Structural and antioxidative properties of royal jelly protein by partial enzymatic hydrolysis

The objective of this study was to investigate the structural and antioxidative properties of royal jelly protein(RJP)at different degrees of hydrolysis(DH)by partial enzymatic hydrolysis. RJP was hydrolyzed by alcalase for 0 min, 15 min, 1 h, 5 h and 8 h to obtain hydrolysates at DH of 5.34%, 11.65%, 15.19%, 21.38% and 23.91%, respectively. With the increased DH, the RJP hydrolysates showed elevated antioxidative activities. The molecular weight of RJP hydrolysates was significantly decreased but their primary backbone kept unchanged. Analysis of circular dichroism spectra revealed that the enzymolysis reduced the content of α-helix but increased the contents of β-sheet, β-turn and random coil. Meanwhile, the surface hydrophobicity and fluorescence intensity of RJP hydrolysates were decreased and a red shift occurred. As the enzymolysis continued, the surface morphology of RJP was gradually changed from a sheet-like structure into microparticles. Changes in antioxidative activities and structures generally followed a DH-dependent manner, however these changes became insignificant for samples at DH beyond 20%. Taking into consideration of both effectiveness and productivity, the optimum enzymatic duration was determined at 5 h.

Battle Royale Optimization-Based Resource Scheduling Scheme for Cloud Computing Environment

Cloud computing(CC)is developing as a powerful and flexible computational structure for providing ubiquitous service to users.It receives interrelated software and hardware resources in an integrated manner distinct from the classical computational environment.The variation of software and hardware resources were combined and composed as a resource pool.The software no more resided in the single hardware environment,it can be executed on the schedule of resource pools to optimize resource consumption.Optimizing energy consumption in CC environments is the question that allows utilizing several energy conservation approaches for effective resource allocation.This study introduces a Battle Royale Optimization-based Resource Scheduling Scheme for Cloud Computing Environment(BRORSS-CCE)technique.The presented BRORSS-CCE technique majorly schedules the available resources for maximum utilization and effectual makespan.In the BRORSS-CCE technique,the BRO is a population-based algorithm where all the individuals are denoted by a soldier/player who likes to go towards the optimal place and ultimate survival.The BRORSS-CCE technique can be employed to balance the load,distribute resources based on demand and assure services to all requests.The experimental validation of the BRORSS-CCE technique is tested under distinct aspects.The experimental outcomes indicated the enhancements of the BRORSS-CCE technique over other models.

Battle Royale Optimization with Fuzzy Deep Learning for Arabic Sentiment Classification

Aspect-Based Sentiment Analysis(ABSA)on Arabic corpus has become an active research topic in recent days.ABSA refers to a fine-grained Sentiment Analysis(SA)task that focuses on the extraction of the conferred aspects and the identification of respective sentiment polarity from the provided text.Most of the prevailing Arabic ABSA techniques heavily depend upon dreary feature-engineering and pre-processing tasks and utilize external sources such as lexicons.In literature,concerning the Arabic language text analysis,the authors made use of regular Machine Learning(ML)techniques that rely on a group of rare sources and tools.These sources were used for processing and analyzing the Arabic language content like lexicons.However,an important challenge in this domain is the unavailability of sufficient and reliable resources.In this background,the current study introduces a new Battle Royale Optimization with Fuzzy Deep Learning for Arabic Aspect Based Sentiment Classification(BROFDL-AASC)technique.The aim of the presented BROFDL-AASC model is to detect and classify the sentiments in the Arabic language.In the presented BROFDL-AASC model,data pre-processing is performed at first to convert the input data into a useful format.Besides,the BROFDL-AASC model includes Discriminative Fuzzy-based Restricted Boltzmann Machine(DFRBM)model for the identification and categorization of sentiments.Furthermore,the BRO algorithm is exploited for optimal fine-tuning of the hyperparameters related to the FBRBM model.This scenario establishes the novelty of current study.The performance of the proposed BROFDL-AASC model was validated and the outcomes demonstrate the supremacy of BROFDL-AASC model over other existing models.

