The role of turmerones on curcumin transportation and P-glycoprotein activities in intestinal Caco-2 cells.

The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases.

Immunostimulatory activities of polysaccharide extract isolated from Curcuma longa.

Several curcuminoids and sesquiterpenoids isolated from Curcuma longa (CL) have been shown to have many pharmacological activities. In the present study, the immunomodulatory activities of the polar fractions of CL hot water extracts were investigated using human peripheral blood mononuclear cells (PBMC). Our results showed that the high polarity fraction of the hot water extract exhibited stimulatory effects on PBMC proliferation as shown in [methyl-(3)H]-thymidine incorporation assay. In an attempt to isolate the active components responsible for the activities, further partition with ethyl acetate, n-butanol and ethanol, progressively were performed. The cytokine productions (TGF-beta, TNF-alpha, GM-CSF, IL-1alpha, IL-5, IL-6, IL-8, IL-10, IL-13, etc.) have been modulated by a polysaccharide-enriched fraction as shown in ELISA and cytokine protein array. The proportion of CD14 positive stained PBMC was increased by such fraction. The composition of monosaccharide of the active fraction has been determined by GC-MS and gel permeation chromatography. The immunostimulatory effects of C. longa polysaccharides on PBMC were shown for the first time. The findings revealed the potential use of C. longa crude extract (containing curcuminoids and polysaccharides) as an adjuvant supplement for cancer patients, whose immune activities were suppressed during chemotherapies.

Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa.

The rhizome of Curcuma longa (CL) has been commonly used in Asia as a potential candidate for the treatment of different diseases, including inflammatory disorders and cancers. The present study evaluated the anti-proliferative activities of the isolated compounds (three curcuminoids and two turmerones) from CL, using human cancer cell lines HepG2, MCF-7 and MDA-MB-231. The immunomodulatory activities of turmerones (alpha and aromatic) isolated from CL were also examined using human peripheral blood mononuclear cells (PBMC). Our results showed that the curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) and alpha-turmerone significantly inhibited proliferation of cancer cells in dose-dependent manner. The IC(50) values of these compounds in cancer cells ranged from 11.0 to 41.8 microg/ml. alpha-Turmerone induced MDA-MB-231 cells to undergo apoptosis, which was confirmed by annexin-V and propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant decrease of procaspases-3, -8 and -9 in alpha-turmerone treated cells. Both alpha-turmerone and aromatic-turmerone showed stimulatory effects on PBMC proliferation and cytokine production. The anti-proliferative effect of alpha-turmerone and immunomodulatory activities of ar-turmerone was shown for the first time. The findings revealed the potential use of CL crude extract (containing curcuminoids and volatile oil including turmerones) as chemopreventive agent.

Antitussive and central respiratory depressant effects of Stemona tuberosa.

AIMS OF THE STUDY: Stemona alkaloids with distinctly different chemical skeletons are recently reported as the active components in the antitussive herb Baibu derived from the root-tubers of Stemona tuberosa. This study aims to determine if alkaloids of this herb contribute equally to the antitussive functions, act on the same sites of cough reflex, and play any role in inducing central respiratory depressant effects. MATERIALS AND METHODS: Antitussive potency of four major alkaloids was evaluated on guinea pigs with citric acid aerosol to induce cough. The action sites of the alkaloids on cough reflex pathway were tested with electrical stimulation of the superior laryngeal nerve in guinea pigs. The central respiratory effects of croomine were also tested on guinea pigs. RESULTS: Croomine, neotuberostemonine and stemoninine showed similar antitussive potency, while tuberostemonine showed much weaker antitussive potency. Neotuberostemonine, tuberostemonine and stemoninine acted on the peripheral cough reflex pathway, while croomine acted on the central part. Croomine also showed obvious central respiratory depressant effects. CONCLUSIONS: The four major Stemona alkaloids in Stemona tuberosa do not contribute equally to antitussive potency in guinea pigs. Neotuberostemonine, tuberostemonine and stemoninine target on peripheral cough reflex pathway. Croomine acts on central sites in the cough reflex pathway and demonstrates central respiratory depressant effects, which can partly account for the adverse reactions reported for the herb.

