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.

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.

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.

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.

Schisandrin B protects against carbon tetrachloride toxicity by enhancing the mitochondrial glutathione redox status in mouse liver.

Previous studies in our laboratory have demonstrated the effect of Schisandrin B (Sch B),an active ingredient of the fruit of Schisandra chinensis, on enhancing the hepatic glutathione antioxidant system in mice, as evidenced by the hepatoprotection against carbon tetrachloride (CCl4) toxicity. In the present study, the mechanism involved in the hepatoprotection afforded by Sch B treatment was investigated. Treating female Balb/c mice with 1, 3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of glutathione reductase (GRD), at a dose of 2 mmol/kg (i.p.) did not abrogate the hepatoprotective action of Sch B in CCl4-treated mice. The result indicates that the increased activity of hepatic GRD is not ascribable to the hepatoprotective action of Sch B. In control mice, the same Sch B treatment regimen caused an enhancement in hepatic mitochondrial glutathione redox status, as indicated by the significant increase and decrease in reduced and oxidized glutathione levels, respectively. While the CCl4 intoxication greatly impaired mitochondrial glutathione redox status, the beneficial effect of Sch B treatment became more evident after CCl4 challenge. Our results strongly suggest that the mechanism of hepatoprotection afforded by Sch B treatment may involve the enhancement of mitochondrial glutathione redox status.

Cytotoxic effects of sinococuline.

Three alkaloids, aknadinine, 1-nitroaknadinine and sinococuline, isolated from Stephania sutchuenensis were studied for their effects on a fibroblast cell line, eight tumor cell lines and a rat alveolar macrophage culture. Sinococuline is an effective tumor cell growth inhibitor whereas the toxicity of aknadinine and 1-nitroaknadinine towards all tested cells is low. A dose-dependent decrease in cell viability and in the uptake of tritiated-thymidine, -leucine and -uridine by these cells was observed when they were grown in the presence of sinococuline for 24 h. Exposure to sinococuline in vitro also altered the macrophage function by reducing the production of tumor necrosis factor and reactive nitrogen intermediates. Human leukaemic HL60 cells and mouse fibroblast L929 cells were used to study the underlying mechanism of cytotoxicity and apoptosis seem to be the mode of death induced by sinococuline

Methylenedioxy group as determinant of schisandrin in enhancing hepatic mitochondrial glutathione in carbon tetrachloride-intoxicated mice.

As a preliminary approach to exploring whether the methylenedioxy group of the dibenzocyclooctadiene skeleton of schisandrins plays an important role in hepatic mitochondrial-reduced glutathione (GSH) stimulatory activity, we examined the effects of three schisandrins, namely schisandrin A (Sch A), schisandrin B (Sch B), and schisandrin C (Sch C), on carbon tetrachloride (CCl4) hepatotoxicity and hepatic mitochondrial GSH status in mice. Pretreating mice with Sch A at a daily oral dose of 1 mmol/kg for 3 days did not protect against CCl4 hepatotoxicity, whereas pretreatment with Sch B or Sch C at the same dosage regimen produced almost complete protection. The hepatoprotection afforded by Sch B or Sch C pretreatment was associated with significant increases in the hepatic mitochondrial GSH level and glutathione reductase (EC 1.6.4.2) activity. Our results indicate that the methylenedioxy group of the dibenzocyclooctadiene skeleton of schisandrin is an important structural determinant in the stimulation of hepatic mitochondrial GSH, particularly under conditions of CCl4 intoxication.

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.

Cytotoxicity assessment of Ma-huang (Ephedra) under different conditions of preparation.

Ma-huang is a traditional Chinese medicinal herb derived from EPHEDRA: sinica Stapf and other EPHEDRA: species, used to treat asthma, nose and lung congestion, and fever with anhidrosis. It contains 0.5-2.5% by weight of total alkaloids, of which ephedrine accounts for 30 to 90%. Recently, large amounts of ma-huang were used as a source of ephedrine in many dietary supplements formulated for weight reduction, because ephedrine has been found effective in inducing weight loss in diet-restricted obese patients. However, indiscriminate consumption of ma-huang-containing products has resulted in many cases of poisoning, some of which were fatal. The objective of this study is to investigate the relative toxicity of ma-huang extracted under different conditions. The toxicities of various extracts were assayed using MTT colorimetry on a battery of cell lines, while ephedrine alkaloids were analyzed with HPLC. The results are summarized as follows. (1) The cytotoxicity of all ma-huang extracts could not be totally accounted for by their ephedrine contents, suggesting the presence of other toxins in the extracts. (2) Grinding was a significant condition enhancing the toxicity of the extracts. (3) The relatively high sensitivity of the Neuro-2a cell line to the toxicity of ma-huang extracts suggests that the toxic principles were acting on neuronal cells. (4) One condition to produce a ma-huang extract with high ephedrine-to-toxins ratio would be to boil the whole herb for two h.

