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Context: Zerumbone (ZER) is known to exhibit anticancer properties on various cancer cells both in vitro and in vivo. However, the anti-angiogenesis effect of ZER on liver cancers in vivo is not yet addressed clearly. Aims: This study aimed to investigate the in vivo antiproliferative and antiangiogenesis effects of ZER using rats with diethylnitrosamine-induced hepatocellular carcinoma (HCC). Materials and Methods: The antiproliferative and anti-angiogenesis effects of ZER were determined using the hepatosomatic index, vascular endothelial growth factor (VEGF) immunoassay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, histopathology, and immunohistochemistry analysis. Results: Nontreated rats with HCC had higher median liver weight than those treated with ZER or suramin. The expression of VEGF, matrix metalloprotease, and Ki-67 that were high in nontreated HCC rats was down-regulated with ZER or suramin treatments. Statistical Analysis Used: Statistical analyses were performed using the Statistical Package for Social Science version 21.0 (SPSS Inc, IBM, Maryland, USA). The data were expressed as the mean ± standard deviation and analyzed using a one-way analysis of variance. P < 0.05 was considered statistically significant. Conclusion: ZER has the potential to be developed as the pro-apoptotic and antiangiogenic agent in the treatment of HCC. [ABSTRACT FROM AUTHOR]

The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host. • High population of rats can transmit infectious diseases that are difficult to treat. • It's imperative to control the population of rats using immunocontraception. • A self-protein ZP3 localized in female ovaries plays vital role during sperm-ovum fertilization. • An immunization with ZP3 protein will induce a generalize host immune response. • Inducement of infertility in female rats could control the rate of population. [ABSTRACT FROM AUTHOR]

This study investigated the healing effects of topical application of zerumbone, a well-known anti-inflammatory compounds loaded on nanostructured lipid carrier gel (Carbopol 940) (ZER-NLCG) on excisional wounds in streptozotocin-induced diabetic rats. Diabetic rats with inflicted superficial skin wound were topically treated with ZER-NLCG, empty NLCG, and silver sulfadiazine cream (SSDC) once daily for 21 days. Wound tissue samples were analyzed for proinflammatory cytokines, namely, interleukin-6 (IL-6), interleukin-1 β (IL-1 β ), and tumor necrosis factor- α (TNF- α ), hydroxyproline contents, catalase, superoxide dismutase activities, and lipid peroxidation level, and were subjected to histopathological analysis, respectively. Among the treated groups, ZER-NLCG was the most effective at decreasing proinflammatory cytokine level and inflammatory cell infiltration while increasing antioxidant enzyme activities, hydroxyproline content, and granulation of wound tissues of diabetic rats. ZER-NLCG is a potent formulation for the enhancement of wound healing in diabetic rats through its anti-inflammatory, antioxidant, and tissue repair activities., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Shaymaa Fadhel Abbas Albaayit et al.)

Context and Objective: Zerumbone has been reported to exert anti-microbial effects, but the mechanism by which the compound exerts its action is not known. Thus, this study aimed to investigate the mechanism of action of zerumbone against methicillin-resistance Staphylococcus aureus (MRSA), using the atomic force microscopy (AFM), scanning electron microscopy (SEM), and flow cytometry techniques., Methods: MRSA (NCTC 13277) cell viability was determined using the microplate AlamarBlue assay. AFM and SEM were used to determine the morphology of zerumbone-treated MRSA cells. Flow cytometric analysis was used to determine the effect of zerumbone on bacterial membrane permeability and membrane potential, using the propidium iodide (PI) staining method, membrane potential-sensitive fluorescence probe, and DiBAC4(3) dye. DCFDA dye was used to determine the generation of reactive oxygen species (ROS) by MRSA., Results: Zerumbone significantly inhibited MRSA growth with a minimum inhibitory concentration (MIC) of 125 µg/ml. The AFM analysis showed that zerumbone caused leakage of cytoplasmic content from the bacterial cells. Ultrastructure analysis showed small colonies of the bacteria with pores on the membrane surface. There were increases in zerumbone-treated MRSA PI and DiBAC4(3) fluorescence, indicating an increase in cell membrane permeability and a decrease in membrane potential that culminated in the loss of membrane structural integrity and bacterial death. Based on DCFDA dye analysis, zerumbone also reduced ROS production by MRSA., Conclusions: Zerumbone exerts anti-MRSA effects by causing membrane depolarization, increasing membrane permeability, and finally disrupting cell membrane and bacterial killing., Competing Interests: The authors have no conflict of interests to declare.