Authentication of the anti-tumor herb Baihuasheshecao with bioactive marker compounds and molecular sequences.

Baihuasheshecao (Hedyotis diffusa), a Chinese herb for cancer treatment, is frequently adulterated by a related species Hedyotis corymbosa. DNA sequencing of the complete internal transcribed spacer region was applied to differentiate H. diffusa from H. corymbosa and other closely related species. The molecular data showed that four out of seven herb samples of Baihuasheshecao were adulterants. Chemical analyses by TLC and HPLC were used to authenticate H. diffusa and H. corymbosa. Two marker compounds were identified exclusively in H. diffusa: 6-O-(E)-p-coumaroyl scandoside methyl ester (compound 1) and 10(S)-hydroxypheophytin a (compound 2). Both compounds showed moderate anti-proliferation effect on PC3 human androgen-independent prostate cancer cells, while compound 2 also showed strong anti-proliferation effect on LNCaP human androgen-sensitive prostate cancer cells. Accordingly, these bioactive marker compounds could be applied to verify the authenticity and assess the quality of Baihuasheshecao.

Quality evaluation of Radix Stemonae through simultaneous quantification of bioactive alkaloids by high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors.

A high-performance liquid chromatography coupled with diode array detection and evaporative light scattering detection (HPLC-DAD-ELSD) method was developed to simultaneously quantify six major bioactive alkaloids belonging to different structure types in Radix Stemonae, Bai-Bu in Chinese, a traditionally used antitussive and insecticidal medicinal material in China and other countries of Southeast Asia. Diode array detector (DAD) with the wavelengths at 307 and 260 nm was used to monitor the conjugated system of protostemonine (2) and maistemonine (4), respectively, whereas evaporative light scattering detector (ELSD) was employed to detect croomine (1), stemoninine (3), neotuberostemonine (5) and tuberostemonine (6), the other four analytes with no or poor chromophores. The assay was validated to be sensitive, precise and accurate, with a detection limit of 3.64-0.04 microg/mL depending on the individual analytes. The overall intra- and inter-day variations were less than 9.3%, and the overall recoveries higher than 91.2%, respectively. The correlation coefficients of the calibration curves were better than 0.996 for all analytes. The newly established method was successfully utilized to determine six major components in the genuine sources of Radix Stemonae: Stemona japonica, S. sessilifolia and S. tuberosa. Significant variations of contents of these components were demonstrated in samples of these three species. This simple, rapid, low-cost and reliable method is suitable for the routine quality control of herbal medicines containing bioactive components with different structure types such as Radix Stemonae.

Leachianone A as a potential anti-cancer drug by induction of apoptosis in human hepatoma HepG2 cells.

The Chinese herbal medicine Radix Sophorae is widely applied as an anti-carcinogenic/ anti-metastatic agent against liver cancer. In this study, Leachianone A, isolated from Radix Sophorae, possessed a profound cytotoxic activity against human hepatoma cell line HepG2 in vitro, with an IC(50) value of 3.4microg/ml post-48-h treatment. Its action mechanism via induction of apoptosis involved both extrinsic and intrinsic pathways. Its anti-tumor effect was further demonstrated in vivo by 17-54% reduction of tumor size in HepG2-bearing nude mice, in which no toxicity to the heart and liver tissues was observed. In conclusion, this is the first report describing the isolation of Leachianone A from Radix Sophorae and the molecular mechanism of its anti-proliferative effect on HepG2 cells.

Lignans from Dysosma versipellis with inhibitory effects on prostate cancer cell lines.

