Simultaneous quantification of 14 ginsenosides in Panax ginseng C.A. Meyer (Korean red ginseng) by HPLC-ELSD and its application to quality control

A new method of high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous quantification of 14 major ginsenosides, which are the marker compounds of Panax ginseng C.A. Meyer (Korean red ginseng). Various types of ginseng samples were extracted, and the amounts of the 14 ginsenosides (Rg1, Re, Rf, Rh1, Rg2, Rb1, Rc, Rb2, Rb3, Rd, Rg3, Rk1, Rg5, and Rh2) were determined by reverse-phase HPLC-ELSD using digoxin as an internal standard. The mobile phase consisted of a programmed gradient of aqueous acetonitrile. Calibration curves for each ginsenoside were determined for the quantification. The method was validated for linearity, precision, accuracy, limit of detection, and limit of quantification. This quantification method was applied to several finished ginseng products including white ginseng, red ginseng powder, and red ginseng concentrate. The amounts of the 14 ginsenosides in the various ginseng samples could be analyzed simultaneously. This validated HPLC method is expected to provide a new basis for the quality assessment of ginseng products.     

Antioxidant effects of flavonoid from Croatian Cystus incanus L. rich bee pollen

Oxidant/antioxidant status, estrogenic/anti-estrogenic activity and gene expression profile were studied in mice fed with Cystus incanus L. (Cistaceae) reach bee pollen from location in Central Croatia’s Dalmatia coast and offshore islands. Seven phenolic compounds (out of 13 tested) in bee pollen sample were detected by high performance liquid chromatography (HPLC) analysis. Phenolics detected in C. incanus L. bee pollen belong to flavonol (pinocembrin), flavanols (quercetin, kaempferol, galangin, and isorhamnetin), flavones (chrysin) and phenylpropanoids (caffeic acid). Bee pollen as a food supplement (100 mg/kg bw mixed with commercial food pellets) compared to control (commercial food pellets) modulated antioxidant enzymes (AOE) in the mice liver, brain and lysate of erythrocytes and reduced hepatic lipid peroxidation (LPO). Bee pollen induced 25% of anti-estrogenic properties while no estrogenic activity was found. Differential gene expression profile analyses after bee pollen enriched diet identify underexpressed gene Hspa9a, Tnfsf6 (liver) and down-regulated gene expression of Casp 1 and Cc121c (brain) which are important in the apoptosis pathway and chemotaxis. These results indicate that used bee pollen possess a noticable source of compounds with health protective potential and antioxidant activity.     

Inhibitory effects of methanol extract of plum (Prunus salicina L., cv. ‘Soldam’) fruits against benzo(α)pyrene-induced toxicity in mice

This study was carried out to investigate the chemopreventive effects of immature plum extracts. The methanol extract of immature plums (plum 1), that are picked at 20–40 days before final harvest, has remarkably inhibited the growth of hepatoma HepG2 cells. The effects of immature plum extracts on hepatotoxicity in benzo(α)pyrene (B(α)P, carcinogen)-treated mice were investigated. Male ICR mice were pretreated with immature plum extracts (2.5 or 5 g/kg bw/day, for 5 days, i.p.) before treatment with B(α)P(0.5 mg/kg bw, i.p., single dose). The activities of serum aminotransferase, cytochrome P450 (CYPs) and the hepatic content of lipid peroxide were increased on B(α)P-treatment group than control, but those levels were significantly decreased by the pretreatment of immature plum extracts. The primary CYPs involved in the metabolism and bioactivation of B(α)P are CYP1A1. The pretreatment of immature plum extracts inhibited the induction of CYP1A1 expression. The activities of glutathione peroxidase, superoxide dismutase and catalase were decreased by the pretreatment of immature plum extracts more than with B(α)P alone. Whereas, the hepatic content of glutathione and glutathione S-transferase activity depleted by B(α)P was significantly increased (p > 0.05). These results suggest that immature plum extracts may counteract toxic effects of carcinogens, such as B(α)P, and therefore possess the chemopreventive efficacy.     