Over the last few decades, there have been several global outbreaks of severe respiratory infections. The causes of these outbreaks were coronaviruses that had infected birds, mammals and humans. The outbreaks predominantly caused respiratory tract and gastrointestinal tract symptoms and other mild to very severe clinical signs. The current coronavirus disease-2019 (COVID-19) outbreak, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly spreading illness affecting millions of people worldwide. Among the countries most affected by the disease are the United States of America (USA), India, Brazil, and Russia, with France recording the highest infection, morbidity, and mortality rates. Since early January 2021, thousands of articles have been published on COVID-19. Most of these articles were consistent with the reports on the mode of transmission, spread, duration, and severity of the sickness. Thus, this review comprehensively discusses the most critical aspects of COVID-19, including etiology, epidemiology, pathogenesis, clinical signs, transmission, pathological changes, diagnosis, treatment, prevention and control, and vaccination., Competing Interests: The authors declare no conflicts of interest related to this work/review article., (© 2021 Rahman et al.)

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., (© 2021. The Author(s).)

Context: Clausena excavata Burm. f is a plant used in folklore medicine for the treatment of various ailments in South East Asia. The plant parts contain chemical components that are cytotoxic to many cancer cells., Objective: The study investigated the cytotoxic effects of ethyl acetate, methanol and chloroform C. excavata leaf extracts on the non-small-lung cancer, NCI-H460, cell line., Methods: Based on the 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay, among extracts, ethyl acetate C. excavata leaf extract (EACE) was the most potent anti-NCI-H460 cells, with IC50 value of 47.1 ± 6.1 μg/ml. The effects of EACE on NCI-H460 cells were also determined by clonogenic, 4', 6-diamidino-2-phenylindole (DAPI), and annexin-V-fluorescein isothiocyanate/propidium iodide-PI flow cytometric assays. Reactive oxygen species (ROS) production and apoptotic gene expressions was determined via flow cytometry and real-time quantitative PCR, respectively., Results: EACE-treated NCI-H460 cells after 48 h underwent apoptosis as evident by loss of cell viability, cell shrinkage, and chromatin condensation. The results also showed EACE mediated increase in ROS production by the NCI-H460 cells. After 48 h treatment, EACE increased the pro-apoptotic BAX and decreased the anti-apoptotic Bcl-2, Survivin and c-Myc gene expressions., Conclusions: EACE is a potential anti-lung cancer by increasing cancer cell ROS production and apoptosis., Competing Interests: We wish to confirm that there is no known conflict of interests associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Background: Thymoquinone (TQ), an active compound isolated from Nigella sativa , has been proven to exhibit various biological properties such as antioxidant. Although oral delivery of TQ is valuable, it is limited by poor oral bioavailability and low solubility. Recently, TQ-loaded nanostructured lipid carrier (TQ-NLC) was formulated with the aim of overcoming the limitations. TQ-NLC was successfully synthesized by the high-pressure homogenization method with remarkable physiochemical properties whereby the particle size is less than 100 nm, improved encapsulation efficiency and is stable up to 24 months of storage. Nevertheless, the pharmacokinetics and biodistribution of TQ-NLC have not been studied. This study determined the bioavailability of oral and intravenous administration of thymoquinone-loaded nanostructured lipid carrier (TQ-NLC) in rats and its distribution to organs., Materials and Methods: TQ-NLC was radiolabeled with technetium-99m before the administration to the rats. The biodistribution and pharmacokinetics parameters were then evaluated at various time points. The rats were imaged at time intervals and the percentage of the injected dose/gram (%ID/g) in blood and each organ was analyzed., Results: Oral administration of TQ-NLC exhibited greater relative bioavailability compared to intravenous administration. It is postulated that the movement of TQ-NLC through the intestinal lymphatic system bypasses the first metabolism and therefore enhances the relative bioavailability. However, oral administration has a slower absorption rate compared to intravenous administration where the AUC 0-∞ was 4.539 times lower than the latter., Conclusion: TQ-NLC had better absorption when administered intravenously compared to oral administration. However, oral administration showed greater bioavailability compared to the intravenous route. This study provides the pharmacokinetics and biodistribution profile of TQ-NLC in vivo which is useful to assist researchers in clinical use., Competing Interests: Prof. Dr Rasedee Abdullah reports a pending patent: Thymoquinone-loaded nanostructured lipid carriers (TQ-NLC) and uses thereof, Malaysian Patent No. P12012001818. The authors report no other potential conflicts of interest in this work., (© 2020 Zakarial Ansar et al.)

The current rampant coronavirus infection in humans, commonly known as COVID-19, a pandemic that may cause mortality in humans, has been declared a global emergency by the World Health Organization (WHO). The morbidity and mortality rates due to the pandemic are increasing rapidly worldwide, with the USA most affected by the disease. The source COVID-19 is not absolutely clear; however, the disease may be transmitted by either by COVID-19-positive individuals or from a contaminated environment. In this review, we focused on how the COVID-19 virus is transmitted in the community. An extensive literature search was conducted using specific keywords and criteria. Based on the published report, it is concluded that COVID-19 is primarily transmitted human-to-human via oral and respiratory aerosols and droplets with the virus-contaminated environment play a lesser role in the propagation of disease. Healthcare providers and the elderly with comorbidities are especially susceptible to the infection., (© 2020 The Author(s).)

