Anti-Diabetic Effect of Lactobacillus Paracasei Isolated from Malaysian Water Kefir Grains.

The prevalence of type 2 diabetes mellitus (T2DM) is alarming because it is always linked to the increase in chronic diseases, mortality, and socioeconomic burden. Water kefir has a wide range of functional and probiotic characteristics attributed to the microorganisms present in the kefir grains. The present study aims to evaluate the in vivo anti-diabetic potential of the isolated Lactobacillus paracasei from Malaysian water kefir grains (MWKG) which was reported to have excellent probiotic properties and high antioxidant activities as reported previously. High-fat diet/streptozotocin (HFD/STZ) induction was used to obtain a T2DM model followed by treatment with the isolated L. paracasei from MWKG. The levels of glucose, insulin, and in vivo liver antioxidants were quantified after 14 weeks. Gene expression analysis of the liver was also carried out using microarray analysis, and several genes were selected for validation using quantitative real-time PCR. Insulin tolerance test demonstrated that the L. paracasei isolated from the MWKG alleviated T2DM by improving the area under the curve of the insulin tolerance test whereby low-dose and high-dose concentrations treated groups showed 2424.50 ± 437.02 mmol/L·min and 2017.50 ± 347.09 mmol/L·min, respectively, compared to untreated diabetic mice which was 3884.50 ± 39.36 mmol/L·min. Additionally, treatment with the isolated L. paracasei from MWKG regulated the expression of several genes related to glucose homeostasis and lipid metabolism in diabetic mice. These results suggested that the isolated L. paracasei from MWKG could be a potential dietary supplement for T2DM.

Timeline of SARS-CoV-2 Transmission in Sabah, Malaysia: Tracking the Molecular Evolution.

BACKGROUND: The COVID-19 pandemic poses an unprecedented public health challenge in Malaysia. The impact of COVID-19 varies between countries, including geographically divided states within a country. The deadly transmission of COVID-19 has taken a heavy toll in Sabah, Malaysia's third most populous state, contributing nearly 10% to the recorded national death toll as of 31 December 2022. Although several SARS-CoV-2 genome sequences have been analysed in Malaysia, molecular epidemiology data from Sabah focusing on the diversity and evolution of SARS-CoV-2 variants are still lacking. This study examines the major SARS-CoV-2 variants and emerging mutations from Sabah, the Malaysian Borneo, which is geographically divided from West Malaysia by the South China Sea. METHODS: A total of 583 COVID-19 samples were subjected to whole genome sequencing and analysed with an additional 1123 Sabah COVID-19 sequences retrieved from the GISAID EpiCoV consortium. Nextclade and Pangolin were used to classify these sequences according to the clades and lineages. To determine the molecular evolutionary characteristics, Bayesian time-scaled phylogenetic analysis employing the maximum likelihood algorithm was performed on selected SARS-CoV-2 genome sequences, using the Wuhan-Hu-1 sequence as a reference. RESULTS: Sabah was affected starting from the second COVID-19 wave in Malaysia, and the early sequences were classified under the O clade. The clade was gradually replaced during subsequent waves by G, GH, GK and GRA, with the latter being dominant as of December 2022. Phylogenetically, the Delta isolates in this study belong to the three main subclades 21A, 21J and 21I, while Omicron isolates belong to 21M, 21L and 22B. The time-scaled phylogeny suggested that SARS-CoV-2 introduced into Sabah originated from Peninsular Malaysia in early March 2020, and phylodynamic analysis indicated that increased viral spread was observed in early March and declined in late April, followed by an evolutionary stationary phase in June 2020. CONCLUSION: Continuous molecular epidemiology of SARS-CoV-2 in Sabah will provide a deeper understanding of the emergence and dominance of each variant in the locality, thus facilitating public health intervention measures.

Physicochemical characterization, cytotoxic effect and toxicity evaluation of nanostructured lipid carrier loaded with eucalyptol.

