Seven quassinoids from Fructus Bruceae with cytotoxic effects on pancreatic adenocarcinoma cell lines.

Our previous studies showed that the alcohol extract of the fruit of Brucea javanica (Fructus Bruceae) possessed significant cytotoxicity in pancreatic adenocarcinoma cell lines. A bioassay-guided fractionation and purification resulted in the isolation and characterization of seven quassinoids including brusatol, bruceine D, bruceine H, yadanzioside A, yadanzioside G, javanicoside C and bruceantinoside A. Among them, brusatol exhibited the most potent in vitro antipancreatic cancer action, with IC(50) values of 0.36 µm and 0.10 µm on PANC-1 and SW1990 cell lines, respectively. This is the first report on the antipancreatic adenocarcinoma activity of brusatol.

Role of neuronal nitric oxide synthase in colonic distension-induced hyperalgesia in distal colon of neonatal maternal separated male rats.

BACKGROUND: Nitric oxide (NO) is implicated in the pathogenesis of irritable bowel syndrome (IBS) but the underlying mechanism is unclear. Thus, the aim of the present study is to examine the role of NO synthase (NOS) expression in the distal colon of neonatal maternal separation (NMS) model rats employed in IBS studies. METHODS: Male neonates of Sprague-Dawley rats were randomly assigned into NMS and normal control (N) groups. Rats of NMS group were subjected to 3 h daily maternal separation on postnatal day 2-21. Rats were administrated non-selective NOS inhibitor l-NAME (100 mg kg(-1) ), selective neuronal NOS (nNOS) inhibitor 7-NINA (10mgkg(-1) ), selective inducible NOS (iNOS) inhibitor, endothelial NOS (eNOS) inhibitor (10mgkg(-1) ) or Vehicle (Veh; distilled water) intraperitoneally 1h prior to the experiment for the test and control groups, respectively. KEY RESULTS: The amount of NO was significantly higher in the NMS Veh rats compared with unseparated N rats. Western-blotting and real-time quantitative PCR studies showed that protein and mRNA expression of nNOS were higher in the NMS group than that in the N rats; whereas no significant change in iNOS and eNOS was found in either groups. Neonatal maternal separation Veh rats showed low pain threshold and increased electromyogram (EMG) activity in response to colonic distension stimuli. l-NAME and 7-Nitroindazole monosodium salt (7-NINA) increased pain threshold pressure and attenuated EMG activity in the NMS rats. In addition, l-NAME and 7-NINA substantially reduced oxidative marker malondialdehyde level in NMS rats. CONCLUSIONS & INFERENCES: Neonatal maternal separation increased the NO generation by nNOS upregulation that interact with reactive oxygen species contributing to the visceral hypersensitivity in IBS.

Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells.

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.

Ethyl acetate fraction of the root of Rubia cordifolia L. inhibits keratinocyte proliferation in vitro and promotes keratinocyte differentiation in vivo: potential application for psoriasis treatment.

Psoriasis is a skin disease associated with hyperproliferation and aberrant differentiation of keratinocytes. Our previous studies have identified the root of Rubia cordifolia L. as a potent antiproliferative and apoptogenic agent in cultured HaCaT cells (IC(50) 1.4 microg/ml). In the present study, ethanolic extract of Radix Rubiae was fractioned sequentially with hexane, ethyl acetate (EA), n-butanol and water. EA fraction was found to possess most potent antiproliferative action on HaCaT cells (IC(50) 0.9 microg/ml). Mechanistic study revealed that EA fraction induced apoptosis on HaCaT cells, as it was capable of inducing apoptotic morphological changes. Annexin V-PI staining assay also demonstrated that EA fraction significantly augmented HaCaT apoptosis. In addition, EA fraction decreased mitochondrial membrane potential in a concentration- and time-dependent manner. The standardized EA fraction was formulated into topical gel and its keratinocyte-modulating action was tested on mouse tail model. EA fraction dose-dependently increased the number and thickness of granular layer and epidermal thickness on mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present preclinical study confirms that EA fraction is a promising antipsoriatic agent warranting further development for psoriasis treatment.

In vitro antidiabetic activities of five medicinal herbs used in Chinese medicinal formulae.