Evaluation of the potential protective effects of ad libitum black grape juice against liver oxidative damage in whole-body acute X-irradiated rats

Aims

The aim of this study was to evaluate the potential protective effects of ad libitum black grape (Vitis labrusca) juice against liver oxidative damage in whole-body acute X-irradiated rats.

Main methods

Animals were fed ad libitum and drank voluntarily black grape juice or placebo (isocaloric glucose and fructose solution) for 6 days before and 15 days following a 6 Gy X-irradiation from a 200 kV machine.

Key findings

Irradiated animals receiving placebo showed a significant increase in the concentration of thiobarbituric acid-reactive substances (TBARS), a marker of lipid peroxidation, as well as a significant decrease in both Cu/Zn superoxide dismutase (Cu/ZnSOD) and glutathione peroxidase (GPx) activity and reduced glutathione concentration (GSH). Black grape juice supplementation resulted in a reversal of lipid peroxidation, Cu/ZnSOD activity, and GSH concentration, towards values not significantly differing from those in non-irradiated, placebo-supplemented rats. Poly(ADP-ribose) polymerase (PARP-1) and Cu/ZnSOD changes in protein expression were observed for irradiated rats. No change in p53 expression or DNA fragmentation was found.

Significance

Ad libitum black grape juice intake is able to restore the liver primary antioxidant system against adverse effects due to whole-body acute X-irradiation in rats after 15 days post-irradiation. The results support using antioxidant supplements as a preventive tool against radiation-induced harm.     

Effect of Linum usitatissimum L. (linseed) oil on mild and moderate carpal tunnel syndrome: a randomized,double-blind,placebo-controlled clinical trial

Background

Carpal tunnel syndrome is known as the most common entrapment neuropathy. Conservative treatments cannot reduce the symptomatic severity satisfactorily; therefore, effectiveness of Linum usitatissimum L. (linseed) oil on carpal tunnel syndrome, as a complementary treatment, was evaluated in the current study. Linseed oil is a well-known preparation in Iranian traditional medicine and its analgesic, anti-inflammatory and anti-oxidative effects have been shown in previous studies.

Methods

A randomized, double-blind, placebo-controlled clinical trial was conducted. One hundred patients (155 hands) with idiopathic mild to moderate carpal tunnel syndrome aged between 18 and 65 years old were randomized in two parallel groups. These two groups were treated during 4 weeks with topical placebo and linseed oil. In addition, a night wrist splint was prescribed for both groups. Symptomatic severity and functional status were measured using Boston Carpal Tunnel Questionnaire. In addition, median sensory nerve conduction velocity, motor distal latency, sensory distal latency and compound latency as electrodiagnostic parameters were measured at baseline and after the intervention period.

Results

After the intervention, significant improvement was observed regarding Boston Carpal Tunnel Questionnaire symptomatic severity and functional status mean differences (p <0.001) in the linseed oil group compared with those in the placebo group. Also, regarding the mean differences of both groups, significant improvement of nerve conduction velocity of the median nerve was seen in the linseed oil group by a value of 2.38 m/sec (p < 0.05). However, motor distal latency and sensory distal latency of the median nerve showed no between-group significant changes (p = 0.14 for both items). Finally, compound latency was improved slightly in the case group, comparing mean differences between the groups (p <0.05). No significant adverse events were reported from using linseed oil.

Conclusions

It seems that linseed oil could be effective in the management of mild and moderate carpal tunnel syndrome, especially in improving the severity of symptoms and functional status. In addition, its effect on electerodiagnostic parameters, especially on the nerve conduction velocity, can be considered as a valuable point.     

