Activation of mitochondrial-associated apoptosis signaling pathway and inhibition of PI3K/Akt/mTOR signaling pathway by voacamine suppress breast cancer progression.

BACKGROUND: Breast cancer is one of the malignant tumors with the highest morbidity and mortality rate. Numerous efficient anti-breast cancer drugs are being derived from the development of natural products. Voacamine (VOA), a bisindole alkaloid isolated from Voacanga africana Stapf, possesses various pharmacological and biological activities. PURPOSE: In this study, we investigated the efficacy of VOA against breast cancer cells and elucidated the underlying mechanisms in vitro and in vivo. METHODS: Human breast cancer cell line MCF-7 and mouse breast cancer cell line 4T1 were used to study the underlying anti-cancer mechanisms of VOA. The proliferation was detected by MTT, colony formation, cell proliferation and wound-healing migration assays. Flow cytometry was utilized to determine the level of reactive oxygen species (ROS) cell-cycle, apoptosis and mitochondrial membrane potential. The target proteins were analyzed by Western blot. Molecular docking was performed and scored by AutoDock. Subcutaneous cancer models in mice were established to evaluate the anticancer effects in vivo. RESULT: Our results demonstrated that VOA selectively suppressed breast cancer MCF-7 and 4T1 cells proliferation with IC50 values of 0.99 and 1.48 µM, and significantly inhibited the migration and colony formation of tumor cells. Furthermore, the cell cycle was arrested in the S phase with the decreased expression levels of CDK2, Cyclin A and Cyclin E. Additionally, exposure to VOA dose-dependently brought about dose-dependently the loss of mitochondrial membrane potential (Δψm) and amassment of reactive oxygen species (ROS), resulting in the initiation of the intrinsic apoptotic pathway. Western blot analysis unveiled that VOA significantly activated mitochondrial-associated apoptosis and obviously suppress the PI3K/Akt/mTOR pathway via modulation of related protein expression levels in both tumor cell lines. In tumor-bearing mouse models, administration of VOA dose-dependently inhibited the tumor growth without causing apparent toxicities. CONCLUSION: These findings revealed the novel properties of VOA in promoting apoptosis of breast cancer cells by activating mitochondrial-associated apoptosis signaling pathway and inhibiting PI3K/Akt/mTOR signaling pathway and significantly decreasing tumor size without detecting appreciable toxicity. In summary, the present results demonstrated VOA could be an encouraging drug candidate to cure breast cancer, exhibiting an effective method to exploit unique drugs from natural components.

Fish Oil Is More Potent than Flaxseed Oil in Modulating Gut Microbiota and Reducing Trimethylamine-N-oxide-Exacerbated Atherogenesis.

Trimethylamine-N-oxide (TMAO) is a risk factor for atherosclerosis. We compared the potency of fish oil with flaxseed oil in reducing TMAO-exacerbated atherogenesis. Five groups of ApoE-/- mice were given one of five diets, namely, a low-fat diet, a Western high fat diet (WD), a WD plus 0.2% TMAO, and two WDs containing 0.2% TMAO with 50% lard being replaced by flaxseed oil or fish oil. TMAO accelerated atherosclerosis and disturbed cholesterol homeostasis. Compared with flaxseed oil, fish oil was more effective in inhibiting TMAO-induced atherogenesis by lowering plasma cholesterol and inflammatory cytokines. Both oils could reverse TMAO-induced decrease in fecal acidic sterols. Fish oil promoted fecal output of neutral sterols and downregulated hepatic cholesterol biosynthesis. Fish oil was more effective than flaxseed oil in promoting the growth of short-chain fatty acid-producing bacteria and lowering microbial generation of lipopolysaccharide. In conclusion, fish oil is more potent than flaxseed oil to ameliorate TMAO-exacerbated atherogenesis.

Comprehensive Quantitative Analysis of 32 Chemical Ingredients of a Chinese Patented Drug Sanhuang Tablet.