Fructus Corni, Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae are commonly used in traditional Chinese medicine for diabetes treatment. They are also the component herbs of an antidiabetic foot ulcer formula with demonstrated clinical efficacy. Although some of these herbal extracts were previously shown to possess in vivo antidiabetic effects (i.e. lowering blood glucose levels), the underlying mechanisms remain elusive. The objective of this study is to investigate the possible antidiabetic mechanisms of these individual herbs, using a systematic study platform which includes four in vitro tissue models: glucose absorption into intestinal brush border membrane vesicles (BBMV), gluconeogenesis by rat hepatoma cell line H4IIE, glucose uptake by human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1. All tested herbs showed significant in vitro antidiabetic effects in at least two models. Fructus Schisandrae Chinensis, Poria, Rhizoma Alismatis and Rhizoma Dioscoreae showed significant inhibitory effects in the BBMV glucose uptake assay. All tested herbs showed significant stimulatory effects to the glucose uptake of Hs68 and 3T3-L1 cells, except Poria and Rhizoma Dioscoreae which were not effective to Hs68 and 3T3-L1 respectively. However, none of the tested herbs inhibited hepatic gluconeogenesis. In conclusion, the five herbs exhibited distinct antidiabetic mechanisms in vitro and hence our investigations provided scientific evidence to support the traditional usage of these herbs for diabetic treatment in medicinal formulae.

Improvement of Ca balance by Fructus Ligustri Lucidi extract in aged female rats.

UNLABELLED: This study aimed to demonstrate and delineate the mechanism of action of Fructus Ligustri Lucidi (FLL) involved in improving Ca balance in aged female rats. FLL could enhance the apparent Ca absorption rate and reduce Ca excretion, via its actions on modulating the levels of calciotropic hormones and CaBPs expression. INTRODUCTION: Fructus Ligustri Lucidi (FLL) is a herb classically used for the treatment of age-related symptoms in China. The present study aimed to determine if FLL could improve calcium balance in aged female rats and delineate its mechanism of action in vivo. METHODS: Aged Sprague-Dawley rats (11 months of age) were orally administered with ethanol extract of FLL or its vehicle and fed with diets containing different levels of Ca (low Ca diet, LCD, 0.1% Ca; medium Ca diet, MCD, 0.6% Ca; high Ca diet, HCD, 1.2% Ca) for 12 weeks. Serum, urine and feces were collected for biochemical markers and Ca balance determination. mRNA expressions of calcium binding proteins (CaBPs) were determined by real time RT-PCR. The effects of diets and herb were analyzed by both one-way and two-way ANOVA. RESULTS: FLL significantly reduced fecal Ca excretion and induced apparent Ca absorption rate in rats fed with MCD and HCD. FLL increased serum 1,25(OH)(2)D(3) level and duodenal CaBP-9k mRNA expressions in all rats. Renal CaBP-28k mRNA expressions were induced in rats fed with MCD and HCD upon FLL treatment. CONCLUSIONS: Our study demonstrated that FLL improved Ca balance in aged female rats by increasing serum 1,25 (OH)(2)D(3) level and vitamin D-dependent CaBPs expression.

Pharmacological investigations of the anti-diabetic effect of Cortex Moutan and its active component paeonol.

Cortex Moutan (CM, root bark of Paeonia suffruticosa Andr.) is one of the common herbs found in anti-diabetic traditional Chinese medicine formulae. To study the potential anti-diabetic mechanisms of CM, four in vitro models (intestinal brush border membrane vesicles (BBMV), rat hepatoma cell line H4IIE, human skin fibroblasts cell line Hs68 and mouse adipocytes 3T3-L1) were used. CM showed significant in vitro anti-diabetic effects by inhibiting glucose uptake of BBMV and enhancing glucose uptake into Hs68 and 3T3-L1 cells. Using bioassay-guided fractionation, paeonol was confirmed to be one of the active constituents for inhibiting BBMV glucose uptake. With neonatal-streptozotocin diabetic rats, paeonol (200 and 400mg/kgbody wt.) was found to improve oral glucose tolerance in vivo. To the best of our knowledge, this is the first report on the anti-diabetic effect of paeonol.

Influence of an anti-diabetic foot ulcer formula and its component herbs on tissue and systemic glucose homeostasis.