中华蜜蜂王浆抗菌肽Acc-royalisin基因原核表达及多克隆抗体制备

以含中华蜜蜂王浆抗菌肽Acc-royalisin基因的该蜂头部cDNA文库质粒为模板,用PCR方法扩增出其前体片段,并克隆入表达载体pGEX-4T-2中,在大肠杆菌BL21中与谷胱甘肽S-转移酶(GST)融合进行表达,得到大小为36ku,占细胞总蛋白16.3%,与GST多克隆抗体有免疫反应的表达产物;用割胶回收获得的目的蛋白注射新西兰大白兔,采集血清制备多克隆抗体,该抗体与纯化的GST-Acc-royalisin融合蛋白经ELISA反应显示出较高效价,经Westernblot印迹分析获得特异性条带.这为进一步利用免疫方法检测王浆的防腐性能和Acc-royalisin生物制品质量,以及蜜蜂抗病性奠定了基础.

Differential volatile organic compounds in royal jelly associated with different nectar plants

The aim of this work was to distinguish volatile organic compound（VOC） profiles of royal jelly（RJ） from different nectar plants. Headspace solid-phase microextraction（HS-SPME） was used to extract VOCs from raw RJ harvested from 10 nectar plants in flowering seasons. Qualitative and semi-quantitative analysis of VOCs extracts were performed by gas chromatography-mass spectrometry（GC-MS）. Results showed that VOC profiles of RJ from the samples were rich in acid, ester and aldehyde compound classes, however, contents of them were differential, exemplified by the data from acetic acid, benzoic acid methyl ester, hexanoic acid and octanoic acid. As a conclusion, these four VOCs can be used for distinguishing RJ harvested in the seasons of different nectar plants.

Major royal jelly proteins accelerate onset of puberty and promote ovarian follicular development in immature female mice

The major royal jelly proteins(MRJPs)are the central constituents responsible for the specific activities of royal jelly.Here MRJPs via oral administration daily for 45 consecutive days were evaluated the effects on the reproductive parameters in immature female mice(FM).Neonatal FM were divided into four groups fed MRJPs with doses of 0,125,250 and 500 mg/kg/body weight(M125,M250 and M500).The results in M125,M250 and M500 showed that the times of estrus were accelerated by 10.7%,15.5%and 10.7%,the secondary follicles number were increased by 50.7%,78.8%and 38.6%,the Graafian follicles were increased by 600.0%and 774.0%and 150.0%,respectively.M500 induced multi-oocyte follicles.The serum estradiol levels of the three groups were increased by 47.1%,64.9%and 31.1%,the action of MRJPs raising hormone secretion level is mainly via upregulating expression of ERˇgene.Antioxidant parameters of ovarian tissue showed that the malondialdehyde levels in M125 and M250 were decreased,the superoxide dismutase activities and glutathione peroxidase activities in M125 and M250 were increased.In conclusion,MRJPs may accelerate onset of puberty and promote follicular development in FM.Our findings would facilitate better understanding of the benefit effect of MRJPs as the key ingredient in royal jelly on promoting fertility performance.

Royal-jelly-based apitherapy can attenuate damages to male reproductive parameter following nicotine administration

Background:Nicotine administration can generate severe oxidative stress and lipid peroxidation.Royal jelly,with its antioxidant properties,acts as a scavenger of reactive oxygen species.This study describes the apitherapy effects of royal jelly on testicular damage following nicotine administration.Methods:Forty-eight male BALB/c mice were divided into 8 groups(n=6):saline,3 different doses of royal jelly(100,150,and 200 mg/kg body weight(BW)per day),nicotine(1.5 mg/kg),and 3 different groups of Nic+Roy(1.5 mg/kg of Nic+100,150,and 200 mg/kg BW per day of royal jelly).Nicotine was administrated intraperitoneally,and royal jelly was prescribed orally for 10 consecutive days.Serum levels of hormones(testosterone,luteinizing hormone,and follicle-stimulating hormone),total antioxidant capacity,nitric oxide(NO)status,malondialdehyde levels,sperm DNA fragmentation,sperm parameters,histopathological changes(H&E staining),immunohistochemistry against apoptotic proteins,and gene expression of Bcl-2,p53,Caspase-3,and Nrf2(real-time PCR)were assessed to evaluate the molecular and histological changes.Results:Hormone levels,sperm parameters,and status of antioxidants were decreased significantly(p<.05)following nicotine administration.Moreover,royal jelly treatment normalized hormonal and antioxidant characteristics,decreased apoptotic gene expression,increased Nfr2 gene expression,and restored histopathological alteration to the physiological status significantly(p<.05).Conclusion:Royal jelly upregulates the antioxidant status,inhibits the mitochondrialdependent apoptosis pathway,and increases the rate of proliferation.This therapeutic agent effectively protected the testis against nicotine-associated damages by antioxidant and anti-apoptotic effects.

