588929
research-article2015
ICTXXX10.1177/1534735415588929Integrative Cancer TherapiesYang et al
Original Article
Antitumor Effects of Purified Protosappanin B Extracted From Lignum Sappan
Integrative Cancer Therapies 1–9 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1534735415588929 ict.sagepub.com
Xihua Yang, DE1, Liansheng Ren, BS1, Shengwan Zhang, BS2, Lili Zhao, MM1, and Jing Wang, MAgr1
Abstract Hypothesis. To assess the antitumor effects of protosappanin B extracted from Lignum Sappan. Study Design. Lignum Sappan was sequentially extracted by boiling water and ethyl acetate. The resulting extract was separated by column chromatography, to yield protosappanin B. The compound was then identified by thin-layer chromatography, highperformance liquid chromatography, elemental analysis, and spectrometry (infrared and ultraviolet). The effects on tumor cell viability and growth of purified protosappanin B were evaluated in vitro by trypan blue exclusion and MTT assays, respectively. And the effects of protosappanin B were assessed in vivo, on H22 mouse liver cancer cell invasion and the survival of tumor-bearing mice. Results. Protosappanin B (2 mg/mL) reduced the viability of human bladder cancer T24 cells and mouse bladder cancer BTT cells in a time-dependent manner (P < .05) and significantly inhibited the growth of the human colon cancer cell lines HCT-116 and SW-480. IC50 values of 21.32, 26.73, and 76.53 µg/mL were obtained for SW480, HCT-116, and BTT cells, respectively, after 48 hours of treatment with protosappanin B. In addition, pretreatment of H22 cells with protosappanin B (final concentration = 6.25 mg/mL) resulted in complete inhibition of tumor formation in KM mice. Furthermore, protosappanin B (200 and 300 mg/kg) significantly increased the survival of BTT tumor-bearing T739 mice, at a rate comparable to that of 1 mg/kg mitomycin. Conclusion. Protosappanin B extracted from Lignum Sappan exerts marked antitumor effects both in vitro and in vivo. Keywords Lignum Sappan, protosappanin B, antitumor effects
Introduction Lignum Sappan (Sappan wood) is the dry heartwood of Caesalpinia sappan L, a legume recorded in the Pharmacopoeia of China 2010.1 Sappan wood promotes blood circulation, removes blood stasis, and alleviates pain. Recent studies have shown that Lignum Sappan extract exerts significant effects, such as cytotoxicity, growth inhibition, and apoptosis induction, on multiple tumor cell lines,2 including human gastric cancer cells,3 the human promyelocytic leukemia cell line HL-60,4 the chronic myelogenous leukemia cell line K562,5 transplanted H22 tumor cells,6 and the ovarian cancer cell line SKOV3.7 Several chemical compounds have been isolated from Lignum Sappan and are classified according to molecular skeleton into 5 different types: hematoxylins, protosappanins, homoisoflavonoids, sappan chalcones, and phenylpropanoids. Hematoxylins include brazilein,8 brazilin,8 and 3′-O-methylbrazilin9; protosappanins comprise protosappanins A10 and B,11 10-O-methylprotosappanin,12 isoprotosappanin B,13 and protosappanins E1 and E2 (polymers of
hematoxylin and protosappanin components).14 Previous studies have shown that sappan chalcones, brazilein, and 3-deoxysappanchalcone inhibit HL-60 and SWO-38 tumor cell growth; specifically, brazilein inhibits topoisomerase I activity,15 while the mechanisms underlying the antitumor activities of the other compounds are still unclear. Brazilin and protosappanin B are 2 major components of Lignum Sappan, and their structures are clearly defined and used as indicators of traditional Chinese medicine quality in the Pharmacopoeia of China.1 Our previous studies showed that brazilin significantly induces bladder tumor cell (T24) death,2,16 in agreement with other data,17 showing that this compound induces myeloma cancer cell (U266) death over 1
Shanxi Medical University, Taiyuan, China Shanxi University, Taiyuan, China
2
Corresponding Author: Liansheng Ren, Affiliated Tumor Hospital, Shanxi Medical University, Taiyuan, 030000 China. Email: [email protected]
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a short time period and reduces the risk of metastasis. However, protosappanin B has not been evaluated for similar effects. Therefore, the aim of this study was to assess the antitumor activity of protosappanin B extracted from Lignum Sappan on tumor cells in vitro and in vivo.
Materials and Methods Cell Lines and Animals The human bladder cancer cell line T24 was from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China. Mouse bladder cancer BTT cells were a kind gift from Professor Xiaofeng Yang (Department of Urology, the First Affiliated Hospital of Shanxi Medical University, China). Human colon cancer SW-480 and HCT116 cells were a kind gift from Professor Xudong Zhang (Newcastle University, Australia). The mouse liver cancer cell line H22 was obtained from the Laboratory Animal Center of Hebei Medical University (China). Seventy-five female KM mice (4 weeks, 18-22 g) were purchased from the Animal Laboratory of Shanxi Cancer Institute (Production License No. SCXK [Jin] 2012-0001). Fifty female SPF T739 mice (20-24 g) were purchased from Beijing HuaFuKang Biotechnology, China (Production License No. SCXK [Jing] 2009-0004). All animals were housed under specific pathogen-free conditions, with cages and bedding sterilized at 121°C for 20 minutes. Drinking water was sterilized by 60Co irradiation. All animals were allowed access to food and water ad libitum. The protocol for animal study (No. 2012001) was approved by the Laboratory Animal Management Committee of the Shanxi Cancer Institute, in accordance with the guidelines by National Research Council Guide for the Care and Use of Laboratory Animals in China.
Extraction and Purification of Protosappanin B Isolation and Purification of Protosappanin B. A total of 1 kg sappan wood (Wansheng Traditional Chinese Medication Decoction Pieces, Anhui, China) was macerated, immersed in distilled water, and boiled for 60 minutes for extraction. This process was repeated 3 times, and all extracts were combined, concentrated, and filtered through an 80 to 120 µm membrane under normal pressure. Then, an equal volume of petroleum ether was added to the filtrate for lipid removal, a procedure repeated twice. Subsequently, an equal volume of ethyl acetate was added to the aqueous phase, and the ethyl acetate phase isolated; this was repeated 3 times. All aqueous phases were combined, dried under reduced pressure, and the resulting powder was collected. For the purification of protosappanin B, 150 g of 160 to 200 mesh silica gel (Qingdao Marine Chemical Factory, Qingdao, China) was packed in a chromatographic column (4 cm × 40 cm). Then, 15 g of powdered sappan extract were
dissolved in 100 mL ethyl acetate and thoroughly mixed with 30 g silica gel. The solvent was removed by distillation under vacuum, and the sample loaded onto the chromatographic column. Fractions were eluted with ethyl acetate/ petroleum ether/acetic acid (20:100:1) and analyzed by thin-layer chromatography (TLC) using chloroform/acetone/formic acid (8:4:1) as developing solvent. The eluent was changed to ethyl acetate/petroleum ether/acetic acid (20:60:1) when brazilin spots appeared and the distillate was collected (distillate 1). Distillate 1 was reapplied to the chromatographic column, eluted with ethyl acetate/petroleum ether/acetic acid (20:100:1), and analyzed by TLC. The distillates containing the same components after development were combined, dried under reduced pressure, and the powder was collected as pure protosappanin B. Identification of Protosappanin B. Protosappanin B was identified by TLC and high-performance liquid chromatography (HPLC). In TLC analyses, purified protosappanin B samples and the reference compound protosappanin B (Chengdu Mansite Biological Technology, Chengdu, China) were dissolved in ethanol and applied to a TLC plate (Qingdao Marine Chemical Factory, Qingdao, China), which was placed in a chromatography tank filled with the developing solvent chloroform/acetone/formic acid (8:4:1). The Rf values of samples were evaluated under ultraviolet light. For HPLC analyses, an appropriate amount of purified protosappanin B was dissolved in methanol/water (18:82) to obtain a solution at 0.2 mg/mL. HPLC was performed on a Waters 1525 high-performance liquid chromatograph (Waters, Milford, MA), and protosappanin B amounts were obtained by normalization. Determination of Protosappanin B Structure. The structure of the purified protosappanin B was confirmed using elemental analysis (DR × 300 MHz NMR equipment, Bruker, Billerica, MA), and ultraviolet (UV-2550 ultraviolet absorption spectrometry, Shimadzu, Tokyo, Japan), and infrared (Fourier transform infrared spectroscopy, 380, NICOLET, Jacksonville, FL) spectrometry.
