A novel gastroretentive porous microparticle for anti-Helicobacter pylori therapy: Preparation, in vitro and in vivo evaluation

Gastroretentive drug delivery system is a promising option for the treatment of Helicobacter pylori infection, which can prolong gastric residence time and supply high drug concentration in the stomach. In the present study, a low density system of metronidazole-loaded porous Eudragit^® RS microparticle with high drug loading capacity (>25%) was fabricated via electrospray method. The porous structure and size distribution of microparticles were affected by polymer concentration and flow rate of solution. FTIR and XRD analyses indicated that drug has been entrapped into the porous microparticles. In addition, sustained release profiles and slight cytotoxicity in vitro were detected. Gamma scintigraphy study in vivo demonstrated that ¹³¹I-labeled microparticles retained in stomach for over 8 h, and about 65.50% radioactive counts were finally detected in the region of interest. The biodistribution study confirmed that hotspot of radioactivity was remaining in the stomach. Furthermore, metronidazole-loaded porous microparticles can eradicate H. pylori completely with lower dose and administration frequency of antibiotic compared with pure drug, which were also more helpful for the healing of mucosal damages. These results suggest that prepared porous microparticle has the potential to provide better treatment for H. pylori infection.     

Cellulose acetate phthalate microparticles containing Vibrio cholerae: steps toward an oral cholera vaccine

Abstract

Oral cholera vaccine (OCV) has been recommended in some endemic areas and epidemic situations since 1999. Although safe and effective vaccines are currently on the market, the burden of transport and storage remains an issue. Herein, we report an approach to develop an alternative OCV in the form of a gastro-resistant powder. Heat-killed Vibrio cholerae (VC) was encapsulated with a spray-drying technique at different temperatures. Cellulose acetate phthalate (Aquacoat® CPD) was chosen as the core polymer and the addition of alginate was studied. The microparticles (MPs) produced were characterized by surface morphology, particle size, drug loading, antigenicity and gastro resistance. The MPs obtained were 6?µm in size and had appropriate drug content, ranging from 8.16 to 8.64%. Furthermore, antigenicity was maintained, never dropping below 85%, and enteric properties were achieved for all the formulations. Next, an in vivo study was carried out with Aquacoat® CPD MP prepared at 80?°C with and without alginate. Two different doses were assayed, 30 and 60?mg, and compared to the VC suspension. The evoked immune responses showed that alginate containing MPs, especially at the 30?mg dose, displayed values that were very similar to those of VC. In conclusion, spray-dried alginate VC MPs seem to be a promising step toward a powder-form cholera vaccination.     

Dexamethasone-containing biodegradable superparamagnetic microparticles for intra-articular administration: Physicochemical and magnetic properties, in vitro and in vivo drug release

Compared with traditional drug solutions or suspensions, polymeric microparticles represent a valuable means to achieve controlled and prolonged drug delivery into joints, but still suffer from the drawback of limited retention duration in the articular cavity. In this study, our aim was to prepare and characterize magnetic biodegradable microparticles containing dexamethasone acetate (DXM) for intra-articular administration. The superparamagnetic properties, which result from the encapsulation of superparamagnetic iron oxide nanoparticles (SPIONs), allow for microparticle retention with an external magnetic field, thus possibly reducing their clearance from the joint. Two molecular weights of poly(lactic-co-glycolic acid) (PLGA) were used, 12 and 19 kDa. The prepared batches were similar in size (around 10 μm), inner morphology, surface morphology, charge (neutral) and superparamagnetic behaviour. The SPION distribution in the microparticles assessed by TEM indicates a homogeneous distribution and the absence of aggregation, an important factor for preserving superparamagnetic properties. DXM release profiles were shown to be quite similar in vitro (ca. 6 days) and in vivo, using a mouse dorsal air pouch model (ca. 5 days).     