Effect of solvent on the crystal morphology of royal demolition explosive

Important crystal faces that dominate the crystal morphology of royal demolition explosive （RDX） in vacuum were analyzed with the attachment energy （AE） method. Molecular dynamics （MD） simulations were used to calculate the interaction energies between these crystal faces and different solvent molecules for an attachment energy correction. Growth habits in the presence of different solvents were generated. The results showed that some crystal faces in solutions became morphologically more important than that in vacuum while others became less important. Thus, crystal shape and surface property changed a lot with the variation of crystal faces. The results from calcula- tion were in agreement with those from the re-crystallization experiment, which indicated that cyclohexanone （CH） was a promising solvent to modify the crystal morphology of RDX for obtaining products with regular shape and high purity, while butyrolactone （BL） played a great role in improving the surface electrostatic property of RDX.

Effects of environmental factors on seedling growth and anthocyanin content in Betula ‘Royal Frost’ leaves

Determining the influences of environmental factors on seedling growth and leaf color may improve our understanding of the chromogenic mechanisms in leaves and the biosynthesis of anthocyanin. To determine the optimum conditions for Betula 'Royal Frost' seedling cultivation, we assessed seedling growth and anthocyanin content under natural conditions. The temperature had a significant influence on height but not on radial growth. Between June and September, the anthocyanin content was significantly positively correlated with light intensity and air moisture and significantly negatively correlated with temperature. In single-factor experiments to determine the variation in anthocyanin content in the greenhouse, anthocyanin content increased with increasing soil water content and at first increased, then decreased with increasing pH of the irrigation water and temperature, but decreased with increasing light intensity. K2SO4 can increase the anthocyanin content. So to maintain leaf color, seedlings should be cultivated with < 200 mu mol m(-2) s(-1) light intensity at 20 A degrees C and soil moisture content between 60 and 90%, and the irrigation water should be neutral or alkaline. Additionally, spraying with K2SO4 can be beneficial.

Profile Analysis of the Proteome of the Egg of the High Royal Jelly Producing Bees (Apis mellifera L.)

The protein composition of the egg development in the high royal jelly producing bees （Apis mellifera L.） was investigated. This pioneer study was to separate and quantify the proteins in the egg of the high royal jelly producing worker bees （Apis mellifera L.） by using two-dimensional gel electrophoresis along with their three-day development. The results showed that 160, 195, and 176 proteins, with a wide range of molecular weight （17-80 KDa） and relatively narrow scope of pI （4. 00-8.40） could be detected on day 1, day 2, and day 3, respectively, during the developmental process of the egg. Meanwhile 44 protein spots were constantly detected along with the egg development. Among them 36% were in the uptrend along with the egg development, 14% were in the downtrend, and 39% were of the largest expressed volume on day 2. In addition, the specific proteins were expressed on day 1, day 2, and day 3 （89, 77, and 80, respectively）. Besides the coexistent and specific proteins, 24 proteins were expressed on day 1 and day 2, but silenced on day 3, 49 proteins were expressed on day 2 and day 3, but silenced on day 1, only 3 proteins were expressed on day 1 and day 3, but silenced on day 2. The result indicates that egg development is a sequential and complex gene controlled process, where the eggs of day 2 express the most active proteins. The coexistent proteins suggest that it is conservative and indispensable for this event. These specific proteins suggest that the different developmental stage needs specific proteins to regulate it.

