Derivatives of Deoxypodophyllotoxin Induce Apoptosis through Bcl-2/Bax Proteins Expression.

BACKGROUND: Deoxypodophyllotoxin, isolated from the Traditional Chinese Medicine Anthriscus sylvestris, is well-known because of its significant anti-tumor activity with strong toxicity in vitro and in vivo. OBJECTIVE: In this article, a series of deoxypodophyllotoxin derivatives were synthesized and their anti-tumor effectiveness was evaluated. METHODS: The anti-tumor activity of deoxypodophyllotoxin derivatives was investigated by the MTT assay method. Apoptosis percentage was measured by flow cytometer analysis using Annexin-V-FITC. RESULTS: The derivatives revealed obvious cytotoxicity in the MTT assay by decreasing the number of late cancer cells. The decrease of Bcl-2/Bax could be observed in MCF-7, HepG2, HT-29, and MG-63 using Annexin V-FITC. The ratio of Bcl-2/Bax in the administration group was decreased, which was determined by the ELISA kit. CONCLUSION: The derivatives of deoxypodophyllotoxin could induce apoptosis in tumor cell lines by influencing Bcl-2/Bax.

Yan-Hou-Qing formula attenuates ammonia-induced acute pharyngitis in rats via inhibition of NF-κB and COX-2.

BACKGROUND: Yan Hou Qing (YHQ) is a Chinese medicinal formula designed to alleviate sore throat symptoms, but underlying mechanism of YHQ treatment for pharyngitis is poorly defined up to now. METHODS: In this study, the modulation of YHQ on pharyngitis is investigated in ammonia-induced acute pharyngitis rat models. After treatment with YHQ or dexamethasone respectively for five consecutive days, all rats were sacrificed for biomolecular and histopathologic study. Protein expressions of MAPKs, NF-κB, COX-2 and 5-LOX in pharyngitis tissue were evaluated by western blot analysis and the levels of TNF-α, IL-6, prostaglandin (PG) E2, leukotrienes (LT)-B4 and LT-D4 in pharyngeal tissue were measured via ELISA assay. Evans blue (EB) dye exudation test was performed parallelly to assess the integrity of pharyngeal tissue. RESULTS: Compared with normal control group, EB dye exudation, and inflammatory cytokines in the model group were significantly increased, and the pharynx tissue was obviously infiltrated by inflammatory cells. YHQ treatment improved the inflammatory infiltrate in pharyngeal tissue, and reduced EB dye exudation in AP rat models. The up-regulated TNF-α and IL-6 in pharyngeal tissue of AP were significantly reduced by YHQ through inhibition of phosphorylation of p38, Erk and NF-κB. YHQ treatment also reversed the increased level of PGE2 through down-regulation of COX-2. CONCLUSIONS: YHQ formula attenuated the pharyngitis related symptoms via suppression of COX-2 and phosphorylation of p38, Erk and NF-κB (p65).

Therapeutic assessment of fractions of Gastrodiae Rhizoma on chronic atrophic gastritis by 1H NMR-based metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodiae Rhizoma (GR), a well-known and commonly-used TCM (Traditional Chinese Medicine) for treating headache, dizziness, tetanus, epilepsy, and etc., has been proven to relieve chronic atrophic gastritis (CAG). Due to its complex ingredients, the active fractions responsible for the treatment of CAG remain largely unknown. AIM OF THE STUDY: To explore the underlying material and interpret its underlying mechanism, the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG was studied based on the 1H NMR metabolomics. MATERIALS AND METHODS: The rat model of CAG was established by autoimmune method. The modeled CAG rats were then treated with 4 polar parts (T1-4 in descending polarity, corresponding to water, n-butanol, ethyl acetate and petroleum ether extracts, respectively) of Gastrodiae Rhizoma for 21 consecutive days. The stomach and serum samples were collected and then subjected to histopathology observation, biochemical measurement (MDA, SOD, GSH, NO, XOD and pepsin), 1H NMR metabolic profiling and multivariate/univariate statistical analysis. RESULTS: The results showed that T1 had the best therapeutic effect, T2 the second, and T3 and T4 the poorest with no obvious therapeutic effect, demonstrating that the effective components of Gastrodiae Rhizoma should be compounds of high polarity. T1 achieved good therapeutic effects due to the anti-inflammatory and anti-oxidant activities, and by rectifying the disturbed energy and amino acid metabolism in CAG model. CONCLUSION: This integrated metabolomics approach proved the validity of the therapeutic effect of extract from different polar parts of Gastrodiae Rhizoma on autoimmune CAG, providing new insights into the underlying mechanisms, and demonstrating the feasibility of metabolomics to evaluate efficacy of herbal drug, which is often difficult by traditional means.