BACKGROUND: Eucalyptol is an active compound of eucalyptus essential oil and was reported to have many medical attributes including cytotoxic effect on breast cancer cells. However, it has low solubility in aqueous solutions which limits its bioavailability and cytotoxic efficiency. In this study, nanostructured lipid carrier loaded with eucalyptol (NLC-Eu) was formulated and characterized and the cytotoxic effect of NLC-Eu towards breast cancer cell lines was determined. In addition, its toxicity in animal model, BALB/c mice was also incorporated into this study to validate the safety of NLC-Eu. METHODS: Eucalyptol, a monoterpene oxide active, was used to formulate the NLC-Eu by using high pressure homogenization technique. The physicochemical characterization of NLC-Eu was performed to assess its morphology, particle size, polydispersity index, and zeta potential. The in vitro cytotoxic effects of this encapsulated eucalyptol on human (MDA MB-231) and murine (4 T1) breast cancer cell lines were determined using the MTT assay. Additionally, acridine orange/propidium iodide assay was conducted on the NLC-Eu treated MDA MB-231 cells. The in vivo sub-chronic toxicity of the prepared NLC-Eu was investigated using an in vivo BALB/c mice model. RESULTS: As a result, the light, translucent, milky-colored NLC-Eu showed particle size of 71.800 ± 2.144 nm, poly-dispersity index of 0.258 ± 0.003, and zeta potential of - 2.927 ± 0.163 mV. Furthermore, the TEM results of NLC-Eu displayed irregular round to spherical morphology with narrow size distribution and relatively uniformed particles. The drug loading capacity and entrapment efficiency of NLC-Eu were 4.99 and 90.93%, respectively. Furthermore, NLC-Eu exhibited cytotoxic effects on both, human and mice, breast cancer cells with IC50 values of 10.00 ± 4.81 µg/mL and 17.70 ± 0.57 µg/mL, respectively at 72 h. NLC-Eu also induced apoptosis on the MDA MB-231 cells. In the sub-chronic toxicity study, all of the studied mice did not show any signs of toxicity, abnormality or mortality. Besides that, no significant changes were observed in the body weight, internal organ index, hepatic and renal histopathology, serum biochemistry, nitric oxide and malondialdehyde contents. CONCLUSIONS: This study suggests that the well-characterized NLC-Eu offers a safe and promising carrier system which has cytotoxic effect on breast cancer cell lines.

Metagenomic and phytochemical analyses of kefir water and its subchronic toxicity study in BALB/c mice.

BACKGROUND: In recent years, researchers are interested in the discovery of active compounds from traditional remedies and natural sources, as they reveal higher therapeutic efficacies and improved toxicological profiles. Among the various traditional treatments that have been widely studied and explored for their potential therapeutic benefits, kefir, a fermented beverage, demonstrates a broad spectrum of pharmacological properties, including antioxidant, anti-inflammation, and healing activities. These health-promoting properties of kefir vary among the kefir cultures found at the different part of the world as different media and culture conditions are used for kefir maintenance and fermentation. METHODS: This study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays. RESULTS: The 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir was Lactobacillus hilgardii followed by Lactobacillus harbinensis, Acetobacter lovaniensis, Lactobacillus satsumensis, Acetobacter tropicalis, Lactobacillus zeae, and Oenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples. CONCLUSIONS: This study will help to intensify the knowledge on the water kefir microbial composition, available phytochemicals and its toxicological and antioxidant effects on BALB/c mice since there are very limited studies on the water kefir grain fermented in Malaysia.

Anti-Metastatic and Anti-Angiogenic Effects of Curcumin Analog DK1 on Human Osteosarcoma Cells In Vitro.

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.

Selected Kefir Water from Malaysia Attenuates Hydrogen Peroxide-Induced Oxidative Stress by Upregulating Endogenous Antioxidant Levels in SH-SY5Y Neuroblastoma Cells.