Complications of diabetes impose major public health burdens worldwide. The positive effect of a Radix Astragali-based herbal preparation on healing diabetic foot ulcers in patients has been reported. Formula 1 is also referred as the 'Herbal drink to strengthen muscle and control swelling'. This formula contains six Chinese medical herbs, including Radix Astragali, Radix Rehmanniae, Rhizoma Smilacis Chinensis, Rhizoma Atractylodis Macrocephalae, Radix Polygoni Multiflori Preparata, and Radix Stephania Tetrandrae. Three of these herbs (Radix Astragali, Radix Rehmanniae, Rhizoma Atractylodis Macrocephalae) are commonly used in different anti-diabetic formulae of Chinese medicine. The objective of the current study is to use an interdisciplinary approach to test the hypothesis that Formula 1 and its components influence tissue and systemic glucose homeostasis. In vitro and in vivo models have been established including: (1) glucose absorption into intestinal brush border membrane vesicles (BBMV); (2) gluconeogenesis by H4IIE hepatoma cells; (3) glucose uptake by 3T3-L1 adipocytes and Hs68 skin fibroblasts; (4) normalization of glycaemic control in a diabetic rat model. The results of in vitro studies indicated that all herbal extracts can modify cellular glucose homeostasis. Since Formula 1 and Rhizoma Smilacis Chinensis extracts demonstrated potent effects on modifying glucose homeostasis in multiple tissues in vitro, they were further studied for their anti-diabetic activities in vivo using a streptozotocin (STZ)-induced diabetic rat model. The results showed that Formula 1 and Rhizoma Smilacis Chinensis extracts did not significantly improve oral glucose tolerance or basal glycaemia in diabetic rats. In conclusion, the anti-diabetic foot ulcer Formula 1 contains ingredients active in modifying tissue glucose homeostasis in vitro but these biological activities could not be associated with improved glycaemic control of diabetes in vivo.

A new labdane diterpene from Leonurus heterophyllus.

A new labdane diterpene, heteronone B (1), together with a known labdane diterpene, heteronone A (2), have been isolated from the aerial part of Leonurus heterophyllus. Their structures were established mainly by 1D and 2D NMR analysis and the stereochemistry of 2 was confirmed by single-crystal X-ray diffraction analysis.

In vitro immunomodulatory activities of a newly concocted traditional Chinese medicine formula: VI-28.

Previous studies have suggested that Vigconic VI-28, an anti-aging traditional Chinese medicine (TCM) formula containing Radix Ginseng and Cornu Cervi Pantotrichum, possesses immunological efficacy. This in vitro study further investigated the immunomodulatory effects of the hot water extracts of VI-28. The study included (1) colorimetric 5-bromo-2'-deoxy-uridine proliferation ELISA for estimating mitogenicity in human peripheral blood mononuclear cells (PBMC), (2) immunofluorescence staining for measuring the expression of IL-2 receptor alpha (CD25) on lymphocytes, (3) cytometric bead array (CBA) for quantifying cytokine liberation from PBMC, and (4) intracellular immunophenotyping for macrophage phagocytosis and hydrogen peroxide (H(2)O(2)) production from monocytes. The results demonstrated that VI-28 (1) could dose-dependently inhibited the proliferation of unstimulated and lipopolysaccharide-activated PBMC but enhanced the proliferation of phytohemagglutinin-activated PBMC at concentrations of <1 mg/mL, (2) significantly augmented the expression of CD25 on lymphocytes at concentrations of 0.4 mg/mL or above (p < 0.05), (3) dose dependently (0.1-1.0 mg/mL) activated macrophage phagocytosis and monocyte synthesis of H(2)O(2) and (4) significantly increased the production of cytokines IL-8, IL-10, IL-12 and IL-1beta at various concentrations of VI-28 (p < 0.05). The results suggest that VI-28 is a potential immunomodulator which probably acts through the activation of lymphocytes and monocytes.

Apoptotic activity of isomalabaricane triterpenes on human promyelocytic leukemia HL60 cells.

