Two New Cytotoxic Maytansinoids Targeting Tubulin from Trewia nudiflora.

Two new maytansinoids, N-methyltreflorine (1: ) and methyltrewiasine (2: ), were isolated from the dried fruits of Trewia nudiflora, together with three known congeners (3:  - 5: ). Their structures were elucidated by spectroscopic methods, and the absolute configuration of 1: and 2: was determined by X-ray crystallographic analysis. Compounds 1:  - 5: exhibited strong cytotoxicity against human tumor cell lines, including HeLa, MV-4 - 11, and MCF-7, with IC50 values ranging from 0.12 to 11 nM. Compounds 1: and 4: also showed inhibitory activity against the MCF-7/ADR cell line with IC50 values of 13 and 28 nM, respectively. Compounds 1: and 2: significantly inhibited tubulin polymerization in vitro with IC50 values of 3.6 and 3.2 µM, respectively.

Metabolic profiling investigation of Fritillaria thunbergii Miq. by gas chromatography-mass spectrometry.

Thunberg fritillary bulb (the dry bulbs of Fritillaria thunbergii Miq.), a traditional Chinese Medicine, is widely applied as an expectorant and antitussive. In this investigation, the primary metabolites of bulbs, flowers, leaves, and stems of F. thunbergii were analyzed by gas chromatography-mass spectrometry. Principal component analysis, partial least squares-discriminate analysis, orthogonal projection to latent structures-discriminate analysis, and heat map analysis showed that there were dissimilar metabolites, and a negative correlation between amino acids and saccharides in different analytes. Furthermore, carbodiimide, tryptophan, glucose-6-phosphate, xylose, 2-piperidinecarboxylic acid, monoamidomalonic acid, phenylalanine, and histidine were found to play an important role in the plant metabolism net of F. thunbergii.

Systematic Understanding of the Mechanism of Salvianolic Acid A via Computational Target Fishing.

Salvianolic acid A (SAA) is one of the most abundant water-soluble and potent anti-oxidative compounds isolated from Danshen, a traditional Chinese medicine. A systematic overview of its mechanism of action is yet to be performed. In the present study, the druggability of SAA was measured using the TCMSP server, and potential targets of SAA were identified by PharmMapper and DRAR-CPI. Intersecting targets were then assessed by GeneMANIA and GO pathway analysis, and drug-target-pathway networks were constructed to give a visual view. The results showed that SAA has good druggability, and 13 putative protein targets were identified. Network analysis showed that these targets were associated with cancer, metabolism and other physiological processes. In summary, SAA is predicted to target multiple proteins and pathways to form a network that exerts systematic pharmacological effects.

[Identification of alkaloids and flavonoids in all parts of Fritillaria thunbergii using LC-LTQ-Orbitrap MSn].

Alkaloids and flavonoids in flowers, flower buds, stems, leaves, and bulbs of Fritillaria thunbergii were identified by LC-LTQ-Orbitrap MSn.Alkaloids were identified by ACQUITY UPLC BEH C18（2.1 mm×50 mm, 1.7 µm ) chromatographic column with a mobile phase of 10 mmol•L⁻¹ ammonium formate-acetonitrile and gradient elution in positive MS scan mode.Meanwhile, flavonoids were analyzed by Agilent-Zorbax SB C18 (4.6 mm×250 mm, 5 µm) chromatographic column with a mobile phase of 0.2% acetic acid-acetonitrile and gradient elution in negative MS scan mode.Combined with literature reports, chemical constituents were identified and determined by accurate molecular weights and fragment ion peaks in the ESI-MS/MS spectra based on high resolution mass spectrometer.In all parts of F.thunbergii, 37 alkaloids including 7 alkaloids (zhebeininoside, peimisine, peimine, peiminine, ebeiedinone/puqiedinone, ebeiedine/ puqiedine, peimisine-N-oxide) were simultaneously analyzed.Moreover, 16 flavonoids including quercetin, kaempferol and their glycosides were identified.The results indicated that the aerial parts had the similar alkaloids as the bulbs on the whole.Meanwhile, it had a series of flavonoids undetected in the bulbs.Our results provided the scientific basis for the development and utilization of aerial parts of F.thunbergii.

One-pot ß-cyclodextrin-assisted extraction of active ingredients from Xue-Zhi-Ning basing its encapsulated ability.