Vernonia amygdalina (VA) is a medicinal tropical herb for diabetes and malaria and believed to be beneficial for joint pains. The antiosteorthritis effects of VA leaf in cartilage explant assays and on postmenopausal osteoarthritis (OA) rat model were investigated. The VA reduced the proteoglycan and nitric oxide release from the cartilage explants with interleukin 1β (IL-1β) stimulation. For the preclinical investigation, ovariectomized (OVX) female rats were grouped (n = 8) into nontreated OA, OA + diclofenac (5 mg/kg), OA + VA extract (150 and 300 mg/kg), and healthy sham control. Monosodium iodoacetate was injected into the knee joints to accelerate OA development. After 8 weeks, the macroscopic, microscopic, and histological images showed that the OA rats treated with VA 300 mg/kg and diclofenac had significantly reduced cartilage erosions and osteophytes unlike the control OA rats. The extract significantly down-regulated the inflammatory prostaglandin E2, nuclear factor κβ, IL-1β, ADAMTS-5, collagen type 10α1, and caspase3 in the OVX-OA rats. It up-regulated the anti-inflammatory IL-10 and collagen type 2α1 mRNA expressions, besides reducing serum collagenases (MMP-3 and MMP-13) and collagen type II degradation biomarker (CTX-II) levels in these rats. The VA (containing various caffeoyl-quinic acids, flavanone-O-rutinoside, luteolin, apigenin derivative and vernonioside D) suppressed inflammation, pain, collagenases as well as cartilage degradation, and improved cartilage matrix synthesis to prevent OA., (© 2019 John Wiley & Sons, Ltd.)

Clausena excavata Burm f. is used by traditional healers to treat cancer patients in South East Asia. The use of the plant and its compounds is based on Asian folklore with little or no scientific evidence supporting the therapeutic efficacy The current study aimed to determine the effect of pure clausenidin isolated from C. excavata on caspase-8-induced cell death as well as angiogenesis in the HepG2 hepatocellular carcinoma cell line. Caspase-8 and extrinsic death receptor protein expression was determined using spectrophotometry and protein profile arrays, respectively. Ultrastructural analysis of clausenidin-treated cells was conducted using transmission electron microscopy. In addition, anti-angiogenic effects of clausenidin were investigated by Western blot analysis. Clausenidin significantly (p<0.05) increased the activity of caspase-8 and expression of protein components of the death inducing signaling complex (DISC) in HepG2 cells. Ultrastructural analysis of the clausenidin-treated HepG2 cells revealed morphological abnormalities typical of apoptosis. Furthermore, clausenidin significantly (p<0.05) decreased the expression of vascular endothelial growth factor (VEGF). Therefore, clausenidin is a potential anti-angiogenic agent which may induce apoptosis of hepatocellular carcinoma cells., (Creative Commons Attribution License)

Background: Ardisia crispa Thunb. D.C is used mostly in some parts of the Asian region by traditional practitioners to treat certain diseases associated with oxidative stress and inflammation including cancer and rheumatism. In Malaysia, it is popularly known as 'Mata Ayam' and local traditional practitioners believed that the root of the plant is therapeutically beneficial., Methods: The cytotoxic effect of hydromethanolic extract of A. crispa and its solvents partitions (ethyl acetate and aqueous extracts) against breast cancer cells were evaluated by using MTT assay. The cells were treated with concentration of extracts ranging from 15.63 μg/mL- 1000 μg/mL for 72 h. The quantification of phenolic and flavonoid contents of the extracts were carried out to determine the relationship between of phytochemical compounds responsible for cytotoxic and antioxidative activities. The antioxidant capacity was measured by DPPH and ABTS free radical scavenging assay and expressed as milligram (mg) Trolox equivalent antioxidant capacity per 1 g (g) of tested extract., Results: The hydromethanolic and ethyl acetate extracts showed moderate cytotoxic effect against MCF-7 with IC 50 values of 57.35 ± 19.33 μg/mL, and 54.98 ± 14.10 μg/mL, respectively but aqueous extract was inactive against MCF-7. For MDA-MB-231, hydromethanolic, ethyl acetate and aqueous extracts exhibited weak cytotoxic effects against MDA-MB-231 with IC 50 values more than 100 μg/mL. The plant revealed high total phenolic content, total flavonoid and antioxidant capacity., Conclusion: The response of different type of breast cancer cell lines towards A. crispa extract and its partitions varied. Accordingly, hydromethanolic and ethyl acetate extracts appear to be more cytotoxic to oestrogen receptor (ER) positive breast cancer than oestrogen receptor (ER) negative breast cancer. However, aqueous extract appears to have poor activity to both types of breast cancer. Besides that, hydromethanolic and ethyl acetate extracts exhibit higher TPC, TFC and antioxidant capacity compared to aqueous extract. Synergistic effect of anticancer and antioxidant bioactives compounds of A. crispa plausibly contributed to the cytotoxic effects of the extract.