Effectiveness of royal jelly supplementation in glycemic regulation:A systematic review

BACKGROUND Royal jelly(RJ)has been observed to have therapeutic properties in diabetic individuals,including the reduction of high blood sugar.This systematic review synthesized existing evidence to investigate the effectiveness of RJ supplementation in managing measures of blood glucose.AIM To determine the effectiveness of RJ supplementation on glycemic responses in healthy and non-insulin dependent diabetic adults,as well as animal models of diabetes.METHODS This was a systematic review employing the PRISMA strategy.Peer-reviewed,published articles were extracted from several databases using key words related to target population,intervention and outcome and hand-selected for inclusion.Included articles proceeded to data extraction phase,where information on target parameters and effectiveness of treatment was summarized.Following this,the risk of bias for each included study was evaluated.Then,the long-term and immediate effectiveness of RJ supplementation in glycemic control were assessed using the Grading of Recommendations,Assessment,Development and Evaluations(GRADE)tool,which rates the quality of evidence.RESULTS Of 168 articles extracted from database searching,eighteen were included for analysis in this systematic review.Across the studies,studied populations,intervention styles and outcome measures were largely heterogeneous.Despite this,the results in studies indicate a general trend of positive effect of RJ in glycemic regulation in vitro and in vivo.Additionally,some dose-dependent glycemic effects were observed,along with some large effect sizes.The risk of bias for human and animal studies is generally low-unclear risk,although lack of blinding is a serious concern in both categories.Overall,as per the GRADE tool,the quality of evidence is low,and very low for long-term and immediate effectiveness of RJ,respectively.A major limitation affecting evidence quality is the heterogeneity among included studies.Fasting blood glucose and glucose clearance appear to be most affected by RJ supplementation.CONCLUSION Quality of evidence suggesting that RJ is an effective modulator of glycemic regulation is low for long-term effects of RJ,and very low for immediate effects.

Landscapes of Royal Botanic Garden Edinburgh

Built in the middle and late 17th century, the Royal Botanic Garden Edinburgh located in Scotland is one of the famous five botanic gardens in the world and one of the earliest botanic gardens in British history. This study sorted out the historical development and changes of Royal Botanic Garden Edinburgh for more than 300 years, and then made a comprehensive introduction and analysis of the botanic garden from three aspects of touring, education and scientific research. Finally, the rock garden, the peat garden, the Chinese hillside, the alpine house, glasshouses and the Queen Mother’s Memorial Garden were analyzed, in order to explore the historical, landscape and scientific value of Royal Botanic Garden Edinburgh and to seek the effective way of landscape construction and development of the botanic garden.

Investigating the Effect of Supplementary Feeding on Carbohydrate Composition and Quantity of Royal Jelly

One of the main practices followed by beekeepers during the production of royal jelly (RJ) is the artificial sugar feeding. In this study, the effect of carbohydrate supplementary feeding on the composition of the three main sugars (fructose, glucose, sucrose) and on the final quantity of the product was explored using one-way ANOVA and non-parametric tests. Also, the correlations among the parameters were examined. The average yield per colony for non-supplemented colonies (Group A) was 12.8 g, while the average content of fructose, glucose and sucrose was 4.32%, 3.78%, and 0.04%, respectively. For the colonies fed at the grafting day one time (Group B), these values were 12.76 g, 3.11%, 3.19% and 3.71%, and for the colonies fed from the insertion until the collection day (Group C), 12.81 g, 3.05%, 3.12% and 3.54% respectively. It should also be noted that the sucrose content in all samples from supplemented colonies was found greater than 1.97%. The statistical tests highlighted the impact of artificial feeding on fructose and glucose contents, while the produced quantity remained uninfluenced. Finally, the Spearman (rho) coefficient test showed statistically significantly negative correlation between the monosaccharides (fructose, glucose) and sucrose.

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin α3 and Integrin β1 mRNA Expression, via Akt/mTOR/HIF-1α Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

Analysis of Style of Rockery Building and Stone Stacking Technique in the Royal Garden of the Song Dynasty: A Case Study of Genyue Royal Garden

Rockery building and stone stacking is a technique commonly used in building of classical gardens.Its emergence allows the fairy thought of"one pond and three mountains"to be reflected in different classical gardens.In the Northern Song Dynasty,the style of rockery building and stone stacking in the royal garden was an artistic climax in the history of the garden,and the technique of rockery building and stone stacking in Genyue Royal Garden was considered as the beginning of specialization.Through sorting out the records of the royal gardens in the Song Dynasty,this paper summarized the content of studying the royal gardens of the Song Dynasty,and systematically sorted out the development history of the techniques and styles of the royal gardens of the Song Dynasty and applied them into construction of some gardens.

