Supercritical fluid extraction assisted isolation of sesquiterpene lactones with antiproliferative effects from Centipeda minima.

Pseudoguaianolide sesquiterpene lactones minimolides A (1), B (2), C (3) and D (4) and two guaianolide sesquiterpene lactones minimolides E (5) and F (6), along with seven known ones (7-13), were isolated from the supercritical fluid extract of Centipeda minima. The structures of these compounds were elucidated by extensive spectroscopic methods (IR, UV, HRESIMS, 1D-NMR and 2D-NMR), and the complete structure and stereochemistry of 1 was further confirmed by X-ray diffraction analysis. Compounds 1, 5-8,11 and 13 displayed inhibitory activity against human nasopharyngeal cancer cells (CNE) with IC(50) values ranging from 1.1 to 20.3 µM. Compound 13 containing both α-methylene-γ-lactone and α, ß-unsaturated cyclopentenone moieties exhibited even stronger inhibitory activity than that of cisplatin (positive control) through cell cycle arrest at G2/M phase. Isolation of six sesquiterpene lactones from Centipeda minima highlighted the potential of supercritical fluid extraction for enrichment of minor constituents for phytochemical study.

Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells.

Deoxyelephantopin (ESD), a naturally occurring sesquiterpene lactone present in the Chinese medicinal herb, Elephantopus scaber L. exerted anticancer effects on various cultured cancer cells. However, the cellular mechanisms by which it controls the development of the cancer cells are unavailable, particularly the human nasopharyngeal cancer CNE cells. In this study, we found that ESD inhibited the CNE cell proliferation. Cell cycle arrest in S and G2/M phases was also found. Western blotting analysis showed that modulation of cell cycle regulatory proteins was responsible for the ESD-induced cell cycle arrest. Besides, ESD also triggered apoptosis in CNE cells. Dysfunction in mitochondria was found to be associated with the ESD-induced apoptosis as evidenced by the loss of mitochondrial membrane potential (ΔΨm), the translocation of cytochrome c, and the regulation of Bcl-2 family proteins. Despite the Western blotting analysis showed that both intrinsic and extrinsic apoptotic pathways (cleavage of caspases-3, -7, -8, -9, and -10) were triggered in the ESD-induced apoptosis, additional analysis also showed that the induction of apoptosis could be achieved by the caspase-independent manner. Besides, Akt, ERK and JNK pathways were found to involve in ESD-induced cell death. Overall, our findings provided the first evidence that ESD induced cell cycle arrest, and apoptosis in CNE cells. ESD could be a potential chemotherapeutic agent in the treatment of nasopharyngeal cancer (NPC).

6-O-Angeloylenolin induced cell-cycle arrest and apoptosis in human nasopharyngeal cancer cells.

High incidence of lymph node spread and distant metastasis make poor prognosis in human nasopharyngeal carcinoma (NPC). Therefore, better treatments for NPC are needed. This study investigated the anticancer activity of 6-O-angeloylenolin, a plant-derived sesquiterpene lactone, on human nasopharyngeal cancer (CNE) cells. 6-O-Angeloylenolin was found to significantly inhibit the proliferation of CNE cells. The rate of inhibition was comparable to that of cisplatin, a well known chemotherapeutic agent used to treat NPC. Further mechanistic studies revealed that 6-O-angeloylenolin caused cell-cycle arrest in the S and G2/M phases and, subsequently, the induction of apoptosis. Rapid repressions of cyclin D1, cyclin D3, p27, cdc25c and p-cdc25c (Ser216) were observed after 1-h treatment, followed by decreases in the expression of CDK4, cdc2 and p-cdc2 (Tyr15) after 12h. Down-regulations of p-Rb (Ser780), p21(Waf1/Cip1), cyclin A, and cyclin E were also detected as later events. Two early events that marked the occurrence of apoptosis were phosphatidylserine exposure and mitochondria membrane potential depletion, which occurred after 12h of treatment, while a sub-G1 peak was also detected after 36-h treatment. Apoptosis induction was further confirmed by other apoptotic features, including nuclear fragmentation, and PARP cleavage. Moreover, 6-O-angeloylenolin caused the release of cytochrome c and AIF to the cytosol by regulating the expression of the Bcl-2 family proteins. However, pretreatment of the general caspase inhibitor failed to attenuate the apoptosis induction effect, suggesting that apoptosis induction of 6-O-angeloylenolin was independent of caspase activation. While 6-O-angeloylenolin also triggered the activation of Akt, ERK and JNK, only the JNK inhibitor significantly decreased the extent of cell death and apoptosis in CNE cells. Taken together, these results suggest the potential applicability of 6-O-angeloylenolin as a candidate for NPC treatment.

