Cytotoxic sesquiterpenoids from the ethanol extract of fruits of Celastrus orbiculatus

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Journal of Ethnopharmacology 117 (2008) 175–177

Ethnopharmacological communication

Cytotoxic sesquiterpenoids from the ethanol extract of fruits of Celastrus orbiculatus Jing Xu a , Yuan-Qiang Guo a,∗ , Xian Li b , Kun Wei c , Xiao-Jun Zhao d,∗ a

b

Department of Pharmaceutical Sciences, Nankai University, Tianjin 300071, PR China Research Department of Natural Medicine, Shenyang Pharmaceutical University, Shenyang 110016, PR China c School of Chemical Science and Technology, Yunnan University, Yunnan 650091, PR China d College of Chemistry & Life Sciences, Tianjin Normal University, Tianjin 300387, PR China Received 18 September 2007; received in revised form 11 December 2007; accepted 26 January 2008 Available online 6 February 2008

Abstract Aim of the study: To investigate the cytotoxic compounds against human melanoma A375-S2 and human cervical carcinoma Hela cell lines from the fruits of Celastrus orbiculatus. Materials and methods: The ethanol extract of the fruits of C. orbiculatus was found to exhibit significant cytotoxic activity. After partition, the column chromatography and Semi-preparative HPLC were used for activity guided fractionation from the active petroleum ether-soluble part. NMR and ESI-MS spectra for structure analysis were recorded on spectrometers, respectively. The cytotoxic activities of the isolated compounds were determined using the MTT assay. Results: Four sesquiterpenoids (1–4) were isolated by activity guided fractionation from the ethanol extract of the fruits of C. orbiculatus. Compounds 1–3 exhibited promising cytotoxicities against the Hela cells and A375-S2 cells. The cytotoxic activities of compounds 1–3 seemed to be dose-dependent, which exhibited more potent inhibition for the proliferation of two cell lines when increased the concentrations of every compounds. Among these compounds, compound 2 showed most cytotoxicities against either Hela cells or the A375-S2 cells. Conclusions: The cytotoxic activity of the ethanol extract of fruits of C. orbiculatus against human melanoma A375-S2 and human cervical carcinoma Hela cells due to its content of sesquiterpenoids, supports its potential therapeutic value for the treatment of tumor. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Celastrus orbiculatus; Sesquiterpenoids; Cytotoxic activity

1. Introduction Celastrus orbiculatus Thunb. (Celastraceae), which is widely distributed in PR China, has been used as a folk medicine in China for the treatment of tissue agglomeration and early tumor, and as a sedative and hypnotic (Editorial commission of Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, 1999; Jiangsu New Medical College, 1977). Recently, celastrol, a titerpenoid from this plant showed cytotoxicity (Zhang et al., 2005) and its extract was active against sarcoma 180 cells (Zhang et al., 2006). Bioassay guided isolation indicated the presence of sesquiterpenoids in this plant, which showed antifeedant, multi-drug

∗

Corresponding authors. Tel.: +86 22 23503375; fax: +86 22 23503375. E-mail address: [email protected] (Y.-Q. Guo).

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.01.028

resistance, and anti-inflammation activity (Guo et al., 2003, 2004, 2006; Jin et al., 2002; Kim et al., 1998, 1999; Wang et al., 1997). In addition, several flavonones from this plant and their anti-inflammatory activity have also been reported (Hwang et al., 2001; Min et al., 1999). However, up to the present, there has been no report on the cytotoxic constituents of the ethanol extract of fruits from Celastrus orbiculatus against human melanoma A375-S2 and human cervical carcinoma Hela cells. In our search for natural products with cytotoxic activity from medicinal plants, much attention was given to Chinese folk medicine for the treatment of tissue agglomeration, early tumor and inflammation. Though the extract of Celastrus orbiculatus showed cytotoxic activity reported in the literature, there are no further phytochemical investigations aimed at isolating cytotoxic compounds. We found that the ethanol extract obtained from the fruits of Celastrus orbiculatus (Celastraceae) exhibited significant cytotoxicity against