In Vitro Inhibitory Effects of Protosappanin B on Tumor Cells T24 and BTT cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) containing 0.05 g/L streptomycin (North China Pharmaceutical Group Corp, Shijiazhuang, China), 0.05 g/L penicillin (North China Pharmaceutical Group Corp), 0.8 g/L NaHCO3, 3.6 g/L HEPES (Sangon Biotech (Shanghai) Co, Ltd, Shanghai, China), and 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials, Hangzhou, China); HCT-116 and SW-480 cells were cultured in DMEM (Thermo-Fisher Biological and Chemical Products, Beijing, China) containing 100 U/mL
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Yang et al streptomycin, 100 U/mL penicillin, and 10% FBS. The cells were cultured at 37°C in a humid environment containing 5% CO2. Logarithmic phase cells were harvested and digested with 0.25% trypsin (Sangon Biotech Co, Ltd, Shanghai, China) to obtain cell suspensions for use in subsequent experiments. Evaluation of Protosappanin B’s Inhibitory Effects on T24 and BTT Cells by the Trypan Blue Exclusion Assay. T24 and BTT cell densities were adjusted to 2 × 106/mL with normal saline (NS). Then, 500 µL of the cell suspension were added to 500 µL protosappanin B dissolved in NS (final concentration = 2 mg/mL) or 500 µL NS (control group). Samples were assessed in duplicate and incubated as described above. Exactly 100 µL of treated cell suspension were collected at 0 (baseline control), 20, 40, 60, and 100 minutes, and mixed with an equal volume of trypan blue (Sigma, St Louis, MO). The numbers of dead (dark bluestained) and viable (nonstained) cells in 4 large grids of the hemocytometer were recorded under an optical microscope (100× magnification; Olympus, Tokyo, Japan). The death rates of T24 and BTT cells were calculated as follows: cell death rate = dead cell count/(dead cell count + viable cell count) × 100%. Evaluation of Protosappanin B’s Inhibitory Effects on HCT116, SW-480, and BTT Cells by MTT Assay. HCT-116, SW-480, and BTT cells were seeded into 96-well plates (4 × 103 cells/well) in 100 µL culture medium. The plate was then incubated as described above. After 24 hours, the culture medium was replaced by 200 µL fresh medium containing protosappanin B or brazilin at final concentrations of 12.5, 25, 50, 100, and 200 µg/mL; an equal volume of culture medium was added to control wells. Culture medium alone was used for blank adjustment, and 5 replicates were setup for each group. After 48 hours of incubation, 20 µL MTT (5 mg/mL, Solarbio, Beijing, China) were added to each well and further incubated for 4 hours. The culture medium was then carefully removed before adding 150 µL dimethyl sulfoxide (Beijing Chemical Works, Beijing, China). The plate was shaken at low speed for 10 minutes to allow complete dissolution of the formazan crystals, and optical density (OD) was measured with an enzyme-linked immunosorbent assay reader at 570 nm (OD570). Growth inhibition ratios and IC50 values of protosappanin B and brazilin for HCT-116, SW-480, and BTT cells were calculated as follows: Growth inhibition ratio (%) = (1 − mean OD570 in the treatment group/mean OD570 in the control group) × 100%; lgIC50 = Xm − I(P − (3 − Pm − Pn)/4), where Xm is lg maximum dose; I represents lg(maximum dose/ adjacent dose); P is the sum of positive reaction rates; Pm represents the maximum positive reaction rate; and Pn is the minimum positive reaction rate.
Effects of Protosappanin B on H22 Tumor Cell Invasion KM mice were randomly divided into 15 groups (n = 5) to be treated with low protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), moderate protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), high protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), mitomycin (Zhejiang Hisun Pharmaceutical Co, Zhejiang, China; 10 minutes, 20 minutes, and 40 minutes), and NS (control group, 10 minutes, 20 minutes, and 40 minutes). Frozen H22 cells were thawed and inoculated (2.5 × 106/ mL, 0.2 mL) into the abdominal cavity of KM mice. Ascites were collected using aseptic techniques at day 7 after inoculation. Then, the cell density was adjusted to 2.5 × 107/mL with NS before addition of protosappanin B (final concentrations of 6.25, 3.12, and 1.56 mg/mL), mitomycin (final concentration of 0.66 mg/mL), or NS (1:9). Afterwards, cells were incubated at 37°C and washed at 10, 20, and 40 minutes to remove the drugs and resuspended in the same volume of NS prior to inoculation into the abdominal cavity of mice in a total volume of 0.2 mL. Mice were observed twice daily (morning and afternoon) after inoculation, and body weights were measured every 3 days. The number of tumors formed and survival time were assessed over a 60-day observation period (60 days was recorded as the maximum survival time). For animals found dead in the morning, survival time was recorded as 1 day; for those found dead in the afternoon, survival time was recorded as 1.5 days. Postmortem examinations were performed on all mice at the end of the observation period, and the ascites and organ metastasis were examined. Tumor formation rate and mean survival time in each group were calculated, and the rate of extended survival was estimated to identify the shortest effective time and minimal effective concentration of the drugs.
Effects of Protosappanin B on the Survival of Tumor-Bearing T739 Mice Suspensions of cultured BTT cells (107/mL) in the logarithmic phase were prepared and injected subcutaneously (0.1 mL) into the right axilla of T739 mice. The mice were sacrificed by cervical dislocation when the tumor diameter reached approximately 1.5 to 2 cm. Euthanized mice were immersed in 75% ethanol for 2 minutes before tumor tissue collection using aseptic techniques. Then, tumor tissues were homogenized to obtain single-cell suspensions, washed twice with NS, and resuspended at 5 × 106/mL prior to inoculation (0.2 mL) into the abdominal cavity of mice. After 24 hours, the animals were randomly divided into 5 groups: control, mitomycin (1 mg/kg), protosappanin B (100 mg/kg), protosappanin B (200 mg/kg), and protosappanin B (300 mg/kg) groups (n = 10). The drugs or NS
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Table 1. High-Performance Liquid Chromatography Analysis of the Purified Protosappanin B. Peak
Retention Time (minutes)
Area (µV s)
Purity (%)
24.413
5 704 748
100.00
1
(0.2 mL) were injected intraperitoneally into each animal, daily for 6 consecutive days. The survival time was measured over a 60-day observation period (60 days was recorded as the maximum survival time) for each group. Survival times were compared among groups, and the rates of extended survival were calculated.
Statistical Analysis The SPSS 17.0 software (SPSS, Chicago, IL) was used for all statistical analyses. Data among groups were compared with one-way analysis of variance (one-way ANOVA) followed by least significant difference (LSD) tests as post hoc. T test was used for comparison between 2 groups. Data are shown as mean ± standard deviation (SD). P < .05 was considered statistically significant.
Results Identification of Protosappanin B TLC and HPLC Analyses. Protosappanin B, purified from Lignum Sappan extract, was quantitatively analyzed by TLC using a commercial protosappanin B standard. The Rf value of the purified protosappanin B was 0.32, identical to that obtained for the reference compound; this result confirmed the identity of the purified protosappanin B, whose purity exceeded 99% by HPLC analysis (Table 1 and Figure 1). Elemental Analysis of Carbon, Hydrogen, and Oxygen Contents. Elemental analyses showed an error rate not exceeding ±3%, suggesting that the carbon, hydrogen, and oxygen contents of the purified protosappanin B were in accordance with the theoretical values of the reference protosappanin B (C16H16O6; Table 2). Ultraviolet and Infrared Spectroscopic Data. Three maximum absorption peaks were observed at λmaxMeOH of 209 nm, 260 nm, and 286 nm in the ultraviolet (UV) spectrum, representing the E- and B-bands of aromatic compounds. The absorption features were in accordance with protosappanin B standard’s data; most important, the UV spectra of both purified and reference protosappanin B were identical (Figures 2 and 3). The infrared spectrum showed a wide, strong νO-H peak at 3381.15 cm−1 and a specific νC-O peak at 1296.25 cm−1,
indicating the presence of phenolic hydroxyl groups in the purified protosappanin B; the νC-O peaks at 1164.83 cm−1 and 1025.13 cm−1 represented tertiary alcohol hydroxyl and primary hydroxyl groups, respectively; the ν-CH2 peak at 2933.88 cm−1 and δCH2 peak at 1436.10 cm−1 suggested the presence of methylene groups; peaks at 1613.27 and 1499.36 cm−1 represented the skeletal vibration of a benzene ring, with a peak at 816.95 cm−1 for 2 adjacent hydrogens on the benzene ring, and that at 883.15 cm−1 suggesting an isolated hydrogen on the benzene ring. Importantly, the infrared spectra of both purified and reference protosappanin B were identical (Figures 4 and 5). These results clearly demonstrated that the compound purified from Lignum Sappan in the present study was protosappanin B, with molecular formula of C16H16O6 and molecular weight of 304.09. The chemical formula of protosappanin B is shown in Figure 6.
