Development and Validation of Quantitative (1)H NMR Spectroscopy for the Determination of Total Phytosterols in the Marine Seaweed Sargassum.

Knowledge of phytosterol (PS) contents in marine algae is currently lacking compared to those in terrestrial plants. The present studies developed a quantitative (1)H NMR method for the determination of the total PSs in Sargassum. The characteristic proton signal H-3α in PSs was used for quantification, and 2,3,4,5-tetrachloro-nitrobenzene was used as an internal standard. Seaweed samples could be recorded directly after total lipid extraction and saponification. The results showed that the PS contents in Sargassum fusiforme (788.89-2878.67 mg/kg) were significantly higher than those in Sargassum pallidum (585.33-1596.00 mg/kg). The variable contents in both species suggested that fixed raw materials are very important for future research and development. Orthogonal projection to latent structures discriminant analysis was carried out in the spectral region of δ 3.00-6.50 in the (1)H NMR spectrum. S. fusiforme and S. pallidum could be separated well, and the key sterol marker was fucosterol.

The antiangiogenic activity of Kushecarpin D, a novel flavonoid isolated from Sophora flavescens Ait.

AIMS: Kushecarpin D (KD) is a novel flavonoid isolated from the traditional Chinese herbal medicine Kushen (the dried root of Sophora flavescens Ait). As part of our continuous effort to explore Chinese traditional medicinal herbs and to identify novel natural anticancer products, the antiangiogenic properties of KD were examined in vitro using a human umbilical vein endothelial cell line (ECV304). MAIN METHODS: The SRB and Trypan Blue exclusion assays were used to evaluate the effect of KD on cell proliferation. The antiangiogenic activities of KD were evaluated through studies of cell migration, cell adhesion, and tube formation. DCFH-DA and DHE fluorescent assays were used to detect the reactive oxygen species (ROS) levels. Catalase activity was detected using the colorimetric ammonium molybdate method. Cell cycle and apoptosis were measured using flow cytometry and the Hoechst 33258 staining assay. KEY FINDINGS: The results indicated that KD showed antiangiogenic activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. ROS levels were down-regulated and catalase activity was up-regulated after treatment with KD. The cell cycle was arrested at the G2/M phase, while no apoptosis was observed using the Hoechst 33258 staining assay or following the flow cytometric analysis of the sub-G1 proportion. SIGNIFICANCE: The antiangiogenic properties of KD, in combination with its anti-proliferative effect and ability to induce cell cycle arrest without inducing apoptosis, make it a good candidate for development as antitumor agent. However, further studies are essential to elucidate its mechanism of action.

A novel flavonoid isolated from Sophora flavescens exhibited anti-angiogenesis activity, decreased VEGF expression and caused G0/G1 cell cycle arrest in vitro.

Kushen, the dried root of Sophora flavescens Ait, is a traditional Chinese herbal medicine. Kushen alkaloids have been developed in China as anticancer drugs, and more potent antitumor activities have been identified in kushen flavonoids than in kushen alkaloids. In this study, the anti-angiogenic properties of (2S)-7,2',4'-triihydroxy-5-methoxy-8-dimethylallyl flavanone (Compound 1, a novel flavonoid isolated from Kushen), were examined using the human umbilical vein endothelial cell line (ECV304) in vitro. The results indicated that compound 1 shows anti-angiogenesis activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. Further studies indicated that compound 1 blocks cell cycles in the G0/G1 phase without inducing apoptosis, and down regulates vascular endothelial growth factor (VEGF) expression. The free radical scavenging activity of compound 1 was found through 2',7'-dichlorofluorescin diacetate (DCFH-DA) incubation assay in cells. The anti-angiogenic properties of compound 1 and its antiproliferative effect on endothelial cells without causing apoptosis make it a good candidate for development as a agent against development of tumors.

[Effects of astragali radix extract on matrix metalloproteinase 9 expression and atherosclerotic plaque formation in aorta of apolipoprotein E deficient mice fed with high fat diet].