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human melanoma A375-S2 and human cervical carcinoma Hela cells in vitro using a MTT assay. Activity guided fractionation of the ethanol extract resulted in the isolation of four sesquiterpenoids: 1␤-acetoxy-9␣-cinnamoyloxy-␤dihydroagarofuran (1), 1␤, 2␤, 6␣-triacetoxy-9␣-benzloxy-␤dihydroagarofuran (2), 1␤, 2␤-diacetoxy-6␣-benzoyloxy-9␣cinnamoyloxy-␤-dihydro-agarofuran (3), 6␣, 13-diacetoxy-1␤, 8␤, 9␤-tribenzoyloxy-␤-dihydroagarofuran (4). Here we report for the first time the nature of the isolated constituents of Celastrus orbiculatus responsible for the cytotoxic activity observed. 2. Materials and methods 2.1. Plant material The fruits of Celastrus orbiculatus were collected in Shenyang city, Liaoning Province, PR China and identified by Prof. Yunzhen Guo, Shenyang Pharmaceutical University, Shenyang, PR China. A voucher specimen (No. 200315) was deposited in the Research Department of Natural Medicine, Shenyang Pharmaceutical University. 2.2. Extraction and isolation The air-dried fruits of Celastrus orbiculatus (10 kg) were extracted with 95% ethanol under reflux for 2 h. After removing the solvent in vacuo, the ethanol extract was suspended in water, then partitioned with petroleum ether, chloroform, EtOAc and n-BuOH successively. The petroleum ether-soluble part exhibited the most cytotoxic activity among these partitions. The petroleum ether part (160 g) was subjected to column chromatography on silica gel (200–300 mesh) and eluted with petroleum ether-acetone (100:0–1:1) to provide 9 fractions (Fr. 1–9). Fraction 2 was followed by column chromatography on a semi-preparative Shimadzu CTO-6A HPLC equipped with a Shimadzu Shim-pack PREP-ODS (i.d. 250 mm × 21.6 mm) column (flow rate: 5 mL/min, UV 254 nm) to yield compounds 1 (16 mg), using MeOH–H2 O (80:20) as eluent. The purification of fraction Fr. 4 using the same HPLC procedure as above with the solvent MeOH–H2 O (72:28) resulted in the isolation of compound 2 (14 mg). Fraction 3 was purified by above HPLC using MeOH–H2 O (75:25) to afford compounds 3 (11 mg). Compound 4 was obtained from Fr. 5 on semi-preparative HPLC using MeOH–H2 O (70:30) as eluent. 2.3. Methods of chromatography and structure analysis Silica gel for chromatography was purchased from Qingdao Ocean Chemical Group Co. of PR China. Semi-preparative HPLC separations were performed on the above HPLC system. 1 H NMR and 13 C NMR spectra were obtained on Bruker-ARX300 spectrometer in CDCl3 , using TMS as internal standard. ESI-MS was performed on VG-70SE mass spectrometer. 2.4. Determination of cytotoxic activity The cells were grown and maintained in a humidified incubator at 37 ◦ C and in 5% CO2 atmosphere in RPMI

1640 medium containing 100 g/L fetal bovine serum (FBS) and 10 g/L penicillin–streptomycin–glutamine (PSG). The MTT assay, which is based on the reduction of MTT [3(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] by mitochondrial dehydrogenase to a purple formazan product (Mosmann, 1983), was used to assess the antiproliferative action of the fruit extracts of Celastrus orbiculatus, fractions and pure compounds in human melanoma A375-S2 and human cervical carcinoma Hela cells. The cells were suspended at a density of 2 × 104 cells/mL in RPMI 1640 medium containing 100 g/L FBS and 10 g/L PSG, then 100 ␮L of each was seeded into each well of 96-well microliter plates and incubated for 24 h. Different extracts and pure compounds added to the wells with various concentrations and incubated at 37 ◦ C and 5% CO2 for 48 h. MTT solution (10 ␮L) of 0.5 mg/mL was then added to each well and incubated for another 4 h. After removing the upper solution, the resulting MTT-formazan product was dissolved by the addition of 100 ␮L DMSO solution into each well, followed by mixing and measuring the absorbance at 570 nm using a microplate reader (Bio-Rad, Model 550, USA). The results are expressed as the optical density ratio of the treatment to control. Cells inhibition =