Composite microparticles with in vivo reduction of the burst release effect

The aim of this study was to develop microparticles containing nanoparticles (composite microparticles) for prolonged drug delivery with reduced burst effect in vitro and in vivo. Such composite microparticles were prepared with hydrophobic and biodegradable polymers [poly(ε-caprolactone), poly(lactic-co-glycolic) acid]. Ibuprofen was chosen as the model drug, and microparticles were prepared by the extraction technique with ethyl acetate as the solvent. Nanoparticles and microparticles and an ibuprofen solution (Pedea^®) were administered subcutaneously at the dose of 1 mg of ibuprofen per kg to overnight-fasted rats (male Wistar). Composite microparticles showed prolonged ibuprofen release and less burst effect when compared to simple microparticles (without nanoparticles inside) or nanoparticles both in vitro (PBS buffer) and in vivo. Moreover, ibuprofen was still detected in the plasma after 96 h with composite microparticles. Consequently, it has been demonstrated that composite microparticles were able to reduce burst release and prolong the release of ibuprofen for a long period of time.     

Inhibition of surface crystallisation of amorphous indomethacin particles in physical drug–polymer mixtures

Surface coverage may affect the crystallisation behaviour of amorphous materials. This study investigates crystallisation inhibition in powder mixtures of amorphous drug and pharmaceutical excipients. Pure amorphous indomethacin (IMC) powder and physical mixtures thereof with Eudragit^® E or Soluplus^® in 3:1, 1:1 and 1:3 (w/w) ratios were stored at 30 °C and 23 or 42% RH. Samples were analysed during storage by X-ray powder diffraction, thermogravimetric analysis, differential scanning calorimetry, and scanning electron microscopy (SEM). IMC Eudragit^® mixtures showed higher physical stability than pure IMC whereas IMC Soluplus^® mixtures did not.     

The effect of polymer properties on direct compression and drug release from water-insoluble controlled release matrix tablets

The objective of this study was to identify and evaluate key polymer properties affecting direct compression and drug release from water-insoluble matrices. Commonly used polymers, such as Kollidon^® SR, Eudragit^® RS and ethyl cellulose, were characterized, formulated into tablets and compared with regard to their properties in dry and wet state. A similar site percolation threshold of 65% v/v was found for all polymers in dry state. Key parameters influencing polymer compactibility were the surface properties and the glass transition temperature (T_g), affecting polymer elasticity and particle size-dependent binding. The important properties observed in dry state also governed matrix characteristics and therefore drug release in wet state. A low T_g (Kollidon^® SR < Eudragit^® RS) decreased the percolation threshold, particle size effect and tortuosity, but increased permeability and sensitivity to heat/humidity treatment. Hence, lower permeability and higher stability are benefits of a high-T_g polymer (ethyl cellulose). However, release retardation was observed in the same order as matrix integrity (Eudragit^® RS < ethyl cellulose < Kollidon^® SR), as the high permeability was counteracted by PVP in case of Kollidon^® SR. Therefore, the T_g and composition of a polymer need to be considered in polymer design and formulation of controlled-release matrix systems.     

Studies on the in vitro genotoxicity of baytubes,agglomerates of engineered multi-walled carbon-nanotubes (MWCNT)

The increasing production and expanding application of nanoparticles in multiple aspects of life necessitate reliable safety assessment. In this context we here report on the evaluation of the potential genotoxicity of baytubes^®, i.e. agglomerates of multi-walled carbon-nanotubes (MWCNT). Testing for chromosome aberrations was done in V79 cells and for gene mutations in the Salmonella microsome test. Baytubes^® were formulated in deionised water at 10 mg/ml and treated with ultrasound for 30 min at 25 °C. Particle size distribution was determined under the incubation conditions in the in vitro studies.     

Controlled non-invasive transdermal iontophoretic delivery of zolmitriptan hydrochloride in vitro and in vivo

The objective was to investigate the transdermal delivery kinetics of zolmitriptan from an iontophoretic patch system in Yorkshire swine in vivo. Preliminary in vitro experiments showed that cumulative drug transport during a 6-h current application (0.25 mA cm⁻²) was independent of patch load (263.7 ± 92.7, 357.2 ± 85.9, 374.9 ± 74.3 and 335.9 ± 27.7 μg cm⁻² for 7.5, 15, 45 and 90 mg patch loads, respectively; ANOVA, p < 0.05); the steady-state flux was ∼92 μg cm⁻² h⁻¹. The in vivo studies used multistep current profiles to demonstrate (i) rapid drug uptake and (ii) the effect of superposing a bolus input on basal drug levels. In both studies, zolmitriptan was detected in the blood after 2.5 min; drug levels were 7.1  1.7 and 10.4 ± 3.5 ng ml⁻¹ at t = 30min in Studies 1 and 2, respectively. In Study 2, increasing current intensity from 0.2 to 1.4 mA (0.05-0.35 mA cm⁻²) at t = 180 min caused zolmitriptan levels to rise from 9.38 ± 0.93 ng ml⁻¹ at t = 180 min to 13.57 ± 1.85 ng ml⁻¹ at t = 190 min; a ∼50% increase in 10 min. Extrapolation of these results to humans suggests the feasibility of delivering therapeutic amounts of zolmitriptan at faster rates than those from existing dosage forms.     

