Herbal mixtures consisting of puerarin and either polyenylphosphatidylcholine or curcumin provide comprehensive protection against alcohol-related disorders in P rats receiving free choice water and 15% ethanol in pure water.

Chronic alcohol drinking has been associated with the development of a number of abnormalities, including neuron-behavioral disorders, liver, pancreas, and heart-related diseases and inflammation and immune disorders. Because diverse mechanisms are involved in the development of these disorders, the commonly used receptor- or enzyme-specific drugs do not provide comprehensive protection against the adverse effects of alcoholism. This study describes possible therapeutic potency of puerarin (PU) from kudzu root, polyenylphosphatidylcholine from soy (SPCh), and curcumin (CU) from turmeric against alcohol's addiction-related and inflammatory-related abnormalities in alcohol-preferring P rats receiving free choice water and 15% ethanol in water. P-rats were fed once daily either the vehicle (for control) or different doses of PU, SPCh, CU, PU + SPCh, or PU + CU. The rats were divided in two groups: one received water alone, and the other free choice water and ethanol. Four rats from each group were fitted with electroencephalogram (EEG) electrodes for EEG recording. After 70 days of alcohol drinking, alcohol was withdrawn for 2 weeks, and the withdrawal symptoms were assessed. This study showed that alcohol drinking for 70 days (1) caused liver inflammation characterized by elevated tumor necrosis factor-alpha, interleukin-1beta, and matrix metalloproteinase-9 expression and (2) dysregulated lipopolysaccharide (LPS)-induced pleurisy. Alcohol withdrawal after 70 days of drinking generated severe withdrawal symptoms including seizure-type EEG activity. PU suppressed the addiction-mediated abnormalities but did not affect the inflammation-related abnormalities, while SPCh or CU suppressed only the inflammation-related abnormalities in alcohol-drinking rats subjected to LPS-induced pleurisy. A combination of PU with SPCh or CU suppressed both the addiction-related and inflammation-related abnormalities of alcohol drinking. Therefore, a mixture consisting of PU and either SPCh or CU may provide alternative therapy for alcohol-related disorders.

Effects of chronic ethanol drinking on the blood brain barrier and ensuing neuronal toxicity in alcohol-preferring rats subjected to intraperitoneal LPS injection.

AIMS: Although alcohol drinking impairs the blood-brain barrier (BBB), the underlying mechanism is not fully understood. Thus, the effects of chronic ethanol drinking on the BBB were studied in vivo. METHODS: Alcohol-preferring rats were given for 70 days free choice water and 15% ethanol. Then, they received LPS by i.p. injection. Efflux of [(14)C]sucrose or [(14)C]dextran was measured by their microinjection into the brain. Endothelial cells and neurons were isolated from the brain and analysed for mitogen-activated protein kinase (MAPK) and the tight-junction (TJ) protein phosphorylation, NFkappaB activation, mRNA levels of TJ proteins, inducible nitric oxide synthase, tumour necrosis factor alpha, interleukin-1 beta (IL-1beta), IL-10, CASPASE-8, and DNA damage. RESULTS: LPS transiently increased [(14)C]sucrose efflux in water drinking, while it caused a lasting increase in [(14)C]sucrose and [(14)C]dextran efflux in ethanol-drinking rats. The time-course of changes in the TJ correlated with (i) an increase in extracellular signal-regulated kinase (ERK), p38(mapk) Jun-N-terminal Kinase (JNK), and TJ protein phosphorylation, (ii) RelA-p50 and p50-p50 activation, and (iii) a decrease in the TJ proteins' mRNA levels in endothelial cells and neurons. Apoptotic cells were detected in water drinking and LPS (WC-LPS) neurons at 24 h after LPS exposure. Neurons from Et-LPS rats did not exhibit apoptosis. CONCLUSIONS: LPS injection in WC-LPS rats transiently disrupted the BBB. Lack of JNK activation and CASPASE-8 may be responsible for lack of apoptosis in endothelial cells and vice versa in neurons. Chronic alcohol drinking in ethanol drinking and LPS (Et-LPS) rats augmented and dysregulated the LPS-induced BBB abnormalities but suppressed apoptosis in neurons.

Kudzu root extract suppresses voluntary alcohol intake and alcohol withdrawal symptoms in P rats receiving free access to water and alcohol.

Alcohol-preferring (P) rats, given free choice to drink water or 15% alcohol, drank 7-10 g of alcohol/kg/day, giving blood alcohol values ranging from 16 to 24 mg/dL. Body weight and food and total fluid intake values in control and alcohol-drinking P rats did not differ significantly, while water intake was inversely related to the amount of alcohol consumed. Alcohol withdrawal after 50 days of alcohol drinking caused withdrawal symptoms such as hypersensitivity, poor landing coordination, and tremors. A daily 0.5, 0.75, and 1.0 g/kg dose of kudzu root (KdR) did not affect body weight and food and water intake values in control (no alcohol) P rats. Subchronic feeding of relatively higher KdR doses (0.75 and 1.0 g/kg) caused a 25-30% reduction in weight gain. The 0.5 g/kg KdR dose caused a 50-60% reduction in alcohol consumption, abolished the development of alcohol withdrawal symptoms, but did not affect blood alcohol levels. The higher KdR doses did not further reduce alcohol consumption. Alcohol suppressed the weight-reducing effects of KdR. The KdR extract used in this study contained 150 mg/g of puerarin, 13 mg/g of daidzin, 4 mg/g of daidzein, 3 mg/g of genistin, 0.2 mg/g of genistein, and 1 mg/g of glycetin. Blood and liver samples contained mostly puerarin and a trace amount of daidzein that may have been formed by the hydrolysis of daidzin by liver enzymes. An important observation was that brain samples obtained from KdR-fed or alcohol + KdR-fed rats did not contain any of the KdR isoflavones. Thus, KdR isoflavones suppressed alcohol drinking and withdrawal symptoms without entering the brain.