Dillenia suffruticosa, which is locally known as Simpoh air, has been traditionally used to treat cancerous growth. The ethyl acetate extract of D. suffruticosa (EADs) has been shown to induce apoptosis in MCF-7 breast cancer cells in our previous study. The present study aimed to elucidate the molecular mechanisms involved in EADs-induced apoptosis and to identify the major compounds in the extract. EADs was found to promote oxidative stress in MCF-7 cells that led to cell death because the pre-treatment with antioxidants α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of the extract (P<0.05). DCFH-DA assay revealed that treatment with EADs attenuated the generation of intracellular ROS. Apoptosis induced by EADs was not inhibited by the use of caspase-inhibitor Z-VAD-FMK, suggesting that the cell death is caspase-independent. The use of JC-1 dye reflected that EADs caused disruption in the mitochondrial membrane potential. The related molecular pathways involved in EADs-induced apoptosis were determined by GeXP multiplex system and Western blot analysis. EADs is postulated to induce cell cycle arrest that is p53- and p21-dependent based on the upregulated expression of p53 and p21 (P<0.05). The expression of Bax was upregulated with downregulation of Bcl-2 following treatment with EADs. The elevated Bax/Bcl-2 ratio and the depolarization of mitochondrial membrane potential suggest that EADs-induced apoptosis is mitochondria-dependent. The expression of oxidative stress-related AKT, p-AKT, ERK, and p-ERK was downregulated with upregulation of JNK and p-JNK. The data indicate that induction of oxidative-stress related apoptosis by EADs was mediated by inhibition of AKT and ERK, and activation of JNK. The isolation of compounds in EADs was carried out using column chromatography and elucidated using the nuclear resonance magnetic analysis producing a total of six compounds including 3-epimaslinic acid, kaempferol, kaempferide, protocatechuic acid, gallic acid and β-sitosterol-3-O-β-D-glucopyranoside. The cytotoxicity of the isolated compounds was determined using MTT assay. Gallic acid was found to be most cytotoxic against MCF-7 cell line compared to others, with IC50 of 36 ± 1.7 μg/mL (P<0.05). In summary, EADs generated oxidative stress, induced cell cycle arrest and apoptosis in MCF-7 cells by regulating numerous genes and proteins that are involved in the apoptotic signal transduction pathway. Therefore, EADs has the potential to be developed as an anti-cancer agent against breast cancer.

Ethnopharmacological Relevance: Dillenia suffruticosa (Family: Dilleniaceae) or commonly known as "Simpoh air" in Malaysia, is traditionally used for treatment of cancerous growth including breast cancer., Aim of the Study: D. suffruticosa root dichloromethane extract (DCM-DS) has been reported to induce G0/G1 phase cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 breast cancer cells. The present study was designed to investigate the involvement of p53/p21 and mitochondrial pathway in DCM-DS-treated MCF-7 cells as well as to identify the bioactive compounds responsible for the cytotoxicity of DCM-DS., Materials and Methods: Extraction of D. suffruticosa root was performed by the use of sequential solvent procedure. GeXP-based multiplex system was employed to investigate the expression of p53, p21, Bax and Bcl-2 genes in MCF-7 cells treated with DCM-DS. The protein expression was then determined using Western blot analysis. The bioactive compounds present in DCM-DS were isolated by using column chromatography. The structure of the compounds was elucidated by using nuclear magnetic resonance spectroscopy. The cytotoxicity of the isolated compounds towards MCF-7 cells was evaluated by using MTT assay. The percentage of betulinic acid (BA) in DCM-DS was determined by HPLC analysis., Results: The expression of p53 was significantly up-regulated at protein level. The expression of p21 at both gene and protein levels was significantly up-regulated upon treatment with DCM-DS, suggesting that the induction of G0/G1 phase cell cycle arrest in MCF-7 cells was via p53/p21 pathway. Bcl-2 protein was down-regulated with no change at the mRNA level, postulating that post-translational modification has occurred resulting in the degradation of Bcl-2 protein. Overall, treatment with DCM-DS increased the ratio of Bax/Bcl-2 that drove the cells to undergo apoptosis. A total of 3 triterpene compounds were isolated from DCM-DS. Betulinic acid appears to be the most major and most cytotoxic compound in DCM-DS., Conclusion: DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via p53/p21 pathway. In addition, DCM-DS induced apoptosis by increasing the ratio of Bax/Bcl-2. Betulinic acid, which is one of the major compounds, is responsible for the cytotoxicity of the DCM-DS. Therefore, BA can be used as a marker for standardisation of herbal product from D. suffruticosa. DCM-DS can also be employed as BA-rich extract from roots of D. suffruticosa for the management of breast cancer., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis.

