Analysis of bioactive compounds in cinnamon leaves and preparation of nanoemulsion and byproducts for improving Parkinson's disease in rats.

Introduction: Cinnamomum osmophloeum Kanehira (C. osmophloeum), a broad-leaved tree species of Taiwan, contains phenolic acids, flavonoids, and phenylpropanoids such as cinnamaldehyde and cinnamic acid in leaves. Many reports have shown that the cinnamon leaf extract possesses anti-inflammatory, hypoglycemic, hypolipidemic and neuroprotective functions. This study aims to analyze bioactive compounds in C. osmophloeum (cinnamon leaves) by UPLC-MS/MS and prepare hydrosol, cinnamon leaf extract and cinnamon leaf nanoemulsion for comparison in improving Parkinson's disease (PD) in rats. Methods: After extraction and determination of total phenolic and total flavonoid contents, cinnamaldehyde and the other bioactive compounds were analyzed in cinnamon leaves and hydrosol by UPLC-MS/MS. Cinnamon leaf nanoemulsion was prepared by mixing a suitable proportion of cinnamon leaf extract, soybean oil, lecithin, Tween 80 and deionized water, followed by characterization of particle size and polydispersity index by dynamic light scattering analyzer, particle size and shape by transmission electron microscope, encapsulation efficiency, as well as storage and heating stability. Fifty-six male Sprague-Dawley rats aged 8 weeks were divided into seven groups with group 1 as control (sunflower oil) and group 2 as induction (2 mg/kg bw rotenone in sunflower oil plus 10 mL/kg bw saline), while the other groups including rotenone injection (2 mg/kg bw) followed by high-dose of 60 mg/kg bw (group 3) or low-dose of 20 mg/kg bw (group 4) for tube feeding of cinnamon leaf extract or cinnamon leaf nanoemulsion at the same doses (groups 5 and 6) every day for 5 weeks as well as group 7 with rotenone plus hydrosol containing 0.5 g cinnamon leaf powder at a dose of 10 mL/kg bw. Biochemical analysis of brain tissue (striatum and midbrain) was done to determine dopamine, α-synuclein, tyrosine hydroxylase, superoxide dismutase, catalase, glutathione peroxidase and malondialdehyde contents by using commercial kits, while catalepsy performed by bar test. Results and discussion: An extraction solvent of 80% ethanol was found to be the most optimal with a high yield of 15 bioactive compounds being obtained following UPLC analysis. A triple quadrupole tandem mass spectrometer with electrospray ionization mode was used for identification and quantitation, with cinnamaldehyde present at the highest amount (17985.2 µg/g). The cinnamon leaf nanoemulsion was successfully prepared with the mean particle size, zeta potential, polydispersity index and encapsulation efficiency being 30.1 nm, -43.1 mV, 0.149 and 91.6%, respectively. A high stability of cinnamon leaf nanoemulsion was shown over a 90-day storage period at 4 and heating at 100 for 2 h. Animal experiments revealed that the treatments of cinnamon leaf extract, nanoemulsion and hydrosol increased the dopamine contents from 17.08% to 49.39% and tyrosine hydroxylase levels from 17.07% to 25.59%, while reduced the α-synuclein levels from 17.56% to 15.95% in the striatum of rats. Additionally, in the midbrain of rats, an elevation of activities of superoxide dismutase (6.69-16.82%), catalase (8.56-16.94%), and glutathione peroxidase (2.09-16.94%) was shown, while the malondialdehyde content declined by 15.47-22.47%. Comparatively, the high-dose nanoemulsion exerted the most pronounced effect in improving PD in rats and may be a promising candidate for the development of health food or botanic drug.

Quantification of Xanthone and Anthocyanin in Mangosteen Peel by UPLC-MS/MS and Preparation of Nanoemulsions for Studying Their Inhibition Effects on Liver Cancer Cells.