Kefir, a fermented probiotic drink was tested for its potential anti-oxidative, anti-apoptotic, and neuroprotective effects to attenuate cellular oxidative stress on human SH-SY5Y neuroblastoma cells. Here, the antioxidant potentials of the six different kefir water samples were analysed by total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), and 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) assays, whereas the anti-apoptotic activity on hydrogen peroxide (H2O2) induced SH-SY5Y cells was examined using MTT, AO/PI double staining, and PI/Annexin V-FITC assays. The surface and internal morphological features of SH-SY5Y cells were studied using scanning and transmission electron microscopy. The results indicate that Kefir B showed the higher TPC (1.96 ± 0.54 µg GAE/µL), TFC (1.09 ± 0.02 µg CAT eq/µL), FRAP (19.68 ± 0.11 mM FRAP eq/50 µL), and DPPH (0.45 ± 0.06 mg/mL) activities compared to the other kefir samples. The MTT and PI/Annexin V-FITC assays showed that Kefir B pre-treatment at 10 mg/mL for 48 h resulted in greater cytoprotection (97.04%), and a significantly lower percentage of necrotic cells (7.79%), respectively. The Kefir B pre-treatment also resulted in greater protection to cytoplasmic and cytoskeleton inclusion, along with the conservation of the surface morphological features and the overall integrity of SH-SY5Y cells. Our findings indicate that the anti-oxidative, anti-apoptosis, and neuroprotective effects of kefir were mediated via the upregulation of SOD and catalase, as well as the modulation of apoptotic genes (Tp73, Bax, and Bcl-2).

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro.

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012-2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC50) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.

The prevalence risk of anxiety and its associated factors among university students in Malaysia: a national cross-sectional study.

BACKGROUND: Anxiety disorder is one of the most common mental health problems worldwide, including Malaysia, and this issue has gained concern and attention from many, including experts and authorities globally. While average levels of stress and worry may help to motivate students to perform well in their studies, excessive feelings will increase their level of anxiety. METHODS: A cross-sectional study was conducted at selected government and private universities throughout Malaysia. A total of 1851 students participated in this study. The students were asked to complete self-administered questionnaires, including socio-demographic, academic, and psychosocial characteristics. The Generalized Anxiety Disorder-7 (GAD-7) questionnaire was used to measure the prevalence risk of anxiety among the students. Chi-square analysis was conducted to find the relationship between the variables and anxiety, and multivariate logistic regression analysis was used to identify the predictors. RESULTS: The response rate was 97.90%, where 1821 out of 1860 students participated in the study. The prevalence risk of anxiety in this study was recorded at 29%. The data revealed that academic year, financial support for the study, alcohol consumption, poor sleep quality, body mass index (BMI), having a good friend in the university, having doubt regarding the future, actively involved in the society, and having problems with other students and lecturer(s) were significantly associated with risk of anxiety; with the academic year as the primary predictor. CONCLUSIONS: The findings highlight the current prevalence risk of anxiety among university students in Malaysia. The outcome of this study can serve as the evident baseline data and help with the development of specific interventions in addressing and managing the issue appropriately.

Pineapple Vinegar Regulates Obesity-Related Genes and Alters the Gut Microbiota in High-Fat Diet (HFD) C57BL/6 Obese Mice.