Thymoquinone (TQ) has been shown to exhibit antitumor properties. Thymoquinone-loaded nanostructured lipid carrier (TQ-NLC) was developed to improve the bioavailability and cytotoxicity of TQ. This study was conducted to determine the cytotoxic effects of TQ-NLC on breast cancer (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa). TQ-NLC was prepared by applying the hot high pressure homogenization technique. The mean particle size of TQ-NLC was 35.66 ± 0.1235 nm with a narrow polydispersity index (PDI) lower than 0.25. The zeta potential of TQ-NLC was greater than -30 mV. Polysorbate 80 helps to increase the stability of TQ-NLC. Differential scanning calorimetry showed that TQ-NLC has a melting point of 56.73°C, which is lower than that of the bulk material. The encapsulation efficiency of TQ in TQ-NLC was 97.63 ± 0.1798% as determined by HPLC analysis. TQ-NLC exhibited antiproliferative activity towards all the cell lines in a dose-dependent manner which was most cytotoxic towards MDA-MB-231 cells. Cell shrinkage was noted following treatment of MDA-MB-231 cells with TQ-NLC with an increase of apoptotic cell population (P < 0.05). TQ-NLC also induced cell cycle arrest. TQ-NLC was most cytotoxic towards MDA-MB-231 cells. It induced apoptosis and cell cycle arrest in the cells.

Background: Waterlily Mango (Mangifera indica L.) is thought to be antioxidant-rich, conferred by its functional phytochemicals., Methods: The potential anticancer effects of the ethanolic kernel extract on breast cancer cells (MDA-MB-231 and MCF-7) using MTT, anti-proliferation, neutral red (NR) uptake and lactate dehydrogenase (LDH) release assays were evaluated. Cytological studies on the breast cancer cells were also conducted, and phytochemical analyses of the extract were carried out to determine the likely bioactive compounds responsible for such effects., Results: Results showed the extract induced cytotoxicity in MDA-MB-231 cells and MCF-7 cells with IC50 values of 30 and 15 μg/mL, respectively. The extract showed significant toxicity towards both cell lines, with low toxicity to normal breast cells (MCF-10A). The cytotoxic effects on the cells were further confirmed by the NR uptake, antiproliferative and LDH release assays. Bioactive analyses revealed that many bioactives were present in the extract although butylated hydroxytoluene, a potent antioxidant, was the most abundant with 44.65%., Conclusions: M. indica extract appears to be more cytoxic to both estrogen positive and negative breast cancer cell lines than to normal breast cells. Synergistic effects of its antioxidant bioactives could have contributed to the cytotoxic effects of the extract. The extract of M. indica, therefore, has potential anticancer activity against breast cancer cells. This potential is worth studying further, and could have implications on future studies and eventually management of human breast cancers.

Background: Dillenia suffruticosa root dichloromethane extract (DCM-DS) has been reported to exhibit strong cytotoxicity towards breast cancer cells. The present study was designed to investigate the cell cycle profile, mode of cell death and signalling pathways of DCM-DS-treated human caspase-3 deficient MCF-7 breast cancer cells., Methods: Dillenia suffruticosa root was extracted by sequential solvent extraction. The anti-proliferative activity of DCM-DS was determined by using MTT assay. The mode of cell death was evaluated by using inverted light microscope and Annexin-V/PI-flow cytometry analysis. Cell cycle analysis and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry. MCF-7 cells were co-treated with antioxidants α-tocopherol and ascorbic acid to evaluate whether the cell death was mainly due to oxidative stress. GeXP-based multiplex system was employed to investigate the expression of apoptotic, growth and survival genes in MCF-7 cells. Western blot analysis was performed to confirm the expression of the genes., Results: DCM-DS was cytotoxic to the MCF-7 cells in a time-and dose-dependent manner. The IC50 values of DCM-DS at 24, 48 and 72 hours were 20.3 ± 2.8, 17.8 ± 1.5 and 15.5 ± 0.5 μg/mL, respectively. Cell cycle analysis revealed that DCM-DS induced G0/G1 and G2/M phase cell cycle arrest in MCF-7 cells at low concentration (12.5 and 25 μg/mL) and high concentration (50 μg/mL), respectively. Although Annexin-V/PI-flow cytometry analysis has confirmed that DCM-DS induced apoptosis in MCF-7 cells, the distinct characteristics of apoptosis such as membrane blebbing, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies were not observed under microscope. DCM-DS induced formation of ROS in MCF-7 cells. Nevertheless, co-treatment with antioxidants did not attenuate the cell death at low concentration of DCM-DS. The pro-apoptotic gene JNK was up-regulated whereby anti-apoptotic genes AKT1 and ERK1/2 were down-regulated in a dose-dependent manner. Western blot analysis has confirmed that DCM-DS significantly up-regulated the expression of pro-apoptotic JNK1, pJNK and down-regulated anti-apoptotic AKT1, ERK1 in MCF-7 cells., Conclusion: DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via multiple signalling pathways. It shows the potential of DCM-DS to be developed to target the cancer cells with mutant caspase-3.