Inhibitory Effects of Protosappanin B on Tumor Cells In Vitro Inhibitory Effects of Protosappanin B on T24 and BTT Cell Viability. Compared with the controls, purified protosappanin B (2 mg/mL) effectively induced the death of T24 and BTT cells in vitro in a time-dependent manner (P < .05), suggesting that protosappanin B is highly cytotoxic to these cancer cells (Tables 3 and 4). Inhibitory Effects of Protosappanin B on HCT-116, SW-480, and BTT Cells In Vitro. Protosappanin B reduced HCT-116, SW-480, and BTT cell viability in vitro. The OD values in the MTT assay of HCT-116, SW-480, and BTT cells treated with protosappanin B, at concentrations not exceeding 50 µg/mL, were all significantly higher than those in the brazilin group (P < .05); this indicated a concentrationdependent effect of protosappanin B (Figure 7). IC50 values of 21.32 µg/mL, 26.73 µg/mL, and 76.53 µg/ mL were obtained for protosappanin B toward SW-480, HCT-116, and BTT cells, respectively, after 48 hours of treatment; meanwhile, the IC50 values of brazilin were 7.79 µg/mL, 12.35 µg/mL, and 8.76 µg/mL for SW-480, HCT116, and BTT cells, respectively, after 48 hours of treatment.
Effects of Protosappanin B on H22 Tumor Cell Invasion in KM Mice Characteristics of mouse disease, including increased abdominal circumference and weight, were found at day 6 after H22 cell inoculation. Instances of animal death were first recorded at days 11, 14, and 19 in the 10-, 20-, and 40-minute treatment groups, respectively. Postmortem examinations revealed approximately 5 to 10 mL of
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Figure 1. High-performance liquid chromatogram of purified protosappanin B. Table 2. Elemental Analysis of Carbon, Hydrogen, and Oxygen Contents of the Purified Protosappanin B. Sample
Carbon
Hydrogen
Oxygen
Purified protosappanin B Reference protosappanin B
63.08% 63.16%
5.27% 5.26%
31.65% 31.58%
Figure 2. Ultraviolet absorption spectrum of purified protosappanin B.
Figure 3. Ultraviolet absorption spectrum of the reference protosappanin B.
blood-containing ascites in the abdomen. Tumor formation rates were 100% in the 10-, 20-, and 40-minute groups, while mean survival times of 15.10 ± 5.04 days, 18.40 ± 2.61 days, and 22.50 ± 4.00 days, were obtained, respectively (Table 5). Interestingly, mice showed no characteristic symptoms of illness after inoculation with high protosappanin B dose (6.25 mg/mL) pretreated H22 cells, with no mortality recorded throughout the observation period. In addition, postmortem examination of these mice revealed no ascites or organ metastasis. The tumor formation rate was 0% in the 10-, 20-, and 40-minute groups, with a mean survival time of 60.00 ± 0.00 days (Table 5). After inoculation with moderate protosappanin B dose (3.12 mg/mL) pretreated H22 cells, instances of death were first recorded at days 11, 19, and 21 in the 10-, 20-, and 40-minute groups, respectively. Postmortem examination of the surviving mice at day 60 showed no ascites or organ metastasis, while 5 to 10 mL of blood-containing ascites were found in the abdomen of mice that died during the observation period. Tumor formation rates were 100%, 80%, and 60% in the 10-, 20-, and 40-minute groups, respectively, with mean survival times of 20.40 ± 8.27 days, 32.10 ± 16.34 days, and 41.30 ± 19.73 days, respectively (Table 5). After inoculation with low protosappanin B dose (1.56 mg/mL) pretreated H22 cells, instances of death were first recorded at days 19, 14, and 26 in the 10-, 20-, and 40-minute groups, respectively. Postmortem examination of the only surviving mouse at day 60 revealed small amounts of blood-containing ascites in the abdomen, while 5 to 10 mL of blood-containing ascites were found in the abdomen of
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Figure 4. Infrared absorption spectrum of purified protosappanin B.
Figure 5. Infrared absorption spectra of the reference protosappanin B.
Figure 6. Chemical structural formula of purified protosappanin B.
the animals that died during the observation period. Tumor formation rates were 100% in the 10-, 20-, and 40-minute groups, and mean survival times of 21.60 ± 2.97 days, 24.20 ± 12.6 days, and 35.50 ± 14.11 days, respectively, were obtained (Table 5). Mice showed no characteristic symptoms of illness after inoculation with mitomycin pretreated H22 cells, and all mice survived throughout the experiment. Postmortem examination revealed no ascites, while liver metastasis was found in 1 mouse. The tumor formation rates were 0%, 20%, and 0% in the 10-, 20-, and 40-minute groups, respectively, with mean survival time of 60.00 ± 0.00 days in all 3 groups (Table 5).
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Yang et al Table 3. Death Rates (%) of T24 Cells After Treatment With Protosappanin B In Vitro. Group Control group Protosappanin B group
0 minutes
20 minutes
40 minutes
60 minutes
80 minutes
100 minutes
1.40 ± 0.34 1.42 ± 0.25
2.25 ± 0.39 19.06 ± 4.26*
2.79 ± 0.66 46.76 ± 7.83*
3.18 ± 0.61 58.83 ± 8.02*
3.61 ± 0.96 69.47 ± 3.64*
3.88 ± 0.55 79.58 ± 4.13*
*P < .05, versus the control group.
Table 4. Death Rates (%) of BTT Cells After Treatment With Protosappanin B In Vitro. Group Control group Protosappanin B group
0 minutes
20 minutes
40 minutes
60 minutes
80 minutes
100 minutes
2.55 ± 0.69 2.58 ± 0.54
3.31 ± 0.81 55.7 ± 2.66*
3.66 ± 1.45 66.48 ± 4.14*
3.91 ± 0.54 69.88 ± 2.65*
3.79 ± 0.92 76.38 ± 5.41*
2.76 ± 0.60 81.12 ± 6.11*
*P < .05, versus the control group.
Taken together, these findings indicated that both protosappanin B and mitomycin increased the survival time of tumor-bearing mice. The mean survival times in the high protosappanin B dose (10, 20, and 40 minutes) and mitomycin (10, 20, and 40 minutes) groups were significantly higher than control values (P < .05); the high protosappanin B dose extended animal survival by 297.35%, 226.09%, and 166.67%, respectively, in the 10-, 20-, and 40-minute groups (Table 5).
Protosappanin B Extends Survival of TumorBearing T739 Mice The administration of protosappanin B or mitomycin into the abdominal cavity of BTT tumor-bearing T739 mice significantly increased the mean survival times compared with the control group (P < .05; Table 6).
Discussion
Figure 7. Inhibitory effects of protosappanin B on tumor cells (n = 5): (A) HCT-116 cells; (B) SW-480 cells; (C) BTT cells. *P < .05, versus the brazilin group.