Mangosteen peel, a waste produced during mangosteen processing, has been reported to be rich in xanthone and anthocyanin, both of which possess vital biological activities such as anti-cancer properties. The objectives of this study were to analyze various xanthones and anthocyanins in mangosteen peel by UPLC-MS/MS for the subsequent preparation of both xanthone and anthocyanin nanoemulsions to study their inhibition effects on liver cancer cells HepG2. Results showed that methanol was the optimal solvent for the extraction of xanthones and anthocyanins, with a total amount of 68,543.39 and 2909.57 µg/g, respectively. A total of seven xanthones, including garcinone C (513.06 µg/g), garcinone D (469.82 µg/g), γ-mangostin (11,100.72 µg/g), 8-desoxygartanin (1490.61 µg/g), gartanin (2398.96 µg/g), α-mangostin (51,062.21 µg/g) and ß-mangostin (1508.01 µg/g), as well as two anthocyanins including cyanidin-3-sophoroside (2889.95 µg/g) and cyanidin-3-glucoside (19.72 µg/g), were present in mangosteen peel. The xanthone nanoemulsion was prepared by mixing an appropriate portion of soybean oil, CITREM, Tween 80 and deionized water, while the anthocyanin nanoemulsion composed of soybean oil, ethanol, PEG400, lecithin, Tween 80, glycerol and deionized water was prepared as well. The mean particle size of the xanthone extract and nanoemulsion were, respectively, 22.1 and 14.0 nm as determined by DLS, while the zeta potential was -87.7 and -61.5 mV. Comparatively, xanthone nanoemulsion was more effective than xanthone extract in inhibiting the growth of HepG2 cells, with the IC50 being 5.78 µg/mL for the former and 6.23 µg/mL for the latter. However, the anthocyanin nanoemulsion failed to inhibit growth of HepG2 cells. Cell cycle analysis revealed that the proportion of the sub-G1 phase followed a dose-dependent increase, while that of the G0/G1 phase showed a dose-dependent decline for both xanthone extracts and nanoemulsions, with the cell cycle being possibly arrested at the S phase. The proportion of late apoptosis cells also followed a dose-dependent rise for both xanthone extracts and nanoemulsions, with the latter resulting in a much higher proportion at the same dose. Similarly, the activities of caspase-3, caspase-8 and caspase-9 followed a dose-dependent increase for both xanthone extracts and nanoemulsions, with the latter exhibiting a higher activity at the same dose. Collectively, xanthone nanoemulsion was more effective than xanthone extract in inhibiting the growth of HepG2 cells. Further research is needed to study the anti-tumor effect in vivo.

Improved Analytical Method for Determination of Furan and Its Derivatives in Commercial Foods by HS-SPME Arrow Combined with Gas Chromatography-Tandem Mass Spectrometry.

Owing to the presence of significant levels of toxic furan compounds reported globally in commercial foods by various food authorities, the objectives of this study were to develop an analytical method for determination of furan and its 10 derivatives in commercial foods using headspace-solid phase microextraction (HS-SPME)-Arrow coupled with gas chromatography-tandem mass spectrometry. Furan and its 10 derivatives were separated within 10 min by employing an HP-5MS capillary column with d4-furan as the internal standard for quantitation. The most optimal sample weight and extraction time for various commercial food samples, respectively, ranged from 1 to 5 g and 10-15 min depending on the sample variety. For extraction, carboxen/poly(dimethylsiloxane) (CAR/PDMS) cellulose was used with the temperature at 30 °C, equilibration time of 15 min, and desorption time of 3 min. The limit of detection ranged from 0.001 to 1.071 ng/g, while the limit of quantitation ranged from 0.003 to 3.571 ng/g. A high precision and accuracy were obtained for this method. The total furan content in commercial foods ranged from nd to 40 725.85 ng/g, in which the mean contents were the highest for brewed coffee (35 082.26 ng/g) and canned coffee (25 152.22 ng/g), while the lowest were for potato chip and cookies (0.57-1.48 ng/g), donut (1.50 ng/g), milk (0.34-30.38 ng/g), and oat (6.56 ng/g).

A Comparative Study on Analysis of Ginsenosides in American Ginseng Root Residue by HPLC-DAD-ESI-MS and UPLC-HRMS-MS/MS.