A new monoepoxylignan, dysosmarol (1), along with eight known compounds, podophyllotoxin (2), 4'-demethylpodophyllotoxin (3), deoxypodophyllotoxin (4), 4'-demethyldeoxypodophyllotoxin (5), diphyllin (6), kaempferol, quercetin, and beta-sitosterol, were isolated from the roots of Dysosma versipellis. The structure of 1 was elucidated by spectroscopic methods. Aryltetralin lignans 2-4 showed the most potent inhibitory activities against the growth of androgen-sensitive (LNCaP) and androgen-independent (PC-3) human prostate cancer cell lines, with IC50 values in the ranges 0.030-0.056 and 0.032-0.082 microM, respectively. A quantitative HPLC analysis showed that compound 2 occurred at the highest concentration in the plant (37.21 mg/g) followed by compound 4 (5.01 mg/g) and compound 3 (2.75 mg/g). Furthermore, D. versipellis roots contain a similar content of compound 2 as compared with the rhizomes and roots of Podophyllum hexandrum, a commercial source of the lignan. Thus, cultivation of D. versipellis in suitable locations may serve as an alternative source for podophyllotoxin (2) production.

Proteomic characterization of Sophoraflavone J-induced apoptosis in HepG2 cells.

Chinese herb Radix sophorae is widely applied as an anticarcinogenic/antimetastatic agent against liver cancers. In the current study, Sophoraflavone J, a flavonoid constituent enriched in the root of Radix sophorae, induced apoptosis in human hepatoma HepG2 cells via the intrinsic mitochondrial death pathway. The molecular mechanism of the cytotoxic effect was further investigated by a comparative proteomic approach. Differentially expressed proteins identified included membrane proteins/antigens, structural proteins, transcriptional factors, glycolytic enzymes, heat-shock chaperon proteins, ROS-related proteins and proteosomes, etc. These findings were further validated by Western blot analysis and real-time PCR. Preliminary experiments to characterize the roles of these proteins were conducted. Our data suggested that Sophoraflavone J treatment triggered nutrient depletion and generation of ROS in cells, which subsequently led to mitochondrial dysfunction and apoptosis.

Antitussive effects of Stemona tuberosa with different chemical profiles.

The root tubers of Stemona tuberosa, Stemona japonica and Stemona sessilifolia are recognized by the Pharmacopoeia of the People's Republic of China as authentic sources of the herb Radix Stemonae (Baibu). Careful anatomical analyses of these three species, whose identities were confirmed by flowering and fruiting samples, revealed that the root tubers of Stemona tuberosa could be distinguished from those of the other two species by the presence of scattered fibers in the cortex and pith and by the absence of thickened striations on the surface of velamen cells. HPLC analyses demonstrated that the total alkaloid profiles could be grouped into four types represented as the major component by stenine-type Stemona alkaloids such as tuberostemonine (4) and neotuberostemonine (3), or by non-stenine types such as croomine (1) and stemoninine (2). Nevertheless, all these samples demonstrated different degrees of antitussive properties in guinea pigs. These results suggested that non-stenine-type of Stemona alkaloids also contributed to the antitussive properties. The variations in chemical profiles among herb samples add difficulty in ensuring quality control in botanical products.

Alkaloids and chemical diversity of Stemona tuberosa.

Phytochemical investigation of the chemical components of Stemona tuberosa led to the isolation of two new alkaloids named tuberostemonine K (1) and tuberospironine (2), together with the known tuberostemonine (3). The new structures of 1 and 2 were elucidated through extensive spectroscopic analyses, while the molecular structure of 3 was confirmed by X-ray analysis. A gradient reversed-phase HPLC-ELSD method was established for the investigation of the chemical diversity of S. tuberosa from 13 localities, and four types of chemical variation featured by the major components 3, neotuberostemonine (4), croomine (5), and stemoninine (6), respectively, were observed.

Analyses of Stemona alkaloids in Stemona tuberosa by liquid chromatography/tandem mass spectrometry.

Alkaloid profiles in Stemona tuberosa were found to be highly variable. Six Stemona alkaloids isolated from the plant were subjected to on-line high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and tandem mass spectrometry (MS/MS) analyses. Their fragmentation patterns and products were useful for their characterization. The LC/MS fingerprints of these alkaloids, though variable among samples, could provide an overall characterization of the authenticity and quality of this species and help to differentiate it from S. japonica and S. sessilifolia, as all three species are recognized as genuine sources of the herb Radix Stemonae in the Pharmacopoeia of the People's Republic of China.

Pheophorbide a, a major antitumor component purified from Scutellaria barbata, induces apoptosis in human hepatocellular carcinoma cells.

