The roots of C. lanceolata, three years old, were collected in Jilin Province (Jilin, China). The specimens were identified and authenticated by Dr. Wei Li, Jilin Agricultural University. A voucher specimen (No. 20140311) has been deposited in the herbarium of the same college. Cyclophosphamide (CTX) was provided by Jiangsu Hengrui Pharmaceutical Co., Ltd. Hematoxylin and eosin (H&E) kits were acquired from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The Hoechst 33258 dye kit was obtained from Shanghai Beyotime Co., Ltd. (Shanghai, China). ELISA kits for mouse, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), were purchased from R&D Systems (Minneapolis, USA). Rabbit monoclonal anti-Bax, anti-Bcl-2 and anti-VEGF antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Other chemicals were all of analytical grade from Beijing Chemical Factory.

Mouse H22 hepatocarcinoma cells (H22) were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China), and maintained in the peritoneal cavity of male ICR mice, as previously described [24].

Male ICR mice, 22–25 g, were purchased from the Experimental Animal Holding of Jilin University with Certificate of Quality No. SCXK (JI) 2011-0004 (Jilin, China). Animals were housed individually in cages in a temperature-controlled room with a 12-hour light/dark cycle. After one week of acclimation with free access to regular rodent chow and water, the mice were used for further experiments. The experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). All of the animal experimental procedures were approved by the Ethical Committee for Laboratory Animals of Jilin Agricultural University on 15 June 2013 (Approval NO. JLAU-ECLA-20140822).

The fresh roots of C. lanceolata were steamed in an autoclave at 105 °C for 1 h and then dried at room temperature (25 °C) to obtain steamed C. lanceolata (SCL). The SCL powders were extracted with 10 volumes of methanol using ultrasound-assisted extraction at a temperature of 40 °C for 90 min. After extracting 3 times, the obtained filtrate was concentrated under reduced pressure in a rotary evaporator to give a crude extract and to make up the solution for the animal experiment.

After an acclimatization period of one week, to establish the murine solid tumor H22 transplanted model, corresponding ascites tumor cells (0.2 mL, 2 × 10⁶ cells per mouse) were subcutaneously injected into the right axillary region of the mice in all groups (Day 7). Twenty four hours after inoculation (Day 8), the animals were randomly divided into five groups (n = 10) and treated for 14 days as follows: (i) the model group: animals received saline intragastrically; (ii) the CTX group (positive control group): animals received CTX (25 mg/kg) by intraperitoneal injection; (iii) the low and high dosage SCL-treated groups: animals received SCL test solution (200 and 400 mg/kg) intragastrically. The mice weights were recorded before and after each drug administration. Twenty four hours after the last administration of tested drug on the 22nd day of the experiment, blood samples were collected from the mice’s eyes, and serum was harvested by centrifugation. Then, all of the mice were sacrificed, and the whole bodies, the segregated tumor, thymus and spleen of the mice were weighed immediately. The tumor inhibitory rate was calculated by the following formula: tumor inhibitory rate % = (tumor weight of model group − tumor weight of tested group)/tumor weight of model group × 100. The volume of the solid tumor was measured with a digital caliper every other day. The values obtained were calculated according to the equation: V (mm³) = A × B²/2, where A and B represent the largest diameter and the smallest diameter, respectively. The tumor tissues of mice were collected for pathological sectioning with H&E and Hoechst 33258 staining. The experimental design is shown in Figure 1.

To measure the effect of SCL on survival time, thirty male ICR mice were inoculated with H22 tumor cells prepared by intraperitoneal inoculation, so as to observe the mice for a longer time period. The treatment was performed for 40 days, and the survival time of animals was monitored and recorded daily. The test continued for 40 days, and those that lived more than 40 days were defaulted as 40 days. The percent survival (%) was calculated using the following equation: percent survival (%) = ((10 − number of mice died in each group)/10) × 100.

A specific two-sided ELISA assay was performed to quantify serum levels of TNF-α, IFN-γ, IL-2, IL-6 and VEGF according to the manufacturer’s instructions. The absorbance was measured at 450 nm in an ELISA reader (Bio-Rad, CA, USA).

Hoechst 33258 staining was performed as previously described with some modifications [25]. Briefly, at the end of the experiments, the mice were euthanized, and the transplanted tumors were dissected out and fixed in 10% neutral buffered formalin solution. We randomly chose three tumors from each group. Then, these samples were cut into 5-μm sections and stained by Hoechst 33258 (10 μg/mL). After being washed by PBS three times, stained nuclei were visualized under UV excitation and photographed under a fluorescent microscope (Olympus BX-60, Tokyo, Japan).