Sanhuang Tablet (SHT) is a Chinese patented drug commonly used for the treatment of inflammations of the respiratory tract, gastrointestinal tract, and skin. It contains a special medicinal composition including the single compound berberine hydrochloride, extracts of Scutellariae Radix and Rhei Radix et Rhizoma, as well as the powder of Rhei Radix et Rhizoma. Despite advances in analytical techniques, quantitative evaluation of a Chinese patented drug like SHT remains a challenge due to the complexity of its chemical profile. In this study, ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to simultaneously quantify 29 non-sugar small molecule components of SHT (11 flavonoids, two isoflavonoids, one flavanone, five anthraquinones, two dianthranones, five alkaloids, two organic acids and one stilbene). Three major saccharide components, namely fructose, glucose, and sucrose, were also quantitatively determined using high performance liquid chromatography-charged aerosol detector (HPLC-CAD) on an Asahipak NH2P-50 4E amino column. The established methods were validated in terms of linearity, sensitivity, precision, accuracy, and stability, and then successfully applied to analyze 27 batches of commercial SHT products. A total of up to 57.61% (w/w) of SHT could be quantified, in which the contents of the determined non-saccharide small molecules varied from 5.91% to 16.83% (w/w) and three saccharides accounted for 4.41% to 48.05% (w/w). The results showed that the quality of the commercial products was inconsistent, and only four of those met Chinese Pharmacopoeia criteria.

Comprehensive quantitative analysis of Chinese patent drug YinHuang drop pill by ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry.

YinHuang drop pill (YHDP) is a new preparation, derived from the traditional YinHuang (YH) decoction. Since drop pills are one of the newly developed forms of Chinese patent drugs, not much research has been done regarding the quality and efficacy. This study aims to establish a comprehensive quantitative analysis of the chemical profile of YHDP. ultra high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was used to identify 34 non-sugar small molecules including 15 flavonoids, 9 phenolic acids, 5 saponins, 1 iridoid, and 4 iridoid glycosides in YHDP samples, and 26 of them were quantitatively determined. Sugar composition of YHDP in terms of fructose, glucose and sucrose was examined via a high performance liquid chromatography-evaporative light scattering detector on an amide column (HPLC-NH2P-ELSD). Macromolecules were examined by high performance gel permeation chromatography coupled with ELSD (HPGPC-ELSD). The content of the drop pill's skeleton component PEG-4000 was also quantified via ultra-high performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD). The results showed that up to 73% (w/w) of YHDP could be quantitatively determined. Small molecules accounted for approximately 5%, PEG-4000 represented 68%, while no sugars or macromolecules were found. Furthermore, YHDP showed no significant differences in terms of daily dosage, compared to YinHuang granules and YinHuang oral liquid; however, it has a higher small molecules content compared to YinHuang lozenge.

[Chemical variation in Aurantii Fructus before and after processing based on UHPLC-Q-TOF-MS].

To explore the processing mechanism of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong by analysing the chemical variation between raw and processed Aurantii Fructus with different methods based on UHPLC-Q-TOF-MS. The total ion chromatograms detected in positive and negative ion modes, and ion peak area ratio before and after processing were taken as variation indexes in the comparison. The results indicated that fermented Aurantii Fructus could produce three new ingredients, namely eriodictyol-7-glucoside, hesperetin-7-O-glucoside and 5-demethylnobiletin. At the same time, it could significantly increase the content of naringenin and hesperetin components, and could increase the content of such limonin derivatives as sudachinoid A, obacunoic acid and limoninand nomilinic acid. This suggests that the fermentation processing method of Aurantii Fructus decoction pieces used in Guangdong province and Hong Kong is of important significance for enhancing biological activity and bioavailability, and improving the clinical efficacy of Aurantii Fructus decoction pieces, and so is worth further protection and promotion.

Simultaneous determination of ten compounds in rat plasma by UPLC-MS/MS: Application in the pharmacokinetic study of Ma-Zi-Ren-Wan.

Ma-Zi-Ren-Wan (MZRW) is a classic Chinese formula which has been used to treat human constipation in China for over 2000 years. In order to make good and rational use of this formula in the future, this paper presents the first attempt to track the pharmacokinetic features of MZRW in rat using rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Ten chemical components of MZRW, namely, rhein, emodin, aloe emodin, hesperidin, naringin, amygdalin, albiflorin, paeoniflorin, magnolol and honokiol, were simultaneously determined in rat plasma after a single oral administration (10g/kg body weight) of MZRW to rats. Geniposide and liquiritin were used as internal standards. The separation was performed on a Waters ACQUITY BEH C18 column (100mm×2.1mm, 1.7µm). The detection was conducted by multiple-reaction monitoring (MRM) in negative ionization mode. Two highest abundant MRM transitions without interference were optimized for each analyte. This method was well validated in terms of linearity, precision, accuracy, recovery, matrix effect and stability. All calibration curves had good linearity (r(2)>0.995) over the concentration range from 3.9 to 125.0ng/mL for emodin, 3.9-500.0ng/mL for amygdalin, 2.0-4000.0ng/mL for naringin and hesperidin, 3.9-2000.0ng/mL for magnolol, 7.8-2000.0ng/mL for rhein and 3.9-4000.0ng/mL for albiflorin, paeoniflorin, aloe emodin and honokiol. The intra-day and inter-day precision (relative standard deviation) was within 15%, the accuracy (relative error) ranged from -13.6% to 15.1%, and the lower limit of quantification in plasma ranged between 2.0ng/mL and 7.8ng/mL. Extraction recovery, matrix effect and stability were satisfactory. The validated method was successfully applied to a pharmacokinetic study of these ten compounds after oral administration of MZRW to rats. The pharmacokinetic parameters of each compound can facilitate clinical studies in the future.