Ginseng (Panax quinquefolius), a popular herbal and nutritional supplement consumed worldwide, has been demonstrated to possess vital biological activities, which can be attributed to the presence of ginsenosides. However, the presence of ginsenosides in ginseng root residue, a by-product obtained during processing of ginseng beverage, remains unexplored. The objectives of this study were to develop a high-performance liquid chromatography-photodiode array detection-mass spectrometry (HPLC-DAD-ESI-MS) and an ultra-high-performance-liquid-chromatography-tandem mass spectrometry (UPLC-HRMS-MS/MS) method for the comparison of ginsenoside analysis in ginseng root residue. Results showed that by employing a Supelco Ascentis Express C18 column (150 × 4.6 mm ID, particle size 2.7 µm) and a gradient mobile phase of deionized water and acetonitrile with a flow rate at 1 mL/min and detection at 205 nm, a total of 10 ginsenosides, including internal standard saikosaponin A, were separated within 18 min and detected by HPLC-DAD-ESI-MS. Whereas with UPLC-HRMS-MS/MS, all the 10 ginsenosides were separated within six minutes by using an Acquity UPLC BEH C18 column (50 × 2.1 mm ID, particle size 1.7 µm, 130 Å) and a gradient mobile phase of ammonium acetate and acetonitrile with column temperature at 50 °C, flow rate at 0.4 mL/min and detection by selected reaction monitoring (SRM) mode. High accuracy and precision was shown, with limit of quantitation (LOQ) ranging from 0.2−1.9 µg/g for HPLC-DAD-ESI-MS and 0.269−6.640 ng/g for UPLC-HRMS-MS/MS. The contents of nine ginsenosides in the ginseng root residue ranged from

A Comparative Study on Improving Streptozotocin-Induced Type 2 Diabetes in Rats by Hydrosol, Extract and Nanoemulsion Prepared from Cinnamon Leaves.

Cinnamomoum osmophloeum Kanehira (C. osmophloeum) contains various biologically active antioxidant compounds such as flavonoids, phenolic acids and cinnamaldehyde. Type 2 diabetes mellitus is a chronic disease of metabolic abnormality caused by insulin deficiency or resistance. The objectives of this study were to analyze various bioactive compounds in C. osmophloeum leaves by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and compare the effects of hydrosol, extract and nanoemulsion prepared from C. osmophloeum leaves on improving type 2 diabetes in rats. Our results show that a total of 15 bioactive compounds in C. osmophloeum leaves, including quercetin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, rutin, caffeic acid, benzoic acid, 5-O-caffeoylquinic acid, kaempferol 3-ß-D-glucopyranoside, trans-cinnamic acid, coumarin, cinnamyl alcohol, p-coumaric acid, eugenol, kaempferol and cinnamaldehyde, were separated within 14 min for subsequent identification and quantitation by UPLC-MS/MS. The nanoemulsion was successfully prepared by mixing C. osmophloeum leaf extract, soybean oil, lecithin, Tween 80 and deionized water in an appropriate proportion with a mean particle size, polydispersity index, zeta potential and encapsulation efficiency of 36.58 nm, 0.222, -42.6 mV and 91.22%, respectively, while a high storage and heating stability was obtained. The animal experiment results reveal that the high-dose nanoemulsion was the most effective in reducing both fasting blood glucose and oral glucose tolerance test value, followed by low-dose nanoemulsion, high-dose extract, low-dose extract and leaf powder in hydrosol. A similar trend was shown in reducing serum insulin and the homeostatic model assessment of insulin resistance index. In addition, the contents of serum biochemical parameters, including total cholesterol, triglyceride, aspartate aminotransferase, alanine aminotransferase, uric acid, urea nitrogen and creatinine, were reduced, with the high-dose nanoemulsion showing the most pronounced effect. Collectively, the high-dose nanoemulsion may possess great potential to be developed into a hypoglycemic health food or botanic drug.

Preparation of Catechin Nanoemulsion from Oolong Tea Leaf Waste and Its Inhibition of Prostate Cancer Cells DU-145 and Tumors in Mice.

Anti-cancer activity of catechin nanoemulsions prepared from Oolong tea leaf waste was studied on prostate cancer cells DU-145 and DU-145-induced tumors in mice. Catechin nanoemulsions composed of lecithin, Tween-80 and water in an appropriate proportion was prepared with high stability, particle size of 11.3 nm, zeta potential of -67.2 mV and encapsulation efficiency of 83.4%. Catechin nanoemulsions were more effective than extracts in inhibiting DU-145 cell growth, with the IC50 being 13.52 and 214.6 µg/mL, respectively, after 48 h incubation. Furthermore, both catechin nanoemulsions and extracts could raise caspase-8, caspase-9 and caspase-3 activities for DU-145 cell apoptosis, arresting the cell cycle at S and G2/M phases. Compared to control, catechin nanoemulsion at 20 µg/mL and paclitaxel at 10 µg/mL were the most effective in reducing tumor volume by 41.3% and 52.5% and tumor weight by 77.5% and 90.6% in mice, respectively, through a decrease in EGF and VEGF levels in serum.