Four isomalabaricane triterpenes were isolated from marine sponge Geodia japonica [W.H. Zhang, C.T. Che, Isomalabaricane-type nortriterpenoids and other constituents of the marine sponge Geodia japonica, J. Nat. Prod. 64 (2001) 1489-1492. ] and their cytotoxicity was evaluated using a human promyelocytic leukemia HL60 cell line. Of the four triterpenes tested, geoditin A was the most cytotoxic to HL60 cells [IC50=3 microg/ml (<6.6 microM)], followed by stellettins A and B, whereas geoditin B exhibited relatively weak cytotoxicity. The treated cells manifested nuclear changes characteristic for apoptosis, and associated with dissipation of mitochondrial membrane potential, activation of caspase 3, and decrease of cytoplasmic proliferating cell nuclear antigen (PCNA), as demonstrated by fluorescence and immunofluorescence microscopy. When the HL60 cells were exposed to geoditin A ranging from 1.25 to 25 microg/ml, a dose-dependent increase of reactive oxygen species, a progressive dissipation of mitochondrial membrane potential, and an increase in annexin V-FITC binding were measured by flow cytometry. Taken together our results suggest that geoditin A markedly induced reactive oxygen species, decreased mitochondrial membrane potential and mediated a caspases 3 apoptosis pathway.

Effect of chronic hypoxia on glutathione status and membrane integrity in the pancreas.

BACKGROUND: Our recent study has shown that chronic hypoxia could upregulate significantly a local renin-angiotensin system in the pancreas. The activation of such a local renin-angiotensin system may provide an alternate mechanism that leads to the generation of reactive radical species in the pancreas during chronically hypoxic exposure. The present study aims at elucidating the antioxidant status in the pancreas during varying degrees of chronic hypoxia. METHODS: Sprague-Dawley rats were exposed to an isobaric hypoxic (10% oxygen) chamber for a period up to 28 days. The glutathione status and membrane integrity of the pancreas were studied with a time course of chronic hypoxia (3, 7, 14, 21 and 28 days). The effect of chronic hypoxia on changes of oxidative states in the pancreas was assessed based on the measurements of glutathione, malondialdehyde, alpha-amylase and DNA fragmentation using biochemical assays. RESULTS: Pancreatic glutathione was decreased drastically after 3-day hypoxia and its level was almost completely recovered after 7-day hypoxia. Malondialdehyde was not affected while DNA fragmentation was increased significantly in a time-dependent manner during the course of chronic hypoxia. Membrane integrity of the pancreatic cells was improved, as evidenced by the decrease of plasma alpha-amylase during the time-course study of chronic hypoxia. CONCLUSION: Pancreatic glutathione was depleted only in the early period of chronic hypoxia followed by a rapid recovery, suggesting that adaptive response of the pancreas may occur during chronic hypoxia. The enhancement of glutathione-dependent antioxidant capacity during chronic hypoxia prevented oxidative damage to the membrane of the pancreatic cells.

Growth inhibitory activity of lemnabourside on human prostate cancer cells.

Induction of apoptosis and androgen ablation are two major approaches for treating human prostate carcinoma. In a study of the bioactive components of the soft coral Nephthea chabroli, we found that lemnabourside is a 5alpha-reductase inhibitor, as shown by its ability to inhibit the conversion of testosterone into the more potent dihydrotestosterone in rat prostate homogenate. The compound also inhibited the incorporation of tritiated thymidine into human prostate androgen-dependent carcinoma LNCaP cells, and thus blocking the cell proliferation (IC50 = 37.5 microM). The expression of prostate marker genes, including 5alpha-reductase, prostate-specific antigen, prostatic acid phosphatase and androgen receptor, and the anti-apoptotic bcl-2 gene were markedly reduced, but the transcription of apoptosis-related caspase 3 gene showed a dose-dependent increase in lemnabourside-treated LNCaP cells. Immunofluorescent microscopy and flow cytometric analysis further demonstrated apoptotic changes in these cells. Taken all results together, a relatively weak 5alpha-reductase inhibitory activity on LNCaP cells (EC50 > 250 microM), and a similar growth inhibitory activity on both androgen dependent- and independent-prostate cells (IC50 approximately 37.5 microM) indicated that caspase-3 apoptosis pathway is one of the possible antiproliferative activities mediated by lemnabourside.

Cycloartane glycosides from Cimicifuga dahurica.