Bioactive constituents from Salacia cochinchinensis.

Two new isopimarane diterpenoids, named 1α-hydroxy-7-oxoisopimara-8, 15-diene (1), 11ß-hydroxy-7-oxoisopimara-8(14), 15-diene (2), together with six known compounds (3-8), were isolated from the medicinal plant Salacia cochinchinensis. All isolates were assayed for their cytotoxicity and α-glucosidase inhibitory activity. Results suggested compounds 1, 3 possessed significant cytotoxic activity against HepG2, HL60, and Hela cell lines with IC50 values ranging from 0.23 to 0.35 µM, and compounds 7, 8 exhibited noticeable α-glucosidase inhibitory ability with IC50 values of 0.25 and 0.31 µM, respectively.

[UPLC-Q-TOF-MS analysis on chemical constituents of classical prescription Huangqi Guizhi Wuwu Tang standard decoction].

UPLC-Q-TOF-MS technology was used to analyze the chemical constituents from classical prescription Huangqi Guizhi Wuwu Tang standard decoction. Acquity HSS T3 column(2.1 mm × 100 mm, 1.8 µm) was used as the chromatographic column, with 0.1% formic acid solution-0.1% formic acid acetonitrile as the mobile phase for gradient elution. The volume flow rate was 0.4 mL·min~(-1) and the column temperature was 40 ℃. Mass spectrometry data of Huangqi Guizhi Wuwu Tang standard decoction were collected in positive and negative ion modes. The chemical constituents from classical prescription Huangqi Guizhi Wuwu Tang standard decoction were analyzed and identified by Masslynx 4.1 software combined with SciFinder database, comparison with reference mate-rials, mass spectrometry data analysis and reference to relevant literature. A total of 110 compounds were analyzed and identified, including 33 flavonoids, 14 monoterpene glycosides, 8 triterpenoids, 8 gingerols, 17 phenylpropanoids, 12 organic acids, 7 amino acids and 11 other compounds. The results of this study provide an experimental basis for the further research on the substance basis and quality control of Huangqi Guizhi Wuwu Tang standard decoction.

Bioactive glycosides from Salacia cochinchinensis.

Four new triterpene glycosides, named salaciacochinosides A-D (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds 2α,3ß,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-ß-d-glucopyranoside (5), racemiside (6), alangiplatanoside (7), acantrifoside E (8), and syringin (9). The structures of the four new triterpenoids were characterized by chemical methods and MS, IR, 1D and 2D NMR spectral analyses. The α-glucosidase inhibitory activities of the nine compounds were assessed, compounds 6 and 7 showed remarkable α-glucosidase inhibitory activities, with IC50 values of 0.44 and 0.75 µM, respectively. Compounds 1-5 exhibited moderate α-glucosidase inhibitory activities, and compounds 8 and 9 showed none α-glucosidase inhibitory activity in our current experiments.

[A new phenolic glycoside from honey-fried Eriobotrya japonica].

A total of twelve compounds were isolated from the ethyl acetate of the water extract of honey-fried Eriobotrya japonica through column chromatography over silica gel,Sephadex LH-20,RP-18,and preparative HPLC. Their structures were established by MS,1 D NMR and 2 D NMR data as japonicanoside A( 1),nerolidol-3-O-α-L-rhamnopyranosyl-( 1→2)-ß-D-glucopyranoside( 2),nerolidol-3-O-α-L-rhamnopyranosyl-( l→4)-α-L-rhamnopyranosyl-( 1 → 2)-[α-L-( 4-trans-feruloyl)-rhamnopyranosyl-( 1 → 6) ]-ß-D-glucopyranoside( 3),( +)-catechin( 4),(-)-epicatechin( 5),kaempferol 3-O-α-L-rhamnopyranoside( 6),quercitrin( 7),quercetin-3-O-ß-D-galactopyranoside( 8),quercetin-3-O-ß-glucopyranoside( 9),vanillin( 10),protocatechuic aldehyde( 11),and maltol( 12). Among them,1 is a new phenolic glycoside.

[Chemical constituent analysis for seeds of Celosia argentea by UPLC-ESI-Q-TOF-MS].