Antiproliferative effects of volatile oils from Centipeda minima on human nasopharyngeal cancer CNE cells.

Volatile oils from Centipeda minima extracted by steam distillation (SD) and supercritical fluid extraction (SFE) were investigated for their antiproliferative effects on the human nasopharyngeal cancer CNE cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay results showed that CNE cells were more susceptible to the SFE oil than to the SD oil. The IC50 values of the SFE oil were 56.6, 8.7 and 5.2 microg/mL after 24-, 48- and 72-h of treatment, respectively, whereas those of the SD oil were 123.5 microg/mL (24 h), 97.1 microg/mL (48 h) and 83.3 microg/mL (72 h). Mechanistic investigation revealed that SFE oil induced CNE cell death via induction of apoptosis by regulating the expression of the Bcl-2 family of proteins, resulting in the dysfunction of mitochondria, leading to the release of cytochrome c into the cytosol, which then activated caspase-9, and subsequently cleaved caspases-3 and -7. This study provided strong evidence for the anti-NPC potential of the SFE oil from C. minima.

Toxicological study of a Chinese herbal medicine, Wikstroemia indica.

Wikstroemia indica, a commonly used herbal medicine in China, is thought to be poisonous. However, based on the experience of herbal doctors, it is claimed that the toxicity can be reduced by decocting for 3-5 hours. In this study, the acute toxicities of W. indica (crude drug), and its aqueous and ethanolic extracts were evaluated in mice. The cytotoxicities of both extracts were also assessed using the MTT and LDH assays. Also, in order to evaluate the influence of decoction time on the toxicity of W. indica extracts, the cytotoxicities of W. indica decoctions prepared by boiling the herb for 1, 3, 5, 8, and 10 hours, respectively, were tested. Neither the herb nor its aqueous and ethanolic extracts showed obvious acute toxicity in mice, with maximal tolerance doses higher than 18.7, 11.7, and 25.0 g/kg p.o., respectively. The cytotoxicities of both extracts were low with CC50 values higher than 250 microg/mL. The five W. indica decoctions also showed low cytotoxicity, with CC50 values higher than 1000 microg (crude drug)/mL. Moreover, the result of statistical analysis indicated that the cytotoxicities of the five W. indica decoctions did not exhibit significant differences. The results obtained suggest that W. indica is a relatively nontoxic herb and longer decoction times for reducing the toxicity of the herb seems unnecessary.

Antiproliferative activities of five Chinese medicinal herbs and active compounds in Elephantopus scaber.