A new cycloartane bisdesmoside and two new trinorcycloartane glycosides, along with four known cycloartane compounds, were isolated from the rhizomes of Cimicifuga dahurica (Ranunculaceae). The structures of the new compounds were elucidated as 3-O-alpha-L-arabinopyranosyl cimigenol 15-O-beta-D-glucopyranoside, 24-hydroxy-12beta-acetoxy-25,26,27-trinorcycloartan-16,23-dione 3beta-O-alpha-L-arabinopyranoside, and 16alpha,24alpha-dihydroxy-12beta acetoxy-25,26,27-trinor-16,24-cyclocycloartan-23-one 3beta-O-alpha-L-arabinopyranoside by extensive NMR methods, FAB-MS, and hydrolysis.

Two unusual oleanane saponins from Anemone anhuiensis.

From the rhizomes of Anemone anhuiensis (Ranunculaceae), two new triterpene saponins, anhuienosides A (1) and B (2), were isolated. These compounds are glycosylated at C-23 and their structures were elucidated as hederagenin 23-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranoside (1) and 23-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl hederagenin 28-O-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl ester (2) on the basis of chemical and spectral evidence.

Effects of schisandrin B pretreatment on tumor necrosis factor-alpha induced apoptosis and Hsp70 expression in mouse liver.

Tumor necrosis factor-alpha (TNFalpha) could cause apoptosis in hepatic tissue of D-galactosamine sensitized mice, as evidenced by the increase in the extent of DNA fragmentation. The hepatic apoptosis induced by TNFalpha was associated with hepatocellular damage as assessed by plasma alanine aminotransferase activity. Schisandrin B (Sch B) pretreatment at daily doses ranging from 0.5 to 2 mmol/kg for 3 days caused a dose-dependent protection against TNFalpha-induced apoptosis in mice. The hepatoprotection was accompanied by a parallel reduction in the extent of hepatocellular damage. The same Sch B pretreatment regimens increased hepatic Hsp70 level in a dose-dependent manner. The relevance of Sch B-induced increase in Hsp70 expression to the prevention of TNFalpha-triggered hepatic apoptosis remains to be elucidated.

A pair of 24-hydroperoxyl epimeric dammarane saponins from flower-buds of Panax ginseng.

Further investigation on the saponins of the flower-buds of Panax ginseng C. A. Meyer has resulted in the isolation and structural elucidation of a pair of new 24-epimers of dammarane type saponins named ginsenoside I and II. The structures of the epimers were characterized on the basis of chemical and spectral evidence as 3-O-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-20-S-O-beta-D-glucopyranosyl-3beta, 12beta,20(S)-trihydroxy-24xi-hydroperoxydammar-25-ene, except for their C-24 configurations. Ginsenoside I is a new triterpene glycoside, and ginsenoside II is a known compound first isolated from a natural plant.

Antisweet saponins from Gymnema sylvestre.

Three new oleanane-type triterpene glycosides (1-3), along with the sodium salt of alternoside II (4), were isolated from an ethanol extract of the leaves of Gymnema sylvestre. The structures of these new saponins were identified as 21 beta-O-benzoylsitakisogenin 3-O-beta-D-glucopyranosyl(1-->3)-beta-D-glucuronopyranoside (1), the potassium salt of longispinogenin 3-O-beta-D-glucopyranosyl(1-->3)-beta-D-glucuronopyranoside (2), and the potassium salt of 29-hydroxylongispinogenin 3-O-beta-D-glucopyranosyl(1-->3)-beta-D-glucuronopyranoside (3). The aglycon of 3, gymnemagenol (3a), was characterized as 3 beta,16 beta,28, 29-tetrahydroxyolean-12-ene. Structure elucidation was accomplished by interpretation of NMR (DQF-COSY, HMQC, and HMBC) results, FABMS, and hydrolysis. Saponin 1 and the sodium salt of alternoside II (4) exhibited antisweet activity.

Four new oleanane saponins from Anemone anhuiensis.

Four new oleanane triterpene saponins, anhuienosides C-F, together with three known saponins, were isolated from the rhizomes of Anemone anhuiensis (Ranunculaceae). The structures of anhuienosides C-F were elucidated as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-beta-D-xylopyranosyl oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, respectively.