Production of polyhydroxyalkanoates (PHA) using sludge from different wastewater treatment processes and the potential for medical and pharmaceutical applications.

In this study, seven strains of bacteria with polyhydroxyalkanoates (PHA)-producing ability (i.e. Bacillus cereus, Pseudomonas putida, Bacillus pumilus, Pseudomona huttiensis, Yersinia frederiksenii, Aeromonas ichthiosmia, and Sphingopyxis terrae) were isolated from various waste treatment plants in Hong Kong. Simultaneous wastewater treatment and PHA accumulation were successfully achieved in the bioreactors using isolated bacteria from different sludges. At the organic loading less than 13,000 ppm, more than 95% of chemical oxygen demand (COD) was removed by the isolated strains before the decrease of PHA accumulation. In addition, more than 95% of nitrogen removal was achieved by all isolated strains. In the bioreactors inoculated with single strains, the highest yields of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyvalerate) (PHV) were obtained in A. ichthiosmia (84 mg PHB/g) and B. cereus (69 mg/g), respectively. For the mixed culture, the highest yields of PHB and PHV were increased by 55% and 45% in the system inoculated with B. pumilus and A. ichthiosmia. The biologically synthesized PHA also showed the potential applications in drug delivery and tissue engineering. PHA-nanoparticles loaded with pyrene were successfully prepared by recombinant Escherichia coli. The results of in vitro drug release and biocompatibility tests revealed that nanoparticles could be used as safer dray carriers with high loading capacity and efficiency. After 20 days, the cells successfully grew on 90% of the PHA-aortic valve.

Cortex Fraxini (Qingpi) Protects Rat Pheochromocytoma Cells against 6-Hydroxydopamine-Induced Apoptosis.

Parkinson's disease (PD) is a chronic neurodegenerative disorder having close relationship with oxidative stress induced by reactive oxygen species (ROS). Cortex Fraxini (QP) is a kind of traditional Chinese medicinal herb with antioxidant properties. It may be a potential candidate for preventing the development of chronic neurodegenerative diseases. Thus, the key objective of the current study was to investigate the neuroprotective effect of QP water extract on 6-hydroxydopamine (6-OHDA) induced apoptosis in rat pheochromocytoma (PC12) cells. It was found that QP water extract possesses strong antioxidant property with SC50 = 0.15 mg/mL. Total phenolic content of QP water extract was found to be 200.78 ± 2.65 mg GAE/g. QP water extract's free radical scavenging capacity was demonstrated by reversing the increased level of intracellular ROS induced by 6-OHDA, using 2',7'-dichlorodihydrofluorescein diacetate. Moreover, QP water extract (0.5 mg/mL) could remarkably increase the viability of PC12 cells treated with 6-OHDA. The protective effect of QP water extract was found to be via inhibiting MEK/ERK pathway and reversing PI3-K/Akt/GSK3ß pathway. The current results suggest that QP might be a potential candidate for preventing the development of neurodegenerative diseases, such as PD.

Emodin elicits cytotoxicity in human lung adenocarcinoma A549 cells through inducing apoptosis.

This study investigated the mechanism of the cytotoxic effect of emodin, an active anthraquinone, on human lung adenocarcinoma A549 cells. In vitro growth inhibition and suppression on colony forming were used to evaluate the effects of emodin on A549 cells. Emodin's ability in changing the expressions of apoptosis-related genes was studied by real-time RT-PCR. Emodin could significantly inhibit the growth of A549 cells with IC50 = 16.85 µg/ml (~60 µM). It also concentration dependently inhibited the colony-forming ability of A549 cells with IC50 = 7.60 µg/ml (~30 µM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in A549 cells treated with emodin. Emodin (72 h) treatment could up-regulate the gene expression of FASL (p < 0.05) and down-regulate the gene expression of C-MYC (p < 0.01), but induce no significant changes in the gene expressions of MCL1, GAPDH, BAX and CCND1. These results suggest that emodin could induce growth inhibition and apoptosis in A549 cells through modifying the extrinsic apoptotic pathways and the induction of cell cycle arrest.

Emodin induces cytotoxic effect in human breast carcinoma MCF-7 cell through modulating the expression of apoptosis-related genes.