Comprehensive quantitative analysis of Shuang-Huang-Lian oral liquid using UHPLC-Q-TOF-MS and HPLC-ELSD.

Shuang-Huang-Lian oral liquid (SHL) is a well-known Chinese patent drug containing three herbal medicines: Radix Scutellariae, Flos Lonicerae Japonicae and Fructus Forsythiae. It is usually used to treat acute upper respiratory tract infection caused by virus or bacteria. Although the licensing of botanical drug Veregen approved by FDA has indicated the importance of quantitative analysis in quality control of herbal medicines, quantitative evaluation of a Chinese patent drug like SHL remains a challenge due to the complex chemical profile. In this study, 15 small molecular components of SHL (four flavonoids, six quinic acid derivatives, three saponins and two phenylethanoid glycosides) were simultaneously determined using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The contents of the three major saccharides, namely fructose, glucose and sucrose were quantified using high performance liquid chromatography-evaporative light scattering detector on an amino column (HPLC-ELSD). The macromolecules were quantified by precipitating in 80% ethanol, drying the precipitate, and then weighing. The established methods were validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to analyze 12 batches of commercial products of SHL produced by four different manufacturers. The results indicated that 57.52-78.11% (w/w) of SHL could be quantitatively determined (non-saccharide small molecules: 1.77-3.75%, monosaccharides: 0.93-20.93%, macromolecules: 2.63-5.76% and sucrose: 49.20-65.94%). This study may provide a useful way to comprehensively evaluate the quality of SHL.

Histochemical evaluation of alkaloids in rhizome of Coptis chinensis using laser microdissection and liquid chromatography/mass spectrometry.

Traditional macroscopic and microscopic identification methods of medicinal materials are economical and practical, but usually experience-based due to few chemical supports. Here histochemical evaluation on bioactive components of Coptidis Rhizoma (CR) in anatomic sections using laser microdissection and liquid chromatography-mass spectrometry (LMD-LC-MS) was developed to correlate the inner quality and outer features of materials from different growing areas. Results of a total 33 peaks representing potential different alkaloids were detected and 8 common peaks were identified as the major alkaloids, namely magnoflorine, thalifendine, columbamine, epiberberine, jatrorrhizine, coptisine, palmatine, and berberine. Six major alkaloids were quantified in the top and middle sections of raw materials and in their tissues and cells at the same time. Histochemical analyses showed consistent results with direct determination in raw materials and explained the reason why top sections of all samples contained higher contents of alkaloids by giving out attributions of each alkaloid in different anatomic sections. Besides, results manifested the distribution and accumulation rules of alkaloids in diverse tissues and cells of CR. This study demonstrates an effective and scientific way to correlate bioactive components and morphological features of medicinal materials, which is beneficial to future research, agriculture and application.

A novel and rapid HPGPC-based strategy for quality control of saccharide-dominant herbal materials: Dendrobium officinale, a case study.

Qualitative and quantitative characterization of natural saccharides, especially polysaccharides, in herb materials remains a challenge due to their complicated structures and high macromolecular masses. Currently available methods involve time-consuming and complicated operations, and present poor specificity. Here, a novel and rapid high-performance gel permeation chromatography (HPGPC)-based approach is described for quality assessment of saccharide-dominant herbal materials by simultaneous qualitative and quantitative analysis of saccharide components. Dendrobium officinale, one of the rarest tonic herbs worldwide, was employed as the model herb in this study. First, a HPGPC fingerprint based on the molecular weight distribution of its carbohydrate components was established for qualitative identification of D. officinale. Then, HPGPC-guided dominant holistic polysaccharide marker was separated using ultra-filtration followed by HPGPC determination for quantitative evaluation of D. officinale. The experimental results suggest that this method is more efficient, stable, and convenient compared with the currently available methods for authentication and quality evaluation of D. officinale, and we expect the method will have similar advantages when used for quality control of other saccharide-dominant herbal materials and products.

Transformation of ginsenosides from notoginseng by artificial gastric juice can increase cytotoxicity toward cancer cells.

