Screening Antitumor Bioactive Fraction from Sauromatum giganteum (Engl.) Cusimano & Hett and Sensitive Cell Lines with the Serum Pharmacology Method and Identification by UPLC-TOF-MS

Sauromatum giganteum (Engl.) Cusimano & Hett Tuber are used in Chinese folklore medicine for treatment of neoplasms. However, the claim has not been scientifically validated. The aim of the study is to screen the antitumor bioactive fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber and sensitive tumor cell lines using a cytotoxicity assay in vitro and tumor transplantation method in vivo, to support its use in folk medicine. The petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction were successively extracted by turn by the maceration under reflux assay. Screening of antitumor bioactive fraction and sensitive cell lines were measured by MTT assay and the serum pharmacology method, and in vivo the antitumor activities of the active fraction was evaluated by using S180 or H22 tumor-bearing mice model and Kunming mice. The active constituents of ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett were characterized by UPLC-TOF-MS. Compared with control groups, mice serum containing ethyl acetate fraction had a inhibition effect on SMMC-7721 cell, SGC-7901 cell, MCF-7 cell, HeLa cell, A549 cell, HT-29, and MDA-MB-231, respectively, but mice serum containing other four fractions had no different with that of control group. The inhibition capabilities of mice serum containing ethyl acetate fraction on the seven cell lines in descending order is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231. In vivo the inhibition rate of 106, 318, 954 mg/kg·d ethyl acetate fraction dry extract to sarcoma S180 is 15.22%, 26.15% and 40.24%, respectively, and life prolonging rate to hepatoma H22 is 33.61%, 40.16% and 55.74%. A total of 14 compounds were identified in the ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett. The results of the experimental studies proved the antitumor activity of Sauromatum giganteum (Engl.) Cusimano & Hett and supported the traditional use of this plant. These data indicate the potential for the use of ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber in tumor therapy, anti-tumor activity on cancer cell line in descending order is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231.


Introduction
Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. In recent years, the increase in the number of cancer cases has motivated the growth of cancer research [1]. A large number of natural products and dietary components have been evaluated as potential chemopreventive agents, and herbal remedies used in traditional folk medicine provide a largely unexplored source of potential novel drugs [2]. The traditional Chinese medicine have a long time clinical practice, so it is a very safe and effective cure [3].
Sauromatum giganteum (Engl.) Cusimano & Hett is a herbal plant, a synonym is Typhonium giganteum Engl. or Typhonium giraldii (Baroni) Engl. or Typhonium stoliczkae Engl., the dried root tuber of which is named Baifuzi in Chinese and recorded in Chinese pharmacopoeia as a traditional Chinese medicine [4,5]. It has the effect of "dispelling wind-phelgm", and been used for the folklore treatment of cancer for a long time in Northeast of China.
There are a few research on Sauromatum giganteum (Engl.) Cusimano & Hett, Chen XS et al. reported the chemical components of Typhonium giganteum Engl. tuber [6] and synthesis method of a new cerebroside isolated from Typhonium giganteum Engl [7,8], and Chi S reported that Baifuzi reduces transient ischemic brain damage through an interaction with the STREX domain of BKCa channels [4], and the cerebrosides [9] from baifuzi, a novel potential blocker of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells [5], is the active compounds of activation of BKCa channels [10].
As concerns experimental research on the anti-tumor activity, only Li Q et al. reported that SFE-CO2 extract from Typhonium giganteum Engl. Tubers induced apoptosis in human hepatoma SMMC-7721 cells [11]. Ma YL reported the up-regulation of TRAIL/TRAIL-R1 and TRAIL-R2 by Lignans of Rhizoma Typhonii could be involved in the induction of apoptosis in SGC-7901 cells [12]. Shan BE found Rhizoma typhonii extract has immunoenhancing activity to human T cell and macrophage, through stimulating the killer cell and phagocytosis of tumor cell and allo-antigen, which could be used clinically for modulating immune responses and for treating tumor and other diseases [13]. Wang SQ reported the effect of aqueous extract of Typhonium giganteum on genes expression in hepatocellular carcinoma SMMC-7721 cells [14].
Study of traditional Chinese medicine is very difficult because the chemical components are very complex. "Serum pharmacology" was first presented by Tashino [15], a Japanese scholar, in 1984. The theory stated that only chemical components of a Chinese herb absorbed in blood could exert their activity on diseases [16]; therefore, the serum pharmacology method provides a good research approach for traditional Chinese medicine [17][18][19][20], which could avoid the interference of the chemical components not absorbed in blood [21,22]. This method not only reveals the mechanism of the active ingredient's action due to its excellent controllability of experimental conditions and detection convenience, but also prevents the interference from crude herbal, so it is more scientific and real. Until now, serum-pharmacology has been widely utilized to explore herbal or traditional Chinese formulations because of its advantage. In addition, it has been widely applied in the research of natural medicine in China [23][24][25][26] and East Asian countries in recent years [27].
The antitumor bioactive solvent fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber and cytotoxicity on different tumor cell lines has not been revealed. In this paper, the serum pharmacology method and MTT assay in vitro and tumor transplantation method in vivo were adopted to determine the antitumor bioactive fraction of Sauromatum giganteum (Engl.) Cusimano & Hett tuber, sensitive cell lines and antitumor activity in vivo. The results might provide a scientific explanation for the folk or traditional application of Sauromatum giganteum (Engl.) Cusimano & Hett in cancer therapy.