OBJECTIVE: To explore the effects of astragali radix extract on the expressions of matrix metalloproteinase 9 (MMP-9) and the formation of atherosclerotic plaque in aortic atherosclerotic plaques of apolipoprotein E-deficient mice (ApoE-/-). METHODS: Male 8-week-old ApoE-/- mice fed with high fat diet were randomly divided into four groups (n=12 each): control group (saline 0.2 ml/d), atorvastatin group (atorvastatin 10 mg×kg(-1)×d(-1)), low-dose astragali radix extract group (1.25 g×kg(-1)×d(-1)) and high-dose astragali radix extract group (5 g×kg(-1)×d(-1)). After 12 weeks, serum oxLDL was measured by the method of ELISA. The formation of atherosclerotic plaque was determined in HE and oil red O stained aortic slice. The expressions of macrophage and MMP-9 in the aortic plaque were detected by immune fluorescence and immunohistochemistry staining method. RESULTS: Similarly as atorvastatin, astragali radix extract significantly decreased the level of serum oxLDL in ApoE-/-1 mice in a dose-dependent manner. The level of oxLDL in the high-dose astragali radix extract group [(5.2±6.1) µg/ml] was significantly lower than that in the control group [(15.8±5.4) µg/ml, P<0.01]. The area of atherosclerosis plaques was smaller (17.24%±4.22% vs. 49.87%±9.37%, P<0.01) and the penetration degree of plaques in the arterial wall was relieved in the high-dose astragali radix extract group compared to those in the control group (P<0.01). The expressions of Mac3 in atherosclerosis plaques of the high-dose astragali radix extract group was also significantly lower than in the control group (P<0.01). The mean absorbance value of the expression of MMP-9 in the high-dose astragali radix extract group (0.0154±0.0014)was significantly lower than that in the control group (0.0263±0.0065) (P<0.01). CONCLUSIONS: Similar as atorvastatin, astragali radix extract can dose-dependently inhibit the expression of MMP-9 and the formation of the atherosclerotic plaque in ApoE-/- mouse, probably by reducing the serum oxLDL, inhibiting macrophage infiltration, migration and secretion of MMP-9.

Anti-atherosclerotic function of Astragali Radix extract: downregulation of adhesion molecules in vitro and in vivo.

BACKGROUND: Atherosclerosis is considered to be a chronic inflammatory disease. Astragali Radix extract (ARE) is one of the major active ingredients extracted from the root of Astragalus membranaceus Bge. Although ARE has an anti-inflammatory function, its anti-atherosclerotic effects and mechanisms have not yet been elucidated. METHODS: Murine endothelial SVEC4-10 cells were pretreated with different doses of ARE at different times prior to induction with tumor necrosis factor (TNF)-α. Cell adhesion assays were performed using THP-1 cells and assessed by enzyme-linked immunosorbent assay, western blotting and immunofluorescence analyses to detect the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), phosphorylated inhibitor of κB (p-iκB) and nuclear factor (NF)-κB. We also examined the effect of ARE on atherosclerosis in the aortic endothelium of apolipoprotein E-deficient (apoE(-/-)) mice. RESULTS: TNF-α strongly increased the expression of VCAM-1 and ICAM-1 accompanied by increased expression of p-iκB and NF-κB proteins. However, the expression levels of VCAM-1 and ICAM-1 were reduced by ARE in dose- and time-dependent manners, with the strongest effect at a dose of 120 µg/ml incubated for 4 h. This was accompanied by significantly decreased expression of p-iκB and inhibited activation of NF-κB. Immunofluorescence analysis also revealed that oral administration of ARE resulted in downregulation of adhesion molecules and decreased expression of macrophages in the aortic endothelium of apoE(-/-) mice. ARE could suppress the inflammatory reaction and inhibit the progression of atherosclerotic lesions in apoE(-/-) mice. CONCLUSION: This study demonstrated that ARE might be an effective anti-inflammatory agent for the treatment of atherosclerosis, possibly acting via the decreased expression of adhesion molecules.

[Study on the chemical constituents of Phlomis younghusbandii].