Recent studies have shown that Lignum Sappan extract significantly inhibits multiple tumor cells,4 for example, inducing apoptosis in the ovarian cancer cell line SKOV3.18 Furthermore, brazilin in Lignum Sappan extract has been shown to induce bladder cancer T24 cell death, as well as apoptosis and G2/M arrest via inactivation of histone deacetylase in multiple myeloma U266 cells.17 Brazilin and protosappanin B are 2 major components of Lignum Sappan.19,20 They are both polyphenols with similar structures and chemical characteristics and may have similar biological activity, especially for antitumor effect. The chemical similarity between protosappanin B and brazilin make the isolation of these components problematic. In the present study, 160 to 200 mesh silica gel was used for the chromatographic separation, with ethyl acetate/ petroleum ether/acetic acid (20:100:1) as eluent in TLC tracking analysis. The distillates were combined and dried to obtain purified (>99%) protosappanin B.
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Table 5. Survival Extension of Protosappanin B and Mitomycin in H22 Tumor-Bearing KM Mice (Mean ± SD). n Group
Baseline
End
Concentration (mg/mL)
Tumor Formation Rate (%)
Mean Survival Time (days)
Rate of Extended Survival (%)
5 5 5
0 0 0
0 0 0
100 100 100
15.1 ± 5.04 18.4 ± 2.61 22.5 ± 4.00
— — —
5 5 5
5 5 5
6.25 6.25 6.25
0 0 0
60.0 ± 0.00* 60.0 ± 0.00* 60.0 ± 0.00*
297.35 226.09 166.67
5 5 5
0 1 2
3.12 3.12 3.12
100 80 60
20.4 ± 8.27 32.1 ± 16.34 41.3 ± 19.73
35.10 74.46 83.56
5 5 5
0 0 1
1.56 1.56 1.56
100 100 100
21.6 ± 2.97 24.2 ± 12.6 35.5 ± 14.11
43.05 31.52 57.78
5 5 5
5 5 5
0.66 0.66 0.66
0 20 0
60.0 ± 0.00* 60.0 ± 0.00* 60.0 ± 0.00*
297.35 226.09 166.67
Control 10 minutes 20 minutes 40 minutes High-dose protosappanin B 10 minutes 20 minutes 40 minutes Moderate-dose protosappanin B 10 minutes 20 minutes 40 minutes Low-dose protosappanin B 10 minutes 20 minutes 40 minutes Mitomycin 10 minutes 20 minutes 40 minutes
*P < .05, compared with the corresponding control group.
Table 6. Survival Extension of Protosappanin B in Tumor-Bearing T739 Mice (Mean ± SD). n Group
Baseline
End
Drug Dose (mg/kg/d)
Drug Administration Time (days)
Mean Survival Time (days)
Survival Extension (%)
10 10 10 10 10
0 2 7 8 8
— 100 200 300 1
6 6 6 6 6
19.28 ± 4.94 22.64 ± 5.49* 43.81 ± 10.21* 51.38 ± 11.31* 52.44 ± 8.83*
— 17.43 127.23 166.49 177.99
Control Low-dose protosappanin B Medium-dose protosappanin B High-dose protosappanin B Mitomycin *P < .05, compared with the control group.
We used trypan blue exclusion assays to assess the inhibitory effects of 2 mg/mL protosappanin B on target cells following exposure for 20 to 100 minutes in vitro. Protosappanin B significantly reduced the viability of human bladder cancer T24 cells and colon cancer SW-480 cells, in a time-dependent manner. MTT assay was used to evaluate the effects of different concentrations (12.5, 25, 50, 100, and 200 µg/mL) of protosappanin B on the proliferation of human colon cancer cell lines HCT-116 and SW-480, and the mouse bladder cancer cell line BTT, following 48 hours of exposure. Protosappanin B significantly inhibited the proliferation of all 3 cell lines, with IC50 values of 26.73 µg/mL, 21.32 µg/mL, and 76.53 µg/mL, respectively. These results suggest that protosappanin B has
different antitumor efficacy in different tumor cells. Protosappanin B is most effective against SW480 and least toward BTT, implying a selective antitumor component. To confirm the inhibitory effects induced on tumor cells by short-term treatment with protosappanin B in vivo, mouse liver cancer H22 cells were pretreated with varying concentrations of protosappanin B for different time periods and inoculated into recipient KM mice. No tumor formation was observed following inoculation of H22 cells pretreated with 6.25 µg/mL protosappanin B for 10, 20, and 40 minutes; thus, the tumor inhibition rate was 100%. When the concentration of protosappanin B used to pretreat H22 cells for 40 minutes was decreased to 3.12 µg/mL and 1.73 µg/mL, survival was extended by 83.56% and 57.78%,
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Yang et al respectively, in agreement with the above in vitro data. These findings suggest that protosappanin B inhibits tumor cells both in vitro and in vivo. Mitomycin treatment also resulted in significant tumor-suppressive effects; however, postmortem examination of the animals revealed liver metastasis in one mouse of the 20-minute group. Comprehensive analysis of the results in these 2 groups showed that high-dose protosappanin B is superior to mitomycin. In vivo experiments conducted in the present study showed that intraperitoneal injection of 200 µg/kg and 300 µg/kg (daily for 6 days) of protosappanin B significantly inhibits tumor growth in tumor-bearing animals, resulting in survival extension by 166.49% and 127.23%, with no apparent toxicity. These data suggest that treatment with protosappanin B is safe and effective. Besides, the antitumor effect of protosappanin B is dose-dependent.
Conclusions In summary, in vitro and in vivo data presented here show that protosappanin B significantly inhibits the viability of the human bladder cancer cell line T24, mouse bladder cancer cell line BTT, and human colon cancer cell lines SW-480 and HCT-116, reducing cell growth. In addition, protosappanin B inhibits H22 cell invasion in animals and extends the survival of tumor-bearing mice. Therefore, protosappanin B is a component of Lignum Sappan with antitumor activity; however, the mechanisms underlying these effects remain to be elucidated. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Special Fund for Experimental Animals from Shanxi Science and Technology Department, China (No. 2013K02), and Shanxi Fundamental Resources Platform of Science and Technology from Shanxi Science and Technology Department, China (No. 2013091011).
References 1. National Pharmacopoeia Committee. People’s Republic of China Pharmacopoeia. Beijing, China: Chinese Medical Science and Technology Press; 2010:153. 2. Zhao LL, Ren LS, Wang GP. Study on anticancer active principle in lignum Caesalpinia sappan. Cancer Res Clin. 2012;24:157-160. 3. Wang X, Zhao SL, Xu JG, Zhao YF. Study on proliferative inhibition and apoptosis of human gastric cancer cell with treatment of Caesalpinia sappan L. extract. Chin Remedies Clin. 2007;7:843-845.