Removal of polycyclic aromatic hydrocarbons from water by magnetic activated carbon nanocomposite from green tea waste.

This study aims to synthesize a magnetic activated carbon nanocomposite from green tea leaf waste (MNPs-GTAC) for evaluation of adsorption efficiency of 4 priority polycyclic aromatic hydrocarbons (PAHs). MNPs-GTAC contained spherically-shaped MNPs with cubic spinel structure, surface area at 118.8 m2/g, particle size at 8.6 nm and saturation magnetization at 34.2 emu/g. PAH adsorption reached a plateau at an MNPs-GTAC dose of 50 or 60 mg/L, pH of 2-4 and ionic strength of 0.1-10%, with PAH reduction in the presence of humic acid being compensated by addition of 0.1% sodium chloride. Kinetics was rapid attaining 80% removal within 5 min and the pseudo-second-order rate decreased in this order: Benzo[a]anthracene>Chrysene>Benzo[b]fluoranthene>Benzo[a]pyrene. Isotherm modeling revealed a Langmuir type-2 shape with the maximum adsorption capacity being 28.08, 22.75, 19.14 and 15.86 mg/g for Benzo[b]fluoranthene, Benzo[a]pyrene, Chrysene and Benzo[a]anthracene, respectively. Temperature study showed the PAH adsorption to be an endothermic and spontaneous process with increased randomness at solid-solution interface. Acetonitrile could completely recover the adsorbed PAH and MNPs-GTAC was successfully recycled 5 times with a minimum loss. Application to mineral water showed 86-98% and 72-89% removal for PAHs spiked respectively at 0.1 and 1 mg/L, while a complete removal was attained in tap and river waters.

Camelia oil and soybean-camelia oil blend enhance antioxidant activity and cardiovascular protection in hamsters.

OBJECTIVES: The aim of this study was to examine the effects of several vegetable oils and blended oil composed of soybean and camellia oils on blood lipid reduction and antioxidative activity. METHODS: Forty male hamsters were fed an AIN-93 G diet for 1 wk, followed by dividing into five groups: control group-1 was fed a low-fat diet containing 5% oil for 6 wk, and the other four groups were fed high-fat diets with group-2 containing 14% palm oil, group-3 containing 14% camellia oil, group-4 containing 14% soybean oil, and group-5 containing 14% blended oil (8.4% soybean oil and 5.6% camellia oil) along with 0.2% cholesterol and 0.1% bile acid. RESULTS: High-fat diets raised serum triacylglycerol, total cholesterol, and aspartate aminotransferase in hamsters without affecting alanine aminotransferase. Compared with palm oil-containing diet, the other three high-fat diets reduced serum total cholesterol, low-density lipoprotein cholesterol, and the ratio of low-density lipoprotein to high-density lipoprotein cholesterol with an opposite trend for liver total cholesterol. However, compared with the control group, the serum high-density lipoprotein cholesterol level was raised for all four high-fat diets. The higher the degree of oil unsaturation, the higher the serum thiobarbituric acid reactive substances and the lower the liver triacylglycerol level and activities of fatty acid synthase, glucose 6-phosphate dehydrogenase, and malic enzymes. Both soybean and blended oils lowered the antioxidative activity of liver. CONCLUSION: Camellia and blended oils were more efficient than soybean oil in elevating serum high-density lipoprotein cholesterol and decreasing the ratio of low-density lipoprotein to high-density lipoprotein cholesterol in hamsters.

Functional components in Scutellaria barbata D. Don with anti-inflammatory activity on RAW 264.7 cells.

The objectives of this study were to determine the variety and amount of various functional components in Scutellaria barbata D. Don as well as study their anti-inflammatory activity on RAW 264.7 cells. Both ethanol and ethyl acetate extracts were shown to contain the functional components including phenolics, flavonoids, chlorophylls, and carotenoids, with the former mainly composed of phenolics and flavonoids, and the latter of carotenoids and chlorophylls. Both extracts could significantly inhibit (p < 0.05) the production of lipopolysaccharide-induced nitric oxide, prostaglandin E2, interlukin-6, and interlukin-1ß, as well as the expressions of phosphor extracellular signal-regulated kinase and phosphor-c-Jun N-terminal kinase (p-JNK), but failed to retard tumor necrosis factor-α expression. Both ethanol and ethyl acetate extracts had a dose-dependent anti-inflammatory activity on RAW 264.7 cells. Furthermore, the anti-inflammatory efficiency can be varied for both ethanol and ethyl acetate extracts, which can be attributed to the presence of different varieties and amounts of functional components, as mentioned above. This finding suggested that S. Barbata extract may be used as an anti-inflammatory agent for possible future biomedical application.

