Chengdu Institute of Biology, Chinese Academy of Sciences.

Extraction and Isolation
The air-dried and powdered roots (30.0 kg) were extracted three times with 75% ethanol (100 L × 3, each 3 days).The resulting solution was filtered, combined, and concentrated under reduced pressure to give 3.0 Kg of brown residue.The 75% ethanol extract (3.0 Kg) was partitioned between H 2 O and ethyl acetate.The ethyl acetate part (2.2 Kg) was separated by silica gel column (6.0 Kg, 150 × 40 cm) eluted with petroleum ether and ethyl acetate (10:1 to 1:1) to yield four fractions (Fr.1-4).

Cell Culture
HGC27 cells were purchased from American Type Culture Collection (ATCC).All cells grew adherently.HGC27 cells were cultured in RPMI 1640 media, and MRC-5 cells were cultured in a minimal essential medium (Gibco) (MA, USA).All media contained 10% endotoxin-free, heat-inactivated FBS (Gibco) and were maintained at 37 • C in a humidified atmosphere of 5% CO 2 .

Colony Formation Assay
Colony formation assay was performed as described previously.Briefly, 500 cells per well were seeded in 12-well plates and then incubated for 7 d.The colonies were fixed in methanol for 20 min and stained with 2% crystal violet for 20 min.Then, the stain solution was removed using tap water, and the cells were air-dried.Finally, the number of colonies was counted.

Cell Apoptosis Assay
HGC27 cells were cultured in a 6-well plate at a density of 700,000 cells/well and incubated in a cell incubator for 24 h.When HGC27 cells were in the logarithmic growth phase, HGC27 cells were treated with 10 µM or 20 µM compound 10 for 48 h.Then, cells were collected by trypsin digestion without EDTA and washed twice with cold PBS.An amount of 5 µL Annexin V/Alexa Fluor 488 solution was added to the cell suspension and incubated in the dark at room temperature for 5 min.Then, 10 µL PI and 400 µL PBS solution were added.The stained cell suspension was transferred to a flow tube, and the proportion of apoptotic cells was detected using BD Facscanto II, (Piscataway, NJ, USA), and the experimental results were statistically analyzed using Flowjo.

Transwell Migration Assay
Before the experiment, the cells were starved by culturing in a serum-free medium for 4 h.Then, cells were collected, and 30,000 cells were cells were treated with 3 µM and 6 µM Compound 10 inoculated into 500 µL serum-free medium in the upper chamber of Transwell (Corning, NY, USA), and 500 µL culture medium with 10% FBS was added into the lower chamber.After 24 h of incubation, the culture medium was discarded.Cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 15 min, then stained with 0.4% crystal violet (Beyotime, China) for 15 min.The cells were observed and photographed under a light microscope.

Western Blot Analysis
HGC27 cells which were treated with concentrations 10 µM and 20 µM compound 10 were lysed by RIPA lysis buffer (Beyotime, Shanghai) for 20 min.After centrifugation at 13,000 rpm for 25 min at 4 • C, the sediment was removed, and the supernate was mixed with 5 × Loading Buffer boiled for 5 min at 100 • C. Next, equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA).After blocked with 5% non-fat milk for 2 h at room temperature (RT), the membranes were incubated with the specific primary antibodies (Signalway Antibody, Nanjing, China) overnight at 4 • C.After washing three times with Tris Buffered saline Tween 20 (TBST), the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ProteinTech, Wuhan, China) for 1 h at RT.The immune-blotting signals were detected using Beyo ECL Plus electrochemiluminescence reagent (Beyotime, Shanghai, China).Next, equal amounts of proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA).

Conclusions
Phytochemical investigation of the 75% ethanol extract of the root bark of M. alba led to the isolation and identification of three new compounds (1, 11, and 12) and 32 known ones (2-10, 13-35).All compounds were tested for their cytotoxic activities against gastric cancer HGC27 cells.As a result, compounds 5, 8, and 30 exhibited certain cytotoxic activities with the IC 50 values ranging from 33.76 ± 2.64 µM, 28.94 ± 0.72 µM, and 10.24 ± 0.89 µM, respectively.Compound 10 showed the most significant cytotoxic activity with an IC 50 value of 6.08 ± 0.34 µM.Then, compound 10 was subjected to further experiments to explore its anti-tumor mechanism.It was found that compound 10 has inhibitory effects on proliferation and migration of human cancer HGC27 cells.In addition, the experimental results indicated that compound 10 was able to induce apoptosis through upregulation of Bax protein expression and downregulation of Caspase-3 and Bcl-2 protein expression.This study was the first time to preliminarily explore the mechanism of compound 10 in inhibiting gastric cancer cell HGC27 in vitro.In conclusion, the present study provides a reference for the phytochemical and biological activities of Morus alba L.

Figure 3 .
Figure 3. Cells apoptosis and migration assay.(a) Visual observation of cell clonogenicity.(b) Statistical analysis of cell clonogenicity.Data are mean ± SD (n = 3).Differences between treatment groups and control groups are determined, * p < 0.05, ** p < 0.01 as compared with control.(c) Apoptosis caused by 10 in HGC27 cells.(d) Treatment groups cells apoptosis rates were compared with control, * p < 0.05.Data are expressed as mean ± SD (n = 3).(e) The migration of HGC27 cells.(f) Quantitative evaluations of cell migration induced by 10 in the transwell assay, treatment groups were compared with control, * p < 0.05, ** p < 0.01.Data are expressed as mean ± SD (n = 3).Note: control: blank control group.

Figure 3 .
Figure 3. Cells apoptosis and migration assay.(a) Visual observation of cell clonogenicity.(b) Statistical analysis of cell clonogenicity.Data are mean ± SD (n = 3).Differences between treatment groups and control groups are determined, * p < 0.05, ** p < 0.01 as compared with control.(c) Apoptosis caused by 10 in HGC27 cells.(d) Treatment groups cells apoptosis rates were compared with control, * p < 0.05.Data are expressed as mean ± SD (n = 3).(e) The migration of HGC27 cells.(f) Quantitative evaluations of cell migration induced by 10 in the transwell assay, treatment groups were compared with control, * p < 0.05, ** p < 0.01.Data are expressed as mean ± SD (n = 3).Note: control: blank control group.

Figure 4 .
Figure 4. Compound 10 influences the expression of relevant proteins.Detection of Caspase-3, Bcl-2 and Bax proteins expression level in HGC27cells by Western Blot; the treatment groups were compared with control, * p < ** p < 0.01.Data were expressed as mean ± SD (n = 3).Note: control: blank control group.

Figure 2. The 1 H-1 H COSY and key HMBC correlations of compounds 1, 11, and 12. Table 1. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data of 1 in CD3OD (δ in ppm, J in Hz). No. δH (J in Hz) δC No. δH (J in Hz) δC
Compound 11 was obtained as a yellow powder.The molecular formula of 11 was

Table 2 .
25 NMR and13C NMR data for the compounds 11 and 12 (δ in ppm, J in Hz).determined as C25H 26 O 7 according to its HR-ESI-MS at m/z 439.1749 [M + H] + (calcd.for C 25 H 27 O 7 , 439.1751), indicating 13 degrees a recorded at 600 (150) MHz in DMSO.b recorded at 600 (150) MHz in CD 3 OD.(*) refers to overlapped signals.Compound 12 was obtained as a yellow powder.The molecular formula of 12 was