Bio-Guided Isolation of the Cytotoxic Terpenoids from the Roots of Euphorbia kansui against Human Normal Cell Lines L-O2 and GES-1

The dried roots of Euphorbia kansui (kansui) have been used for centuries in China as a herbal medicine for edema, ascites, and asthma. The 95% ethanol extract showed a significant inhibition of cell proliferation against human normal cell lines L-O2 and GES-1. Bioassay-guided separation of the 95% ethanol extract from the roots of E. kansui led to the isolation of 12 diverse terpenoids whose structures were identified by 1H, 13C NMR spectroscopy and ESI-MS as kansuinine A (1), kansuinine B (2), kansuinine C (3), kansuiphorin C (4), 3-O-(2′E,4′Z-decadienoyl)-20-O-acetylingenol (5), 3-O-(2′E,4′Edecadienoyl)-20-O-acetylingenol (6), 3-O-(2′E,4′Z-decadienoyl)-20-deoxyingenol (7), 3-O-benzoyl-20-deoxyingenol (8), 5-O-benzoyl-20-deoxyingenol (9), kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5–11) exhibited a relatively lower IC50 value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb.

, kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5-11) exhibited a relatively lower IC 50 value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb.

Introduction
With the development of science and technology, and the increasing health awareness and life expectancy of humans, the safety problem of traditional Chinese medicines (TCM) is attracting more and more attention. The dried roots of Euphorbia kansui T.N. Liou ex T.P. Wang, known as "kansui," which were recorded in Shennong-Bencao and have been used for centuries in China as a herbal medicine for edema, ascites, and asthma [1,2]. Recently, it was found that kansui has excellent prospects for the treatment of cancer [2][3][4][5][6], pancreatitis [7][8][9], and intestinal obstruction [10,11]. However, kansui causes severe skin, oral, and gastrointestinal irritation, hepatic injury and tumor promoting toxicity, which have seriously restricted its clinical application [12][13][14][15][16]. So, outweighing all other considerations is the need to understand the toxicity mechanism of kansui, and further to reduce its toxicity without affecting its therapeutic action.

Cytotoxicity Activity on Human Normal Cell Lines of Extracts and Compounds from Kansui
As shown in Table 4, the EtOH and EtOAc extract exhibited significant inhibition of cell proliferation against two human normal cell lines L-O2 and GES-1 with dose-dependent relationship. The IC 50 values of EtOH extract on L-O2 and GES-1 were 42.02 and 30.67 µg/mL, respectively. And the cytotoxicity of EtOAc extract on L-O2 and GES-1 were stronger than that of EtOH extract. The IC 50 values of EtOAc extract on L-O2 and GES-1 were 27.08 and 21.89 µg/mL, respectively. The water extract showed no obvious cytotoxicity against these two human normal cell lines.  Compared with the control group, compounds 5, 7, 8 and 10 in the concentration range of 0.78-12.5 µg/mL, compounds 6 and 11 in the concentration range of 0.39-6.25 µg/mL, and compound 9 in the concentration range of 1.56-25 µg/mL, showed significant inhibition activity on GES-1 cell lines growth with a dose-dependent relationship (Figure 2). And compounds 5, 6, 7, 10 and 11 in the concentration range of 0.78-12.5 µg/mL, compound 9 in the concentration range of 1.56-25 µg/mL, and compound 8 in the concentration range of 3.125-50 µg/mL, had significant inhibition activity on L-O2 cell lines growth with a dose-dependent relationship (Figure 3). Whereas, other compounds did not show the obvious cytotoxicity activity in the concentration range of 3.125-50 µg/mL. Considering their structure, the results indicated that ingenane-type diterpenoids (5-9) except compound 4 and 8-ene-7-one triterpenoids (10 and 11) in kansui possessed stronger gastrointestinal toxicity and hepatotoxicity than other compounds. More interestingly, the IC 50 values of 5-11 and the kansui extracts on GES-1 cell lines were less than those on L-O2 cell lines, which suggested that the gastrointestinal toxicity of kansui is stronger than its hepatotoxicity. This result coincided with the clinical toxic performance of kansui [14][15][16].