Determination of oral bioavailability of curcuminoid dispersions and nanoemulsions prepared from Curcuma longa Linnaeus.

BACKGROUND: Curcuminoid from Curcuma longa Linnaeus has been demonstrated to be effective in anti-cancer and anti-inflammation. The objectives of the present study were to prepare curcuminoid dispersion and nanoemulsion from C. longa and determine their oral bioavailabilities in rats. RESULTS: After curcuminoid extraction using 99.5% ethanol, bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC) and curcumin were separated within 10 min by high-performance liquid chromatography using an Eclipse XDB-C18 column (Agilent, Palo Alto, CA, USA) and a gradient mobile phase of 0.1% aqueous formic acid and acetonitrile, with a flow rate of 1 mL min-1 , column temperature of 35 °C and detection wavelength of 425 nm. Curcuminoid nanoemulsion at a particle size of 12.1 nm and encapsulation efficiency 98.8% was prepared using lecithin, Tween 80 and water. A pharmacokinetic study in rats revealed that the parameters including Tmax , Cmax , t1/2 and the area under the curve were higher for curcuminoid nanoemulsions than for curcuminoid dispersion at the same dose employed for gavage administration, whereas, for intravenous injection, an opposite trend was shown. The oral bioavailabilities of BDMC, DMC, curcumin and total curcuminoids in nanoemulsion and dispersion were 34.39 and 4.65%, 39.93 and 5.49%, 47.82 and 9.38%, and 46 and 8.7%, respectively. CONCLUSION: The results of the present study demonstrate a higher oral bioavailability after incorporation of curcuminoid into nanoemulsion, facilitating its application as a botanic drug. © 2017 Society of Chemical Industry.

Preparation of curcuminoid microemulsions from Curcuma longa L. to enhance inhibition effects on growth of colon cancer cells HT-29.

The objectives of this study were to extract curcuminoid from Curcuma longa L. (C. longa), a vital medicinal plant demonstrated to possess many biological activities, and prepare the curcuminoid extract and microemulsion for studying the inhibition mechanism of HT-29 colon cancer cells. Results showed that a total of 3 curcuminoids including curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC), were separated within 10 min by using an Eclipse XDB-18 column and a gradient mobile phase of 0.1% formic acid solution (A) and acetonitrile (B). The curcuminoid microemulsion composed of soybean oil, Tween 80, ethanol and water was prepared with a high stability and mean particle size of 10.9 nm, zeta-potential of -65.2 mV and encapsulation efficiency of 85.7%. Both curcuminoid extract and microemulsion were effective in inhibiting HT-29 cell growth with the IC50 being 3.83 and 2.51 µg mL-1 after 24 h incubation, respectively, but further reduced to 2.23 and 1.94 µg mL-1, after 48 h incubation. Both treatments could decrease the proportion of both viable and necrosis cells and increase the proportion of both early and late apoptosis cells in a dose-dependent manner, with the cell cycle arrested at the S phase. Also, both treatments could up-regulate p53 expression and down-regulate cyclin A and CDK2 expressions through a p21-independent pathway. In addition, the expressions of Bax and cytochrome C as well as the activities of caspase-8, caspase-9 and caspase-3 increased for the curcuminoid extract, while the curcuminoid microemulsion showed the same trend with the exception that an insignificant change (p > 0.05) in Bax expression was observed. Collectively, this study demonstrated that the curcuminoid microemulsion prepared from C. longa may possess great potential for the treatment of colon cancer in the future.

Preparation of coffee oil-algae oil-based nanoemulsions and the study of their inhibition effect on UVA-induced skin damage in mice and melanoma cell growth.

