Pressurised hot water extraction (PHWE) of natural colorants from the heartwood of Xylocarpus Moluccensis and its dyeing characteristics on different fabrics / Nursyamirah Abd Razak

A remarkable growth of public awareness towards environmental pollution caused by the synthetic dyes resulted in an increased demand of natural dyes which led to the exploration of potential plants as source for natural dyes. Traditionally, natural dyes were extracted by soaking or boiling method which requires long extraction time and plenty of water. Hence, there is a demand to exploit suitable efficient techniques to extract natural dyes from the plant materials. One of the potential plants is Xylocarpus moluccensis (Nyireh batu) species which can be found in mangroves forest around Peninsular Malaysia. In this study, a pressurised hot water extraction (PHWE) technique was employed to selectively extract natural dyes from the Xylocarpus moluccenisheartwood at different elevated temperature from 50 °C to 150 °C. This technique is more efficient and environmental friendly method and may increase productivity of natural dyes for dyeing fabrics. The dye extracts were then used to dye fabrics such as cotton, silk and viscose rayon with the addition of 3% mordant such as paddy husk ash (PHA). vinegar and alum. Each of dyed fabrics was analysed to determine its dyeability and dyeing properties. Based on the experiment, colour attained (h°) on cotton and viscose rayon was varied from 3° to 48° and 6° to 48° respectively. However, the h° of silk fabrics ranges between 40° to 48° (brownish colour). PHA and vinegar were observed to give high colour intensity (C*) and colour strength (K/S) to dyed silk compared to alum. In contrast, cotton and viscose rayon had better colour strength (K/S) and colour intensity (C*) when mordanted with alum compared to natural mordant. Based on colour fastness test, viscose rayon had the best colour fastness to washing and perspiration compared to cotton and silk although the K/S and the C*were low. The colorant compounds were extracted by soaking with methanol and dichloromethane followed by purification on a polyamide solid phase extraction cartridge prior togas chromatography mass spectrometry (GC-MS) and Fourier Transform Infra Red (FT-IR) spectroscopy analysis. It was found that,the heartwood extracts contained mainly quinoline and anthraquinone moieties in their molecules

Induction of apoptosis and regulation ofMicroRNA expression by (2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)- cyclohexanone (BHMC) treatment on MCF-7 breast cancer cells

(2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) is a synthetic curcumin analogue, which has been reported to possess anti-tumor, anti-metastatic, and anti-invasion properties on estrogen receptor (ER) negative breast cancer cells in vitro and in vivo. However, the cytotoxic effects of BHMC on ER positive breast cancer cells were not widely reported. This study was aimed to investigate the cytotoxic potential of BHMC on MCF-7 cells using cell viability, cell cycle, and apoptotic assays. Besides, microarray and quantitative polymerase chain reaction (qPCR) were performed to identify the list of miRNAs and genes, which could be dysregulated following BHMC treatment. The current study discovered that BHMC exhibits selective cytotoxic effects on ER positive MCF-7 cells as compared to ER negative MDA-MB-231 cells and normal breast cells, MCF-10A. BHMC was shown to promote G2/M cell cycle arrest and apoptosis in MCF-7 cells. Microarray and qPCR analysis demonstrated that BHMC treatment would upregulate several miRNAs like miR-3195 and miR-30a-3p and downregulate miRNAs such as miR-6813-5p and miR-6132 in MCF-7 cells. Besides, BHMC administration was also found to downregulate few tumor-promoting genes like VEGF and SNAIL in MCF-7. In conclusion, BHMC induced apoptosis in the MCF-7 cells by altering the expressions of apoptotic-regulating miRNAs and associated genes