OBJECTIVE: To study the chemical constituents of Phlomis younghusbandii. METHODS: Column chromatography was used in the isolation procedure. The structures of isolated compounds were elucidated by spectral data RESULTS: Eight compounds were isolated and their structures were identified as barlerin (1), sesamoside (2), phloyoside II (3), salidroside (4), shanzhiside methyl ester (5), acteoside (6), luteolin-7-O-glucoside (7), daucosterol (8). CONCLUSION: Compound 4 is firstly isolated from Phlomis genus. Compound 4 and 6 are obtained from this plant for the first time.

[Clinical observation on injection of small dose of morphine into Neiguan (PC 6) for treatment of chest pain in the patient with acute myocardial infarction].

OBJECTIVE: To probe into an effective injection way of Morphine for treatment of chest pain of acute cardiac infarction. METHODS: Ninety cases of myocardial infarction were randomly divided into 3 groups, an acupoint-injection group, an intravenous injection group and a hypodermic injection group, 30 cases in each group. The acupoint-injection group were treated with injection of 2 mg Morphine into bilateral Neiguan (PC 6) respectively, and the intravenous injection group with intravenous injection of 5 mg Morphine and the hypodermic injection group with hypodermic injection of 5 mg Morphine, and other treatments were same in the 3 groups. The analgesic effects were assessed with visual analogue scale (VAS) 5, 10, 30, 60 and 180 minutes after treatment and the complications were observed. RESULTS: There were no significant differences among the 3 groups before treatment in the VAS score (P > 0.05). The analgesic effect in the acupoint-injection group was better than those in other two groups 5 min, 30 min and 180 min after treatment (all P < 0.01). The incidence rate of nausea and vomiting of 0.3% in the acupoint-injection group was significantly lower than 40.0% in the intravenous injection group and 20.0% in the hy podermic injection group (all P < 0.01). CONCLUSION: Injection of small dose of Morphine into Neiguan (PC 6) has a definite therapeutic effect on chest pain of acute myocardial infarction with earlier analgesic effect, smaller dose of Morphine, longer analgesic duration and less complications.

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.

Characterization of C-glycosyl quinochalcones in Carthamus tinctorius L. by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.

C-Glycosyl quinochalcones are unique components in Carthamus tinctorius L. The reported C-glycosyl quinochalcones have the same quinochalcone skeleton with a hydroxyl group at the 5'-position and a glucose linked to this position with a carbon-carbon bond. In this study, the standard hydroxysafflor yellow A and water-extracted fraction of Carthamus tinctorius L. were analyzed by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOFMS) in both positive and negative ion modes. The fragmentation pathways of C-glycosyl quinochalcones were interpreted and validated by accurate mass measurement. Their fragmentation showed a special cleavage at the C-C bond except for the typical internal cleavage at the sugar moiety of other C-glycosyl flavonoids. In positive ion mode, cleavage of the 5'-glucose produced an [M+H-162](+) ion by a neutral loss, while cleavage of the 5'-glucose in negative ion mode led to an [M-H-163](-.) ion by radical cleavage. The cleavage from the carbonyl group produced fragment ions containing an A or a B ring. The fragment ions containing an A ring were common product ions of seven compounds in both ion modes, and fragment ions containing the B ring were used to judge the different substituent groups at the 3'-position. The fragmentation patterns of seven structurally related C-glycosyl quinochalcones were analyzed systematically and the formation of the fragment ions in two modes is explained in detail in this report. UPLC/Q-TOFMS is an effective tool for characterizing a complex sample, which gives higher resolution separation and generates accurate mass measurement of the product ions.

Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells.

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the DeltaPsi(m). These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.

[The effect of icariin and astragalosid I on the proliferation and differentiation of bone marrow stromal cells].