Oleanane saponins from Gymnema sylvestre.

Six oleanane-type saponins, along with two known triterpene saponins, were isolated from the leaves of Gymnema sylvestre. The structures of the oleanane triterpene glycosides were characterized as longispinogenin 3-O-beta-D-glucuronopyranoside, 21 beta-benzoylsitakisogenin 3-O-beta-D-glucuronopyranoside, 3-O-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, oleanolic acid 3-O-beta-D-xylopyranosyl(1-->6)-beta-D-glucopyranosyl(1-->6)-beta-D- glucopyranoside, 3-O-beta-D-xylopyranosyl(1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester and 3-O-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl oleanolic acid 28-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl ester on the basis of hydrolysis and spectral evidence, including 1D- and 2D-NMR (TOCSY, ROESY, HMQC and HMBC) and FABMS analyses.

Anti-proliferative effect of ginseng saponins on human prostate cancer cell line.

Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. In a study of the anti-proliferative activity of ginsenosides using human prostate carcinoma LNCaP cell line, ginsenoside Rg3 displayed growth inhibitory activity. The cells lost its adherent property after incubation in the presence of 250 microM of ginsenoside for 48h. The expression of biomarker genes, including prostate specific antigen (PSA), androgen receptor (AR) and 5alpha-reductase (5alphaR), and that of the proliferating cell nuclear antigen (PCNA), were suppressed. Ginsenoside Rg3 induced classic apoptotic morphology and interfered with the expression of apoptosis-related genes, bcl-2 and caspase-3, in LNCaP cells, as demonstrated by fluorescence microscopy, flow cytometry and reverse transcriptase-polymerase chain reaction. Taken our results together, we suggested that ginsenoside Rg3 activated the expression of cyclin-kinase inhibitors, p21 and p27, arrested LNCaP cells at G1 phase, and subsequently inhibited cell growth through a caspase3-mediated apoptosis mechanism.

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.

Evaluation of the mutagenic and cytostatic potential of aristolochic acid (3,4-methylenedioxy-8-methoxy-10-nitrophenanthrene-1-carboxylic acid) and several of its derivatives.

Aristolochic acid (1), a constituent of Aristolochia species, has been used for medicinal purposes since the Graeco-Roman period. Following the observation that the compound was mutagenic and carcinogenic, it was removed from pharmaceutical products. Consistent with previous reports, we have found that 1 serves as a direct-acting mutagen in Salmonella typhimurium strains TA100, TA102, TA1537 and TM677, but was not active in the nitroreductase-deficient strains TA98NR and TA100NR. However, aristolic acid (2), a compound that differs in structure only by the absence of the nitro group, was also found to be a direct-acting mutagen in Salmonella strains TA98, TA100, TA102, TA1537, and TM677, as well as strains TA98NR and TA100NR. Both compounds (1 and 2) were active mutagens when evaluated with cultured Chinese hamster ovary cells. Thus, in contrast to previous suggestions, the nitro group at position 10 is not required to induce a mutagenic response. Also, a series of structural relatives (the methyl esters of 1 and 2 (3 and 4, respectively), aristolochic acid-D (5), aristolactam (6), aristolactam A-II (7), and aristolactam-N-beta-D-glucoside (8)) were evaluated for mutagenic potential with Salmonella typhimurium strain TM677 and found to be inactive. Since compounds 3 and 4 were found to be active mutagens with Salmonella typhimurium strains TA98, TA100, TA102 and TA1537 (sufficient quantities of compounds 5-8 were not available for testing), differential sensitivity of the tester strains unrelated to mutagenic potential is suggested. Further, compounds 1, 2, and 6-8 were evaluated for potential to inhibit growth with cultured KB or P388 cells. P388 cells were substantially more sensitive, and compound 1 was the most active of the materials tested (ED5 = 0.58 microM). Compound 6 also demonstrated appreciable activity (ED50 = 4.2 microM), as did compound 8 (ED50 = 6.0 microM). It therefore appears that phenanthrene-ring substituents, in addition to the nitro group at position 10, serve important roles for biological potential. In considering the carcinogenic event induced by aristolochic acid, these functionalities should also be taken into account.