Effects of Urtica dioica L. seed on lipid peroxidation,antioxidants and liver pathology in aflatoxin-induced tissue injury in rats

This study was carried out to investigate the hepatoprotective and antioxidant properties of Urtica dioica L. seeds (UDS) extract against aflatoxin (AF)-exposure in rats. The preventive potential and antioxidant capacity of the plant’s extract was evaluated by liver histopathological changes, measuring serum marker enzymes, antioxidant defense systems and lipid peroxidation (Malondialdehyde, MDA) content in some tissues of rats. Eighteen rats were randomly divided into one of three experimental groups: control, AF-treated group and AF+UDS-treated group. Rats in control group were fed with a diet without AF. Rats in AF-treated group and AF+UDS-treated group received approximately 25 μgr of AF/rat/day. AF+UDS groups also received 2 mL of UDS oils/rat/day by gavage for 90 days. Administration of UDS extract restored the AF-induced imbalance between MDA and antioxidant system towards near normal particularly in liver. Hepatoprotection by UDS is further substantiated by the almost normal histologic findings in AF+UDS-treated group as against degenerative changes in the AF-treated rats. It is concluded that UDS has a hepatoprotective effect in rats with aflatoxicosis, probably acting by promoting the antioxidative defense systems.     

Immortalized hepatocytes as in vitro model systems for toxicity testing: the comparative toxicity of menadione in immortalized cells, primary cultures of hepatocytes and HTC hepatoma cells

Immortalized differentiated liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be valuable for in vitro toxicity testing in the pharmaceutical, chemical, food and cosmetics industries. Immortalized rat hepatocyte cell lines have been produced by transfection of SV40 DNA by electroporation or calcium phosphate precipitation. Their utility has been assessed by studying the toxicity of a model compound, menadione, and by measuring the activities of DT-diaphorase and NADPH cytochrome c reductase. Enzyme activities and toxicity were compared in freshly isolated hepatocytes, the immortalized cell lines and dedifferentiated HTC hepatoma cells. In HTC cells DT-diaphorase activity was 100-fold elevated compared with freshly isolated hepatocytes. In only one cell line, C2.1.2. (produced by calcium phosphate precipitation), was DT-diaphorase activity increased (twofold) compared with freshly isolated hepatocytes. Menadione caused loss of viability at similar concentrations (40–80 μ ) in the immortalized cell lines and 24-hr primary cultures of hepatocytes, whereas HTC cells showed loss of viability only with menadione concentrations above 200 μ . The immortalized lines therefore appear to have potential for predicting toxicity and for menadione this can be correlated with the expression of DT-diaphorase.     

In vitro and in vivo safety assessment of edible blue-green algae, Nostoc commune var. sphaeroides Kützing and Spirulina plantensis

Blue-green algae (BGA) have been consumed as food and herbal medicine for centuries. However, safety for their consumption has not been well investigated. This study was undertaken to evaluate in vitro and in vivo toxicity of cultivated Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP). Neither NO nor SP contained detectable levels of microcystin (MC)-LA, MC-RR, MC-LW and MC-LR by LC/MS/MS. Cell viability remained ∼70-80% when HepG2 cells were incubated with 0-500 μg/ml of hexane, chloroform, methanol and water-extractable fractions of NO and SP. Four-week-old male and female C57BL/6J mice were fed an AIN-93G/M diet supplemented with 0%, 2.5% or 5% of NO and SP (wt/wt) for 6 months. For both genders, BGA-rich diets did not induce noticeable abnormality in weight gain and plasma alanine aminotransferase (ALT) and aspartate aminotransferase concentrations except a significant increase in plasma ALT levels by 2.5% NO supplementation in male mice at 6 month. Histopathological analysis of livers, however, indicated that BGA did not cause significant liver damage compared with controls. In conclusion, our results suggest that NO and SP are free of MC and the long-term dietary supplementation of up to 5% of the BGA may be consumed without evident toxic side-effects.     