4. Ren LS, Tang Y, Zhang H, Wang YY. Studies of anti-cancer mechanism of aqueous extract of lignum sappan. Shanxi Med J. 2000;29:201-203. 5. Wang SL, Cai B, Cui CB, Zhang HF, Yao XS, Qu GX. Apoptosis induced by Caesalpinia sappan L. extract in leukemia cell line K562. Chin J Cancer. 2001;20:1376-1379. 6. Peng X. Research on Immunosuppressive and Anti-Tumor Activity of Caesalpinia sappan L. Extract. Beijing, China: Beijing University of Chinese Medicine; 2009. 7. Ren LS, Xu JG, Ma JY, Zhang H, Zhuang BQ, Zhang L. Research on anti-cancer activity of Caesalpinia sappan. Zhongguo Zhong Yao Za Zhi. 1990;15(5):50-51. 8. Dong SK, Nam-In B, Sei RO, Keun YJ, Im SL, HyeongKyu L. NMR assignment of brazilein. Phytochemistry. 1997;46:177-178. 9. Namikoshi M, Nakata H, Yamada H, Nagai M, Saitoh T. Homoisoflavonoids and related compounds. II. Isolation and absolute configurations of 3,4-dihydroxylated homoisoflavans and Brazilins from Caesalpinia sappan L. Chem Pharm Bull. 1987;35:2761-2773. 10. Nagai M, Lee S. Protosappanin A, a novel biphenyl compound from Sappan lignum. Chem Pharm Bull. 1986;34:1-6. 11. Nagai M, Nagumo S. Protosappanin B, a new dibenzoxocin derivative from Sappan lignum. Heterocycles. 1986;24: 601-606. 12. Namikoshi M, Nakata H, Saitoh T. Homoisoflavonoids and related compounds: N. A novel dibenzoxocin derivative from Caesalpinia sappan L. Chem Pharm Bull. 1987;35: 3615-3619. 13. Ouyang B. Research on Chemical Constituents of Caesalpinia sappan. Shanghai, China: Shanghai Institute of MateriaMedica, Chinese Academy of Science; 2002. 14. Nagai M, Nagumo S. Protosappanins E-1 and E-2, stereoisomeric dibenzoxocins combined with Brazilin from Sappan lignum. Chem Pharm Bull. 1990;38:1490-1494. 15. Yu B, Hou JB, Lv H. Effect of different extracts of Caesalpinia sappan L. extract on T lymphocytes subsets and myocardial protectively function after rat heart transplantation. Chin J Endemiol. 2004;23:539-542. 16. Zhao LL, Wang GP, Yang XH, Zhang H, Ren LS. Effect of brazilin on proliferation and apoptosis of bladder carcinoma T24 cell line. Cancer Res Clin. 2013;25:516-519. 17. Kim B, Kim SH, Jeong SJ, et al. Brazilin induces apoptosis and G2/M arrest via inactivation of histone deacetylase in multiple myeloma U266 cells. J Agric Food Chem. 2012;60:9882-9889. 18. Zhang XM, Wang GP, Ren LS. Study of Caesalpinia sappan aqueous exact is apoptosis-inducing effects on human ovarian cancer cell SKOV3. Cancer Res Clin. 2010;22:388-390. 19. Lu Y, Bai H, Kong C, Zhong H, Breadmore MC. Analysis of brazilin and protosappanin B in sappan lignum by capillary zone electrophoresis with acid barrage stacking. Electrophoresis. 2013;34:3326-3332. 20. Tong XZ, Zhu H, Shi Y, Xu HT, Wang B, Zhao JH. An LC/ MS/MS method for simultaneous quantitation of two homoisoflavones: protosappanin B and brazilin with hypoglycemic activity in rat plasma and its application to a comparative pharmacokinetic study in normal and streptozotocin-treated rats. J Ethnopharmacol. 2013;148:682-690.
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research-article2015
ICTXXX10.1177/1534735415588929Integrative Cancer TherapiesYang et al
Original Article
Antitumor Effects of Purified Protosappanin B Extracted From Lignum Sappan
Integrative Cancer Therapies 1–9 © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1534735415588929 ict.sagepub.com
Xihua Yang, DE1, Liansheng Ren, BS1, Shengwan Zhang, BS2, Lili Zhao, MM1, and Jing Wang, MAgr1
Abstract Hypothesis. To assess the antitumor effects of protosappanin B extracted from Lignum Sappan. Study Design. Lignum Sappan was sequentially extracted by boiling water and ethyl acetate. The resulting extract was separated by column chromatography, to yield protosappanin B. The compound was then identified by thin-layer chromatography, highperformance liquid chromatography, elemental analysis, and spectrometry (infrared and ultraviolet). The effects on tumor cell viability and growth of purified protosappanin B were evaluated in vitro by trypan blue exclusion and MTT assays, respectively. And the effects of protosappanin B were assessed in vivo, on H22 mouse liver cancer cell invasion and the survival of tumor-bearing mice. Results. Protosappanin B (2 mg/mL) reduced the viability of human bladder cancer T24 cells and mouse bladder cancer BTT cells in a time-dependent manner (P < .05) and significantly inhibited the growth of the human colon cancer cell lines HCT-116 and SW-480. IC50 values of 21.32, 26.73, and 76.53 µg/mL were obtained for SW480, HCT-116, and BTT cells, respectively, after 48 hours of treatment with protosappanin B. In addition, pretreatment of H22 cells with protosappanin B (final concentration = 6.25 mg/mL) resulted in complete inhibition of tumor formation in KM mice. Furthermore, protosappanin B (200 and 300 mg/kg) significantly increased the survival of BTT tumor-bearing T739 mice, at a rate comparable to that of 1 mg/kg mitomycin. Conclusion. Protosappanin B extracted from Lignum Sappan exerts marked antitumor effects both in vitro and in vivo. Keywords Lignum Sappan, protosappanin B, antitumor effects
Introduction Lignum Sappan (Sappan wood) is the dry heartwood of Caesalpinia sappan L, a legume recorded in the Pharmacopoeia of China 2010.1 Sappan wood promotes blood circulation, removes blood stasis, and alleviates pain. Recent studies have shown that Lignum Sappan extract exerts significant effects, such as cytotoxicity, growth inhibition, and apoptosis induction, on multiple tumor cell lines,2 including human gastric cancer cells,3 the human promyelocytic leukemia cell line HL-60,4 the chronic myelogenous leukemia cell line K562,5 transplanted H22 tumor cells,6 and the ovarian cancer cell line SKOV3.7 Several chemical compounds have been isolated from Lignum Sappan and are classified according to molecular skeleton into 5 different types: hematoxylins, protosappanins, homoisoflavonoids, sappan chalcones, and phenylpropanoids. Hematoxylins include brazilein,8 brazilin,8 and 3′-O-methylbrazilin9; protosappanins comprise protosappanins A10 and B,11 10-O-methylprotosappanin,12 isoprotosappanin B,13 and protosappanins E1 and E2 (polymers of
hematoxylin and protosappanin components).14 Previous studies have shown that sappan chalcones, brazilein, and 3-deoxysappanchalcone inhibit HL-60 and SWO-38 tumor cell growth; specifically, brazilein inhibits topoisomerase I activity,15 while the mechanisms underlying the antitumor activities of the other compounds are still unclear. Brazilin and protosappanin B are 2 major components of Lignum Sappan, and their structures are clearly defined and used as indicators of traditional Chinese medicine quality in the Pharmacopoeia of China.1 Our previous studies showed that brazilin significantly induces bladder tumor cell (T24) death,2,16 in agreement with other data,17 showing that this compound induces myeloma cancer cell (U266) death over 1
Shanxi Medical University, Taiyuan, China Shanxi University, Taiyuan, China
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Corresponding Author: Liansheng Ren, Affiliated Tumor Hospital, Shanxi Medical University, Taiyuan, 030000 China. Email: [email protected]
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a short time period and reduces the risk of metastasis. However, protosappanin B has not been evaluated for similar effects. Therefore, the aim of this study was to assess the antitumor activity of protosappanin B extracted from Lignum Sappan on tumor cells in vitro and in vivo.
Materials and Methods Cell Lines and Animals The human bladder cancer cell line T24 was from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China. Mouse bladder cancer BTT cells were a kind gift from Professor Xiaofeng Yang (Department of Urology, the First Affiliated Hospital of Shanxi Medical University, China). Human colon cancer SW-480 and HCT116 cells were a kind gift from Professor Xudong Zhang (Newcastle University, Australia). The mouse liver cancer cell line H22 was obtained from the Laboratory Animal Center of Hebei Medical University (China). Seventy-five female KM mice (4 weeks, 18-22 g) were purchased from the Animal Laboratory of Shanxi Cancer Institute (Production License No. SCXK [Jin] 2012-0001). Fifty female SPF T739 mice (20-24 g) were purchased from Beijing HuaFuKang Biotechnology, China (Production License No. SCXK [Jing] 2009-0004). All animals were housed under specific pathogen-free conditions, with cages and bedding sterilized at 121°C for 20 minutes. Drinking water was sterilized by 60Co irradiation. All animals were allowed access to food and water ad libitum. The protocol for animal study (No. 2012001) was approved by the Laboratory Animal Management Committee of the Shanxi Cancer Institute, in accordance with the guidelines by National Research Council Guide for the Care and Use of Laboratory Animals in China.