Scutellaria barbata has long been used as a Chinese medicine for the treatment of liver diseases such as hepatitis and hepatocellular carcinoma. In the present study, a bioassay-guided method was used to isolate the most active components from Scutellaria barbata. The anti-proliferative effects on human hepatoma HepG2 and Hep3B cells of each fraction at every stage of the purification were monitored. An active component, which is 97% pure by high performance liquid chromatographic analysis, was isolated. Based on nuclear magnetic resonance (NMR) and mass spectrophotometric (MS) analysis, this active component was identified to be pheophorbide a (C35H36N4O5). Mechanistic studies showed that pheophorbide a induced apoptosis in Hep3B cells, a viral-induced hepatoma cell line. However, it was found to be non-toxic in normal human liver cells WRL-68. DNA fragmentation, sub-G1 cell cycle arrest, as well as suppression of the anti-apoptotic protein Bcl-2, release of cytochrome c to the cytosol, and activation of pro-caspase 3 and pro-caspase 9 were observed when Hep3B cells were treated with 40 microg/mL (i. e., 67.5 microM) pheophorbide a for 48 hours. In conclusion, this is the first report describing the isolation of pheophorbide a from Scutellaria barbata using a bioassay-guided isolation method. The anti-proliferative activity and possible mechanisms of action of pheophorbide a on hepatoma Hep3B cells are also discussed.

Isolation and chemotaxonomic significance of tuberostemospironine-type alkaloids from Stemona tuberosa.

An alkaloid named 6alpha-hydroxycroomine (1) as well as the known croomine (2), both belonging to the tuberostemospironine-alkaloid type, were isolated from Stemona tuberosa as the major components. The structure of 1 was elucidated through extensive spectroscopic analyses. Comparison of the HPLC profiles of the total alkaloids and the crude methanol extract showed that both compounds are naturally occurring. The first isolation of 1 and 2 from S. tuberosa has chemotaxonomic significance, confirming the close relationship between Stemona and Croomia. The trnL sequences of plants from the four genera of Stemonaceae cluster together as a clade, further lending support to retaining them in a single family.

Chemistry and biological activities of caffeic acid derivatives from Salvia miltiorrhiza.

Caffeic acid (3,4-dihydroxycinnamic acid), one of the most common phenolic acids, frequently occurs in fruits, grains and dietary supplements for human consumption as simple esters with quinic acid or saccharides, and are also found in traditional Chinese herbs. Caffeic acid derivatives occur as major water-soluble components of Salvia miltiorrhiza, including caffeic acid monomers and a wide variety of oligomers. This review provides up-to-date coverage of this class of phenolic acids in regard to structural classification, natural resources, chemical and biosyntheses, analytical methods and biological activities including antioxidant, anti-ischemia reperfusion, anti-thrombosis, anti-hypertension, anti-fibrosis, antivirus and antitumor properties. Special attention is paid to both structural classification and biological activities. The structural diversity and the pronounced biological activities encountered in the caffeic acid derivatives of S. miltiorrhiza indicate that this class of compounds is worthy of further studies that may lead to new drug discovery.

Antitussive activity of Stemona alkaloids from Stemona tuberosa.

Bioactivity-directed fractionation of the crude extract of Stemona tuberosa led to the isolation and characterization of four new stenine-type Stemona alkaloids, namely tuberostemonine J ( 2), tuberostemonine H ( 3), epi-bisdehydrotuberostemonine J ( 4) and neostenine ( 5), together with the known neotuberostemonine ( 1). These five isolated alkaloids were examined for antitussive activity in guinea pig after cough induction by citric acid aerosol stimulation. In this report, we demonstrated, for the first time, that compounds 1 and 5 showed significant antitussive activities. Further study of the structure-activity relationship on these isolated alkaloids and two synthetic analogues revealed that the saturated tricyclic pyrrolo[3,2,1- jk] benzazepine nucleus is the primary key structure contributing to the antitussive activity and all cis configurations at the three ring junctions are the optimal structure for the antitussive activity of stenine-type Stemona alkaloids.