OBJECTIVE: To observe the effects of icariin and astragalosid I on the proliferative and alkaline phosphatase (ALP) activity of dog bone marrow stromal cells (BMSCs). METHODS: The dog's BMSCs were isolated and cultured in vitro. The 3th generation BMSCs were treated with icariin or astragalosid I at the concentration of 50 ng/ml and compared with BMSCs of BMP-2 group and control group. The growth curves of BMSCs were drawn by 3-(4,5-dimiethylthiazole-2-yl)-2, 5-hiphenyl tetrazolium bromide (MTT) colorimetric assay every day from the 1st to the 8th day to estimate the proliferative ability of BMSCs. The curves of OD value of ALP excreted by BMSCs on the 1st, 3th, 6th, 10th and the 14th day were recorded to estimate the ALP activity of BMSCs. RESULTS: After the pertreatment with icariin and astragalosid I, the BMSCs acquired higher MTF values and higher ALP's OD values as compared with control group and the difference between experiment group and control group was statistically significant (P < 0.05). CONCLUSION: Icariin and Astragealosid I can accelerate the proliferation and ALP excretion of BMSCs. At the same time, the osteogenesis ability of these cells is greatly improved.

[Modulation of Chinese prescription Shenyang on Th1/Th2 cytokines in serum of SD rats with squamous cell carcinoma of the tongue].

PURPOSE: Prospective research demonstrated that Chinese prescription Shenyang could prolong the survival time and improve the survival rate of patients with oral squamous cell carcinoma (SCC). But the mechanism was not clear. The purpose of this study was to investigate effect of Shenyang on the level of Th1/Th2 cytokines in serum of SD rats with SCC of tongue and clarify its mechanism. METHODS: Among 80 SD rats fed by 0.002% 4-nitroquinoline-1-oxide (4NQO) drinking water for 36 weeks, 61 with SCC of tongue had been found and randomly divided into 4 groups, namely Shenyang A, Shenyang B, positive and negative control group. Before and after high and low dosage of Shenyang, acanthopanax senticoside and water had been given for 15 days respectively, peripheral blood serum was collected. Secretion of Th1-type cytokines, such as IFN-gamma, IL-2, TNF-alpha and Th2-type cytokines, such as IL-4, IL-10 were detected by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was made with paired t test. RESULTS: Shenyang significantly enhanced ,the serum levels of Th1-type cytokine, TNF-alpha and IL-2 (P<0.05), and decreased the expression of Th2-type cytokine, IL-4 and IL-10(P<0.05). Serum IFN-gamma had been increased to some extent. CONCLUSION: Reversing Th2 to Th1 cytokines and then enhancing the immunity may be one of the mechanisms in anti-tumor effect of Shenyang. Supported by Science and Technology Development Fund (No.014319359) of Shanghai Municipality.

[Modulation of Chinese regimen granules of Shenyang on peripheral blood lymphocyte subsets of SD rats with SCC of tongue].

OBJECTIVE: Prospective research demonstrated that Chinese regimen granules of Shenyang could prolong survival time and improve survival rate of patients with oral squamous cell carcinoma (SCC). But the mechanism was not clear. The purpose of this study was to investigate Shenyang's effect on peripheral blood lymphocyte subsets of SD rats with SCC of tongue and explore immunological mechanism. METHODS: Among 80 SD rats fed by 0.002% 4-nitroquinoline-1-oxide (4NQO) drinking water for 36 weeks, 61 rats with SCC of tongue had been found and were randomly divided into 4 groups, namely Shenyang A, Shenyang B, positive and blank control groups. Before and after high and normal dosage of Shenyang, acanthopanax senticoside and water had been given for 15 days respectively, peripheral blood lymphocyte subsets were detected with flow cytometry. The data were statistically analyzed with paired t Test. RESULTS: Percentage of CD3+ CD4+ T cell and CD3-CD161a+ NK cell, ratio of CD4+/CD8+ were increased. Percentage of CD3+CD8+ T cell was decreased, and the effect was better than that of acanthopanax senticoside in improving the percentage of CD3-CD161a+ NK cell. CONCLUSION: Among anti-tumor mechanisms of Shenyang it is that corrects disorder of lymphocyte subsets and increases percentage of CD3-CD161a+ NK cell.