Novel microparticle drug delivery systems based on chitosan and Eudragit®RSPM to enhance diltiazem hydrochloride release property

The aim of this study was to enhance the release properties of diltiazem hydrochloride (diltiazem HCl) by using microparticle system. For this reason, microparticle drug delivery systems based on chitosan and Eudragit^®RSPM were developed. The microparticles were prepared by using double-emulsion solvent extraction method and the mean sizes of microparticles were less than 120?µm. The in vitro drug release from microparticles was studied in simulated gastric (pH 1.2) and intestinal media (pH 7.4) than the results were evaluated by kinetically. In vitro diltiazem HCl release from microparticles showed good zero order kinetic. For the microparticles with chitosan, the release of diltiazem HCl at pH 1.2 could be effectively sustained, while the release of diltiazem HCl increased at pH 7.4 when compared to Eudragit^®RSPM microparticles. The highest release percent obtained was 1:1 ratio of drug: polymer at pH 1.2 and 7.4. All results clearly suggest that the release properties of diltiazem HCl were improved by using microparticle systems especially which contain chitosan.     

Literature review on the safety of Toyocerin,a non-toxigenic and non-pathogenic Bacillus cereus var. toyoi preparation

Bacillus cereus var. toyoi is a naturally occurring, non-toxigenic and non-pathogenic strain of B. cereus. Safety studies were conducted on a B. toyoi preparation (Toyocerin^®), including but not limited to enterotoxicity, eye irritation, genotoxicity, acute, subchronic and chronic toxicity studies and human clinical trials. In rabbits, Toyocerin^® did not exhibit enterotoxicity and was only slightly irritating to the eyes. It was non-mutagenic in an Ames assay at up to 10,000 μg/plate and did not exhibit clastogenic activity in a chromosomal aberration test at up to 450 mg/ml. It was non-toxic in acute and repeated-dose (30 and 60 days and 1 year) toxicity studies in rats and mice at up to 3 × 10¹¹ spores/kg bw/day. In an eight-day human clinical trial, Toyocerin^® did not cause any adverse effects in healthy male and female subjects at 1 × 10⁹ and 1 × 10¹⁰ spores/kg bw/day. In feeding trials, Toyocerin^® did not cause any adverse effects in rabbits, pigs, chickens, turkeys and cattle at doses ranging from 8.5 × 10⁷ to 4 × 10⁹ spores/kg bw/day for durations of 2 weeks to 18 months. Taken together, these studies demonstrate that Toyocerin^® is safe at the doses tested.     

Eudragit? Microparticles for the Release of Budesonide: A Comparative Study

This study compares the behaviour of budesonide-containing microparticles made of Eudragit^®RS or Eudragit^®RS/Eudragit^®RL 70:30 (w/w) prepared either by solvent evaporation or spray-drying technique. The loading efficiency of budesonide within microparticles was about 72% for microparticles prepared by solvent evaporation and around 78% for spray-dried microparticles. Thermal analyses were assessed to collect information about the structural stability of budesonide within the polymeric microspheres. The in vitro release was performed using simulating gastric (fasted state simulated gastric fluid) and intestinal (fasted state simulated intestinal fluid) fluids as the receiving solutions. After 3 h the drug release from Eudragit^®RS/Eudragit^®RL microparticles was about 6-fold higher than that obtained in the case of monopolymer microparticles. Using fasted state simulated intestinal fluid the drug was released between 4 and 30% in both types of preparations. Eudragit^®RS microparticles showed a better protection of the drug from gastric acidity than those of Eudragit^®RS/Eudragit^®RL allowing us to propose Eudragit^®RS microparticles as a hypothetical system of colon specific controlled delivery.     