Obesity is a pandemic metabolic syndrome with increasing incidences every year. Among the significant factors that lead to obesity, overconsumption of high-fat food in daily intake is always the main contributor. Functional foods have shown a positive effect on disease prevention and provide health benefits, including counteracting obesity problem. Vinegar is one of the fermented functional beverages that have been consumed for many years, and different types of vinegar showed different bioactivities and efficacies. In this study, we investigated the potential effects of pineapple vinegar as an antiobesity agent on a high-fat diet- (HFD-) induced C57BL/6 obese mice. C57BL/6 mice were treated with pineapple vinegar (1 mL/kg BW and 0.08 mL/kg BW) for 12 weeks after 24 weeks of HFD incubation. Serum biochemistry profiles, antioxidant assays, qPCR, proteome profiler, and 16S metagenomic were done posttreatment. Our data showed that a high concentration of pineapple vinegar (1 mL/kg BW) treatment significantly (p < 0.05) reduced the bodyweight (∼20%), restored lipid profiles, increased the antioxidant activities, and reduced the oxidative stress. Besides, significant (p < 0.05) regulation of several adipokines and inflammatory-related genes was recorded. Through the regulation of gut microbiota, we found a higher abundance of Akkermansia muciniphila, a microbiota reported to be associated with obesity in the high concentration of pineapple vinegar treatment. Collectively, these data established the mechanism of pineapple vinegar as antiobesity in mice and revealed the potential of pineapple vinegar as a functional food for obesity.

Antitumor and Anti-Metastatic Effects of Citral-Loaded Nanostructured Lipid Carrier in 4T1-Induced Breast Cancer Mouse Model.

Cancer nano-therapy has been progressing rapidly with the introduction of many novel drug delivery systems. The previous study has reported on the in vitro cytotoxicity of citral-loaded nanostructured lipid carrier (NLC-Citral) on MDA-MB-231 cells and some preliminary in vivo antitumor effects on 4T1 breast cancer cells challenged mice. However, the in vivo apoptosis induction and anti-metastatic effects of NLC-Citral have yet to be reported. In this study, the in vitro cytotoxic, anti-migration, and anti-invasion effects of NLC-Citral were tested on 4T1 breast cancer cells. In addition, the in vivo antitumor effects of oral delivery of NLC-Citral was also evaluated on BALB/c mice induced with 4T1 cells. In vitro cytotoxicity results showed that NLC-Citral and citral gave similar IC50 values on 4T1 cells. However, wound healing, migration, and invasion assays reflected better in vitro anti-metastasis potential for NLC-Citral than citral alone. Results from the in vivo study indicated that both NLC-Citral and citral have anti-tumor and anti-metastasis effects, whereby the NLC-Citral showed better efficacy than citral in all experiments. Also, the delay of tumor progression was through the suppression of the c-myc gene expression and induction of apoptosis in the tumor. In addition, the inhibition of metastasis of 4T1 cells to lung and bone marrow by the NLC-Citral and citral treatments was correlated with the downregulation of metastasis-related genes expression including MMP-9, ICAM, iNOS, and NF-kB and the angiogenesis-related proteins including G-CSF alpha, Eotaxin, bFGF, VEGF, IL-1alpha, and M-CSF in the tumor. Moreover, NLC-Citral showed greater downregulation of MMP-9, iNOS, ICAM, Eotaxin, bFGF, VEGF, and M-CSF than citral treatment in the 4T1-challenged mice, which may contribute to the better anti-metastatic effect of the encapsulated citral. This study suggests that NLC is a potential and effective delivery system for citral to target triple-negative breast cancer.

Bromelain Enhances the Anti-tumor Effects of Cisplatin on 4T1 Breast Tumor Model In Vivo.

Background: This study aimed to evaluate the antitumor enhancing effect of bromelain consumption on 4T1-challenged mice treated with cisplatin. Methods: Mice challenged with 4T1 triple-negative breast cancer cells received water, bromelain, cisplatin, or bromelain + cisplatin treatment for 28 days. Tumor size was measured, and lung metastasis was evaluated by clonogenic assay. Expression of tumor inflammatory genes of the harvested tumor was quantified by polymerase chain reaction array and ELISA (enzyme-linked immunosorbent assay). Results: All treatments significantly reduced the size of tumor and lung metastasis, with combination treatment showing the best effect. Also, bromelain alone and combination treatment showed downregulation of the expression of tumor inflammatory genes (Gremlin [GREM1], interleukin 1ß [IL-1ß], interleukin-4 [IL-4], nuclear factor κB subunit 1 [NFκB1], and prostaglandin-endoperoxide synthase 2 [PTGS2]), tumor nitric oxide level, and serum IL-1ß, and IL-4 levels. On the other hand, cisplatin treatment increased the expression of selected inflammatory markers. Conclusion: This study suggests that bromelain treatment could potentiate the antitumor effect of cisplatin on triple-negative breast cancer 4T1 cells through modulating the tumor environmental inflammation.