Association of free radicals and the tissue renin-angiotensin system: prospective effects of Rhodiola, a genus of Chinese herb, on hypoxia-induced pancreatic injury.

The renin-angiotensin system has long been recognized as crucial factor in the regulation of the systemic blood pressure and renal electrolyte homeostasis. Numerous studies have demonstrated the presence of a local renin-angiotensin system in a variety of organs. A recent study of the pancreatic renin-angiotensin system showed that chronic hypoxia significantly increased the mRNA expression for angiotensinogen II receptor subtypes AT1b and AT2. The activation of the renin-angiotensin system may play an important role in cellular pathophysiological processes. Angiotensin II enhances the formation of reactive oxygen species via the activation of xanthine oxidase or NAD(P)H oxidase. The reactive oxygen species can cause oxidative damage in the pancreas and other tissues either directly or indirectly via the formation of other radicals such as reactive nitrogen species. Rhodiola therapy may protect hypoxia-induced pancreatic injury in two ways. It prevents hypoxia-induced biological changes by increasing intracellular oxygen diffusion and efficiency of oxygen utilization. Alternatively, it reduces hypoxia-induced oxidative damage by its antioxidant activities. Additional experimental data are required to fully elucidate the mode of action of this herbal drug.

Cucurbitacin contents in Hemsleya dolichocarpa.

Hemsleya dolichocarpa was studied for its cucurbitacin contents during growing period. A colorimetric method was used to determine the total cucurbitacin levels, and a TLC densitometric assay was employed for estimating dihydrocucurbitacin F and its acetate, two active principles in Hemsleya plants. Cucurbitacin contents rise progressively from May to November; the month of November is thus the most desirable time for harvesting plant materials for medicinal uses. It was noticed that the spoiled plant samples gave an intense color reaction while dihydrocucurbitacin F and its acetate levels were not significantly altered. The accuracy of the assays is discussed.

[Triterpene saponins from Pulsatilla cernua].

AIM: To investigate the chemical constituents of the roots of Pulsatilla cernua (Thunb.) Bercht. et Opiz. METHODS: Column chromatography (including D101 macroporous resin, silica gel and ODS) and HPLC were used to separate the chemical constituents whose structures were elucidated by FAB-MS, NMR (1D and 2D) and hydrolysis methods. RESULTS: Six compounds were isolated and established as hederagenin 3-O-alpha-L-rhamnopyranosyl (1-->2)-alpha-L-arabinopyranoside (pulsatilla saponin A, 1), hederagenin 3-O-beta-D-glucopyranosyl (1-->3)-alpha-L-rhamnopyranosyl (1-->2)-alpha-L- arabinopyranoside (2), hederagenin' 3-O-alpha-L-rhamnopyranosyl(1-->2)[beta-D- glucopyranosyl(1-->4)]-alpha-L-arabinopyranoside (pulsatilla saponin D, 3), 3-O-alpha-L-rhamnopyranosyl (1-->2)-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl ester (dipsacoside B, 4), 3-O-alpha-L-rhamnopyranosyl(1-->2) [beta-D-glucopyranosyl(1-->4)]-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-glucopyranosyl ester (cernuoside C, 5) and daucosterol (6), respectively. CONCLUSION: Compound 5 is a new compound and named as cernuoside C. Compounds 2, 4 and 6 were isolated from Pulsatilla cernua for the first time.

[Cycloartane triterpenes and glycosides from Cimicifuga acerina].

AIM: To investigate the chemical constituents from the rhizomes of Cimicifuga acerina (Sieb. et Zucc.) Tanaka. METHODS: Column chromatgraphy (including silica gel and ODS) together with HPLC was used to separate the chemical constituents whose structures were determined by FAB-MS, NMR (1D and 2D) and hydrolysis methods. RESULTS: Five cycloartane triterpenoids were isolated and identified as: (22R)-22-hydroxycimigenol (I), (22R)-22-hydroxy-24-O-acetylhydroshengmanol 3-O-beta-D-xylopyranoside (II), dahurinol (III), 24-epi-24-O-acetyl-7,8-didehydroshengmanol 3-O-beta-D-xylopyranoside (IV), 25-O-acetyl-7,8-didehydrocimigenol 3-O-beta-D-xylopyranoside (V). CONCLUSION: Compound I is a new natural product, compound II is a new compound and compounds IV and V were isolated from this plant for the first time.