To quantify the fragmented and condensed staining, which indicated an apoptotic nucleus in the slides, we randomly chose five regions from the pictures of each tumor. These pictures were blinded and counted by two people, and their mean values were used in the statistical analysis. To avoid interobserver differences, a datum is valid only if the discrepancy between these two observers is less than 10%.

At the end of the experiments, H22 transplanted tumor tissues were fixed in 10% neutral buffered formalin solution. The washed tumor tissues were dehydrated in descending grades of ethanol and cleared in xylene, then embedded in paraffin. Sections were cut at a 5-μm thickness and stained with hematoxylin and eosin (H&E), then subsequently examined using a light microscope for histopathological examination.

Immunohistochemical analysis was performed as previously described [26]. Briefly, the 5 μm-thick paraffin sections were deparaffinized and rehydrated with a series of xylene and aqueous alcohol solutions, respectively. After antigen retrieval in citrate buffer solution (0.01 M, pH 6.0) for 20 min, the slides were washed three times with TBS (0.01 M, pH 7.4) and incubated with 1% bovine serum albumin for 1 h. The blocking serum was tapped off, and the sections were incubated in a humidified chamber at 4 °C overnight with primary antibodies against Bax (1:400), Bcl-2 (1:400) and VEGF (1:200), followed by a secondary antibody for 30 min. Substrate was added to the sections for 30 min followed by DAB staining and hematoxylin counter-staining. The positive staining was determined mainly by a brownish-yellow color in the nucleus of the cells. The immunostaining intensity was analyzed by light microscopy (Olympus BX-60, Tokyo, Japan). The immunohistochemical signal was assessed by estimating the area of the objects and the medium pixel intensity per object, as the optical density (OD). The Bax/Bcl-2 ratio is the optical density ratio of the Bax and Bcl-2 proteins.

Samples were analyzed on an Agilent HPLC system. Separation was achieved on a Hypersil ODS2 column (4.6 mm × 250 mm, 5 μm) from Dalian Elite Analytical Instruments Co. Ltd. The column temperature was set at 30 °C, and the detection wavelength was set 210 nm. The mobile phase consisted of acetonitrile (A) and water (B) with a flow rate of 1.0 mL/min. The gradient elution was programmed as follows: 0–20 min, 11%–16% A; 20–35 min, 16%–28% A; 35–60 min, 28%–60% A.

The HPLC-UV system was interfaced with the MS detector. The pneumatic-assisted electrospray positive ionization (ESI⁺) detection and cracking voltage is 160 V; the atomizing air pressure is 276 kPa (40 psi); the drying temperature is 350 °C; and the drying gas flow rate is 12 L/min.

Statistical analysis was performed using SPSS 16.0. (Chicago, IL, USA). All values were expressed as the means ± standard deviation (SD). The differences between experimental groups were compared by analysis of variance (ANOVA) followed by a t-test. Differences with p < 0.05 were considered statistically significant. Drawings and survival curves were created using GraghPad Prism 6.04. Image-Pro plus 6.0 was used to quantify immunohistochemical analysis and Hoechst 33258 staining.

The antitumor effect of SCL on H22 tumor-bearing mice is summarized in Table 1 and Figure 2A–B. At the end of the study, the average tumor weight in the CTX group was significantly decreased (p < 0.01) compared to the model group. After treatment for 14 days, SCL with 200 and 400 mg/kg prior to the model group caused a significant decrease (p < 0.01). Accordingly, the tumor inhibitory rates of the CTX and SCL-treated groups were 81.73%, 46.08% and 60.87%, respectively.

Figure 2A shows the tumor volume growth curves; the results indicated that the tumor volumes of the mice in the model group increased rapidly during the 14-day duration with their mean volumes reaching more than 2.3 cm³ at Day 15. In contrast, the treatment of SCL and CTX significantly suppressed the tumor growth (p < 0.05). From the ninth day, the average tumor volume in SCL- and CTX-treated mice had increased relatively slowly.