Apoptosis and metastasis inhibitory potential of pineapple vinegar against mouse mammary gland cells in vitro and in vivo.

BACKGROUND: Plant-based food medicine and functional foods have been consumed extensively due to their bioactive substances and health-beneficial effects. Vinegar is one of them due to its bioactivities, which confers benefits on human body. Our previous study has produced pineapple vinegar that is rich in gallic acid and caffeic acid via 2 steps fermentation. There are many evidences that show the effectiveness of these resources in inhibiting the proliferation and metastasis of the cancer cells through several mechanisms. METHODS: Freeze-dried pineapple vinegar was evaluated for its in vitro apoptosis and metastasis inhibitory potential using MTT, cell cycle, Annexin V and scratch assays. The in vivo test using BALB/c mice challenged with 4 T1 cells was further investigated by pre-treating the mice with 0.08 or 2 ml/kg body weight of freshly-prepared pineapple vinegar for 28 days. The tumor weight, apoptotic state of cells in tumor, metastasis and immune response of the untreated and pineapple vinegar treatment group were evaluated and compared. RESULTS: From the in vitro study, an IC50 value of 0.25 mg/mL after 48 h of treatment was established. Annexin V/PI and scratch closure assays showed that pineapple vinegar induced 70% of cell population to undergo apoptosis and inhibited 30% of wound closure of 4 T1 cells. High concentration of pineapple vinegar (2 ml/kg body weight) led to the reduction of tumor weight and volume by 45%as compared to the untreated 4 T1-challenged mice. This effect might have been contributed by the increase of T cell and NK cells population associated with the overexpression of IL-2 andIFN-γ cytokines and splenocyte cytotoxicity. Furthermore, fewer instances of metastasis events were recorded in the pineapple vinegar treatment group and this could be explained by the downregulation of inflammation related genes (iNOS, NF-kB and COX2), metastasis related genes (iCAM, VEGF and MMP9) and angeogenesis related genes (CD26, TIMP1, HGF, MMP3, IGFBP-1 and IGFBP-2). CONCLUSION: The ability of pineapple vinegar to delay cancer progression portrayed its potential as chemopreventive dietry intervention for cancer therapy.

Isolation and Characterization of Lactobacillus spp. from Kefir Samples in Malaysia.

Kefir is a homemade, natural fermented product comprised of a probiotic bacteria and yeast complex. Kefir consumption has been associated with many advantageous properties to general health, including as an antioxidative, anti-obesity, anti-inflammatory, anti-microbial, and anti-tumor moiety. This beverage is commonly found and consumed by people in the United States of America, China, France, Brazil, and Japan. Recently, the consumption of kefir has been popularized in other countries including Malaysia. The microflora in kefir from different countries differs due to variations in culture conditions and the starter media. Thus, this study was aimed at isolating and characterizing the lactic acid bacteria that are predominant in Malaysian kefir grains via macroscopic examination and 16S ribosomal RNA gene sequencing. The results revealed that the Malaysian kefir grains are dominated by three different strains of Lactobacillus strains, which are Lactobacillus harbinensis, Lactobacillusparacasei, and Lactobacillus plantarum. The probiotic properties of these strains, such as acid and bile salt tolerances, adherence ability to the intestinal mucosa, antibiotic resistance, and hemolytic test, were subsequently conducted and extensively studied. The isolated Lactobacillus spp. from kefir H maintained its survival rate within 3 h of incubation at pH 3 and pH 4 at 98.0 ± 3.3% and 96.1 ± 1.7% of bacteria growth and exhibited the highest survival at bile salt condition at 0.3% and 0.5%. The same isolate also showed high adherence ability to intestinal cells at 96.3 ± 0.01%, has antibiotic resistance towards ampicillin, penicillin, and tetracycline, and showed no hemolytic activity. In addition, the results of antioxidant activity tests demonstrated that isolated Lactobacillus spp. from kefir G possessed high antioxidant activities for total phenolic content (TPC), total flavonoid content (TFC), ferric reducing ability of plasma (FRAP), and 1,1-diphenyl-2-picryl-hydrazine (DPPH) assay compared to other isolates. From these data, all Lactobacillus spp. isolated from Malaysian kefir serve as promising candidates for probiotics foods and beverage since they exhibit potential probiotic properties and antioxidant activities.