CONTEXT: The poor prognostic outcome of breast cancer is largely due to its resistance to cancer therapies. Development of therapeutic agents that can inhibit growth and induce apoptosis in breast cancer cells can help solve the problem. Emodin is an active anthraquinone that has been reported to have diverse biological effects. OBJECTIVE: In this study, the anticancer effects of emodin on growth inhibition, apoptosis induction and the expression of apoptosis-related genes in MCF-7 cells were investigated. MATERIALS AND METHODS: Growth inhibition induced by emodin was investigated by the MTS assay and the colony formation assay; while emodin-induced apoptosis was determined by the COMET assay and DNA fragmentation detection. Emodin (35 µM)-induced alterations in the expression of apoptotic-related genes were detected by using real-time PCR. RESULTS: Emodin had significant growth inhibitory effects on MCF-7 cells with IC50 = 7.22 µg/ml (∼30 µM). It also exerted a concentration-dependant inhibitory effect on the colony-forming ability of MCF-7 cells with IC50 = 7.60 µg/ml (∼30 µM). Hallmarks of apoptosis, such as single-strand DNA breakage and DNA fragmentation, were observed in emodin-treated MCF-7 cells. The gene expression of Fas ligand (FASL) was up-regulated (p < 0.01) but those of MCL1, CCND1 and C-MYC were down-regulated (p < 0.05) in emodin-treated MCF-7 cells. DISCUSSION AND CONCLUSION: This study indicated that emodin could induce growth inhibition and apoptosis in MCF-7 cells through the modulation of the expression of apoptosis-related genes. The growth inhibitory effects of emodin might involve both the intrinsic and the extrinsic apoptotic pathways and cell cycle arrest.

Chlorogenic acid exhibits cholesterol lowering and fatty liver attenuating properties by up-regulating the gene expression of PPAR-α in hypercholesterolemic rats induced with a high-cholesterol diet.

Hypercholesterolemia is a major risk factor for the development of cardiovascular disease and nonalcoholic fatty liver disease. Natural compounds have been proved to be useful in lowering serum cholesterol to slow down the progression of cardiovascular disease and nonalcoholic fatty liver disease. In the present study, the hypocholesterolemic and hepatoprotective effects of the dietary consumption of chlorogenic acid were investigated by monitoring plasma lipid profile (total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein) in Sprague-Dawley rats fed with a normal diet, a high-cholesterol diet or a high-cholesterol diet supplemented with chlorogenic acid (1 or 10 mg/kg/day p.o.) for 28 days. Chlorogenic acid markedly altered the increased plasma total cholesterol and low-density lipoprotein but decreased high-density lipoprotein induced by a hypercholesterolemic diet with a dose-dependent improvement on both atherogenic index and cardiac risk factor. Lipid depositions in liver were attenuated significantly in hypercholesterolemic animals supplemented with chlorogenic acid. It is postulated that hypocholesterolemic effect is the primary beneficial effect given by chlorogenic acid, which leads to other secondary beneficial effects such as atheroscleroprotective, cardioprotective and hepatoprotective functions. The hypocholesterolemic functions of chlorogenic acid are probably due to the increase in fatty acids unitization in liver via the up-regulation of peroxisome proliferation-activated receptor α mRNA.

Neuroprotective effects of luteolin against apoptosis induced by 6-hydroxydopamine on rat pheochromocytoma PC12 cells.

CONTEXT: Apoptotic neuronal cell death plays an important role in Parkinson's disease (PD), a progressive neurodegenerative disorder. Luteolin, a flavonoid, has been shown to possess various pharmacological properties including strong antioxidant capacity. OBJECTIVE: This study investigated the neuroprotective effect of luteolin against cytotoxicity induced by 6-hydroxy-dopamine (6-OHDA) (250 µM) in rat pheochromocytoma (PC12) cell line. MATERIALS AND METHODS: The neuroprotective effect of luteolin against 6-OHDA-induced cytotoxicity in PC12 was evaluated by using cell viability test, nuclear staining and flow cytometry. In addition, the apoptotic role of luteolin was unveiled by monitoring mRNA expression of proapoptotic and anti-apoptotic genes. RESULTS: Pretreatment with luteolin (3.13, 6.25, 12.5, 25 or 50 µM) could markedly attenuate 6-OHDA-induced PC12 cell viability loss in a concentration-dependent manner. Cell morphologic analysis and nuclear staining assays showed that luteolin (3.13, 12.5 or 50 µM) protected the cells from 6-OHDA-induced damage. As shown in the flow cytometry assay, the increased apoptotic rate induced by 6-OHDA could be significantly (p < 0.001) suppressed by luteolin (12.5 or 50 µM) pretreatment. The protection of luteolin (50 µM) against 6-OHDA-induced cell damage was shown to be through suppressing the over-expression of Bax gene (p < 0.01), inhibiting the reduction of Bcl-2 gene expression (p < 0.05) and markedly depressing the enhanced Bax/Bcl-2 ratio. Luteolin also downregulated the gene expression level of p53. DISCUSSION AND CONCLUSION: Luteolin has protective effects against 6-OHDA-induced cell apoptosis and might be a potential nutritional supplement which could be used to prevent neurodegenerative diseases such as PD.