Modulation of the effects of methylmercury on rat neurodevelopment by co-exposure with Labrador Tea (Rhododendron tomentosum ssp. subarcticum)

Seafood is an important source of nutrients for many populations worldwide. Unfortunately the contamination of seafood with methylmercury (MeHg) has raised health concerns, particularly for developing infants. The modification of MeHg toxicity by nutrients, including antioxidants, has been reported in both epidemiological and animal exposure studies. We used a rat perinatal exposure model to investigate the modulation of developmental MeHg toxicity by an extract of Rhododendron tomentosum ssp. subarcticum, a plant rich in antioxidants traditionally consumed by Inuit. Sprague-Dawley rat dams were exposed daily to 2 mg MeHg/kg bw and 100 mg R. tomemtosum extract/kg bw administered either separately or jointly, from the first day of gestation until weaning. MeHg exposure was associated with impaired pup growth and survival, decreased grip strength, hyperactivity and altered exploratory behaviour, delayed acoustic startle response, elevated brain N-methyl-d-aspartate receptor (NMDA-R) levels, and increased serum lipid peroxidation. R. tomemtosum extract co-exposure modulated MeHg’s effects on oxidative stress and brain NMDA-R levels and may have affected MeHg pharmacokinetic. However, modulation of these molecular endpoints did not lead to significant alterations of MeHg’s effects on rat neurobehaviour.     

Antioxidant and hepatoprotective activities of phenolic rich fraction of Seabuckthorn (Hippophae rhamnoides L.) leaves

Present study was aimed to investigate antioxidant and hepatoprotective activities of phenolic rich fraction (PRF) of Seabuckthorn leaves on CCl₄ induced oxidative stress in Sprague Dawley rats. Total phenolic content was found to be 319.33 mg gallic acid equivalent (GAE)/g PRF and some of its phenolic constituents, such as gallic acid, myricetin, quercetin, kaempferol and isorhamnetin were found to be in the range of 1.935-196.89 mg/g of PRF as determined by reverse-phase high-performance liquid chromatography (RP-HPLC).Oral administration of PRF at dose of 25-75 mg/kg body weight significantly protected from CCl₄ induced elevation in aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT) and bilirubin in serum, elevation in hepatic lipid peroxidation, hydroperoxides, protein carbonyls, depletion of hepatic reduced glutathione (GSH) and decrease in the activities of hepatic antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST). The PRF also protected against histopathological changes produced by CCl₄ such as hepatocytic necrosis, fatty changes, vacuolation, etc. The data obtained in the present study suggests that PRF has potent antioxidant activity, prevent oxidative damage to major biomolecules and afford significant protection against CCl₄ induced oxidative damage in the liver.     

Protective effects of Forsythia suspensa extract against oxidative stress induced by diquat in rats

Forsythia suspensa extract has been proved as a potential antioxidant in the recent years. The present study was undertaken to obtain the optimal antioxidant fraction in vitro and examine its antioxidative potential against diquat-induced oxidative stress in male Sprague Dawley rats in vivo. In vitro, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging experiment indicated that the CH₂Cl₂ fraction of F. suspensa (FSC) exerted the strongest scavenging activities; forsythoside A, forythialan A and phillygenin from it might be the major antioxidant constituents. In vivo, pretreatment of rats with different doses of FSC (25, 50 and 100 mg/kg bw) and vitamin C (100 mg/kg bw, positive control) for 15 days significantly lowered the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in plasma compared to the negative control group. Also, FSC significantly increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the levels of glutathione (GSH) in plasma, liver and kidney whereas it decreased the levels of malondialdehyde (MDA) in plasma and kidney. Moreover, the protective effect of FSC (100 mg/kg bw) was better than vitamin C. These results revealed that FSC exerted a protective effect against diquat-induced oxidative stress and is worthy of becoming a potential dietary antioxidant.     