Anti-breast cancer effect of eupatorin in vitro and in vivo

Triple-negative breast cancers (TNBC) represent the most aggressive form of breast cancers and their treatments are challenging due to the tumour heterogeneity and chemo-resistance which have been the major obstacle. Chemotherapy using synthesized drugs is commonly used to treat breast cancer. However, this kind of treatment may cause negative side effects to the patients. Recently, eupatorin had been reported to be a potent candidate to inhibit breast cancer cells proliferation. Eupatorin is a product that belongs to a flavonoid family. However, the detailed information on the efficacy and mechanisms of eupatorin as an anti-breast cancer agent in vitro and in vivo is very limited. In this study, the cytotoxic effect, anti-proliferative action, cell cycle arrest and apoptosis induction of eupatorin on the human breast carcinoma cell lines of MDA-MB-231 (estrogen-receptor negative), MCF-7 (estrogen-receptor positive) and on a cell line derived from normal mammary tissue, MCF-10a were studied. In addition, the in vivo antitumour effect of eupatorin was tested on the Balb/c mice challenged with 4T1 murine mammary cancer cells. MTT assay showed that eupatorin and tamoxifen (positive control) had cytotoxicity effects against the cancerous cell lines of MCF-7, MDA-MB-231 and 4T1 cells. At 48 hours, eupatorin at 14.52 μΜ inhibited cell proliferation of MCF-7 and MDA-MB-231 by 50%. However, the IC50 of tamoxifen at 48 hours to inhibit MCF-7 and MDA-MB-231 cells proliferation was 5.23 μM and 7.22 μM, respectively. In contrast, the IC50 value of eupatorin at 48 hours for MCF-10a was significantly (p<0.05) high with 58.09 μM. Through scratch assay, eupatorin (14.52 μM) prohibited the complete closure of scratched area in MDA-MB-231 cells after 24 hours incubation. In addition, Boyden chamber assay revealed that eupatorin at 14.52 μM inhibited the aggressiveness of MDA-MB-231 cells where less than 40% of the cells were migrating and invading the membrane in the Boyden chamber. Moreover, ex vivo model using aortic ring from Balb/c mouse suggested that eupatorin can act as anti-angiogeneic due to the inhibition of aortic sprouting activation in mouse aortic rings assay. In cell cycle analysis using MCF-7 and MDA-MB-231 cells, eupatorin enhanced the accumulation of cells in sub Gθ/G1. At 48 hours, the number of MCF-7 and MDA-MB-231 cells accumulated in sub Gθ/G1 was 27.52 ± 2.06% and 42.75 ± 4.67% respectively. Concurrently, the percentage of early apoptotic of respective MCF-7 and MDA-MB-231 cells were 28.38 ± 0.24% and 64.04 ± 0.66%. In addition, the population of late apoptotic cells of MCF-7 and MDA-MB-231 were 40.26 ± 0.33% and 18.27 ± 0.57%, respectively. In contrast, more than 95% of the untreated cells for both cell types were distributed in Gθ/G1 phase at 48 hours. In gene expression assay, eupatorin up-regulated the pro-apoptotic genes such as Bak1, Bax, cytochrome c, SMAC/Diablo and HIF1A in both cell lines and concurrently down-regulated the anti-apoptotic genes such as VEGFA and Bcl2L11. Furthermore, Western Blot analysis revealed that eupatorin could inhibit cells proliferation in MDA-MB-231 and MCF-7 cells through depletion of Cdc2 and Chk1 protein level respectively followed by the activation of Chk2 protein which lead to the Akt and MAPK signalling pathway blockage. Moreover, caspases activation confirmed that eupatorin initiated apoptosis via intrinsic pathway in MCF-7 and MDA-MB-231 cells due to high fold change number of activated caspase 9 compared to caspase 8. In vivo study showed that eupatorin at the dosage of 20 mg/kg was sufficient to delay the tumour development. Histologic assessment revealed that eupatorin (20 mg/kg) has significantly (p<0.05) enhanced the number of apoptotic cells in tumour. Additionally, clonogenic assay showed that eupatorin (20 mg/kg) has potently reduced the number of invaded 4T1 cells in lung where 30.70 x 103 ± 10.89 blue colonies were detected whereas the untreated lung possessed 400.00 x 103 ± 28.28 blue colonies. Besides, eupatorin (20 mg/kg) also has significantly (p<0.05) increased the NK cells and T-cell responses to 4T1 tumour cells in splenocytes assay. In immunophenotyping assessment, eupatorin (20 mg/kg) enhanced the stimulation of NK1.1+CD3 and CD8+ expression by 4.75 ±0.37% and 8.83 ±0.36% respectively when compared to the untreated (NK1.1+CD3 :3.57% ± 0.13; CD8+ :4.30%±0.07). Cytokine assay revealed that IL-1β was significantly (p<0.05) suppressed to 812.00 ± 57.50 pg/mL while the IFN-γ was enhanced to 1407.41 ± 25.66 pg/mL compared to the untreated (IL-1β :1045.33 ± 257.98 pg/mL; IFN-γ :1185.19 ± 187.86 pg/mL). Western blot analysis showed that eupatorin (20 mg/kg) could delay the tumour progression, invasion and migration through MAPK pathway blocking. Additionally, gene expression assay revealed that eupatorin (20 mg/kg) inhibited the regulation of gene MMP9, TNFα, and IL-1β expression sufficiently.Taken together, eupatorin is a potent candidate as anti-breast cancer agent that has anti-proliferative and anti-metastasis activities on breast cancer cells which should be examined further in clinical study