Preparation and Characterization of Self Nano-Emulsifying Drug Delivery System Loaded with Citraland Its Antiproliferative Effect on Colorectal Cells In Vitro.

Citral is an active compound naturally found in lemongrass, lemon, and lime. Although this pale-yellow liquid confers low water solubility, the compound has been reported to possess good therapeutic features including antiproliferative and anticancer modalities. The self nano-emulsifying drug delivery system (SNEDDS) is a type of liquid-lipid nanocarrier that is suitable for the loading of insolubilized oil-based compound such as Citral. This study reports the design and optimization of a SNEDDS formulation, synthesis and characterization as well as loading with Citral (CIT-SNEDDS). Further assessment of theantiproliferative effects of CIT-SNEDDS towards colorectal cancer cells was also conducted. SNEDDS composed of coconut oil, dimethyl sulfoxide (DMSO) and Tween 80. CIT-SNEDDS was prepared via gentle agitation of SNEDDS with 0.5% Citral for 72 h at room temperature. Physicochemical characterization was performed using several physicochemical analyses. The average particle size of CIT-SNEDDS was16.86 ± 0.15 nm, zeta potential of 0.58 ± 0.19 mV, and polydispersity index (PDI) of 0.23 ± 0.01. In vitro drug release of Citral from CIT-SNEDDS was 79.25% of release, and for Citral the release percentage was 93.56% over 72 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done to determine the cytotoxicity effect of CIT-SNEDDS in human colorectal cancer cell lines HT29 and SW620. The half maximal inhibitory concentrations (IC50) for 72 hof CIT-SNEDDS and Citral on SW620 were 16.50 ± 0.87 µg/mL and 22.50 ± 2.50 µg/mL, respectively. The IC50 values of CIT-SNEDDS and Citral after 72 h of treatment on HT29 were 34.10 ± 0.30 µg/mL and 21.77 ± 0.23 µg/mL, respectively. This study strongly suggests that CIT-SNEDDS has permitted the sustained release of Citral and that CIT-SNEDDS constitutes a potential soluble drug nanocarrier that is effective against colorectal cancer cells.

In vitro and in vivo antitumour effects of coconut water vinegar on 4T1 breast cancer cells.