Effects of purified puerarin on voluntary alcohol intake and alcohol withdrawal symptoms in P rats receiving free access to water and alcohol.

Alcohol preferring (P) rats, given "free choice" of water, exhibited daily intake of 60-75 g of water/kg of body weight. When given "free choice" of water and 15% ethanol, P rats consumed 7-13 g of alcohol/kg. Their water intake decreased proportionally to the alcohol intake, but total fluid intake did not differ significantly. Alcohol withdrawal after 50 days of alcohol drinking caused withdrawal symptoms such as hypersensitivity, poor coordination, and tremors. A daily 50 mg/kg dose of puerarin (PU) caused approximately 50% suppression in alcohol intake, but did not affect body weight and food and total fluid intake in P rats receiving "free choice" of water and 15% ethanol. Alcohol ingestion gradually returned to the control level despite consistent PU intake. However, alcohol intake following alcohol withdrawal was suppressed in PU-fed P rats. PU suppressed the severity of alcohol withdrawal symptoms. Thus, withdrawal symptoms do not occur in PU-fed rats even though their alcohol ingestion is comparable to that in control P rats. Brain, plasma, and liver samples were analyzed for the presence of kudzu root isoflavones, which are mostly PU (>90% of total isoflavones) and a trace amount of daidzin. Liver samples obtained from PU-fed P rats contained 20-30 microg/g of PU. An important observation was that plasma or brain samples obtained from PU-fed or alcohol + PU-fed rats did not contain PU. This study indicated that PU feeding transiently suppressed alcohol intake and abolished withdrawal symptoms at a time when alcohol intake had returned to the control level. The absence of PU in plasma and brain indicates the possibility that some nonspecific mechanism may be involved in the anti-alcoholism effects of PU in P rats.

Quantitative analysis of phytoestrogens in kudzu-root, soy and spiked serum samples by high-pressure liquid chromatography.

A sensitive and reliable HPLC method that allows simultaneous quantification of phytoestrogens extracted from kudzu-root and soy preparations, and serum samples has been developed. Kudzu-root and soy preparations were mixed with 5 microg flavone and 15 microg rutin (internal standards) and the phytoestrogens were extracted by using solid-phase (C18) extraction cartridges. Blank or spiked serum samples were extracted by using either C18 cartridges or trichloroacetic acid-methanol extraction. The extracts were analyzed by the HPLC equipped with a reverse-phase (250 x 4 mm, C18) column and UV, diode-array or MS detector. A linear gradient of acetic acid and acetonitrile provided excellent separation of glycoside and aglycone-phytoestrogens from kudzu root and soy preparations. The C18 cartridge extraction of serum yielded excellent recovery of both glycoside- and aglycone-phytoestrogens, while the trichloroacetic acid-methanol extraction yielded excellent recovery of glycoside but poor recovery of aglycone compounds. UV and MS detectors were suitable for phytoestrogen analysis in plant and serum samples, while the diode-array detector was suitable for generating the UV absorbance curve for phytoestrogens.

Composition, red blood cell uptake, and serum protein binding of phytoestrogens extracted from commercial kudzu-root and soy preparations.

Kudzu-root and soy phytoestrogens have been associated with anti-cancer and anti-intoxication activities. Sales of capsules containing kudzu-root and soy extracts through health food stores and the Internet are unregulated. To compare efficacy, the amount of phytoestrogens present in commercial preparations and their fate in biological samples must be determined. In this study, the levels and composition of phytoestrogens in kudzu-root and soy extracts were studied using high-performance liquid chromatography with ultraviolet light detection. The bioavailability of phytoestrogens was studied by measuring red blood cell (RBC) uptake and serum protein binding ability. Phytoestrogen levels in acidified kudzu-root samples were 5- to 10-fold greater than those in nonacidified samples. Puerarin accounted for 80% of total phytoestrogens in kudzu-root. In soy extract, puerarin was absent while genistin, glycetein, and daidzin or daidzein were the major phytoestrogens. The RBC uptake depended on the phytoestrogen's polarity and molecular length. When serum was dialyzed with phytoestrogen standards in a buffer, the protein binding of phytoestrogens correlated negatively with their polarity. However, when serum was dialyzed with kudzu-root or soy extract, almost all of the phytoestrogens present in the extract bound to serum protein. Therefore, this study suggests differences in the bioavailability of phytoestrogens when they are ingested as purified compounds compared with crude plant extract. The differential composition of phytoestrogens in kudzu-root and soy may account for the differences in their therapeutic activities.