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0 = control, 5 × 10⁻⁸, 10⁻⁷, 2 × 10⁻⁷ or 5 × 10⁻⁷ M) or diisopropylphosphorofluoridate (DFP, 0 = control, 5 × 10⁻⁶, 10⁻⁵, 2 × 10⁻⁵, and 5 × 10⁻⁵ M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 μM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 μM concentrations. No fullerene derivative at 1 μM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

Estrogenic/antiestrogenic activities of a Epimedium koreanum extract and its major components: in vitro and in vivo studies

The estrogenic and antiestrogenic activities of Epimedii Herba, which is a traditional medicinal herb used in Korea and China were investigated in this study. The in vitro estrogen receptor (ER) mediated estrogenic/antiestrogenic activities of an Epimedii Herba extract (Epi ext) and its major components were determined using an estrogen responsive element driven reporter gene assay in MCF-7/ERE and HEK293T cells. The Epi ext exhibited ERα- and ERβ-mediated estrogenic activity with an EC₅₀ of 5.0 and 17.8 μM in HEK293T cells, respectively. Prenylflavonoid glycosides such as icariin (ICA), epimedin A, B, and C did not show any in vitro estrogenic or antiestrogenic activities. Icaritin (ICT) and quercetin exhibited in vitro ER mediated estrogenic activity with a more potent interaction with ERβ. In vivo estrogenic activities of the Epi ext, ICA and ICT were compared using an uterotrophic assay. Although the potency of in vitro estrogenic activity was in the order of ICT > Epi ext > ICA, ICA had the strongest estrogenic activity and next ICT in ovariectomized rats. These results collectively suggest that phytoestrogens possess both estrogenic and antiestrogenic activity, and that the differential expression of these two compounds with opposing activities is dependent on the physiological environment in terms of estrogen level, which may be the case in humans.     

In vitro evaluation of the permeation through reconstructed human epidermis of essentials oils from cosmetic formulations

The permeation of essential oils through SkinEthic^® reconstructed human epidermis, (RHE), was studied in vitro to establish a convenient tool to monitor the kinetics of release of active principles from cosmetic formulations. Twelve days old human epidermis held on polycarbonate disks was revitalized by addition of growth medium and incubated at 37 °C in 5% CO₂ atmosphere for five days prior to investigation. A system of six custom designed glass Franz-type diffusion cells were used for the permeation studies at 34 °C. The diffusion kinetic for 8 selected terpenes (camphor, carvone, 1,8-cineole, linalool, menthol, α-thujone, menthone, t-anethole), chosen as analytical markers of a mixture of plant essential oils contained in a cosmetic formulation, was probed by HS/SPME–GC–MS analysis and elaborated according to Fick's first law to obtain skin permeability coefficients (P_S = 1.51, 1.47, 1.36, 0.80, 0.62, 0.40 and 0.14 × 10⁻³ cm/h, respectively). The method proved to be sensitive, simple and reproducible, and RHE represents a convenient model for safety/quality assessment of cosmetic formulations.     

Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1 μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1 μM significantly enhanced the intracellular accumulation of [³H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1 μM significantly reduced the rate of [³H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [³H]-E₂17βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15 mg/kg) with doxorubicin (1.8 mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2.     

Validation of in vitro screening models for progestagenic activities: Inter-assay comparison and correlation with in vivo activity in rabbits