Extracts and fractions prepared from five medicinal herbs used to treat cancer in Chinese folk medicine were tested for their antiproliferative activities on five cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT results revealed that the ethanolic extracts possessed stronger antiproliferative activities than the corresponding aqueous ones. The bioactive fractions of the ethanolic extracts were mostly found in n-hexane and/or ethyl acetate fractions, which by TLC analysis were shown to contain terpenes and/or phenolic compounds. The ethyl acetate fraction from the ethanolic extract of Elephantopus scaber showed the highest effect with IC50 values of less than 25 microg/mL on three susceptible cell lines, namely, PC-3 (human prostate carcinoma cell), CNE (human nasopharyngeal carcinoma epithelial cell) and HL-60 (human acute promyelocytic leukemia cell). Further isolation and analysis of this fraction led to the identification of three sesquiterpene lactones, two flavonoids and five triterpenes. MTT results showed that the sesquiterpene lactones were the most effective group with IC50 values ranging from 0.9 to 7.5 microM while the flavonoids possessed moderate to strong effect with IC50 values ranging from 17.3 to 42.6 microM. However, the triterpenes displayed weak effect with IC50 values larger than 50 microM. Among the active compounds, deoxyelephantopin, a sesquiterpene lactone, exhibited the strongest effect on the PC-3, CNE and HL-60 cells, with IC50 values of 4.6, 2.6 and 0.9 microM, respectively. Flow cytometric analysis showed that treatment with deoxyelephantopin caused subG-1 population augmentation in PC-3, CNE and HL-60 cells, suggesting apoptosis was induced in these cells.

[Studies on the chemical comstituents of Pithecellobium clypearia].

OBJECTIVE: To study the chemical constituents of Pithecellobium clypearia. METHODS: The compounds were isolated from Pithecellobium clypearia with column chromatography. Their structures were characterized on the basis of their physical and chemical preperties, as well as chromatographic and spectroscopic evidences. RESULTS: A pair of isomeric flavans consisting of 7, 3'-O-di-gallyoltricetiflavan (I) and 7,4'-O-di-gallyoltricetiflavan (II), along with a catechin compound (-)-epigallocatechin-7-O-gallate (III) were isolated from the leaves and twigs of Pithecellobium clypearia. CONCLUSION: Compound I is a new flavan. Compound III is isolated from Pithecellobium genus for the first time, and its 13C-NMR data is firstly given in the present study.

2beta-(Isobutyryloxy)florilenalin, a sesquiterpene lactone isolated from the medicinal plant Centipeda minima, induces apoptosis in human nasopharyngeal carcinoma CNE cells.

Centipeda minima is a medicinal plant reputed in China as a remedy for nasopharyngeal carcinoma (NPC). In this study, bioactivity-guided fractionation of the anti-NPC compound(s) from C. minima led to the isolation of 2beta-(isobutyryloxy)florilenalin (IF), a sesquiterpene lactone. IF showed significant dose- and time- dependent inhibition on the growth of the human nasopharyngeal carcinoma epithelia cells (CNE). It induced apoptosis in CNE cells, as shown by the accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation, caspase-3 activation and PARP cleavage. Such induction was associated with the depletion of mitochondrial membrane potential (DeltaPsim) and the release of cytochrome c to cytosol to regulate the expression of Bcl-2 family proteins. These activities led to the cleavage of caspases and the trigger of cell death process. Overall, IF in C. minima showed potent antiproliferative effect of C. minima on NPC cells, suggesting that the plant deserves more extensive investigation for its potential medicinal application.

Isolation, characterization, molecular cloning and modeling of a new lipid transfer protein with antiviral and antiproliferative activities from Narcissus tazetta.

A fetuin-binding peptide with a molecular mass of about 9kDa (designated NTP) was isolated and purified from the bulbs of Chinese daffodil, Narcissus tazetta var. chinensis L., by gel filtration and high-performance liquid chromatography, after removing the mannose-binding proteins by mannose-agarose column. Molecular cloning revealed that NTP contained an open reading frame of 354bp encoding a polypeptide of 118 amino acids which included a 26-amino-acid signal peptide. An analysis of the deduced amino acid sequence of NTP shows considerable sequence homology to the non-specific lipid transfer proteins (nsLTPs) of certain plants. Model of the three-dimensional (3D) structure of NTP exhibits an internal hydrophobic cavity which can bind lipid-like molecules and transfer a wide range of ligands. As a member of the putative non-specific lipid transfer protein of N. tazetta, NTP did not possess hemagglutinating activity toward rabbit erythrocytes. In a cell-free system, it could arrest the protein synthesis of rabbit reticulocytes. Using the in vitro antiviral assays, NTP could significantly inhibit the plaque formation by respiratory syncytial virus (RSV) and the cytopathic effect induced by influenza A (H1N1) virus, as well as the proliferation of human acute promyelocytic leukemia cells (HL-60).