Antiproliferative Effect of Fraction-Containing Serum on 7 Human Tumor Cell Lines
The effects of the five different fraction-containing serums (petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction, water fraction) on the proliferation of SMMC-7721, SGC-7901, MCF-7, HeLa, A549, HT-29, and MDA-MB-231 cell lines assessed using the MTT assay are shown in Tables 1-8. Compared with the control group (negative control of fetal bovine serum, negative control of mice serum containing water or negative control of mice serum containing olive oil), treated cells with serum containing ethyl acetate fraction had a decline in viability. The inhibition rate was 71.31% on SMMC-7721 cell, 73.89% on SGC-7901 cell, 50.75% on MCF-7 cell, 22.75% on HeLa cell, 23.05% on A549 cell, 34.81% on HT-29, and 12.28% on MDA-MB-231, respectively. Viability of other fraction groups had no different with that of control group. The positive control ADR or mice serum containing 5-FU was very toxic with a significant inhibition rate.  Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S).  Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S). Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S). Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S). Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S). Note: ** p < 0.01 compared with Negative control of fetal bovine serum (control-FBS); ## p < 0.01 compared with negative control mice serum containing olive oil (oil-control-S); ∆∆ p < 0.01 compared with negative control mice serum containing water (water-control-S).

Antiproliferative Effects of Ethyl Acetate Fractions on 7 Cell Lines
Comparing the data on the inhibition rate of mice serum containing ethyl acetate fraction in Tables 1-7  (shown in Table 8), we found the inhibition strength of mice serum containing ethyl acetate fraction on different tumor lines is different. However, the mice serum containing ethyl acetate fraction were from different batches of mice, likely to cause errors in results. Therefore, we prepared the mice serum containing ethyl acetate fraction again using the same batch of mice, and observed the cytotoxicity on 7 tumor cell lines by MTT assay. Table 9 shows the result, and we found that the anti-tumor effect shows the same trend as Table 8. The order from strong to weak is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231.

The Result of Acute Toxicity Test
Lethal dose 50 (LD50) were found to be >1.150 g/kg b.w. (per os) for the petroleum ether fraction, >1.014 g/kg b.w. (per os) for the chloroform fraction, >0.954 g/kg b.w. (per os) for the ethyl acetate fraction, >4.635 g/kg b.w. (per os) for the n-butanol fraction, and >4.7575 g/kg b.w. (per os) for the water fraction in mice. Tested mice of each group did not show any overt signs of toxicity during 24 h and 14 days observation. No mortality was recorded throughout 14 days monitoring.

Effect of Ethyl Acetate Fraction from Sauromatum giganteum (Engl.) Cusimano & Hett Tuber on Growth of Transplanted Tumor S180 and H22 in Mice
We investigated the effect of ethyl acetate fraction on the growth of S180 and H22 xenograft tumor in mice. After the tumor cells were inoculated, all the mice were inoculated successfully, tumorigenicity was 100%. The tumor weight is shown in Table 10. The results indicated that the growth of implanted sarcoma S180 in mice could be significantly inhibited by ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett tuber in a dosedependent manner compared with the negative control group (p < 0.01). The inhibitory rates were 15.22%, 26.15%, and 40.24% at the doses of 106, 318, 954 mg/kg·d Ethyl acetate fraction dry extract, respectively.