Xue-Zhi-Ning (XZN) is a traditional Chinese medicine formula, containing active ingredients with poor solubility in water, which has been demonstrated to be helpful for patients with hyperlipidemia. One-pot ß-cyclodextrin (ß-CD)-assisted extraction of active ingredients from XZN has been carried out to develop an efficient and eco-friendly extraction process. Five active compounds--rubrofusarin gentiobioside, 2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside, emodin, nuciferine and quercetin--were identified by UPLC/DAD/MS and used as indexes to evaluate the process optimized by an orthogonal test. The results showed that addition of ß-CD significantly enhanced the extraction ratios of all five components. The enhancement of extraction ratios was positively correlated with the apparent formation constants between ß-CD and the compounds. The study also showed that the stabilities and dissolution rates of the active ingredients were improved in the presence of ß-CD. This one-pot ß-cyclodextrin-assisted extraction has the potential to be applied in pharmaceutical preparations directly.

GOLPH3 predicts survival of colorectal cancer patients treated with 5-fluorouracil-based adjuvant chemotherapy.

BACKGROUND: Golgi phosphoprotein 3 (GOLPH3) has been validated as a potent oncogene involved in the progression of many types of solid tumors, and its overexpression is associated with poor clinical outcome in many cancers. However, it is still unknown the association of GOLPH3 expression with the prognosis of colorectal cancer (CRC) patients who received 5-fluorouracil (5-FU)-based adjuvant chemotherapy. METHODS: The expression of GOLPH3 was determined by qRT-PCR and immunohistochemistry in colorectal tissues from CRC patients treated with 5-FU based adjuvant chemotherapy after surgery. The association of GOLPH3 with clinicopathologic features and prognosis was analysed. The effects of GOLPH3 on 5-FU sensitivity were examined in CRC cell lines. RESULTS: GOLPH3 expression was elevated in CRC tissues compared with matched adjacent noncancerous tissues. Kaplan-Meier survival curves indicated that high GOLPH3 expression was significantly associated with prolonged disease-free survival (DFS, P = 0.002) and overall survival (OS, P = 0.011) in patients who received 5-FU-based adjuvant chemotherapy. Moreover, multivariate analysis showed that GOLPH3 expression was an independent prognostic factor for DFS in CRC patients treated with 5-FU-based chemotherapy (HR, 0.468; 95%CI, 0.222-0.987; P = 0.046). In vitro, overexpression of GOLPH3 facilitated the 5-FU chemosensitivity in CRC cells; while siRNA-mediated knockdown of GOLPH3 reduced the sensitivity of CRC cells to 5-FU-induced apoptosis. CONCLUSIONS: Our results suggest that GOLPH3 is associated with prognosis in CRC patients treated with postoperative 5-FU-based adjuvant chemotherapy, and may serve as a potential indicator to predict 5-FU chemosensitivity.

Pharmacokinetic interaction between scutellarin and valsartan in rats.

Scutellarin is the main effective constituent of breviscapine, a flavonoid mixture isolated from the dried whole plant of Erigeron breviscapus (Vant.) Hand-Mazz, and valsartan is used as an antihypertensive drug. These two drugs have already been clinically used together to treat diabetic nephropathy (DN) in China, and the combined medications showed some enhanced protection against DN. The aim of this study is to investigate the potential pharmacokinetic interaction between scutellarin and valsartan in rats. Breviscapine injection (20 mg x kg(-1), i.v.) and valsartan (15 mg x kg-, i.g.), either alone or together were given to 18 male Sprague-Dawley rats. Concentrations of scutellarin and valsartan were quantified by HPLC, and pharmacokinetic parameters were calculated by non-compartmental methods. We found that the pharmacokinetic parameters of scutellarin altered significantly after co-administration of oral valsartan. The plasma clearance (CL(p)) and the bile clearance (CL(b)) of scutellarin were reduced significantly in the presence of valsartan. After oral administration of valsartan with or without intravenous scutellarin, however, the pharmacokinetic parameters of valsartan were comparable. In conclusion, our data suggests that the concurrent use of valsartan reduces the biliary excretion of scutellarin, and this may be due to the inhibitory effect of valsartan on the biliary excretion of scutellarin mediated by Mrp2 (Multidrug resistance-associated protein 2).

Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis.

Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS) is a ribosome inactivating protein purified from tubers of the Chinese herbal plant Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen). In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.

Identification and evaluation of apoptotic compounds from Garcinia oligantha.