Extraction and Purification of Protosappanin B Isolation and Purification of Protosappanin B. A total of 1 kg sappan wood (Wansheng Traditional Chinese Medication Decoction Pieces, Anhui, China) was macerated, immersed in distilled water, and boiled for 60 minutes for extraction. This process was repeated 3 times, and all extracts were combined, concentrated, and filtered through an 80 to 120 µm membrane under normal pressure. Then, an equal volume of petroleum ether was added to the filtrate for lipid removal, a procedure repeated twice. Subsequently, an equal volume of ethyl acetate was added to the aqueous phase, and the ethyl acetate phase isolated; this was repeated 3 times. All aqueous phases were combined, dried under reduced pressure, and the resulting powder was collected. For the purification of protosappanin B, 150 g of 160 to 200 mesh silica gel (Qingdao Marine Chemical Factory, Qingdao, China) was packed in a chromatographic column (4 cm × 40 cm). Then, 15 g of powdered sappan extract were
dissolved in 100 mL ethyl acetate and thoroughly mixed with 30 g silica gel. The solvent was removed by distillation under vacuum, and the sample loaded onto the chromatographic column. Fractions were eluted with ethyl acetate/ petroleum ether/acetic acid (20:100:1) and analyzed by thin-layer chromatography (TLC) using chloroform/acetone/formic acid (8:4:1) as developing solvent. The eluent was changed to ethyl acetate/petroleum ether/acetic acid (20:60:1) when brazilin spots appeared and the distillate was collected (distillate 1). Distillate 1 was reapplied to the chromatographic column, eluted with ethyl acetate/petroleum ether/acetic acid (20:100:1), and analyzed by TLC. The distillates containing the same components after development were combined, dried under reduced pressure, and the powder was collected as pure protosappanin B. Identification of Protosappanin B. Protosappanin B was identified by TLC and high-performance liquid chromatography (HPLC). In TLC analyses, purified protosappanin B samples and the reference compound protosappanin B (Chengdu Mansite Biological Technology, Chengdu, China) were dissolved in ethanol and applied to a TLC plate (Qingdao Marine Chemical Factory, Qingdao, China), which was placed in a chromatography tank filled with the developing solvent chloroform/acetone/formic acid (8:4:1). The Rf values of samples were evaluated under ultraviolet light. For HPLC analyses, an appropriate amount of purified protosappanin B was dissolved in methanol/water (18:82) to obtain a solution at 0.2 mg/mL. HPLC was performed on a Waters 1525 high-performance liquid chromatograph (Waters, Milford, MA), and protosappanin B amounts were obtained by normalization. Determination of Protosappanin B Structure. The structure of the purified protosappanin B was confirmed using elemental analysis (DR × 300 MHz NMR equipment, Bruker, Billerica, MA), and ultraviolet (UV-2550 ultraviolet absorption spectrometry, Shimadzu, Tokyo, Japan), and infrared (Fourier transform infrared spectroscopy, 380, NICOLET, Jacksonville, FL) spectrometry.
In Vitro Inhibitory Effects of Protosappanin B on Tumor Cells T24 and BTT cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) containing 0.05 g/L streptomycin (North China Pharmaceutical Group Corp, Shijiazhuang, China), 0.05 g/L penicillin (North China Pharmaceutical Group Corp), 0.8 g/L NaHCO3, 3.6 g/L HEPES (Sangon Biotech (Shanghai) Co, Ltd, Shanghai, China), and 10% fetal bovine serum (FBS; Hangzhou Sijiqing Biological Engineering Materials, Hangzhou, China); HCT-116 and SW-480 cells were cultured in DMEM (Thermo-Fisher Biological and Chemical Products, Beijing, China) containing 100 U/mL
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Yang et al streptomycin, 100 U/mL penicillin, and 10% FBS. The cells were cultured at 37°C in a humid environment containing 5% CO2. Logarithmic phase cells were harvested and digested with 0.25% trypsin (Sangon Biotech Co, Ltd, Shanghai, China) to obtain cell suspensions for use in subsequent experiments. Evaluation of Protosappanin B’s Inhibitory Effects on T24 and BTT Cells by the Trypan Blue Exclusion Assay. T24 and BTT cell densities were adjusted to 2 × 106/mL with normal saline (NS). Then, 500 µL of the cell suspension were added to 500 µL protosappanin B dissolved in NS (final concentration = 2 mg/mL) or 500 µL NS (control group). Samples were assessed in duplicate and incubated as described above. Exactly 100 µL of treated cell suspension were collected at 0 (baseline control), 20, 40, 60, and 100 minutes, and mixed with an equal volume of trypan blue (Sigma, St Louis, MO). The numbers of dead (dark bluestained) and viable (nonstained) cells in 4 large grids of the hemocytometer were recorded under an optical microscope (100× magnification; Olympus, Tokyo, Japan). The death rates of T24 and BTT cells were calculated as follows: cell death rate = dead cell count/(dead cell count + viable cell count) × 100%. Evaluation of Protosappanin B’s Inhibitory Effects on HCT116, SW-480, and BTT Cells by MTT Assay. HCT-116, SW-480, and BTT cells were seeded into 96-well plates (4 × 103 cells/well) in 100 µL culture medium. The plate was then incubated as described above. After 24 hours, the culture medium was replaced by 200 µL fresh medium containing protosappanin B or brazilin at final concentrations of 12.5, 25, 50, 100, and 200 µg/mL; an equal volume of culture medium was added to control wells. Culture medium alone was used for blank adjustment, and 5 replicates were setup for each group. After 48 hours of incubation, 20 µL MTT (5 mg/mL, Solarbio, Beijing, China) were added to each well and further incubated for 4 hours. The culture medium was then carefully removed before adding 150 µL dimethyl sulfoxide (Beijing Chemical Works, Beijing, China). The plate was shaken at low speed for 10 minutes to allow complete dissolution of the formazan crystals, and optical density (OD) was measured with an enzyme-linked immunosorbent assay reader at 570 nm (OD570). Growth inhibition ratios and IC50 values of protosappanin B and brazilin for HCT-116, SW-480, and BTT cells were calculated as follows: Growth inhibition ratio (%) = (1 − mean OD570 in the treatment group/mean OD570 in the control group) × 100%; lgIC50 = Xm − I(P − (3 − Pm − Pn)/4), where Xm is lg maximum dose; I represents lg(maximum dose/ adjacent dose); P is the sum of positive reaction rates; Pm represents the maximum positive reaction rate; and Pn is the minimum positive reaction rate.
Effects of Protosappanin B on H22 Tumor Cell Invasion KM mice were randomly divided into 15 groups (n = 5) to be treated with low protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), moderate protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), high protosappanin B dose (10 minutes, 20 minutes, and 40 minutes), mitomycin (Zhejiang Hisun Pharmaceutical Co, Zhejiang, China; 10 minutes, 20 minutes, and 40 minutes), and NS (control group, 10 minutes, 20 minutes, and 40 minutes). Frozen H22 cells were thawed and inoculated (2.5 × 106/ mL, 0.2 mL) into the abdominal cavity of KM mice. Ascites were collected using aseptic techniques at day 7 after inoculation. Then, the cell density was adjusted to 2.5 × 107/mL with NS before addition of protosappanin B (final concentrations of 6.25, 3.12, and 1.56 mg/mL), mitomycin (final concentration of 0.66 mg/mL), or NS (1:9). Afterwards, cells were incubated at 37°C and washed at 10, 20, and 40 minutes to remove the drugs and resuspended in the same volume of NS prior to inoculation into the abdominal cavity of mice in a total volume of 0.2 mL. Mice were observed twice daily (morning and afternoon) after inoculation, and body weights were measured every 3 days. The number of tumors formed and survival time were assessed over a 60-day observation period (60 days was recorded as the maximum survival time). For animals found dead in the morning, survival time was recorded as 1 day; for those found dead in the afternoon, survival time was recorded as 1.5 days. Postmortem examinations were performed on all mice at the end of the observation period, and the ascites and organ metastasis were examined. Tumor formation rate and mean survival time in each group were calculated, and the rate of extended survival was estimated to identify the shortest effective time and minimal effective concentration of the drugs.