UPLC-TOF-MS Analytic Results
The analysis of active compounds in ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber was carried out by UPLC-TOF-MS. Through comparing the tR values and the MS characteristics of the peaks with reference compounds and the literatures, a total of 14 compounds of extracts in ethyl acetate fraction were identified. The 14 identified compounds are listed as follows: sucrose, adenosine, coniferin, lariciresinol, piresil-4-O-β-D-glucoside, olivil, piresil-4-O-β-D-glucoside (isomer), pinoresinol, tianshic acid, hexadecanedioic acid, linoleic acid, palmitic acid, linolenic acid, linolic acid. The information about the analyzed and identified compounds is summarized in Figure 1 and Table 12.

Animals
Kunming mice of both sexes were used in this study, which were purchased from Changchun Gao-Xin Experimental Animal Center (Changchun, China) with an initial body weight of 18-22 g.
The animals were given water and food ad libitum. The temperature of the animal laboratory was controlled within 20 ± 2 °C, the humidity was 50%-60% with a natural day-night cycle. All animal care and treatments were carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health, U.S.A.
After 3 days of acclimation. Eighty mice were randomly divided into eight groups with ten mice per group (each group with five male mice and five female mice): water control group, oil control group, positive control group (5-FU), petroleum ether fraction group, chloroform fraction group, ethyl acetate fraction group, n-butanol fraction group, and water fraction group.

Administration of Fractions from Sauromatum giganteum (Engl.) Cusimano & Hett
The petroleum ether fraction, chloroform fraction, and ethyl acetate fraction were respectively dissolved in oil, but the n-butanol fraction and water fraction were respectively dissolved in distilled water before administration at saturation.
The animals were fasted for 12 h, but given water ad libitum before administration. The treatment groups of animals (n = 10) were orally administered different fractions, twice a day. The animals the in water control group and oil control group were orally administered distilled water and olive oil respectively, The animals in the positive control group were orally administered 100 mg/kg·d 5-FU, and they were run concurrently with different fraction-treated groups. The animals in the petroleum ether fraction group, chloroform fraction group, ethyl acetate fraction group, n-butanol fraction group, and water fraction group were orally administered at saturation concentrations of 1.150, 1.014, 0.954, 4.635 and 4.7575 g/kg·d in a final volume 0.5 mL. The animals were fasted for 12 h but given water ad libitum before collection of serum.

Preparation of Fraction-Containing Serum
Sixty minutes after the seventh treatment, the blood of the animals was extracted by eyeball extirpating one by one under aseptic condition, and let stand at 4 °C for twelve hours, then centrifuged at 3000× g for 15 min. Both fraction-containing serum and control serum were filtered through a 0.22 μm micropore film (Millipore, Danvers, MA, USA), termed as PEF-S (petroleum ether fraction-containing mice serum), CF-S (chloroform fraction-containing mice serum), EAF-S (ethyl acetate fraction-containing mice serum), BF-S (n-butanol fraction-containing mice serum), WF-S (water fraction-containing mice serum), 5-FU-S (5-FU-containing mice serum), water-control-S (Negative control of serum containing water) and oil-control-S (Negative control of mice serum containing olive oil) and control-FB-S (Negative control of fetal bovine mice serum), respectively, and stored at −80 °C until use [30].

Cytotoxicity Activity of Fraction-Containing Serum on the Human Tumor Cell Lines
The cytotoxicity of 5 different fraction-containing serum on the human tumor cell lines were evaluated using MTT assay. The cells treated with fractions-containing serum were incubated for 72 h after which the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay was carried out as described by Mahmoud, et al. [31,32], but with slight modifications. Cells were plated in 96-well plates (4 × , which is widely used for the treatment of several human cancers, was used as the positive reference as well as 5-FU-S in this study. At the end of 72 h incubation, the medium were discarded and 100 μL of MTT stock solution (1 mg/mL) were added to each well and the plates were further incubated. Four hours later, DMSO (150 μL) was added to each well to solubilize the water-insoluble purple formazan crystals. The amount of MTT-formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 570 nm using microplate reader (Bio-Rad). The percentage of cytotoxic activity compared to the untreated cells was determined using the following equation [33,34]: Cell inhibitory rate (%) = (OD of control cells − OD of treated cells) × 100/OD of control cells

Cytotoxicity Activity of Ethyl Acetate Fraction-Containing Serum on 7 Human Tumor Cell Lines
The assay and operation was same with 3.7, ethyl acetate fraction is an effective anti-tumor bioactive solvent fraction according to our experimental data in 3.7 (Table 8). However, the mice serum containing ethyl acetate fraction were from different batches of mice, likely to cause errors in results. Therefore, using the same batch of mice, we prepared the mice serum containing ethyl acetate fraction again and observed the cytotoxicity on SMMC-7721, SGC-7901, MCF-7, HT-29, HeLa, A549, MDA-MB-231 cell by MTT assay (Table 9).