Four new compounds, oliganthins A-D (1-4), and one known caged xanthone gaudichaudione H (5) were isolated from the stems of Garcinia oligantha. The structures of the new compounds were elucidated by spectroscopic evidences. All of the five compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to produce a fluorescent biosensor capable of detecting caspase-3 activation. All of them induced cell apoptosis at 10 µM or lower concentrations. The apoptotic activity of oliganthins A, B and gaudichaudione H were further confirmed by detecting the cleavage of PARP, which is the substrate of activated caspase-3, in these compounds-treated cells using the method of Western blot. Moreover, the values of IC(50) were measured for all five compounds on HeLa cells using the MTT assay. Among them, gaudichaudione H had the lowest IC(50) value of 0.90 µM, while the other four new compounds had IC(50) values of 1.58, 1.52, 4.15, and 7.82 µM, respectively. These results show that gaudichaudione H has the strongest apoptosis-inducing effect and cell growth inhibition effect among these xanthones and it may have the potential to be developed into a new anticancer agent.

Anti-inflammatory and anti-melanogenic steroidal saponin glycosides from Fenugreek (Trigonella foenum-graecum L.) seeds.

Fenugreek seed ( Trigonella foenum-graecum L.) is used as an herbal medicine for treating metabolic and nutritive dysfunctions. To determine if this plant has other beneficial effects, we tested the inhibitory activities of a methanol (MeOH) extract of fenugreek seed on the production of inflammatory cytokines and melanin synthesis in cultured cell lines in vitro. The MeOH extract inhibited the production of phorbol-12-myristate-13-acetate-induced inflammatory cytokines such as tumor necrosis factor (TNF)-α in cultured THP-1 cells, and also restrained the intracellular synthesis of melanin in murine melanoma B16F1 cells. We isolated three active constituents from fenugreek seed extracts. These were identified as the steroidal saponins 26- O-ß-D-glucopyranosyl-(25 R)-furost-5(6)-en-3 ß,22 ß,26-triol-3- O-α-L-rhamno-pyranosyl-(1'' → 2')-O-[ß-D-glucopyranosyl-(1''' → 6')- O]-ß-D-glucopyranoside 1, minutoside B 2, and pseudoprotodioscin 3. Compounds 1 and 2 strongly suppressed the production of inflammatory cytokines, whereas 3 showed a weaker suppressing effect. Melanogenesis in B16F1 cells was significantly suppressed by 1 and 3, and weakly suppressed by 2. All three compounds showed moderate cytotoxicities. These results indicate that fenugreek extract and its active constituents could protect against skin damage.

Effects of proanthocyanidins from grape seed on treatment of recurrent ulcerative colitis in rats.

The aim of the present study was to investigate the therapeutic effect and mechanism of proanthocyanidins from grape seed (GSPE) in the treatment of recurrent ulcerative colitis (UC) in rats. To induce recurrent colitis, rats were instilled with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (80 mg/kg) into the colon through the cannula in the first induced phase, and then the rats were instilled a second time with TNBS (30 mg/kg) into the colon on the sixteenth day after the first induction UC. Rats were intragastrically administered GSPE (200 mg/kg) per day for 7 days after twice-induced colitis by TNBS. Sulfasalazine at 500 mg/kg was used as a positive control drug. Rats were killed 7 days after GSPE treatment. The colonic injury and inflammation were assessed by macroscopic and macroscopic damage scores, colon weight/length ratio (mg/cm), and myeloperoxidase activity. Then, superoxide dismutase, glutathione peroxidase, inducible nitric oxide synthase (iNOS) activities, and the levels of malonyldialdehyde, glutathione, and nitric oxide in serum and colonic tissues were measured. Compared with the recurrent UC group, GSPE treatment facilitated recovery of pathologic changes in the colon after induction of recurrent colitis, as demonstrated by reduced colonic weight/length ratio and macroscopic and microscopic damage scores. The myeloperoxidase and iNOS activities with malonyldialdehyde and nitric oxide levels in serum and colon tissues of colitis rats were significantly decreased in the GSPE group compared with those in the recurrent UC group. In addition, GSPE treatment was associated with notably increased superoxide dismutase, glutathione peroxidase activities, and glutathione levels of colon tissues and serum of rats. GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response, inhibiting inflammatory cell infiltration and antioxidation damage, promoting damaged tissue repair to improve colonic oxidative stress, and inhibiting colonic iNOS activity to reduce the production of nitric oxide.

[Clinical observation on drug-separated moxibustion at Shenque (CV 8) for treatment of infantile autumn diarrhea].