Effects of Protosappanin B on the Survival of Tumor-Bearing T739 Mice Suspensions of cultured BTT cells (107/mL) in the logarithmic phase were prepared and injected subcutaneously (0.1 mL) into the right axilla of T739 mice. The mice were sacrificed by cervical dislocation when the tumor diameter reached approximately 1.5 to 2 cm. Euthanized mice were immersed in 75% ethanol for 2 minutes before tumor tissue collection using aseptic techniques. Then, tumor tissues were homogenized to obtain single-cell suspensions, washed twice with NS, and resuspended at 5 × 106/mL prior to inoculation (0.2 mL) into the abdominal cavity of mice. After 24 hours, the animals were randomly divided into 5 groups: control, mitomycin (1 mg/kg), protosappanin B (100 mg/kg), protosappanin B (200 mg/kg), and protosappanin B (300 mg/kg) groups (n = 10). The drugs or NS
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Table 1. High-Performance Liquid Chromatography Analysis of the Purified Protosappanin B. Peak
Retention Time (minutes)
Area (µV s)
Purity (%)
24.413
5 704 748
100.00
1
(0.2 mL) were injected intraperitoneally into each animal, daily for 6 consecutive days. The survival time was measured over a 60-day observation period (60 days was recorded as the maximum survival time) for each group. Survival times were compared among groups, and the rates of extended survival were calculated.
Statistical Analysis The SPSS 17.0 software (SPSS, Chicago, IL) was used for all statistical analyses. Data among groups were compared with one-way analysis of variance (one-way ANOVA) followed by least significant difference (LSD) tests as post hoc. T test was used for comparison between 2 groups. Data are shown as mean ± standard deviation (SD). P < .05 was considered statistically significant.
Results Identification of Protosappanin B TLC and HPLC Analyses. Protosappanin B, purified from Lignum Sappan extract, was quantitatively analyzed by TLC using a commercial protosappanin B standard. The Rf value of the purified protosappanin B was 0.32, identical to that obtained for the reference compound; this result confirmed the identity of the purified protosappanin B, whose purity exceeded 99% by HPLC analysis (Table 1 and Figure 1). Elemental Analysis of Carbon, Hydrogen, and Oxygen Contents. Elemental analyses showed an error rate not exceeding ±3%, suggesting that the carbon, hydrogen, and oxygen contents of the purified protosappanin B were in accordance with the theoretical values of the reference protosappanin B (C16H16O6; Table 2). Ultraviolet and Infrared Spectroscopic Data. Three maximum absorption peaks were observed at λmaxMeOH of 209 nm, 260 nm, and 286 nm in the ultraviolet (UV) spectrum, representing the E- and B-bands of aromatic compounds. The absorption features were in accordance with protosappanin B standard’s data; most important, the UV spectra of both purified and reference protosappanin B were identical (Figures 2 and 3). The infrared spectrum showed a wide, strong νO-H peak at 3381.15 cm−1 and a specific νC-O peak at 1296.25 cm−1,
indicating the presence of phenolic hydroxyl groups in the purified protosappanin B; the νC-O peaks at 1164.83 cm−1 and 1025.13 cm−1 represented tertiary alcohol hydroxyl and primary hydroxyl groups, respectively; the ν-CH2 peak at 2933.88 cm−1 and δCH2 peak at 1436.10 cm−1 suggested the presence of methylene groups; peaks at 1613.27 and 1499.36 cm−1 represented the skeletal vibration of a benzene ring, with a peak at 816.95 cm−1 for 2 adjacent hydrogens on the benzene ring, and that at 883.15 cm−1 suggesting an isolated hydrogen on the benzene ring. Importantly, the infrared spectra of both purified and reference protosappanin B were identical (Figures 4 and 5). These results clearly demonstrated that the compound purified from Lignum Sappan in the present study was protosappanin B, with molecular formula of C16H16O6 and molecular weight of 304.09. The chemical formula of protosappanin B is shown in Figure 6.
Inhibitory Effects of Protosappanin B on Tumor Cells In Vitro Inhibitory Effects of Protosappanin B on T24 and BTT Cell Viability. Compared with the controls, purified protosappanin B (2 mg/mL) effectively induced the death of T24 and BTT cells in vitro in a time-dependent manner (P < .05), suggesting that protosappanin B is highly cytotoxic to these cancer cells (Tables 3 and 4). Inhibitory Effects of Protosappanin B on HCT-116, SW-480, and BTT Cells In Vitro. Protosappanin B reduced HCT-116, SW-480, and BTT cell viability in vitro. The OD values in the MTT assay of HCT-116, SW-480, and BTT cells treated with protosappanin B, at concentrations not exceeding 50 µg/mL, were all significantly higher than those in the brazilin group (P < .05); this indicated a concentrationdependent effect of protosappanin B (Figure 7). IC50 values of 21.32 µg/mL, 26.73 µg/mL, and 76.53 µg/ mL were obtained for protosappanin B toward SW-480, HCT-116, and BTT cells, respectively, after 48 hours of treatment; meanwhile, the IC50 values of brazilin were 7.79 µg/mL, 12.35 µg/mL, and 8.76 µg/mL for SW-480, HCT116, and BTT cells, respectively, after 48 hours of treatment.
Effects of Protosappanin B on H22 Tumor Cell Invasion in KM Mice Characteristics of mouse disease, including increased abdominal circumference and weight, were found at day 6 after H22 cell inoculation. Instances of animal death were first recorded at days 11, 14, and 19 in the 10-, 20-, and 40-minute treatment groups, respectively. Postmortem examinations revealed approximately 5 to 10 mL of
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Figure 1. High-performance liquid chromatogram of purified protosappanin B. Table 2. Elemental Analysis of Carbon, Hydrogen, and Oxygen Contents of the Purified Protosappanin B. Sample
Carbon
Hydrogen
Oxygen
Purified protosappanin B Reference protosappanin B
63.08% 63.16%
5.27% 5.26%
31.65% 31.58%
Figure 2. Ultraviolet absorption spectrum of purified protosappanin B.
Figure 3. Ultraviolet absorption spectrum of the reference protosappanin B.
blood-containing ascites in the abdomen. Tumor formation rates were 100% in the 10-, 20-, and 40-minute groups, while mean survival times of 15.10 ± 5.04 days, 18.40 ± 2.61 days, and 22.50 ± 4.00 days, were obtained, respectively (Table 5). Interestingly, mice showed no characteristic symptoms of illness after inoculation with high protosappanin B dose (6.25 mg/mL) pretreated H22 cells, with no mortality recorded throughout the observation period. In addition, postmortem examination of these mice revealed no ascites or organ metastasis. The tumor formation rate was 0% in the 10-, 20-, and 40-minute groups, with a mean survival time of 60.00 ± 0.00 days (Table 5). After inoculation with moderate protosappanin B dose (3.12 mg/mL) pretreated H22 cells, instances of death were first recorded at days 11, 19, and 21 in the 10-, 20-, and 40-minute groups, respectively. Postmortem examination of the surviving mice at day 60 showed no ascites or organ metastasis, while 5 to 10 mL of blood-containing ascites were found in the abdomen of mice that died during the observation period. Tumor formation rates were 100%, 80%, and 60% in the 10-, 20-, and 40-minute groups, respectively, with mean survival times of 20.40 ± 8.27 days, 32.10 ± 16.34 days, and 41.30 ± 19.73 days, respectively (Table 5). After inoculation with low protosappanin B dose (1.56 mg/mL) pretreated H22 cells, instances of death were first recorded at days 19, 14, and 26 in the 10-, 20-, and 40-minute groups, respectively. Postmortem examination of the only surviving mouse at day 60 revealed small amounts of blood-containing ascites in the abdomen, while 5 to 10 mL of blood-containing ascites were found in the abdomen of
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Figure 4. Infrared absorption spectrum of purified protosappanin B.
Figure 5. Infrared absorption spectra of the reference protosappanin B.
Figure 6. Chemical structural formula of purified protosappanin B.
the animals that died during the observation period. Tumor formation rates were 100% in the 10-, 20-, and 40-minute groups, and mean survival times of 21.60 ± 2.97 days, 24.20 ± 12.6 days, and 35.50 ± 14.11 days, respectively, were obtained (Table 5). Mice showed no characteristic symptoms of illness after inoculation with mitomycin pretreated H22 cells, and all mice survived throughout the experiment. Postmortem examination revealed no ascites, while liver metastasis was found in 1 mouse. The tumor formation rates were 0%, 20%, and 0% in the 10-, 20-, and 40-minute groups, respectively, with mean survival time of 60.00 ± 0.00 days in all 3 groups (Table 5).