Coffee grounds, a waste by-product generated after making coffee, contains approximately 15% coffee oil which can be used as a raw material in cosmetics. Algae oil rich in docosahexaenoic acid (DHA) has been demonstrated to possess anticancer and anti-inflammation functions. The objectives of this study were to develop a gas chromatography-mass spectrometry (GC-MS) method for the determination of fatty acids in coffee oil and algae oil and prepare a nanoemulsion for studying its inhibition effect on ultraviolet A-induced skin damage in mice and growth of melanoma cells B16-F10. A total of 8 and 5 fatty acids were separated and quantified in coffee oil and algae oil by GC-MS, respectively, with linoleic acid (39.8%) dominating in the former and DHA (33.9%) in the latter. A nanoemulsion with a particle size of 30 nm, zeta potential -72.72 mV, and DHA encapsulation efficiency 100% was prepared by using coffee oil, algae oil, surfactant (20% Span 80 and 80% Tween 80), and deionized water. Differential scanning calorimetry (DSC) analysis revealed a high stability of nanoemulsion when heated up to 110°C at a pH 6, whereas no significant changes in particle size distribution and pH occurred over a 90-day storage period at 4°C. Animal experiments showed that a dose of 0.1% coffee oil-algae oil nanoemulsion was effective in mitigating trans-epidermal water loss, skin erythema, melanin formation, and subcutaneous blood flow. Cytotoxicity test implied effective inhibition of melanoma cell growth by nanoemulsion with an IC50 value of 26.5 µg/mL and the cell cycle arrested at G2/M phase. A dose-dependent upregulation of p53, p21, cyclin B, and cyclin A expressions and downregulation of CDK1 and CDK2 occurred. Also, both Bax and cytochrome c expressions were upregulated and bcl-2 expression downregulated, accompanied by a rise in caspase-3, caspase-8, and caspase-9 activities for apoptosis execution. Collectively, the apoptosis pathway of melanoma cells B16-F10 may involve both mitochondria and death receptor.

Utilization of Microemulsions from Rhinacanthus nasutus (L.) Kurz to Improve Carotenoid Bioavailability.

Carotenoids have been known to reduce the risk of several diseases including cancer and cardiovascular. However, carotenoids are unstable and susceptible to degradation. Rhinacanthus nasutus (L.) Kurz (R. nasutus), a Chinese medicinal herb rich in carotenoids, was reported to possess vital biological activities such as anti-cancer. This study intends to isolate carotenoids from R. nasutus by column chromatography, identify and quantify by HPLC-MS, and prepare carotenoid microemulsions for determination of absolute bioavailability in rats. Initially, carotenoid fraction was isolated using 250 mL ethyl acetate poured into an open-column packed with magnesium oxide-diatomaceous earth (1:3, w/w). Fourteen carotenoids including internal standard ß-apo-8'-carotenal were resolved within 62 min by a YMC C30 column and gradient mobile phase of methanol-acetonitrile-water (82:14:4, v/v/v) and methylene chloride. Highly stable carotenoid microemulsions were prepared using a mixture of Capryol(TM)90, Transcutol®HP, Tween 80 and deionized water, with the mean particle being 10.4 nm for oral administration and 10.7 nm for intravenous injection. Pharmacokinetic study revealed that the absolute bioavailability of carotenoids in microemulsions and dispersion was 0.45% and 0.11%, respectively, while a much higher value of 6.25% and 1.57% were shown for lutein, demonstrating 4-fold enhancement in bioavailability upon incorporation of R. nasutus carotenoids into a microemulsion system.

Preparation of catechin extracts and nanoemulsions from green tea leaf waste and their inhibition effect on prostate cancer cell PC-3.