BACKGROUND: Coconut water and vinegars have been reported to possess potential anti-tumour and immunostimulatory effects. However, the anti-tumour, anti-inflammatory and immunostimulatory effects of coconut water vinegar have yet to be tested. OBJECTIVE: This study investigated the in vitro and in vivo anti-tumour effects of coconut water vinegar on 4T1 breast cancer cells. METHODS: The 4T1 cells were treated with freeze-dried coconut water vinegar and subjected to MTT cell viability, BrdU, annexin V/PI apoptosis, cell cycle and wound healing assays for the in vitro analysis. For the in vivo chemopreventive evaluation, mice challenged with 4T1 cells were treated with 0.08or 2.00 mL/kg body weight of fresh coconut water vinegar for 28 days. Tumour weight, apoptosis of tumour cells, metastasis and immunity of untreated mice and coconut water vinegar-treated 4T1 challenged mice were compared. RESULTS: Freeze-dried coconut water vinegar reduced the cell viability, induced apoptosis and delayed the wound healing effect of 4T1 cells in vitro. In vivo, coconut water vinegar delayed 4T1 breast cancer progression in mice by inducing apoptosis and delaying the metastasis. Furthermore, coconut water vinegar also promoted immune cell cytotoxicity and production of anticancer cytokines. The results indicate that coconut water vinegar delays breast cancer progression by inducing apoptosis in breast cancer cells, suppressing metastasis and activating anti-tumour immunity. CONCLUSION: Coconut water vinegar is a potential health food ingredient with a chemopreventive effect.

In vitro cytotoxicity and anticancer effects of citral nanostructured lipid carrier on MDA MBA-231 human breast cancer cells.

Very recently, we postulated that the incorporation of citral into nanostructured lipid carrier (NLC-Citral) improves solubility and delivery of the citral without toxic effects in vivo. Thus, the objective of this study is to evaluate anti-cancer effects of NLC-Citral in MDA MB-231 cells in vitro through the Annexin V, cell cycle, JC-1 and fluorometric assays. Additionally, this study is aimed to effects of NLC-Citral in reducing the tumor weight and size in 4T1 induced murine breast cancer model. Results showed that NLC-Citral induced apoptosis and G2/M arrest in MDA MB-231 cells. Furthermore, a prominent anti-metastatic ability of NLC-Citral was demonstrated in vitro using scratch, migration and invasion assays. A significant reduction of migrated and invaded cells was observed in the NLC-Citral treated MDA MB-231 cells. To further evaluate the apoptotic and anti-metastatic mechanism of NLC-Citral at the molecular level, microarray-based gene expression and proteomic profiling were conducted. Based on the result obtained, NLC-Citral was found to regulate several important signaling pathways related to cancer development such as apoptosis, cell cycle, and metastasis signaling pathways. Additionally, gene expression analysis was validated through the targeted RNA sequencing and real-time polymerase chain reaction. In conclusion, the NLC-Citral inhibited the proliferation of breast cancer cells in vitro, majorly through the induction of apoptosis, anti-metastasis, anti-angiogenesis potentials, and reducing the tumor weight and size without altering the therapeutic effects of citral.

Coconut water vinegar ameliorates recovery of acetaminophen induced liver damage in mice.

BACKGROUND: Coconut water has been commonly consumed as a beverage for its multiple health benefits while vinegar has been used as common seasoning and a traditional Chinese medicine. The present study investigates the potential of coconut water vinegar in promoting recovery on acetaminophen induced liver damage. METHODS: Mice were injected with 250 mg/kg body weight acetaminophen for 7 days and were treated with distilled water (untreated), Silybin (positive control) and coconut water vinegar (0.08 mL/kg and 2 mL/kg body weight). Level of oxidation stress and inflammation among treated and untreated mice were compared. RESULTS: Untreated mice oral administrated with acetaminophen were observed with elevation of serum liver profiles, liver histological changes, high level of cytochrome P450 2E1, reduced level of liver antioxidant and increased level of inflammatory related markers indicating liver damage. On the other hand, acetaminophen challenged mice treated with 14 days of coconut water vinegar were recorded with reduction of serum liver profiles, improved liver histology, restored liver antioxidant, reduction of liver inflammation and decreased level of liver cytochrome P450 2E1 in dosage dependent level. CONCLUSION: Coconut water vinegar has helped to attenuate acetaminophen-induced liver damage by restoring antioxidant activity and suppression of inflammation.

DK1 Induces Apoptosis via Mitochondria-Dependent Signaling Pathway in Human Colon Carcinoma Cell Lines In Vitro.

Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-ene-1-one (DK1), was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI) dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays) were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR) and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G0/G1phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA fragmentation and depolarization of the mitochondrial membrane. qRT-PCR results show significant upregulation in the expression of caspase-9 in both HT29 and SW620 cell lines, further supporting that cell death induction by DK1 is via an intrinsic pathway. These outcomes, therefore, demonstrate DK1 as a potential anticancer agent for colon adenocarcinoma due to its anti-apoptotic attributes.

Characterization and toxicity of citral incorporated with nanostructured lipid carrier.

The nanoparticle as a cancer drug delivery vehicle is rapidly under investigation due to its promising applicability as a novel drug delivery system for anticancer agents. This study describes the development, characterization and toxicity studies of a nanostructured lipid carrier (NLC) system for citral. Citral was loaded into the NLC using high pressure homogenization methods. The characterizations of NLC-citral were then determined through various methods. Based on Transmission Electron Microscope (TEM) analysis, NLC-Citral showed a spherical shape with an average diameter size of 54.12 ± 0.30 nm and a polydipersity index of 0.224 ± 0.005. The zeta potential of NLC-Citral was -12.73 ± 0.34 mV with an entrapment efficiency of 98.9 ± 0.124%, and drug loading of 9.84 ± 0.041%. Safety profile of the formulation was examined via in vitro and in vivo routes to study its effects toward normal cells. NLC-Citral exhibited no toxic effects towards the proliferation of mice splenocytes. Moreover, no mortality and toxic signs were observed in the treated groups after 28 days of treatment. There were also no significant alterations in serum biochemical analysis for all treatments. Increase in immunomodulatory effects of treated NLC-Citral and Citral groups was verified from the increase in CD4/CD3 and CD8/CD3 T cell population in both NLC-citral and citral treated splenocytes. This study suggests that NLC is a promising drug delivery system for citral as it has the potential in sustaining drug release without inducing any toxicity.

Subchronic toxicity, immunoregulation and anti-breast tumor effect of Nordamnacantal, an anthraquinone extracted from the stems of Morinda citrifolia L.

BACKGROUND: Morinda citrifolia L. that was reported with immunomodulating and cytotoxic effects has been traditionally used to treat multiple illnesses including cancer. An anthraquinone derived from fruits of Morinda citrifolia L., nordamnacanthal, is a promising agent possessing several in vitro biological activities. However, the in vivo anti-tumor effects and the safety profile of nordamnacanthal are yet to be evaluated. METHODS: In vitro cytotoxicity of nordamnacanthal was tested using MTT, cell cycle and Annexin V/PI assays on human MCF-7 and MDA-MB231 breast cancer cells. Mice were orally fed with nordamnacanthal daily for 28 days for oral subchronic toxicity study. Then, the in vivo anti-tumor effect was evaluated on 4T1 murine cancer cells-challenged mice. Changes of tumor size and immune parameters were evaluated on the untreated and nordamnacanthal treated mice. RESULTS: Nordamnacanthal was found to possess cytotoxic effects on MDA-MB231, MCF-7 and 4T1 cells in vitro. Moreover, based on the cell cycle and Annexin V results, nordamnacanthal managed to induce cell death in both MDA-MB231 and MCF-7 cells. Additionally, no mortality, signs of toxicity and changes of serum liver profile were observed in nordamnacanthal treated mice in the subchronic toxicity study. Furthermore, 50 mg/kg body weight of nordamncanthal successfully delayed the progression of 4T1 tumors in Balb/C mice after 28 days of treatment. Treatment with nordamnacanthal was also able to increase tumor immunity as evidenced by the immunophenotyping of the spleen and YAC-1 cytotoxicity assays. CONCLUSION: Nordamnacanthal managed to inhibit the growth and induce cell death in MDA-MB231 and MCF-7 cell lines in vitro and cease the tumor progression of 4T1 cells in vivo. Overall, nordamnacanthal holds interesting anti-cancer properties that can be further explored.