The In Vivo Anti-Tumor Effect of Curcumin Derivative (2E,6E)-2,6-bis(4-hydroxy-3-methoxybenzylidene) Cyclohexanone (BHMC) on 4T1 Breast Cancer Cells

Curcumin is one of the promising natural products extracted from the rhizomes of curcuma longa and has been extensively investigated by researchers to explore its potential as a chemopreventive and therapeutic agent against several chronic diseases. To further enhance the cytotoxic potential of curcumin, its derivative (2E,6E)-2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (BHMC) has been synthesized and investigated, and its antitumor effect on tested on 4T1 challenged mice. BHMC was recorded with in vitro cytotoxicity on murine 4T1 breast cancer cells with IC50 value 13.66 mM, which was 2 times lower than curcumin after 72 hours of treatment. An in vivo study indicated that BHMC possessed antitumor effect on the 4T1 cells of the challenged mice by induction of apoptosis, antiproliferation, antiinflammation and antimetastasis. This effect is better compared to curcumin treatment at the same evaluated concentration. Thus, BHMC is a potential antitumor agent against breast cancer

Eupatorin suppressed tumour progression and enhanced immunity in a 4T1 murine breast cancer model

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti–breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti–breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1β. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model

The in vivo anti-tumor effect of curcumin derivative (2 E,6 E)-2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (BHMC) on 4T1 breast cancer cells

Curcumin is one of the promising natural products extracted from the rhizomes of curcuma longa and has been extensively investigated by researchers to explore its potential as a chemopreventive and therapeutic agent against several chronic diseases. To further enhance the cytotoxic potential of curcumin, its derivative (2E,6E)-2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (BHMC) has been synthesized and investigated, and its antitumor effect on tested on 4T1 challenged mice. BHMC was recorded with in vitro cytotoxicity on murine 4T1 breast cancer cells with IC50 value 13.66 μM, which was 2 times lower than curcumin after 72 hours of treatment. An in vivo study indicated that BHMC possessed antitumor effect on the 4T1 cells of the challenged mice by induction of apoptosis, antiproliferation, anti-inflammation and antimetastasis. This effect is better compared to curcumin treatment at the same evaluated concentration. Thus, BHMC is a potential antitumor agent against breast cancer

Induction of apoptosis and regulation of MicroRNA expression by (2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) treatment on MCF-7 breast cancer cells

(2E,6E)-2,6-bis-(4-hydroxy-3-methoxybenzylidene)-cyclohexanone (BHMC) is a synthetic curcumin analogue, which has been reported to possess anti-tumor, anti-metastatic, and anti-invasion properties on estrogen receptor (ER) negative breast cancer cells in vitro and in vivo. However, the cytotoxic effects of BHMC on ER positive breast cancer cells were not widely reported. This study was aimed to investigate the cytotoxic potential of BHMC on MCF-7 cells using cell viability, cell cycle, and apoptotic assays. Besides, microarray and quantitative polymerase chain reaction (qPCR) were performed to identify the list of miRNAs and genes, which could be dysregulated following BHMC treatment. The current study discovered that BHMC exhibits selective cytotoxic effects on ER positive MCF-7 cells as compared to ER negative MDA-MB-231 cells and normal breast cells, MCF-10A. BHMC was shown to promote G2/M cell cycle arrest and apoptosis in MCF-7 cells. Microarray and qPCR analysis demonstrated that BHMC treatment would upregulate several miRNAs like miR-3195 and miR-30a-3p and downregulate miRNAs such as miR-6813-5p and miR-6132 in MCF-7 cells. Besides, BHMC administration was also found to downregulate few tumor-promoting genes like VEGF and SNAIL in MCF-7. In conclusion, BHMC induced apoptosis in the MCF-7 cells by altering the expressions of apoptotic-regulating miRNAs and associated genes