(Control absorbance − sample absorbance) Control absorbance × 100%

3. Results and discussion Though the extract of Celastrus orbiculatus showed cytotoxic activity reported in the literature, there are no further phytochemical investigations to identify compounds responsible for the cytotoxic activity. In our screening, the ethanol extract of the fruits of Celastrus orbiculatus was found to exhibit significant cytotoxic activity against Hela and A375-S2 cell lines with IC50 values of 20.6 and 25.0 ␮g/mL, respectively. To isolate the cytotoxic constituents from the ethanol extract of fruits of Celastrus orbiculatus, the petroleum ether-soluble part was subjected to chromatography, as described in the extraction and isolation section. Four compounds (1–4) were isolated by activity guided fractionation. Their 1 H NMR, 13 C NMR and ESI-MS spectra were recorded for the structure elucidation. Compounds 1–4 had similar NMR spectral patterns. The 1 H NMR and 13 C NMR spectra of these compounds exhibited the same characterization of ␤-dihydroagarofuran sesquiterpenoid-type skeleton. However, the position and numbers of substituent groups in every compound indicated different characteristics in their NMR spectra. By comparing their spectral data with those reported in the literature, compounds 1–4 were established to be 1␤-acetoxy9␣-cinnamoyloxy-␤-dihydroagarofuran (Wang et al., 1997), 1␤, 2␤, 6␣-triacetoxy-9␣-benzloxy-␤-dihydroagarofuran (Kim et al., 1999), 1␤, 2␤-diacetoxy-6␣-benzoyloxy-9␣cinnamoyloxy-␤-dihydroagarofuran (Guo et al., 2004), 6␣, 13-diacetoxy-1␤, 8␤, 9␤-tribenzoyloxy-␤-dihydroagarofuran (Guo et al., 2003), respectively. The cytotoxic activities of the isolated compounds were determined using the MTT assay method against two cultured human melanoma A375-S2 and human cervical carcinoma Hela

J. Xu et al. / Journal of Ethnopharmacology 117 (2008) 175–177 Table 1 The IC50 values of compounds 1–4 from the fruits of Celastrus orbiculatus against human melanoma A375-S2 and human cervical carcinoma Hela cell lines Extract/compounds

Ethanol extract 1 2 3 4 5-Fluorouracil

Cytotoxicity IC50 values Hela

A375-S2

20.6 ␮g/mL 13.9 ␮M 3.89 ␮M 18.0 ␮M >100 ␮M 19.6 ␮M

25.0 ␮g/mL 13.2 ␮M 3.64 ␮M 47.7 ␮M >100 ␮M 5.1 ␮M

IC50 values refer to the 50% inhibition concentration (␮g/mL or ␮M), and were calculated from regression lines using five different concentrations with triplicate determinations. 5-Fluorouracil: positive control.

cell lines. As shown in Table 1, compounds 1–3 exhibited promising cytotoxicities against the Hela cells with IC50 values of 13.9, 3.89, 18.0 ␮M, respectively, the A375-S2 cells with IC50 values of 13.2, 3.64, 47.7 ␮M, respectively. The cytotoxic activity of all active compounds 1–3 seemed to be dose-dependent, which exhibited more potent inhibition for the proliferation of two cell lines when increased the concentrations of every compound. Compound 4 was considered inactive against the Hela cells and the A375-S2 cells, due to IC50 values > 100 ␮M. From the above IC50 values, compound 2 showed most cytotoxicities against either Hela cells or the A375-S2 cells. The cytotoxic activity of the ethanol extract of fruits of Celastrus orbiculatus against human melanoma A375-S2 and human cervical carcinoma Hela cells due to its content of sesquiterpenoids, supports its potential therapeutic value for the treatment of tissue agglomeration and early tumor as a folk medicine in China. However, the mechanism of cytotoxic activity of compounds 1–3 against these cell lines is being carried out. References Editorial commission of Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, 1999. Traditional Chinese Medicine,

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