Spatial Characteristics of Royal Gardens in the Early Qing Dynasty: A Case Study of Emperor Yongzheng Enjoying the Happiness of a Family Union in Twelve Lunar Months

Taking the painting Emperor Yongzheng Enjoying the Happiness of a Family Union in Twelve Lunar Months for example, this study explored the spatial characteristics of the architecture and garden reflected by the scroll against the background of the Western learning spreading eastward in the late Ming and early Qing dynasties. Combining with the conjectures put forward by previous scholars on the possible places of the Emperor Yongzheng Enjoying the Happiness of a Family Union in Twelve Lunar Months, with reference to the relevant historical materials, the interior space of the painting was analyzed by restoring the imaginary map and drawing the graph to complete the restoration design of space of the scroll painting. It is hoped that this study can enrich the research on the royal garden in the early Qing Dynasty.

Effect of royal jelly on in vitro fertilization and early embryo development following nicotine treatment in adult female rats

Objective:To scrutinize the protective role of royal jelly as an antioxidant on nicotine-induced changes in malondialdehyde(MDA)level,p53 expression,in vitro fertilization(IVF)rate,and early embryo development in adult female rats.Methods:A total of 56 adult female Wistar rats were divided into 8 groups(n=7 in each group).Group 1 served as an untreated control group,group 2,3 and 4 received nicotine at a dose of 0.50,1.00 and 2.00 mg/kg respectively,group 5 received royal jelly at a dose of 100.00 mg/kg,and group 6,7 and 8 received 0.50,1.00 and 2.00 mg/kg nicotine,respectively,with 100.00 mg/kg body weight royal jelly.Nicotine and royal jelly were administered daily for 49 days in the experimental groups intra-peritoneally and orally,respectively.At the end of the experimental period,p53 expression,IVF rate and early embryo development as well as MDA concentration were measured.Results:The IVF rate,number of cumulus oocytes,two-cell embryos and blastocysts decreased in the nicotine-treated groups in a dose-dependent manner.In addition,p53 mRNA expression and MDA levels increased in the nicotine-treated groups.Royal jelly co-administration led to partial improvement in the aforementioned parameters.Conclusions:Royal jelly may have a repro-protective effect in nicotine-administered female rats in terms of its anti-oxidant and anti-apoptotic properties.

Physical Properties and Exchange System of Seawater in Royal Yacht Club Marina in the Northernmost Gulf of Aqaba

This research was aimed at studying the general environmental status of the marina at Royal Yacht Club (RYC), which is located in the Jordanian Gulf of Aqaba. The physical properties (temperature, salinity and density) were measured at surface and bottom water at 19 sites (T1 - T19). Total hydrocarbon was measured at surface at 12 sites (HC1-HC12). The water exchange and residence time were calculated using data of current speed and direction at 16 sites (C1 - C16). The mean values of temperature, salinity and density at surface and bottom waters in the RYC’s marina were 27.13°C ± 0.03°C, 40.51 ± 0.02 psu and 26.83 ± 0.02 kg&#183;m-3, respectively. The results of physical parameters revealed no significant differences among sites, which are all comparable with open waters. The measurements range of total hydrocarbon in the marina was 1.404 mg/l (at site HC11) to 98.56 mg/l (at HC9). In general, all the high values of total hydrocarbon were found at the corners of the marina and at areas with relatively intense boats’ movements. There was no correlation between distribution of total hydrocarbon and temperature (R2 = 0.048;p = 0.49) in the marina. Therefore, the major factors that influence the distribution of total hydrocarbon within the marina are the current system and boats’ movements as well as the location reference to the gate. The residence time of waters inside the marina was 1.32 days. This flushing time is acceptable to secure good environmental conditions inside the marine to avoid stagnant seawater. The mean value of current speed at the RYC’s marina gate with open water was 5.33 ± 2.63 cm/s, which is relatively comparable with current speed in vicinity of study area. In conclusions, the environmental condition of the RYC’s marina is acceptable in term of the residence time and exchange system with open water. Slightly high values of total hydrocarbon were detected, which can be solved by regular cleaning.