Ethnopharmacological Relevance: The fruit hull of Garcinia mangostana Linn. has been used in traditional medicine for treatment of various inflammatory diseases. Hence, this study aims to investigate the in vitro and in vivo anti-inflammatory effect of β mangostin (βM), a major compound present in Garcinia mangostana., Materials and Methods: The in silico analysis of inflammatory mediators such as cyclooxygenase (COX) and nuclear factor-kappa B (NF-kB) were performed via molecular docking. Further evaluation of anti-inflammatory effect was conducted in lipopolysaccharide (LPS) induced RAW 264.7 macrophages. Suppression of activated NF-kB was analyzed by high content screening. βM triggered inhibition of COX-1 and COX-2 in vitro were studied using biochemical kit. The in vivo model used in this study was carrageenan-induced peritonitis model, where reduction in carrageenan-induced peritonitis is measured by leukocyte migration and vascular permeability. In addition, the evaluation of βM׳s effect on carrageenan induced TNF-α and IL-1β release on peritoneal fluid was also carried out., Results: Treatment with βM could inhibit the LPS-induced NO production but not the viability of RAW 264.7. Similarly, βM inhibited PGE2 production and the cytokines: TNF-α and IL-6. The COX catalyzed prostaglandin biosynthesis assay had showed selective COX-2 inhibition with a 53.0±6.01% inhibition at 20 µg/ml. Apart from this, βM was capable in repressing translocation of NF-kB into the nucleus. These results were concurrent with molecular docking which revealed COX-2 selectivity and NF-kB inhibition. The in vivo analysis showed that after four hours of peritonitis, βM was unable to reduce vascular permeability, yet could decrease the total leukocyte migration; particularly, neutrophils. Meanwhile, dexamethasone 0.5 mg/kg, successfully reduced vascular permeability. The levels of TNF-α and IL-1β in peritoneal fluid was reduced significantly by βM treatment., Conclusion: The current study supports the traditional use of Garcinia mangostana fruit hull for treatment of inflammatory conditions. In addition, it is clear that the anti-inflammatory efficacy of this plant is not limited to the presence of α and γ, but β also with significant activity., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Breast cancer is one of the most dreading types of cancer among women. Herbal medicine has becoming a potential source of treatment for breast cancer. Herbal plant Dillenia suffruticosa (Griff) Martelli under the family Dilleniaceae has been traditionally used to treat cancerous growth. In this study, the anticancer effect of ethyl acetate extract of D. suffruticosa (EADs) was examined on human breast adenocarcinoma cell line MCF-7 and the molecular pathway involved was elucidated., Methods: EADs was obtained from the root of D. suffruticosa by using sequential solvent extraction. Cytotoxicity was determined by using MTT assay, mode of cell death by cell cycle analysis and apoptosis induction by Annexin-FITC/PI assay. Morphology changes in cells were observed under inverted light microscope. Involvement of selected genes in the oxidative stress-mediated signaling pathway was explored using multiplex gene expression analysis., Results: The treatment of EADs caused cytotoxicity to MCF-7 cells in a dose- and time-dependent manner at 24, 48 and 72 hours with IC50 of 76 ± 2.3, 58 ± 0.7 and 39 ± 3.6 μg/mL, respectively. The IC50 of tamoxifen-treated MCF-7 cells was 8 ± 0.5 μg/mL. Induction of apoptosis by EADs was dose- and time- dependent. EADs induced non-phase specific cell cycle arrest at different concentration and time point. The multiplex mRNA expression study indicated that EADs-induced apoptosis was accompanied by upregulation of the expression of SOD1, SOD2, NF-κB, p53, p38 MAPK, and catalase, but downregulation of Akt1., Conclusion: It is suggested that EADs induced apoptosis in MCF-7 cells by modulating numerous genes which are involved in oxidative stress pathway. Therefore, EADs has the potential to act as an effective intervention against breast cancer cells.

Clausena excavata (Lour.), locally known as "Kemantu hitam," is a common plant in Malaysian folklore medicine. This study evaluated the antioxidant properties of the solvent extracts of C. excavata leaves and determined the acute toxicity of methanolic extract C. excavata (MECE) leaves in Sprague-Dawley rats. Harvested leaves were dried and subjected to solvent extraction using petroleum ether, chloroform, ethyl acetate and methanol in succession. The antioxidant activity of each extract was determined using the ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl dihydrazyl (DPPH) radical scavenging activity. The total phenolic content (TPC) and total flavonoids content (TFC) were estimated by Folin-Ciocalteu and ethanolic aluminium chloride method, respectively. The chloroform extract was found to be highest in flavonoid content, while the methanolic extract showed the highest TPC and antioxidant activity. There was no mortality in rats treated with MECE leaves even at a high dose of 5000 mg/kg body weight. However, the MECE leaves produced mild to moderate pathological changes in the liver and kidneys, shown by mild degenerative changes and leucocyte infiltration. The extract did not affect the haematological parameters or relative weights of the liver or kidneys. Overall, the MECE leaves have potent antioxidant activity and are presumed safe to be used orally as health-promoting product at low to moderate doses.