This Paper aimed to analyze and identify the chemical constituents from the seeds of Celosia argentea by UPLC-ESI-Q-TOF-MS. The analysis was performed on an ACQUITY HSS T3 reverse phase column(2.1 mm ×100 mm, 1.8 µm). The mobile phase consisting of 0.1% formic acid acetonitrile and 0.1% aqueous formic acid was used for gradient elution, and the flow rate was 0.4 mL·min~(-1). Mass spectrometry was applied for the qualitative analysis under positive and negative ionization modes and ESI ion source. Data was analyzed by Masslynx 4.1 software, literatures in SciFinder database, and standards. A total of 49 compounds, including 14 triterpenoids, 17 flavonoids, 11 cyclic peptides, 2 phenols, 2 organic acids, and 3 steroids were putatively identified. Among them, 19 compounds were firstly reported from this species. In-depth chemical constituent analysis for the seeds of C. argentea were accomplished here, and the findings could lay a good foundation for its quality control and clarifying the material basis of its efficacy.

Anti-inflammation action of xanthones from Swertia chirayita by regulating COX-2/NF-κB/MAPKs/Akt signaling pathways in RAW 264.7 macrophage cells.

BACKGROUND: Swertia chirayita, has been commonly used under the name "Zang-yin-chen" for the treatment of liver infections, inflammation, abdominal pain, and bacterial infection in traditional Tibetan medicine. However, the bioactive components with anti-inflammatory activities and underlying mechanisms remain poorly evaluated. STUDY DESIGN/METHODS: Repeated column chromatography yielded two main xanthones from petroleum ether (PE) and ethyl acetate fractions of whole plants of S. chirayita, and their structures were determined as bellidifolin (1) and swerchirin (2) on the basis of spectroscopic data and literature analysis. The anti-inflammatory activities and mechanisms of anti-inflammation of these two isolated xanthones were determined via enzyme-linked immunosorbent assay (ELISA) and western blot in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages in vitro. RESULTS: Anti-inflammation assay demonstrated that 1 and 2 inhibit the production of the pro-inflammatory cytokines interleukin-6 (IL-6) and TNF-α in LPS-stimulated RAW 264.7 macrophages. Xanthone 1 also potently inhibited the production of prostaglandin E2 (PGE2) by suppressing the protein expression of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. Western blot showed that the phosphorylation of c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs were remarkably attenuated by 1 in a concentration-dependent manner. Particularly, Compound 1 suppressed the phosphorylation of the inhibitor κB kinase-ß (IKK-ß), Akt, and p65 subunit of nuclear factor-kappaB (NF-κB). CONCLUSION: The potent suppressive effects of 1 from S. chirayita on inflammatory mediators by blocking the expression of COX-2 and phosphorylation of Akt, IKK-ß, MAPK and NF-κB, activation in LPS-stimulated macrophages suggest that 1 can be a preventive therapeutic candidate for the management of inflammatory-mediated immune disorders.

NF-κB and JNK mediated apoptosis and G0/G1 arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. AIM OF THE STUDY: To investigate the cytotoxic mechanism of RG on cancer cells. MATERIALS AND METHODS: RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RESULTS: RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G0/G1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G0/G1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G0/G1 arrest in HeLa cells. CONCLUSION: These results indicated that RG induces mitochondria-mediated apoptosis and G0/G1 cell cycle arrest by activation of JNK signaling as well as inactivation of NF-κB pathway in HeLa cells, which suggests that RG is one of the key active ingredients accounting for the anti-tumor effect of R. yunnanensis.

[Chemical constituents from petroleum ether fraction of Swertia chirayita and their activities in vitro].

The present work is to study the chemical constituents from petroleum ether fraction of Tibetan medicine Swertia chirayita by column chromatography and recrystallization. The structures were identified by physical and chemical properties and spectral data as swerchirin (1), decussatin (2), 1,8-dihydroxy-3,5,7-trimethoxyxanthone (3), 1-hydroxy-3,5,7,8-tetramethoxyxanthone (4), bellidifolin (5), 1-hydroxy-3, 7-dimethoxyxanthone (6), methylswertianin (7), 1-hydroxy-3,5-dimethoxyxanthone (8), erythrodiol (9), oleanolic acid (10), gnetiolactone (11), scopoletin (12), sinapaldehyde (13), syringaldehyde (14), and ß-sitosterol (15). Compounds 3, 4, 9, 11-14 were isolated from S. chirayita for the first time. Compounds 9 and 12 were firstly isolated from the genus Swertia. The cytotoxic activities of compounds 1, 2, 5, 7 and 8 against human pancreatic cancer cell lines SW1990 and BxPC-3,and the protective effects of these compounds against hydrogen peroxide (H2O2)-induced oxidative stress in human endothelium-derived EA.hy926 were investigated in vitro. The results showed no obvious effect at the high concentration of 50 µmol•L⁻¹.

Report: Cytotoxic flavonoids from the young twigs and leaves of Caesalpinia bonduc (Linn) Roxb.