A review of the anticancer and immunomodulatory effects of Lycium barbarum fruit.

The anticancer effects of traditional Chinese medicine (TCM) have attracted the attention of the public vis-à-vis existing cancer therapies with various side effects. Lycium barbarum fruit, commonly known as Gou Qi Zi in China, is a potential anticancer agent/adjuvant. Its major active ingredients, L. barbarum polysaccharides (LBP), scopoletin and 2-O-ß-D-glucopyranosyl-L-ascorbic acid (AA-2ßG), are found to have apoptotic and antiproliferative effects on cancer cell lines. Moreover, LBP also contributes to body's immunomodulatory effects and enhances effects of other cancer therapies. It is not known whether there are any undesirable effects. Further studies on its pharmacological mechanisms and toxicology could facilitate a safe usage of this TCM herb.

Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles.

Puffer fish, Takifugu niphobles, collected from the Hong Kong coastal waters were screened for tetrodotoxin-producing bacteria. A Gram-negative, non-acid-fast, non-sporing and rod shaped bacterial strain (designated as gutB01) was isolated from the intestine of the puffer fish and was shown to produce tetrodotoxin (TTX). Based on the Microbial Identification (MIDI) and 16S-23S rDNA internal transcribed spacer (ITS) phylogenetic analysis, the strain was identified as Raoultella terrigena. The TTX production ability of the strain was confirmed by mouse bioassay, ELISA and mass spectrometry (MALDI-TOF). Our results reiterate that the TTX found in puffer fish was likely produced by the associated bacteria and TTX are widely produced amongst a diversity of bacterial species.

A review of the pharmacological effects of Arctium lappa (burdock).

Arctium lappa, commonly known as burdock, is being promoted/recommended as a healthy and nutritive food in Chinese societies. Burdock has been used therapeutically in Europe, North America and Asia for hundreds of years. The roots, seeds and leaves of burdock have been investigated in view of its popular uses in traditional Chinese medicine (TCM). In this review, the reported therapeutic effects of the active compounds present in the different botanical parts of burdock are summarized. In the root, the active ingredients have been found to "detoxify" blood in terms of TCM and promote blood circulation to the skin surface, improving the skin quality/texture and curing skin diseases like eczema. Antioxidants and antidiabetic compounds have also been found in the root. In the seeds, some active compounds possess anti-inflammatory effects and potent inhibitory effects on the growth of tumors such as pancreatic carcinoma. In the leaf extract, the active compounds isolated can inhibit the growth of micro-organisms in the oral cavity. The medicinal uses of burdock in treating chronic diseases such as cancers, diabetes and AIDS have been reported. However, it is also essential to be aware of the side effects of burdock including contact dermatitis and other allergic/inflammatory responses that might be evoked by burdock.

Evaluation of anti-oxidant capacity of root of Scutellaria baicalensis Georgi, in comparison with roots of Polygonum multiflorum Thunb and Panax ginseng CA Meyer.

In Chinese communities, regular consumption of Chinese-medicated diets (CMD) (usually in the form of soup) is a traditional practice to promote health and prevent disease development. The overall improvement of health conditions is believed to be correlated with the anti-oxidant potentials of these herbs. Huangqin, roots of Scutellaria baicalensis Georgi (Lamiaceae), is one of the herbs commonly used in CMD. In this study, the anti-oxidant capacities of Huangqin extracts (water, ethanol and ether extracts) were evaluated and compared to commonly used CMD herbs, Heshouwu, roots of Polygonum multiflorum Thunb (Polygonaceae) and Renshen (or Ginseng), roots of Panax ginseng CA Meyer (Araliaceae). The anti-oxidant capacities were measured by using both cell-free assay [ferric reducing/anti-oxidant power (FRAP)] and biological methods [2,2'-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay and H(2)O(2)-induced cell damage on H9C2 cells]. Additionally, the total phenolic content was measured using Folin-Ciocalteu methods. Water extract of Huangqin has the highest anti-oxidant activities compared to the ethanol and ether extracts. A positive relationship between the anti-oxidant effects and total phenolic contents of extracts was demonstrated. This shows that Huangqin could be an effective dietary anti-oxidant that can be consumed regularly as a functional food for the prevention of oxidant/free radical-related diseases.