The PR CALUX® cell line is a stably transfected human U2-OS cell line expressing the human PR and a luciferase reporter construct containing three progesterone-responsive elements coupled to a minimal promoter. The validity of this assay has been studied as an alternative to the McPhail assay in rabbits, an in vivo assay to detect progestins. The PR CALUX assay was characterized by its stable expression of PR protein which leads to induction of endogenous PR target genes by progestins. It was found to have a highly selective response to low levels of different progestins, as well as an insignificant response to other nuclear hormone receptor ligands. As an important step in their validation, the PR CALUX bioassay was compared with another earlier described in vitro bioassay, a Chinese Hamster Ovary (CHO) cell-based PR-CHO reporter gene assay as well as with an in vitro PR-binding (PR-BIN) assay, and the in vivo McPhail assay. This was done using 35 (with the most accurate potency determinations in all tests) and 50 (with less reliable potency determinations in some tests) compounds tested in all assays. The correlation scores between PR CALUX and PR-CHO were r² = 0.77, and 0.93, respectively; between PR CALUX and PR-BIN r² = 0.69 and 0.80. Comparison between either the PR CALUX or the PR-CHO transactivation assay and the in vivo McPhail assay revealed very good correlations of r² = 0.68 (n = 35), and 0.85 (n = 50). The transactivation assays can discriminate very potent, from potent, weak and inactive compounds rather easily. Besides testing the biological activity of pure chemicals and pharmaceuticals in vitro, the PR CALUX and PR-CHO transactivation assays proved to be relatively good predictors of in vivo progestagenic activity, allowing the use of these assays as prescreening methods or in vitro alternatives.     

The kinetics of cohesive powder de-agglomeration from three inhaler devices

Purpose

The purpose of the current investigation is to understand the kinetics of de-agglomeration (k_d) of micronised salbutamol sulphate (SS) and lactohale 300 (LH300) under varying air flow rates (30-180 l min⁻¹) from three dry powder inhaler devices (DPIs), Rotahaler^® (RH), Monodose Inhaler^® (MI) and Handihaler^® (HH).

Results

Cumulative fine particle mass vs. time profiles were obtained from the powder concentration, emitted mass and volume percent <5.4 μm, embedded in the particle size distributions of the aerosol at specific times. The rate of de-agglomeration (k_d), estimated from non-linear least squares modelling, increased with increasing air flow rates. The k_dvs. air flow rate profiles of SS and LH300 were significantly different at high air flow rates. The k_d was highest from RH and lowest from MI. Differences in k_d between the devices were related to device mode of operation while the differences between the materials were due to the powder bed structure.

Conclusion

This approach provided a methodology to measure the rate constant for cohesive powder de-agglomeration following aerosolisation from commercial devices and an initial understanding of the influence of device, air flow rate and material on these rate constants.     

Cisplatin,oxaliplatin, and carboplatin unequally inhibit in vitro mRNA translation

DNA is considered the preferential target of platinum containing cytostatics such as cisplatin, oxaliplatin, and carboplatin. Despite profound knowledge on the interaction between platinum drugs and DNA, there is little data on the interaction with mRNA and even less on the potential differences among these antineoplastic agents to inhibit protein synthesis. We therefore established an in vitro translation system using in vitro transcribed mRNA encoding green fluorescent protein (GFP) to evaluate the effects of exposure of GFP mRNA to 0–100 μM of cisplatin, oxaliplatin, or carboplatin. We additionally investigated the interaction between these drugs and mRNA through evaluation of crossing-points during quantitative real-time polymerase chain reactions. In contrast to oxaliplatin or carboplatin, 100 μM cisplatin significantly increased crossing-points by about 3 cycles (P < 0.01) and profoundly attenuated translation of GFP mRNA (P < 0.05). Oxaliplatin showed a trend to reduce GFP mRNA translation, whereas carboplatin entirely failed to influence it.     

Macranthoside B,a hederagenin saponin extracted from Lonicera macranthoides and its anti-tumor activities in vitro and in vivo

Macranthoside B (MB) is a hederagenin saponin extracted from the flower bud of Lonicera macranthoides. In this study, we defined the anticancer effect of MB both in vitro and in vivo using cell proliferation assays and xenograft tumor growth assays. Our data indicate that MB inhibits the proliferation of various kinds of cancer cells with IC₅₀ values in the range of 10–20 μM. Moreover, the volume and weight of xenograft tumors in nude mice treated with 5 mg/kg MB were decreased remarkably compared to those of the vehicle control group. Furthermore, DAPI staining and flow cytometry analysis with Annexin V/PI double staining revealed that more apoptotic cells were observed following MB treatment. In addition, degradation of PARP (poly-ADP-ribose polymerase), and activation of the caspase cascade for intrinsic pathways were observed. We also found that the expression of Bcl-2 protein decreased and the protein level of Bax increased which leading to an increase of the Bax/Bcl-2 ratio. Our results showed that MB exhibited strong anti-tumor effect and mitochondrion-mediated apoptosis induction involved in it.