Multicomponent metabolic profile of notoginseng saponins in artificial gastric juice was qualitatively and quantitatively investigated, showing that ginsenosides were transformed via multiple pathways including deglycosylation, dehydration, hydration, and oxygenation. A total of 83 metabolites was identified by using UPLC-Q-TOF-MS, among which 16 new dammarane glycosides were further characterized by comparing with synthesized authentic compounds. Transformation time-course of notoginseng saponins in artificial gastric juice was quantitatively measured for the first time, showing rapid degradation of primary ginsenosides and concomitant formation of deglycosylation, hydration, and dehydration products. It was further demonstrated that the resultant metabolites exhibited enhanced cytotoxicity toward cancer cells. The extensive metabolism of ginsenosides within a transit time span in stomach, together with the formation of metabolites with diversified chemical structures possessing enhanced biological activities, indicated an important role of transformation in gastric juice in the systematic effects of ginsenosides.

Quality evaluation of commercial Huang-Lian-Jie-Du-Tang based on simultaneous determination of fourteen major chemical constituents using high performance liquid chromatography.

Huang-Lian-Jie-Du-Tang (HLJDT), comprising Coptidis Rhizoma, Scutellariae Radix, Phellodendri Cortex and Gardeniae Fructus, is one of the commonly used Chinese medicine formulas for clearing heat and detoxifying. Quality control of the herbal complex like Chinese medicine formulas still remains a challenge. The successful approval of botanical drug Veregen by FDA indicated the importance of quantitative analysis in quality control of herbal medicines. In this study, an effective quantitative method based on conventional HPLC-DAD was developed for simultaneous determination of fourteen major ingredients (seven alkaloids, four flavonoids, three terpenes) in HLJDT. The established method was well validated in terms of linearity, sensitivity, precision, accuracy and stability and then successfully applied to quality evaluation of commercial HLJDT samples. The developed method can quantitatively determine up to 70% of the chemicals of commercial HLJDT sample and effectively revealed the significant variation in the quality of the commercial HLJDT samples collected from different locations.

Paeoniflorin reduced acute toxicity of aconitine in rats is associated with the pharmacokinetic alteration of aconitine.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the influence of paeoniflorin (major bioactive component of Paeonia lactiflora Pall.) on the pharmacokinetic behavior of aconitine (major toxic and bioactive component of Aconitum carmichaeli Debx.) and potential detoxifying effect of paeoniflorin on the acute toxicity of aconitine, which may provide in depth understanding to the toxicity reduction effect of Paeonia lactiflora to Aconitum carmichaeli. MATERIALS AND METHODS: Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was employed to determine the plasma content of aconitine. Aconitine was administrated by oral to SD rats at the dosage of 200 µg/kg with or without paeoniflorin given by intraperitoneal injection at the dosage of 20 mg/kg. Plasma samples were collected for determination and analysis of pharmacokinetic parameters of aconitine. The LD(50) of aconitine and acute animal death induced by aconitine were examined when aconitine was given alone or jointly with paeoniflorin in ICR mice. RESULTS: A sensitive, accurate, precise, reliable and repeatable UHPLC-MS/MS method was successfully established for determination of the plasma content of aconitine in 12.5 µL plasma sample. The lower limit of quantification of aconitine was 0.01 ng/mL. Compared with the SD rats that were orally administrated with aconitine alone, the rats received aconitine and co-administrated with paeoniflorin by peritoneal injection showed a remarkably lower C(max) (5.69 ng/mL vs 9.66 ng/mL, P<0.05) and delayed T(max) (70 min vs 46 min, P<0.05), as well as a trend of reduction in AUC(0-t) (1082.75 ng-min/mL vs 1650.27 ng-min/mL, P=0.395). The LD(50) values of aconitine coadministered with 120 or 240 mg/kg of paeoniflorin were obviously increased to 2.30 and 2.15 mg/kg against 1.80 mg/kg of aconitine by oral administration alone. Mice treated with paeoniflorin (240 mg/kg) and aconitine (1.8 mg/kg) together revealed a significant decreased death rate than that received aconitine treatment alone (15% vs 50%, P<0.05). CONCLUSIONS: The acute oral toxicity of aconitine in rats was significantly reduced by paeoniflorin; this might result from the alterations of pharmacokinetic behavior of aconitine in the animals by coadministration of paeoniflorin.

Quantitative comparison of ginsenosides and polyacetylenes in wild and cultivated American ginseng.