Figure 2C shows the effect of SCL on life extension in H22 tumor-bearing mice. The results indicated that all of the mice in the model group died within 16 days owing to the significantly fast growth of the H22 transplanted tumor. After treatment with CTX and SCL, half of the mice survived for more than 20 days. The average survival time of ascites H22-bearing mice treated with CTX and SCL at a dosage of 400 mg/kg was 40 and 36 days, respectively. Interestingly, the effect of SCL with 400 mg/kg was almost comparable to that of CTX. The findings clearly demonstrated that SCL treatment greatly prolonged the survival period of H22 tumor-bearing mice.

As shown in Table 1, there was no significant difference among the mean initial body weights of all groups. To evaluate whether SCL administration resulted in any side effect on the immune system, we determined the thymus and spleen indices of the host animals at the end of the study. The results indicated that spleen and thymus indices in the CTX-treated mice were significantly lower than the model group (p < 0.05, p < 0.01), which accounted for the immunosuppressive side effect by CTX during the therapy. However, there was no significant difference between the model group and SCL-treated groups.

Since cytokines play a key role in controlling immune responses and inflammatory reactions, we employed the ELISA assay to investigate the effects of SCL on the production of serum cytokines, including TNF-α, IFN-γ, IL-2 and IL-6, in H22 tumor-bearing mice. As shown in Figure 3A–D, the serum levels of IL-2 and TNF-α were significantly increased in the SCL groups at doses of 200 and 400 mg/kg, respectively (p < 0.05). Mice treated with 400 mg/kg of SCL showed a significant increase in the serum levels of IL-6 and IFN-γ (p < 0.05).

Angiogenesis, an essential process for tumor growth and metastasis, has become an important target for therapeutic intervention in many tumors. VEGF is recognized as a key contributor to the process of angiogenesis [27]. The serum level of VEGF was determined by an ELISA kit according to the manufacturer’s instruction. As shown in Figure 3E, the result indicated that SCL treatment at dosages of 200 and 400 mg/kg markedly decreased the serum level of VEGF (p < 0.05, p < 0.01), suggesting that SCL could repress angiogenesis in the H22 transplanted tumor.

In the present investigation, H&E sections of tumors were examined by microscopy. As shown in Figure 4A, tumor cells from the mice in model group were arranged tightly, having a large nucleus and a clearly apparent nucleolus. However, the tumor cells in SCL treatment groups exerted a loose arrangement and a large necrotic region. Furthermore, different increased degrees of vacuoles and vacuoles number were obviously observed in SCL-treated groups in a dose-dependent manner, which corroborates the significant anti-tumor efficacy of SCL on H22 tumor-bearing mice via inducing cell death.

To elucidate whether SCL treatment induced cell apoptosis in H22 transplanted tumors, Hoechst 33258 staining was performed. As depicted in Figure 4B, H22 tumor cells in the model group were observed as round-shaped nuclei with homogeneous fluorescence intensity, and most cell nuclei exhibited regular contours. After treatment with SCL and CTX for 14 days, significant nuclear fragmentation and condensation was observed in a dose-dependent manner (Figure 4C).

To find out the anti-tumor mechanism of SCL on H22 tumor-bearing mice, we examined the impact of SCL on the anti-apoptotic factor Bcl-2 and the pro-apoptotic factor Bax using immunohistochemical analysis. As shown in Figure 5A, B, high expression of Bcl-2 and low expression of Bax were observed in the tumor tissue sections of the model group. By contrast, SCL treatment decreased Bcl-2 expression and increased Bax expression of H22 tumor tissues in a dose-dependent manner. Interestingly, the ratio of Bax to Bcl-2, a rheostat of cell life or death, was increased in a dose-dependent manner after SCL treatment for 14 days.

VEGF is considered an important growth factor implicated in tumor angiogenesis and can also be used as a tumor marker [27]. As shown Figure 5E, SCL treatment could significantly inhibit the expression of VEGF in a dose-dependent manner, coinciding with the decrease of VEGF level in serum. The above observation is a hint for the possible role of SCL as an angiogenesis inhibitor on H22 tumor-bearing mice.

LC-MS/MS is considered a powerful tool for identifying and quantifying the compounds from natural medicines. As shown in Figure 6, in the present study, under the optimal chromatographic and MS conditions, one polyacetylene (lobetyolin) and six saponins were identified by comparing the retention time and by matching the empirical molecular formula with that of the reported known saponins [28,29,30]. The adduction of [M + NH₄]⁺ would be generated when NH₄OH was added to the mobile phase as a chromatographic modifier. All details of the compounds identified from SCL are summarized in Table 2.