OBJECTIVE: To observe therapeutic effect of drug-separated moxibustion at Shenque (CV 8) for treatment of infantile autumn diarrhea. METHODS: One hundred and thirty-six cases were randomly divided into an obser vation group and a control group, 68 cases in each group. The observation group were treated with drug-separated moxibustion at Shenque (CV 8) and the control group with oral administration of Smecta. The mean diarrhea-stopping time, the negative conversion rate of Human Rotavirus antigen in stool after treatment for 72 h, and the markedly-effective rate and the total effective rate were observed after treatment for 6 days in the twO groups. RESULTS: The markedly-effective rate and the total effective rate were 79.4% and 94.1% in the observation group and 35.3% and 75.0% in the control group, respectively, with very significantly or significantly difference between the two groups (P < 0.01 or P < 0.05); the mean diarrhea-stopping time in the observation group was shorter than that in the control group (P < 0.01); the negative conversion rate of Human Rotavirus antigen in stool after treatment for 72 h was 88.2%0 in the observation group and 69.1% in the control group with a very significantly difference between the two groups (P < 0.01). CONCLUSION: Drug-separated moxibustion at Shenque (CV 8) has a significant therapeutic effect on infantile autumn diarrhea, helps negative conversion of Human Rotavirus antigen in stool and shortens duration of disease.

Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells.

AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma.

[Clinical efficacy of pishen bingbu recipe in patients with sympathetic cervical spondylosis and its impact on heart rate variability].

OBJECTIVE: To investigate the clinical effect of Pishen Bingbu Recipe (PBR) in treating patients with sympathetic cervical spondylosis (SCS) of qi-blood deficient syndrome type and its impact on heart rate variability (HRV). METHODS: Fifty patients were randomized into the control group and the treatment group equally. Both were treated with mecobalamin, vitamin B1, neurotropin, and occipital - jaw band traction in the sitting posture, but to patients in the treatment group, PBR was given additionally. The course of treatment was 60 days. Therapeutic effect and changes of HRV indexes were observed. RESULTS: After treatment, in the treatment group, 5 patients (20%) were clinically cured, treatment was markedly effective in 12 patients (48%), effective in 7 (28%) and ineffective in 1 (4%), while the corresponding data in the control group were 2 (8%), 4 (16%), 18 (72%) and 1 (4%) respectively, demonstrating the efficacy in the treatment group was superior to that in the control group (P <0.05). Before treatment, the HRV indexes in the two groups were insignificantly different respectively (P >0.05). But after treatment, difference between groups was observed in terms of either time domain or frequency domain. Those of time domain were: standard deviation of NN intervals (SDNN, ms) 133.41 +/- 8.61 vs 115.61 +/- 13.49, average standard deviation of 5 min NN intervals (SDANN, ms) 126.90 +/- 9.99 vs 106.20 +/- 8.84, HRV trigonometric index (HRVTI) 35.10 +/- 4.48 vs 25.51 +/- 2.24; and those of frequency domain: low frequency (LF, ms2) 379.90 +/- 159.07 vs 477.70 +/- 396.91 high frequency (HF, ms2) 157.10 +/- 28.18 vs 122.10 +/- 101.90, and LF/HF ratio 2.37 +/- 0.52 vs 4.27 +/- 2.84. All were superior in the treatment group (P < 0.05). CONCLUSION: PBR shows evidently clinical efficacy on SCS, it can significantly improve the functional activities of sympathetic nerve in patients.

Chemical fingerprint and metabolic fingerprint analysis of Danshen injection by HPLC-UV and HPLC-MS methods.

HPLC-UV and HPLC-MS techniques were used in fingerprint analysis of Danshen injection and its raw materials (roots and rhizoma of Salvia miltiorrhiza). HPLC profiles of Danshen injections from a Chinese pharmaceutical factory and their raw materials were established as their characteristic fingerprint and employed to assess their consistency and difference. To develop the representative fingerprint of Danshen injection, 10 batches of samples were analyzed under the same HPLC conditions. The results showed that 10 batches of Danshen injections had very similar HPLC fingerprints. To characterize the major constituents of Danshen injection for quality control, 11 major chromatographic peaks were characterized by their MS spectra and comparison with the reference standards. Through comparison of the HPLC profiles of Danshen injection with its raw material, it was found that they are greatly different, which indicated the changes of major constituents in the course of preparation procedure. In addition, the rat's plasma was analyzed by HPLC-MS technique after intravenous administration of Danshen injection at different time intervals to explore the in vivo metabolism of the major active constituents. Except for protocatechuic aldehyde, the major phenolic acids in Danshen injection appeared in rat's plasma after intravenous administration, but quantity of each phenolic acids was very different from that in Danshen injection. With the administration time prolonged danshensu and salvianolic acid B disappeared quickly, salvianolic D, lithospermic acid and salvianolic A slowly decreased and maintained relatively high concentration after 30 min of intravenous administration. This indicated that polyphenolic acids were significant for biological activity of Danshen injection. It might be concluded that chemical fingerprint combined with metabolic fingerprint is a useful means to control the quality and to clarify the possible mechanism of action of herbal products.