Rapid screening of tetrodotoxin in urine and plasma of patients with puffer fish poisoning by HPLC with creatinine correction.

A rapid and simple detection method for tetrodotoxin (TTX) in urine and plasma of patients with puffer fish poisoning was developed using commercially pre-packed solid-phase extraction (SPE) cartridges (C18 and weak cation exchange columns) and subsequent analyses by HPLC with UV detection. The detection limit of the standard TTX, TTX-spiked urine and plasma samples were all 10 ng/ml and the average TTX recovery in urine and plasma samples after SPE were 90.3 +/- 4.0 and 87.1 +/- 2.9%, respectively. It was noticed that the creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation apparently correlated much better with the severity of poisoning than the urinary TTX concentration without adjusting for variations in concomitant creatinine excretion.

Water extract of Rheum officinale Baill. induces apoptosis in human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study. AIM OF THE STUDY: The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways. MATERIALS AND METHODS: Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay. RESULTS: Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC(50) values 620+/-12.7 and 515+/-10.1 microg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells. CONCLUSIONS: This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.

Two novel species of tetrodotoxin-producing bacteria isolated from toxic marine puffer fishes.

Out of eight dominant discrete bacterial colonies isolated and purified from the toxic marine puffer fishes collected in Hong Kong waters, two novel species of non-sporing, non-acid-fast and chemoorganotrophic bacteria capable of producing tetrodotoxin (TTX, a potent non-protein neurotoxin), as well as one previously reported and confirmed TTX-producing bacterium. They were identified as Microbacterium arabinogalactanolyticum, Serratia marcescens and Vibrio alginolyticus, respectively, all of which are widely distributed in soils, sewage or marine environments. Each bacterial isolate (500 ml broth medium cultured in darkness without aeration for 10 days at 25 degrees C) could produce an amount of toxicity, after extraction and purification, ranging from 78.3 to 105.3 mouse units (MU) in 500 ml of broth medium by mouse bioassay. The principal toxic component in the bacterial cultures was determined to be TTX by thin layer chromatography and mass spectrometry.

Sphaerotilus natans isolated from activated sludge and its production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Sphaerotilus natans is a sheathed bacterium existing in the activated sludge of wastewater treatment plants. It is one of the filamentous bacteria causing the bulking and foaming difficulties of activated sludge. Isolating the strain and culturing it in an axenic environment could not only provide the metabolic knowledge of the strains that would be useful in the development of wastewater treatment methods, but also could enable us to gain an understanding of the mechanism by which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly[3-HB-co-3-HV]) is produced by this strain. This article reports the screening and isolation of the strain from the activated sludge using the Nile blue staining method together with Fourier transform infrared analysis. We investigated the ability of the selected strain to produce poly(3-HB-co-3-HV) copolymer using glucose and peptone, or by adding valeric acid or sodium propionate as precursor. Proper precursor feeding could dramatically enhance its 3HV content in the copolymer P(3HB-co-3HV). By controlling the different feeding times in fed-batch fermentation, different desired copolymers were obtained with 15, 40, and 70% 3HV mole fraction of the copolymer. Polymer properties were analyzed by gas chromatography, differential scanning calorimetry, thermo-gravimetry, and nuclear magnetic resonance analysis.

The annual toxicological profiles of two common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected along Hong Kong coastal waters.

The toxicological profiles of two local common puffer fish, Takifugu niphobles (Jordan and Snyder) and Takifugu alboplumbeus (Richardson), collected in Hong Kong waters were investigated continuously for 14 months (June 1997-August 1998). Their annual spawning seasons (as evident by the enlargement of gonads and presence of eggs in the ovary) were found to be from October to February (four consecutive months) and December to February (two consecutive months), respectively. The toxicities of their internal organs were determined by standard mouse bioassay and expressed in terms of mouse units (MU). The ovary (in both species) and liver (only in T. niphobles) were discovered to be moderately toxic (100-1000 MU/g) in their non-spawning seasons. Whilst all the intestine and skin were weakly toxic (between 10 and 100 MU/g), their flesh, however, was basically non-toxic (less than 10 MU/g) throughout the whole year. The testes, which were only developed in their spawning seasons, were also non-toxic. This study showed that the toxicological profiles of both of the two species had prominent seasonal patterns which were highly related to their spawning seasons. Contrary to the most common belief, both species were discovered to be relatively less toxic during their spawning seasons.