Quantitative comparison of seven ginsenosides in wild and cultivated American ginseng revealed that the Rg(1)/Rd ratio presented a significantly large difference between cultivated and type-I (one of the defined chemotypes) wild American ginseng, facilitating this ratio as a characteristic marker for differentiating these two groups. Similarly, the ratio (Rg(1)+Re)/Rd, and the ratio of protopanaxatriol (PPT)-type ginsenosides to protopanaxadiol (PPD)-type ginsenosides showed a large difference between these two groups. On the other hand, type-II wild samples were found to have high Rg(1)/Rb(1) and Rg(1)/Re ratios and low panaxydol/panaxynol ratio, which is entirely different from Type-I American ginseng, but is very similar to that of Asian ginseng. This not only suggests that the chemotype should be taken into consideration properly when using these parameters for differentiating American and Asian ginseng, but also indicates that type-II wild American ginseng may have distinct pharmacological activities and therapeutic effects.

Soy leaf lowers the ratio of non-HDL to HDL cholesterol in hamsters.

The present study was to examine effect of soy leaf powder (SLP) and soy leaf ethanol extract (SLEE) on serum lipoproteins in hamsters. The control group was fed a semisynthetic diet containing 0.1% cholesterol, while the tested groups were maintained on the same diet but supplemented with 3% SLP or the equivalent amount of SLEE derived from 3% SLP for 4 weeks. SLP supplementation led to a trend of lowering serum total cholesterol (TC) and nonhigh density lipoprotein cholesterol (non-HDL-C), with HDL-C being unaffected, whereas incorporation of SLEE into the diet led to an elevated level of HDL-C and a lower level of non-HDL-C with TC being unchanged. Both SLP and SLEE supplementation caused favorably a decrease in the ratio of non-HDL-C to HDL-C. The present results demonstrate that not only soybean seeds but also soy leaves are cardioprotective, by favorably modulating serum lipid profile.

Difference in flavonoid and isoflavone profile between soybean and soy leaf.

The present study was to compare the flavonoid profile between soybean and soy leaves. Soybean was most abundant in malonyl-genistin followed by malonyl-daidzin, genistin, daidzin, genistein and daidzein in a decreasing order. In contrast, soy leaves contained only trace amounts of malonyl-genistin and genistin, but they had the six unknown flavonoids that were absent in soybean. The six unknown compounds were isolated by using various chromatographic techniques and the structures were identified by studying their varying spectra of ultraviolet (UV), infrared (IR), Mass, 1H-NMR and 13C-NMR. It was found that the six unknown compounds were all kaempferol glycosides namely kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-(2,6-di-O-alpha-rhamnopyranosyl)-beta-galactopyranoside, kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-digalactopyranoside, kaempferol-3-O-diglucopyranoside and kaempferol-3-O-rutinoside. It was concluded that the flavonoids in soy leaves were mainly kaempferol glycosides, whereas those in soybean were mainly isoflavone glycosides and derivatives.

Vascular effects of a soy leaves (Glycine max) extract and kaempferol glycosides in isolated rat carotid arteries.

We have recently purified genistin, and six kaempferol glycosides from a soy leaves ( Glycine max L. Merr.) butanol extract. Here we report the vascular effects of the extract and purified genistin and kaempferol glycosides on contractions induced by different constricting agonists in isolated rat carotid arteries. The butanol extract relaxed artery rings preconstricted by 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F 2 alpha (U46619) or [5 Z,9alpha,11alpha,13 E,15 S]-9,11,15-trihydroxyprosta-5,13-dienoic acid (PGF 2 alpha ) in a dose-dependent manner and this effect was independent of the presence of endothelium. The extract also inhibited the concentration-dependent contraction to U46619 with a slight reduction of the maximal response. The extract produced partial relaxation of both phenylephrine-preconstricted endothelium-intact and -denuded rings. In contrast, the extract had no effect on the contractile response to 50 mM extracellular K (+). None of the six kaempferol glycosides affected vessel tension induced by U46619. A mixture of kaempferol glycosides prepared according to their relative composition in the extract had no effect either. However, kaempferol relaxed U46619- and high K (+)-contracted rings to the same extent. Endothelium played no role in kaempferol-induced relaxation. Genistein induced concentration-dependent relaxation and this effect was attenuated in the endothelium-denuded rings. Genistin caused a smaller relaxant effect. The present results indicate that a butanol extract from soy leaves causes endothelium-independent relaxation in rat carotid artery rings. Kaempferol glycosides, accounting for approximately 48 % of the extract in weight, are not the ingredients responsible for the extract-induced relaxation. Genistein and genistin also caused relaxation, however, the dose range is beyond that of the extract causing relaxation.