Bioassay Directed Identification of Royal Jelly’s Active Compounds against the Growth of Bacteria Capable of Infecting Cutaneous Wounds

Antibiotic-resistant bacteria continue to be of major health concern world-wide. Thus, it is of great interest to study the biological properties and determine active compounds in natural products likely to be used as new health remedies. Therefore, the main objective of this work is to test the antibacterial activity of royal jelly samples, defatted royal jelly samples and their ethyl ether extracts against bacteria capable of infecting cutaneous wounds in humans and animals, and to recognize major bioactive compounds by using bioassay directed identification. The microorganisms used in the study were Staphylococcus aureus (including Methicillin-resistant and sensitive strains), Staphylococcus epidermidis, Micrococcus luteus, Streptococcus uberis, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. The activity of royal jelly samples to inhibit bacterial growth was assessed by using well-difussion tests. Direct bioautography was used to identify bioactivity, and uv-visible spectroscopy and gas chromatography-mass spectrometry were used to identify bioactive compounds. Overall, royal jelly samples showed higher growth inhibition activity against Gram positive bacteria as compared to Gram negative bacteria. The growth of bacterial strains belonging to the Enterococcus and Streptococcus genders was less affected by the presence of royal jelly than bacterial strains of the Staphylococcus gender did. Compounds with antibacterial activity were found in the ethyl ether extract of royal jelly samples. 10-hydroxy-2-decenoic acid was the major component identified in the purified fraction obtained by bioassay guided fractionation of the ethyl ether extract. In conclusion, bioactivity of royal jelly samples is mainly due to their 10-hydroxy-2-decenoic acid content.

Thymoquinone anticancer activity is enhanced when combined with royal jelly in human breast cancer

BACKGROUND Breast cancer is the most common cause of the majority of cancer-related deaths in women,among which triple-negative breast cancer is the most aggressive type of breast cancer diagnosed with limited treatment options.Thymoquinone(TQ),the main bioactive constituent of Nigella sativa,has been extensively studied as a potent anticancer molecule against various types of cancers.Honeybee products such as the royal jelly(RJ),the nutritive secretion fed to honeybee queens,exhibit a variety of biological activities besides its anticancer effect.However,the anticancer activity of the combination of TQ and RJ against breast cancer is still unknown.AIM To investigate cytotoxicity of RJ in FHs 74 Int cells and the anticancer effects of TQ,RJ,and their combinations in the MDA-MB-231 cell line.METHODS Cells were treated with TQ,RJ,and their combinations for 24 h.Using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,we determined the half-maximal inhibitory concentration of TQ.Trypan blue and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were then performed to assess the cell viability in response to different treatment conditions.Cell death and cycle regulation were investigated using propidium iodide deoxyribonucleic acid staining followed by flow cytometry in response to a single dose of TQ,RJ,and their combination.Immunostaining for cleaved caspase 3 and Ki67 expression was used to determine apoptosis induction and changes in cell proliferation.RESULTS TQ alone inhibited cell viability in a dose-dependent manner at concentrations below and above the half-maximal inhibitory concentration.RJ exhibited relatively nontoxic effects against MDA-MB-231 cells and FHs 74 Int small intestinal cells at concentrations below 5μg/mL.High doses of RJ(200μg/mL)had greater toxicity against MDA-MB-231 cells.Interestingly,the inhibition of cell viability was most pronounced in response to 15μmol/L TQ and 5μg/mL RJ.A dose of 15μmol/L TQ caused a significant increase in the PreG1 population,while a more pronounced effect on cell viability inhibition and PreG1 increase was observed in response to TQ and RJ combinations.TQ was the main inducer of caspase 3-dependent apoptosis when applied alone and in combination with RJ.In contrast,no significant regulation of Ki67 expression was observed,indicating that the decrease in cell viability was due to apoptosis induction rather than to inhibition of cell proliferation.CONCLUSION This study is the first to report enhanced anticancer effects of TQ and RJ combination against MDA-MB-231 breast cancer cells,which could confer an advantage for cancer therapy.