Instrumentation
Melting points were determined on a Yanagimoto MP-500 D micro-melting point apparatus and are uncorrected. The UV spectrum was obtained in MeOH on a Shimadzu UV-2401 spectrophotometer. The NMR spectra were recorded on Bruker Avance AV 300 instrument operating at 300 MHz for 1 H and 75 MHz for 13

Plant Material
The roots of Euphorbia kansui T.N. Liou ex T.P. Wang were collected from Red River valley of Baoji (Shanxi Province, China). The crude plant was identified by Professor Chungen Wang (Nanjing University of Chinese Medicine, Nanjing, China). The voucher specimens (No. NJUTCM-20091008) was deposited in the Herbarium of Nanjing University of Chinese Medicine, Jiangsu, China.

Extraction and Isolation
The dried and crushed roots of E. kansui (20.0 kg) were extracted with 95% EtOH under 50 °C water bath, the supernatant was separated every day, and the solvent was removed under reduced pressure, and the residue (2.35 kg) was partitioned with ethyl acetate and water to provide the EtOAc fraction (1.032 kg) and water fraction (1.056 kg). The EtOAc fraction 0.904 kg was subjected to silica gel column chromatography (100 × 10 cm, 200-300 mesh, eluted with petroleum ether and ethyl acetate in increasing polarity). The column chromatographic eluents (500 mL each) were concentrated under reduced pressure and combined according to TLC monitoring into 10 fractions. Fraction 1 was subjected to silica gel column chromatography eluted with petroleum ether-ethyl acetate 100:1, and was further recrystallized to give compound 12 (a large amount). Fractions 2-4 was subjected to silica gel column chromatography eluted with petroleum ether-ethyl acetate from 100:16 to 100:18, and were further recrystallized to give 1 (132 mg), 2 (86 mg) and 3 (46 mg). Fraction 5 was subjected to silica gel column chromatography eluted with petroleum ether-ethyl acetate 100:2, and was further purified by preparative TLC using silica gel (petroleum ether-chloroform-ethyl acetate 5:3:1, R f = 0.83) to give compound 4 (106 mg). Fraction 6 was subjected to silica gel column chromatography eluted with petroleum ether-ethyl acetate 100:5, and was further purified by preparative HPLC (petroleum each well. The plates were shaken gently for 10 min to blend the mixture. The absorbance value of each well was read at 490 nm using a microplate reader. All experiments were performed at least three times. The inhibition rate of cell proliferation was calculated according to the formula: cell inhibition (%) = (1 − sample solution absorbance value/control absorbance value)  100% (1)

Statistical Analysis
The results were expressed as an inhibition ratio and IC 50 (concentration that inhibits 50% of cell growth). The data were analyzed with software SPSS 15.0 (SPSS Inc.: Chicago, IL, USA). The anti-proliferation activity is more significant when the IC 50 is smaller.

Conclusions
In this paper, the 95% EtOH extract of kansui exhibited significant activity against two human normal cell lines GES-1 and L-O2. By bio-guided isolation, 12 terpenoid compounds, including jatrophane-type diterpenoids (1-3) ingenane-type diterpenoids (4-9) and triterpenoids (10)(11)(12), were isolated and identified from the active fraction of kansui. The results showed that five ingenane-type diterpenoids (5-9) and two triterpenoids (10 and 11) exhibited relative lower IC 50 value and thus, stronger potential cytotoxic activity against different human normal cell lines in a dose-dependent fashion. The ingenane-type diterpenoids with 3-unsaturated aliphatic chain, such as compounds 5, 6 and 7, possessed stronger cytotoxic activity. And the cytotoxic activity was slightly decreased if the 3-unsaturated aliphatic chain was replaced by other groups, such as methyl and phenyl groups; and there was the same decrease of cytotoxic activity if the 5-hydroxyl group was replaced by a benzoyl group. And 20-O group had little influence on the cytotoxic activity. The three jatrophane-type diterpenoids (1-3) showed very weak cytotoxic activity against the two human normal cell lines. The triterpenoids with 8-ene-7-one showed stronger cytotoxic activity. For example, compound 10 (kansenone) and 11 (Epi-kansenone) had stronger cytotoxic activity than that of compound 12 (euphol). It is also notable that all identified toxic terpenoids were also reported in the literature to be important pharmaceutical ingredients for anti-tumor, anti-allergy, anti-virus, and antinematodal activity [2][3][4][5]13,[17][18][19]. Therefore, it is essential that the dose of kansui should be controlled within an appropriate range in the clinical application to assure its safe and efficacy.