Nanotechnology has provided new technological opportunities, which could help in challenges confronting stem cell research. Polyamidoamine (PAMAM) dendrimers, a new class of macromolecular polymers with high molecular uniformity, narrow molecular distribution specific size and shape and highly functionalised terminal surface have been extensively explored for biomedical application. PAMAM dendrimers are also nanospherical, hyperbranched and monodispersive molecules exhibiting exclusive properties which make them potential carriers for drug and gene delivery., (© 2013 International Federation for Cell Biology.)

Ethnopharmacological Relevance: Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer., Materials and Methods: To investigate the apoptosis mechanism, we isolated dentatin (DTN) from this plant using a bioassay-guided approach. DTN-induced cytotoxicity was observed with the MTT assay. Acridine orange/propidium iodide staining was used to detect cells in early apoptosis and high content screening (HCS) to observe nuclear condensation, cell permeability, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed with a clonogenic assay, DNA laddering and caspase 3/7 and 9 assays. Reactive oxygen species (ROS) formation, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. The involvement of nuclear factor-kappa B (NF-κB) was analysed with the HCS assay., Results: A significant increase in chromatin condensation in the cell nucleus was observed by fluorescence analysis. Apoptosis was confirmed by the reduced number of colonies in the clonogenic assay and the increased number of cellular DNA breaks in treated cells observed as a DNA ladder. Treatment of MCF-7 cells with DTN encouraged apoptosis with cell death-transducing signals that reduced MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase 9 followed by the executioner caspase 3/7. DTN treatment significantly arrested MCF-7 cells at the G0/G1 phase (p<0.05) and ROS was significantly elevated. Moreover, DTN significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus., Conclusion: Together, the results demonstrated that the DTN isolated from Clausena excavata inhibited the proliferation of MCF-7 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway with involvement of the NF-κB signalling pathway., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The tropical edible red seaweed (Eucheuma cottonii L.) is rich in nutrients and polyphenolic compounds that may suppress cancer through its antioxidant and antiproliferative properties. The study reports on rat mammary tumor suppression and tissue antioxidant status modulation by E. cottonii ethanol extract (ECE). The effect of orally administered ECE (100 mg/kg body-weight) was compared with that of tamoxifen (10 mg/kg body-weight). Rat was induced to develop mammary tumor with subcutaneous injection of LA-7 cells (6 × 10(6) cells/rat). The ECE was more effective than tamoxifen in suppressing tumor growth (27%), improving tissues (plasma, liver, and kidney) malondialdehyde concentrations, superoxide dismutase activity and erythrocyte glutathione concentrations (P < 0.05). Unlike tamoxifen, the ECE displayed little toxicity to the liver and kidneys. The ECE exhibited strong anticancer effect with enzyme modulating properties, suggesting its potential as a suppressing agent for mammary gland tumor.

Background: Nanostructured lipid carriers (NLCs), composed of solid and liquid lipids, and surfactants are potentially good colloidal drug carriers. Thymoquinone is the main bioactive compound of Nigella sativa. In this study, the preparation, gastroprotective effects, and pharmacokinetic (PK) properties of thymoquinone (TQ)-loaded NLCs (TQNLCs) were evaluated., Method: TQNLCs were prepared using hydrogenated palm oil (Softisan® 154), olive oil, and phosphatidylcholine for the lipid phase and sorbitol, polysorbate 80, thimerosal, and double distilled water for the liquid lipid material. A morphological assessment of TQNLCs was performed using various methods. Analysis of the ulcer index, hydrogen concentration, mucus content, and biochemical and histochemical studies confirmed that the loading of TQ into the NLCs significantly improved the gastroprotective activity of this natural compound against the formation of ethanol-induced ulcers. The safety of TQNLC was tested on WRL68 liver normal cells with cisplatin as a positive control., Results: The average diameter of the TQNLCs was 75 ± 2.4 nm. The particles had negative zeta potential values of -31 ± 0.1 mV and a single melting peak of 55.85°C. Immunohistochemical methods revealed that TQNLCs inhibited the formation of ethanol-induced ulcers through the modulation of heat shock protein-70 (Hsp70). Acute hepatotoxic effects of the TQNLCs were not observed in rats or normal human liver cells (WRL-68). After validation, PK studies in rabbits showed that the PK properties of TQ were improved and indicated that the drug behaves linearly. The Tmax, Cmax, and elimination half-life of TQ were found to be 3.96 ± 0.19 hours, 4811.33 ± 55.52 ng/mL, and 4.4933 ± 0.015 hours, respectively, indicating that TQ is suitable for extravascular administration., Conclusion: NLCs could be a promising vehicle for the oral delivery of TQ and improve its gastroprotective properties.

This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F) against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1). The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS) and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin), leading to disruption of mitochondrial membrane potential (MMP), cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.