Green tea is one of the most commonly consumed natural health beverages in Taiwan's market, with the major functional component catechin being shown to possess several biological activities such as antioxidation, anticancer, and prevention of cardiovascular disease. The objectives of this study were to develop a high-performance liquid chromatography-mass spectrometry method to determine the variety and content of catechins in green tea leaf waste, a by-product obtained during processing of tea beverage. In addition, catechin nanoemulsion was prepared to study its inhibition effect on prostate cancer cell PC-3. Results showed that a total of eight catechin standards were separated within 25 minutes by using a Gemini C18 column and a gradient mobile phase of 0.1% formic acid (A) and acetonitrile (B) with flow rate at 1 mL/min, column temperature at 30°C, and detection wavelength at 280 nm. Among various extraction solvents, 50% ethanol generated the highest yield of total catechins from tea leaf waste, of which five catechins were identified and quantified. The catechin nanoemulsion was composed of catechin extract, lecithin, Tween 80, and deionized water in an appropriate proportion, with the mean particle size being 11.45 nm, encapsulation efficiency 88.1%, and zeta potential -66.3 mV. A high stability of catechin nanoemulsion was shown over a storage period of 120 days at 4°C. Both catechin extract and nanoemulsion could inhibit growth of PC-3 tumor cells, with the half maximal inhibitory concentration being 15.4 µg/mL and 8.5 µg/mL, respectively. The PC-3 cell cycle was arrested at S phase through elevation of P27 expression and decline of cyclin A, cyclin B, cyclin-dependent kinase 2, and cyclin-dependent kinase 1 expression. In addition, both catechin extract and nanoemulsion could induce apoptosis of PC-3 cells through decrease in B-cell lymphoma 2 (bcl-2) expression and increase in cytochrome c expression for activation of caspase-3, caspase-8, and caspase-9. Taken together, both caspase-dependent and caspase-independent pathways may be involved in apoptosis of PC-3 cells.

Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus.

The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 µg/mL), demethoxycurcumin (1,147.4 µg/mL), and bisdemethoxycurcumin (190.2 µg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 µg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.

Induction of p53-independent growth inhibition in lung carcinoma cell A549 by gypenosides.

The objectives of this study are to investigate antiproliferative effect and mechanisms of bioactive compounds from Gynostemma pentaphyllum (G. pentaphyllum) on lung carcinoma cell A549. Saponins, carotenoids and chlorophylls were extracted and fractionated by column chromatography, and were subjected to high-performance liquid chromatography-mass spectrometry analyses. The saponin fraction, which consisted mainly of gypenoside (Gyp) XXII and XXIII, rather than the carotenoid and chlorophyll ones, was effective in inhibiting A549 cell growth in a concentration- and a time-dependent manner as evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The estimated half maximal inhibitory concentration (IC50 ) of Gyp on A549 cells was 30.6 µg/ml. Gyp was further demonstrated to induce an apparent arrest of the A549 cell cycle at both the S phase and the G2/M phase, accompanied by a concentration- and a time-dependent increase in the proportions of both the early and late apoptotic cells. Furthermore, Gyp down-regulated cellular expression of cyclin A and B as well as BCL-2, while up-regulated the expression of BAX, DNA degradation factor 35 KD, poly [ADP-ribose] polymerase 1, p53, p21 and caspase-3. Nevertheless, both the treatment of a p53 inhibitor, pifithrin-α, and the small hairpin RNA-mediated p53 knockdown in the A549 cells did not alter the growth inhibition effect induced by Gyp. As a result, the cell cycle arrest and apoptosis of A549 cells induced by Gyp would most likely proceed through p53-independent pathway(s).

Hepatoprotective and anti-oxidant activities of Glossogyne tenuifolia against acetaminophen-induced hepatotoxicity in mice.

The present study investigated the anti-oxidative and hepatoprotective effects of Glossogyne tenuifolia (GT) Cassini, against acetaminophen-induced acute liver injury in BALB/c mice. The extracts of GT by various solvents (hot water, 50% ethanol and 95% ethanol) were compared for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, total phenolic content, and total anti-oxidant capacity. The results showed that hot water (HW) extracts of GT contained high levels of phenolics and exerted an excellent anti-oxidative capacity; thus, these were used in the animal experiment. The male BALB/c mice were randomly divided into control group, acetaminophen (APAP) group, positive control group and two GT groups at low (GT-L) and high (GT-H) dosages. The results showed that mice treated with GT had significantly decreased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). GT-H increased glutathione levels and the ratios of reduced glutathione and oxidized glutathione (GSH/GSSG) in the liver, and inhibited serum and lipid peroxidation. This experiment was the first to determine phenolic compounds, chlorogenic acid and luteolin-7-glucoside in HW extract of GT. In conclusion, HW extract of GT may have potential anti-oxidant capacity and show hepatoprotective capacities in APAP-induced liver damaged mice.

Flavonoids from Gynostemma pentaphyllum exhibit differential induction of cell cycle arrest in H460 and A549 cancer cells.