The extraction, fractionation and recognition of flavonoids from the ethanolic extract of young twigs and leaves of C. bonduc were carried out. In addition, cytotoxic study of the flavonoids on two cancer cell lines, BGC-823 and HeLa was carried our using sulphorhodamine B assay. Seven flavonoids, six of which are being reported for the first time in this plant, were isolated. Their structures were identified by MS and NMR spectroscopic methods. Petroleum ether, ethyl acetate and water fractions exhibited moderate cytotoxic activity against HeLa cells. Five compounds showed cytotoxic activity against HeLa cell in comparison with Paclitaxel, while only one compound showed a good degree of cytotoxic activity against BGC-823 cell in comparison to Paclitaxel. The results obtained showed a structure - activity relationship.

Apoptosis induction and G2/M arrest of 2-methyl-1,3,6-trihydroxy-9,10-anthraquinone from Rubia yunnanensis in human cervical cancer Hela cells.

2-Methyl-1,3,6-trihydroxy-9,10-anthraquinone (MTA), one of the major components isolated from the traditional Chinese medicine Rubia yunnanensis, exhibited inhibitory activity on the proliferation of several human cancer cell lines. The results from an annexin V-FITC (fluoresein-5-isothiocyanate) apoptosis assay and DNA content analysis showed that MTA exerted cytotoxicity via apoptosis induction and G2/M cell cycle arrest in human cervical carcinoma HeLa cells. Further, MTA was found to induce apoptosis of HeLa cells through the mitochondria-mediated pathway. It caused the translocation of Bax to the mitochondria and release of cytochrome c into the cytosol, which caused the cleavage of caspase and poly(ADP-ribose) polymerase and finally triggered the apoptosis. Furthermore, the p53/p21/Cdc2-cyclin B1 signaling was found related to the G2/M arrest caused by MTA. The over-expression of p21 and down-expression of cyclin B1 caused by MTA inactivated the Cdc2-cyclin B1 complex of G2/M checkpoint and finally caused the G2/M arrest in HeLa cells. This study demonstrated that MTA is a potential anti-cancer component of R. yunnanensis, a folk anti-cancer herb used in Yunnan, China.

Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1-AKT interaction.

In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT.

[Cyclopeptides from Rubia schumanniana].

OBJECTIVE: To separate and identify cyclopeptides of tubers of Rubia schumanniana. METHOD: The 70% methanol extracts from tubers of Rubia schumanniana were separated and purified by silica gel, RP-18, Sephedax LH-20 and HPLC. Their structures were identified by spectral analysis. RESULT: Nine cyclopeptides were separated and identified as RA- II (1), RA-V (2), RA-VIII (3), rubiyunnanin C (4), RA-X (5), RY-II (6), RA- I (7), RA-XIII (8) and RA-XIII-OMe (9), respectively. CONCLUSION: All of nine cyclopeptides were separated from R. schumanniana for the first time.

Biologically active arborinane-type triterpenoids and anthraquinones from Rubia yunnanensis.

Twelve new arborinane-type triterpenoids (1-12) and four new anthraquinones (13-16), together with 50 known compounds, were isolated from the roots of Rubia yunnanensis. The structures of 1-16 were elucidated by spectroscopic data analysis and chemical methods. All compounds were evaluated for their cytotoxic, antibacterial, and antifungal activities. Rubiyunnanol C (5) is the first example of an arborinane-type triterpenoid with a double bond at C-8-C-9.

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells.

BACKGROUND AND PURPOSE: Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed. EXPERIMENTAL APPROACH: Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules. KEY RESULTS: RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC(50) values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased. CONCLUSIONS AND IMPLICATIONS: RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.

Apoptosis inducement of bigelovin from Inula helianthus-aquatica on human Leukemia U937 cells.

Inula helianthus-aquatica C. Y. Wu is a traditional medicinal plant used to treat some cancers in folk herbal medicine of Yunnan, China. Bigelovin, a sesquiterpene lactone isolated from this herb, potently inhibits the growth of a panel of eight cancer cell lines, especially in human monoblastic leukemia U937 cells with an IC(50) value of 0.47 microM. Characteristic morphological features of apoptosis were observed in U937 cells treated with bigelovin. Annexin V and nuclear DNA content distribution assays showed that the percentage of Annexin V positive cells increased to 8.86% (24 h) with 1 microM bigelovin treatment, and cells treated with bigelovin at this concentration apparently arrested at G(0)/G(1) phase compared with the control. These data suggested that cytotoxic effect of bigelovin on U937 cells involves induction of apoptosis, and the cell cycle is arrested at G(0)/G(1) phase.