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Yang et al Table 3. Death Rates (%) of T24 Cells After Treatment With Protosappanin B In Vitro. Group Control group Protosappanin B group
0 minutes
20 minutes
40 minutes
60 minutes
80 minutes
100 minutes
1.40 ± 0.34 1.42 ± 0.25
2.25 ± 0.39 19.06 ± 4.26*
2.79 ± 0.66 46.76 ± 7.83*
3.18 ± 0.61 58.83 ± 8.02*
3.61 ± 0.96 69.47 ± 3.64*
3.88 ± 0.55 79.58 ± 4.13*
*P < .05, versus the control group.
Table 4. Death Rates (%) of BTT Cells After Treatment With Protosappanin B In Vitro. Group Control group Protosappanin B group
0 minutes
20 minutes
40 minutes
60 minutes
80 minutes
100 minutes
2.55 ± 0.69 2.58 ± 0.54
3.31 ± 0.81 55.7 ± 2.66*
3.66 ± 1.45 66.48 ± 4.14*
3.91 ± 0.54 69.88 ± 2.65*
3.79 ± 0.92 76.38 ± 5.41*
2.76 ± 0.60 81.12 ± 6.11*
*P < .05, versus the control group.
Taken together, these findings indicated that both protosappanin B and mitomycin increased the survival time of tumor-bearing mice. The mean survival times in the high protosappanin B dose (10, 20, and 40 minutes) and mitomycin (10, 20, and 40 minutes) groups were significantly higher than control values (P < .05); the high protosappanin B dose extended animal survival by 297.35%, 226.09%, and 166.67%, respectively, in the 10-, 20-, and 40-minute groups (Table 5).
Protosappanin B Extends Survival of TumorBearing T739 Mice The administration of protosappanin B or mitomycin into the abdominal cavity of BTT tumor-bearing T739 mice significantly increased the mean survival times compared with the control group (P < .05; Table 6).
Discussion
Figure 7. Inhibitory effects of protosappanin B on tumor cells (n = 5): (A) HCT-116 cells; (B) SW-480 cells; (C) BTT cells. *P < .05, versus the brazilin group.
Recent studies have shown that Lignum Sappan extract significantly inhibits multiple tumor cells,4 for example, inducing apoptosis in the ovarian cancer cell line SKOV3.18 Furthermore, brazilin in Lignum Sappan extract has been shown to induce bladder cancer T24 cell death, as well as apoptosis and G2/M arrest via inactivation of histone deacetylase in multiple myeloma U266 cells.17 Brazilin and protosappanin B are 2 major components of Lignum Sappan.19,20 They are both polyphenols with similar structures and chemical characteristics and may have similar biological activity, especially for antitumor effect. The chemical similarity between protosappanin B and brazilin make the isolation of these components problematic. In the present study, 160 to 200 mesh silica gel was used for the chromatographic separation, with ethyl acetate/ petroleum ether/acetic acid (20:100:1) as eluent in TLC tracking analysis. The distillates were combined and dried to obtain purified (>99%) protosappanin B.
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Integrative Cancer Therapies
Table 5. Survival Extension of Protosappanin B and Mitomycin in H22 Tumor-Bearing KM Mice (Mean ± SD). n Group
Baseline
End
Concentration (mg/mL)
Tumor Formation Rate (%)
Mean Survival Time (days)
Rate of Extended Survival (%)
5 5 5
0 0 0
0 0 0
100 100 100
15.1 ± 5.04 18.4 ± 2.61 22.5 ± 4.00
— — —
5 5 5
5 5 5
6.25 6.25 6.25
0 0 0
60.0 ± 0.00* 60.0 ± 0.00* 60.0 ± 0.00*
297.35 226.09 166.67
5 5 5
0 1 2
3.12 3.12 3.12
100 80 60
20.4 ± 8.27 32.1 ± 16.34 41.3 ± 19.73
35.10 74.46 83.56
5 5 5
0 0 1
1.56 1.56 1.56
100 100 100
21.6 ± 2.97 24.2 ± 12.6 35.5 ± 14.11
43.05 31.52 57.78
5 5 5
5 5 5
0.66 0.66 0.66
0 20 0
60.0 ± 0.00* 60.0 ± 0.00* 60.0 ± 0.00*
297.35 226.09 166.67
Control 10 minutes 20 minutes 40 minutes High-dose protosappanin B 10 minutes 20 minutes 40 minutes Moderate-dose protosappanin B 10 minutes 20 minutes 40 minutes Low-dose protosappanin B 10 minutes 20 minutes 40 minutes Mitomycin 10 minutes 20 minutes 40 minutes
*P < .05, compared with the corresponding control group.
Table 6. Survival Extension of Protosappanin B in Tumor-Bearing T739 Mice (Mean ± SD). n Group
Baseline
End
Drug Dose (mg/kg/d)
Drug Administration Time (days)
Mean Survival Time (days)
Survival Extension (%)
10 10 10 10 10
0 2 7 8 8
— 100 200 300 1
6 6 6 6 6
19.28 ± 4.94 22.64 ± 5.49* 43.81 ± 10.21* 51.38 ± 11.31* 52.44 ± 8.83*
— 17.43 127.23 166.49 177.99
Control Low-dose protosappanin B Medium-dose protosappanin B High-dose protosappanin B Mitomycin *P < .05, compared with the control group.
We used trypan blue exclusion assays to assess the inhibitory effects of 2 mg/mL protosappanin B on target cells following exposure for 20 to 100 minutes in vitro. Protosappanin B significantly reduced the viability of human bladder cancer T24 cells and colon cancer SW-480 cells, in a time-dependent manner. MTT assay was used to evaluate the effects of different concentrations (12.5, 25, 50, 100, and 200 µg/mL) of protosappanin B on the proliferation of human colon cancer cell lines HCT-116 and SW-480, and the mouse bladder cancer cell line BTT, following 48 hours of exposure. Protosappanin B significantly inhibited the proliferation of all 3 cell lines, with IC50 values of 26.73 µg/mL, 21.32 µg/mL, and 76.53 µg/mL, respectively. These results suggest that protosappanin B has
different antitumor efficacy in different tumor cells. Protosappanin B is most effective against SW480 and least toward BTT, implying a selective antitumor component. To confirm the inhibitory effects induced on tumor cells by short-term treatment with protosappanin B in vivo, mouse liver cancer H22 cells were pretreated with varying concentrations of protosappanin B for different time periods and inoculated into recipient KM mice. No tumor formation was observed following inoculation of H22 cells pretreated with 6.25 µg/mL protosappanin B for 10, 20, and 40 minutes; thus, the tumor inhibition rate was 100%. When the concentration of protosappanin B used to pretreat H22 cells for 40 minutes was decreased to 3.12 µg/mL and 1.73 µg/mL, survival was extended by 83.56% and 57.78%,
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Yang et al respectively, in agreement with the above in vitro data. These findings suggest that protosappanin B inhibits tumor cells both in vitro and in vivo. Mitomycin treatment also resulted in significant tumor-suppressive effects; however, postmortem examination of the animals revealed liver metastasis in one mouse of the 20-minute group. Comprehensive analysis of the results in these 2 groups showed that high-dose protosappanin B is superior to mitomycin. In vivo experiments conducted in the present study showed that intraperitoneal injection of 200 µg/kg and 300 µg/kg (daily for 6 days) of protosappanin B significantly inhibits tumor growth in tumor-bearing animals, resulting in survival extension by 166.49% and 127.23%, with no apparent toxicity. These data suggest that treatment with protosappanin B is safe and effective. Besides, the antitumor effect of protosappanin B is dose-dependent.
Conclusions In summary, in vitro and in vivo data presented here show that protosappanin B significantly inhibits the viability of the human bladder cancer cell line T24, mouse bladder cancer cell line BTT, and human colon cancer cell lines SW-480 and HCT-116, reducing cell growth. In addition, protosappanin B inhibits H22 cell invasion in animals and extends the survival of tumor-bearing mice. Therefore, protosappanin B is a component of Lignum Sappan with antitumor activity; however, the mechanisms underlying these effects remain to be elucidated. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Special Fund for Experimental Animals from Shanxi Science and Technology Department, China (No. 2013K02), and Shanxi Fundamental Resources Platform of Science and Technology from Shanxi Science and Technology Department, China (No. 2013091011).
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