Flavonoids, containing mainly kaempferol rhamnohexoside derivatives, were extracted from Gynostemma pentaphyllum (G. pentaphyllum) and their potential growth inhibition effects against H460 non-small cell lung cancer cells was explored and compared to that on A549 cells. The extracted flavonoids were found to exhibit antiproliferation effects against H460 cells (IC50 = 50.2 µg/mL), although the IC50 of H460 is 2.5-fold that of A549 cells (IC50 = 19.8 µg/mL). Further investigation revealed that H460 cells are more susceptible to kaempferol than A549, whereas A549 cell growth is better inhibited by kaempferol rhamnohexoside derivatives as compared with H460. In addition, flavonoids from G. pentaphyllum induced cell cycle arrest at both S and G2/M phases with concurrent modulated expression of the cellular proteins cyclin A, B, p53 and p21 in A549 cells, but not H460. On the contrary, apoptosis and concomitant alteration in balance of BCL-2 and BAX expression as well as activation of caspase-3 were equally affected between both cells by flavonoid treatment. These observations strongly suggest the growth inhibition discrepancy between H460 and A549 following flavonoid treatment can be attributed to the lack of cell cycle arrest in H460 cells and the differences between H460 and A549 cells may serve as contrasting models for further mechanistic investigations.

Polysaccharide Isolated from Zizyphus jujuba ( Hóng Zǎo) Inhibits Interleukin-2 Production in Jurkat T Cells.

Zizyphus jujuba ( Hóng Zǎo), a traditional Chinese herb widely used in many Asian countries, has been shown to possess vital biological activities such as anti-cancer activity. The objective of this study was to evaluate the immunomodulatory effect of deproteinated polysaccharide (DP) isolated from Z. jujuba. The DP isolated from Z. jujuba consisted of two polysaccharide fractions and their molecular weights (MWs) were found to be 143,108 and 67,633 Da, respectively. The DP could significantly decrease interleukin (IL)-2 production in phytohemagglutinin (PHA)-activated Jurkat T cells in a dose-dependent manner after 48 h of incubation, with the inhibition being 47.5%, 61.2%, and 81.7% for DP concentrations of 0.75, 1.75, and 2.5 mg/ml, respectively. Thus, our study showed that DP isolated from Z. jujuba may possess anti-inflammatory activity as it could significantly reduce IL-2 production in activated Jurkat T cells.

In vitro removal of toxic heavy metals by poly(γ-glutamic acid)-coated superparamagnetic nanoparticles.

BACKGROUND: Chelation therapy involving organic chelators for treatment of heavy metal intoxication can cause cardiac arrest, kidney overload, mineral deficiency, and anemia. METHODS: In this study, superparamagnetic iron oxide nanoparticles (SPIONs) modified with an edible biopolymer poly(γ-glutamic acid) (PGA) were synthesized by coprecipitation method, characterized and evaluated for their removal efficiency of heavy metals from a metal solution, and simulated gastrointestinal fluid (SGIF). RESULTS: Instrumental characterization of bare- and PGA-SPIONs revealed 7% coating of PGA on SPIONs with a spherical shape and an iron oxide spinel structure belonging to magnetite. The particle sizes as determined from transmission electron microscopy images were 8.5 and 11.7 nm for bare- and PGA-SPIONs, respectively, while the magnetization values were 70.3 and 61.5 emu/g. Upon coating with PGA, the zeta potentials were shifted from positive to negative at most of the environmental pH (3-8) and biological pH (1-8), implying good dispersion in aqueous suspension and favorable conditions for heavy metal removal. Batch studies showed rapid removal of lead and cadmium with the kinetic rates estimated by pseudo-second-order model being 0.212 and 0.424 g/mg · min, respectively. A maximum removal occurred in the pH range 4-8 in deionized water and 5-8 in SGIF corresponding to most gastrointestinal pH except for the stomach. Addition of different ionic strengths (0.001-1 M sodium acetate) and essential metals (Cu, Fe, Zn, Mg, Ca, and K) did not show any marked influence on lead removal by PGA-SPIONs, but significantly reduced the binding of cadmium. Compared to deionized water, the lead removal from SGIF was high at all pH with the Langmuir monolayer removal capacity being 98.70 mg/g for the former and 147.71 mg/g for the latter. However, a lower cadmium removal capacity was shown for SGIF (23.15 mg/g) than for deionized water (31.13 mg/g). CONCLUSION: These results suggest that PGA-SPIONs could be used as a metal chelator for clinical treatment of metal poisoning.