[Studies on the chemical constituents of Adina pilulifera].

The chemical constituents of Adina pilulifera, which were traditionlly used in the area of Yao minority in Southern China, were isolated and characterized. Two compounds were obtained from the ethyl acetate fraction of ethanol extract of Adina pilulifera, and one compound was obtained from the n-butanol fraction of ethanol extract of Adina pilulifera. Their structure were characterized by spectroscopic analysis and comparison with published data to be sarracenin (1), 2-methyl-5, 7-dihydroxychromone (2) and morroniside (3). Except compound 2, other two compounds were isolated from this plant for the first time, and they were also obtained from the Adina genus for the first time.

[Study on the chemical constituents of Ardisia chinensis].

The chemical constituents of Ardisia chinensis, which traditionally used in the area of Yao minority in Southern China, were analyzed and characterized. Seven compounds were obtained from the ethyl acetate fraction of ethanol extract of Ardisia chinensis. These 7 compounds were characterized by spectroscopic analysis and comparison with published data to be salicylic acid (1), 4-hydroxy-3-methoxy-benzoic acid (2), gallic acid ethyl ester (3), 4-hydroxy-3,5-dimethoxy benzoic acid methyl ester (4), protocatechuic acid (5), gallic acid (6) and catechin (7). All of them were isolated from this plant for the first time, and compound 7 was obtained from this genus for the first time.

Antiviral activity and constituent of Ardisia chinensis benth against coxsackie B3 virus.

Ardisia chinensis Benth is a medicinal plant traditionally used in the area of Yao minority in Southern China. The in vitro antiviral activities of extracts and fractions from Ardisia chinensis were tested by the cytopathic effect (CPE) reduction assay in the present study. As a result, both the aqueous extract and the 95% ethanol extract of Ardisia chinensis showed in vitro antiviral activity against Coxsackie B3 (Cox B3) virus to different extents, and the aqueous extract possessed more potent activity than the ethanol extract. Bioassay-guided fractionation revealed that the antiviral activity of Ardisia chinensis was attributed mainly to its high polar fractions, and finally identified to be a polysaccharide. The Ardisia chinensis polysaccharide (ACP) fractionated from the aqueous extract exhibited a significant antiviral effect against Cox B3 with a 50% inhibitory concentration (IC(50)) of 3.9 microg/mL and a selective index (SI) over 256. Preliminary characterization indicated that ACP is a neutral polysaccharide in which d-glucose is the major component. The average molecular weights of ACP were determined to be 40037 Da (Mw), 28297 Da (Mn) and 33758 Da (Mp) by gel permeation chromatography.

[Studies on antiviral constituents in stems and leaves of Pithecellibium clypearia].

OBJECTIVE: To study the antiviral constituents in the stems and leaves of Pithecellibium clypearia. METHOD: The constituents of P. clypearia were systematically separated with various chromatographic techniques in combination with antiviral activity monitoring. Their structures were elucidated by physical and chemical properties and spectral data. RESULT: Six compounds were isolated from P. clypearia and were identified as: tricetiflavan (5, 7, 3', 4', 5'-pentahydroxylflavan) (1), myricitrin (myricetin-3-O-alpha-L-rhamnopyranoside) (2), quercitrin (quercetin-3-O-alpha-L-rhamnopyranoside) (3), quereetin (4), methyl gallate (5) and gallic acid (6). CONCLUSION: Compound 1 approximately 5 were obtained from this plant for the first time. Compound 4 was found to show an obvious anti-respiratory syncytial virus (RSV) activity.