Evaluation of clinical safety and tolerance of a Lactobacillus reuteri NCIMB 30242 supplement capsule: A randomized control trial

A significant number of human clinical trials have reported no adverse effects associated with consumption of Lactobacillus reuteri (L. reuteri). In the present study, the clinical safety and toxicology of oral ingestion of supplement capsules containing L. reuteri NCIMB 30242 was investigated. A randomized group of 131 subjects received a dose of 2.9 × 10⁹ CFU L. reuteri NCIMB 30242 capsules (n = 67) or placebo capsules (n = 64) twice daily for 9 weeks. Clinical chemistry and hematological parameters of safety were analyzed. The frequency, duration and intensity of adverse events (AE)s and clinical significance of safety parameters were recorded for both groups. No clinically significant differences between the probiotic capsule and placebo capsule treated groups were detected in either the blood clinical chemistry or hematology results. The frequency and intensity of AEs was similar in the two groups. These results demonstrate that administration of a twice daily dose of 2.9 × 10⁹ CFU was safe and well tolerated in the population evaluated over 9 weeks.     

Evaluation of safety and tolerance of microencapsulated Lactobacillus reuteri NCIMB 30242 in a yogurt formulation: A randomized,placebo-controlled,double-blind study

Probiotic organisms have shown promise in treating diseases. Previously, we have reported on the efficacy of microencapsulated Lactobacillus reuteri NCIMB 30242 in a yogurt formulation at lowering serum cholesterol levels in otherwise healthy hypercholesterolemic adults. This study investigates the safety and toxicology of oral ingestion of microencapsulated L. reuteri NCIMB 30242 in a yogurt formulation. A randomized group of 120 subjects received a dose of 5 × 10¹⁰ CFU microencapsulated L. reuteri NCIMB 30242 in yogurt (n = 59) or placebo yogurt (n = 61) twice/day for 6 weeks. Clinical chemistry and hematological parameters of safety were analyzed. Fecal samples were collected at these time points for the analysis of deconjugated bile acids. The frequency, duration and intensity of adverse events (AEs) and clinical significance of safety parameters were recorded for both groups. No clinically significant differences between the probiotic yogurt and placebo yogurt treated groups were detected in either the blood clinical chemistry or hematology results and there was no significant increase in fecal deconjugated bile acids (P > 0.05) between treated and control groups. The frequency and intensity of AEs was similar in the two groups. These results demonstrate the safe use of this formulation in food.     

Toona sinensis Roem leaf extracts improve antioxidant activity in the liver of rats under oxidative stress

Toona sinensis Roem (TS) is an herbal plant widely cultivated in Asia. Recently, several antioxidant compounds in TS leaf (TSL) extracts were chemically identified including quercetin, gallic acid, and others. However, in vivo experiments regarding the antioxidative function of TSL are limited. In this study, Sprague Dawley (SD) rats with oxidative stress were successfully established by intraperitoneal injection of hydrogen peroxide (H₂O₂) (1 mmol/kg BW) and fed with different TSL extracts for in vivo antioxidation evaluation. Among the TSLs tested in this study, TSL6 exhibited the best antioxidative effects which increased the enzyme activities of catalase, cupper/zinc superoxide dismutase (Cu/Zn SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Glutathione S transferase (GST) activities in liver compared to those in TSL-2 and TSL-2P groups. In conclusion, we provide the strong in vivo evidences for the first time that TSL extracts ameliorate the antioxidant enzymes (AOEs) activity in liver and is beneficial for the hepatic detoxification.     