Acute Toxicity Test
Fifty normal kunming mice (20 ± 2 g) of either sex, fed with pellet diets were randomly divided into five groups with ten mice per group (each group with five male mice and five female mice) and administrated with 1.150 g/kg of the petroleum ether fraction, 1.014 g/kg of the chloroform fraction, 0.954 g/kg of the ethyl acetate fraction, 4.635 g/kg of the n-butanol fraction, 4.7575 g/kg of the water fraction orally by gastric gavage. The animals were observed for general behavioral changes, signs of toxicity and mortality continuously for 1 h after treatment, then intermittently for 4 h, and thereafter over a period of 24 h. Further, the mice were observed for up to 14 days following the treatment for any lethality and death [35].

In Vivo Antitumor Effect of Ethyl Acetate Fraction on Mice
Bearing-S180 or H22 Cell Line S180 or H22 tumor cells were harvested and washed three times with normal saline (NS). The cells were pelleted by brief centrifugation at 300× g. The supernatant was aspirated, and the cells were resuspended in NS at a density of 1 × 10 7 cells/mL. The mice were subcutaneously implanted with 2 × 10 6 cells/mouse on the right oxter for S180-tumor-model or peritoneum for H22-tumor-model (day 0). Twenty-four hours after inoculation, fifty mice with S180 cells or fifty mice with H22 cells were randomly divided into 5 groups, respectively.
After tumor implantation for 24 h, mice were administered orally with various doses of ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber (dissolved in oil, twice a day, 106, 318, 954 mg/kg·d Ethyl acetate fraction dry extract) for 7 days. 5-fluorouracil (25 mg/kg·d) was served as a positive drug.
24 h after the last administration, the mice bearing S180 tumor lines were sacrificed and the tumors were excised and weighted. The inhibition rate (IR) of tumor growth was calculated by the

Analytical Conditions of Ethyl Acetate Fraction of Sauromatum giganteum (Engl.) Cusimano & Hett by UPLC-TOF-MS
The chromatographic separation was achieved on Waters Acquity UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column by employing the Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) consisting of a binary solvent manager, a sample manager and a column temperature controller. The mobile phase which consisted of 0.1% formic acid aqueous solution (A)-0.1% formic acid Acetonitrile solution (B) using a gradient elution (Table 13), was pumped at a flow rate of 0.5 mL·min −1 . The temperatures of the column and sample manager were maintained at 40 °C and 4 °C, respectively.
Waters SYNAPT G2-S Mass Spectrometers (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source was employed for detection. The mass spectrometry was operated in negative ionization mode. In order to achieve maximum sensitivity, the mass spectrometric conditions were optimized as follows:cone gas (nitrogen) flow rate, 10 L/h; desolvation gas (nitrogen) flow rate, 800 L/h; capillary voltage, 2500 V; source temperature, 120 °C; desolvation temperature, 500 °C; scan range: m/z 50-1500. Data processing was performed with UNIFI 1.7 software.

Statistical Analysis
The data is represented as the mean ± SD. Statistical significance was calculated using student's t-test. p-values of 5% or less were considered statistically significant.

Conclusions
Five different fraction-containing serums (petroleum ether fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction and water fraction of Sauromatum giganteum (Engl.) Cusimano & Hett) were prepared and investigated for the inhibition activity on tumor cells, respectively. Only the mice serum containing ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber could inhibit the tumor cells proliferation. The inhibition effection of ethyl acetate fraction on the seven cell lines in descending order is SGC-7901 > SMMC-7721 > MCF-7 > HT-29 > A549 > HeLa > MDA-MB-231. Sarcoma S180 and hepatoma H22 tumor-bearing mouse models are adopted in antitumor experiment in vivo [36][37][38][39], We found ethyl acetate fraction of Sauromatum giganteum (Engl.) Cusimano & Hett Tuber could inhibit the S180 growth and prolonged the life span of mice bearing H22 with the dosage increase.