The plant Typhonium flagelliforme, commonly known as "rodent tuber" in Malaysia, is often used as a health supplement and traditional remedy for alternative cancer therapies, including leukemia. This study aimed to evaluate in vitro anti-leukemic activity of dichloromethane extract/fraction number 7 (DCM/F7) from T. flagelliforme tuber on human T4 lymphoblastoid (CEMss) cell line. The DCM extract of tuber has been fractionated by column chromatography. The obtained fractions were evaluated for its cytotoxicity toward CEMss cells as well as human primary blood lymphocytes (PBLs). Assessment of apoptosis produced by the most active fraction was evaluated by various microscopic techniques and further confirmation of apoptosis was done by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Phytochemical screening was done by gas chromatography-mass spectrometry (GC-MS). The results shows that 7 out of 12 fractions showed significant cytotoxicity against the selected cell line CEMss, in which fractions DCM/F7, DCM/F11 and DCM/F12 showed exceptional activity with 3, 5 and 6.2 μg ml(-1), respectively. Further studies in the non-cancerous PBL exhibited significant selectivity of DCM/F7 compared to other fractions. Cytological observations showed chromatin condensation, cell shrinkage, abnormalities of cristae, membrane blebbing, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double-staining of acridine orange (AO)/propidium iodide (PI), SEM and TEM. In addition, DCM/F7 has increased the cellular DNA breaks on treated cells. GC-MS revealed that DCM/F7 contains linoleic acid, hexadecanoic acid and 9-hexadecanoic acid. The present results indicate that T. flagelliforme possess a valuable anti-leukemic effect and was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

Typhonium flagelliforme (TF) is a tropical plant, traditionally used by the ethnic population of Malaysia for the cure of various cancers. This plant had shown to induce antiproliferative effect as well as apoptosis in cancer cells. However, there is no available information to address that TF affects murine leukemia cells in vitro and in vivo. Here, we investigated in vitro and in vivo effects of TF on murine leukemia WEHI-3 cells. It was found that the growth of leukemia cells in vitro was inhibited by the various extracts of TF. Among these fractions, the dichloromethane (DCM) tuber extracts of TF showed the lowest IC(50) (24.0 ± 5.2 μg/ml) and had demonstrated apoptogenic effect when observed under fluorescent microscope. We investigated the in vivo effects of DCM tuber extracts of TF on murine leukemia cells, and the results showed that the counts of immature granulocytes and monocytes were significantly decreased in peripheral blood of BALB/c leukemia mice after the oral administration of DCM tuber extracts of TF for 28 days with three doses (200, 400 and 800 mg/kg). These results were confirmed by observing the spleen histopathology and morphology of enlarged spleen and liver in leukemia mice when compared with the control. Furthermore, the cell death mechanism in the spleen tissue of treated mice was found via apoptosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Ethnopharmacological Relevance: The plant Typhonium flagelliforme (TF), commonly known as 'rodent tuber' in Malaysia, is often used as traditional remedy for cancer, including leukemia., Aim of the Study: We had previously identified morphologically that the linoleic acid rich fraction (DCM/F7) from the tubers of this plant induces selective anti-proliferative effects and apoptosis in CEMss cells. In this present study, we subjected the same DCM/F7 fraction to cell based activity analyses in order to determine the possible mechanism of cell death in leukemic CEMss cells in vitro., Materials and Methods: Extraction of Typhonium flagelliforme tuber has done and fractionation has been done by vacuum liquid column chromatography. The anti-proliferative activity was assayed using MTT and the apoptosis detection was done by Annexin V and DNA laddering assay. Colorimetric caspase assay and immunoblot analysis were employed to detect the expression of protein associated with cell death. Cell cycle analysis was done using flow cytometry., Results: We found that the cancer inhibitory effect of the DCM/F7 fraction in CEMss cells was 3 ± 0.08 μg/ml (IC(50)). An early apoptotic induction in CEMss cells was observed by Annexin V assay, which showed a clear dose-dependent DNA fragmentation being observed in gel electrophoresis at 10 and 20 μg/ml. The DCM/F7 fraction at 3 μg/ml significantly arrested CEMss cells at G0/G1 phase (p<0.05). A constant but increasing pattern-related Sub-G0/G1 index was observed between 12 and 72 h treatment. In relation to this, we further investigated the biochemical events leading to cell death and found that the DCM/F7 fraction increased the cellular levels of caspase-3 and -9 on treated cells. Our results indicated that cytochrome c from mitochondria into the cytosol increased gradually as the DCM/F7 concentration increases, which later lead to the subsequent cleavage of PARP in to 85kDa fragments. On the contrary, Bcl-2 protein was found to decrease concomitantly during treatment., Conclusions: Collectively, results presented in this study demonstrated that the DCM/F7 fraction inhibited the proliferation of leukemia cells, leading to the programmed cell death, which was confirmed to be through the mitochondrial pathway., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P<0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P<0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P<0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P<0.05) lowered. By the TUNEL assay, there were significantly (P<0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)