Chemical composition,antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha Boiss. and Heldr. (var. erythrantha and var. cedretorum P.H. Davis) endemic in Turkey

In the present study, chemical compositions, antimicrobial and antioxidant activities of the essential oils of Sideritis erythrantha var. erythrantha (SE) and Sideritis erythrantha var. cedretorum (SC), which are endemic taxa in Turkey, were investigated. The essential oils obtained by hydrodistillation were analyzed by gas chromatography–mass spectrometry (GC–MS). α-Pinene was the major component of the essential oils of SC and SE. SC essential oil was as effective as antibiotic against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus faecalis (VRE), ampicillin resistant Haemophilusinfluenzae and vancomycin sensitive E. faecalis. Similarly, SE essential oil was also as effective as antibiotic against VRE and ampicillin resistant H. influenzae. Antioxidant activities of the essential oils of SC and SE were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene/linoleic acid and reducing power. Both essential oils exhibited weak antioxidant activity. This is the first report on antimicrobial and antioxidant activities of the essential oils of SC and SE.     

Phloroglucinol: Antioxidant properties and effects on cellular oxidative markers in human HepG2 cell line

Phloroglucinol is an ubiquitous secondary metabolite encountered in a free state or polymerised as phlorotannins in brown macroalgae, and present in higher plants. FRAP and TEAC assays measured the antioxidant properties of phloroglucinol in non-biological conditions. Additionally, the biological effects of phloroglucinol (4–400 μM) were scrutinised using cellular oxidative stress markers, such as the generation of ROS, antioxidant defences (concentration of GSH and activities of GPx, GR and GST), and levels of MDA as a marker for lipid peroxidation. The direct effect was assessed immediately after an incubation period, whereas for the protective effect, the incubation period was followed by 3-h treatment with the pro-oxidant t-BOOH. The results indicated that despite having a higher radical scavenging capacity than Trolox after 30 min, phloroglucinol was not a suitable antioxidant standard for phlorotannins. Regarding the biological effects, phloroglucinol had no impact on cell viability, reduced levels of ROS and increased antioxidant defences in the direct treatment for most concentrations. The results of the protective effect were mitigated as phloroglucinol failed to protect from ROS generation but evoked a significant recovery of the stress-altered cellular antioxidant defences to restful conditions. Additionally, MDA levels were greatly reduced, preventing a radical chain oxidation.     

Prevention and management of diabetic retinopathy in STZ diabetic rats by Tinospora cordifolia and its molecular mechanisms

We investigated the potential of Tinospora cordifolia (TC) in treatment of diabetic retinopathy in STZ-induced rats due to its antihyperglycemic, angiogenic, antiinflammatory and antioxidant effects. The diabetic rats, treated for 24 weeks with TC extract (250 mg/kg), were evaluated for lenticular and fundus changes. Biochemical parameters were estimated and histopathological studies performed. TC significantly reduced blood glucose and glycated hemoglobin in treated rats. It prevented cataract development in treated group. Angiogenic markers VEGF and PKC increased in diabetic retina, which reduced significantly with TC. Anti-inflammatory parameters TNF-α and IL-1β elevated in diabetic group unlike that in treated group. TC also provided defense against depletion of antioxidant enzymes- glutathione and catalase. Histopathological studies revealed thickening of basement membrane of the retinal and glomerular vasculature of diabetic rat, but no basement membrane widening was seen in treated animals. Destruction of pancreatic islet structure was observed in diabetic group, but not in treated. Thus, TC reduces blood glucose and inhibits overexpression of angiogenic and inflammatory mediators, which are distinct markers of diabetic retinopathy. It also prevents retinal oxidative stress and restores antioxidant enzyme levels. These data provide evidence for the safety and potential effect of TC in the management of experimental diabetic retinopathy.     

Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: effect of the incubation medium

Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3-4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca(2+)-containing Krebs-Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams' E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h. Also included was the bicarbonate-free HEPES buffer that does not require carbogen gassing, and is therefore handled more easily. The results clearly demonstrated that the type of incubation medium profoundly affected the functionality of the suspended hepatocytes, changing their sensitivity and response to exogenous damaging effects. While HEPES buffer and Williams' E medium offered the lowest background of spontaneous cell death, bicarbonate-based buffers and media seemed more suitable for obtaining both phases I and II biotransformation. Williams' E medium ensured a constant glutathione content of the cells and a lower level of oxidative stress.